Tccg-biblioteconomia-Inês Conceição Da Silva

UNIVERSIDADE FEDERAL DE GOIÁS
INSTITUTO DE CIÊNCIAS BIOLÓGICAS

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS
BIOLÓGICAS
ALTERAÇÕES CARDIOVASCULARES INDUZIDAS PELO

AUMENTO DA INGESTÃO DE SAL OU DE SACAROSE
MARINA CONCEIÇÃO DOS SANTOS MOREIRA
GOIÂNIA-GO
2017
TERMO DE CIÊNCIA E DE AUTORIZAÇÃO PARA DISPONIBILIZAR AS TESES E
DISSERTAÇÕES ELETRÔNICAS NA BIBLIOTECA DIGITAL DA UFG
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Nome completo do autor: Marina Conceição dos Santos Moreira

Título do trabalho: ALTERAÇÕES CARDIOVASCULARES INDUZIDAS PELO AUMENTO
DA INGESTÃO DE SAL OU DE SACAROSE
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________________________________________ Data: 06 /03 / 2017

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Tese apresentada ao Programa de

Pós-Graduação em Ciências
Biológicas do Instituto de Ciências
Biológicas da Universidade Federal de
Goiás, como requisito parcial para
obtenção do título de Doutor em
Ciências Biológicas.
Área de Concentração: Farmacologia
e Fisiologia
Orientador: Prof. Dr. Gustavo
Rodrigues Pedrino
GOIÂNIA-GO
2017

BANCA EXAMINADORA
_____________________________________________
Prof. Dr. Gustavo Rodrigues Pedrino
Universidade Federal de Goiás
_____________________________________________
Prof. Dra. Fernanda Cristina Alcântara dos Santos
_____________________________________________
Profa. Dra. Ana Cristina Silva Rebelo
_____________________________________________
Profa. Dra. Ângela Adamski da Silva Reis
_____________________________________________
Profa. Dra. Elizabeth Pereira Mendes
Aprovada em: 17 / 01 / 2017.

Dedicatória
Ao meu pai, Benedito Moreira Filho (in memoriam), por que esta é a
realização de seu sonho.
Agradecimentos
À UFG que tanto amo e que é a minha segunda casa há 8 anos. Às agências
de fomento que deram suporte para realização deste trabalho (Fapeg, Capes, CnPq).
Agradeço ao meu orientador, Professor Gustavo, pela oportunidade e confiança
construída ao longo de 6 anos de trabalho.
Agradeço aos meus pais, Benedito e Maria Auxiliadora, pelo esforço e

dedicação com que criaram a mim e ao meu irmão e que nos permitiram o privilégio
de fazer um curso de graduação. Pelos exemplos de honestidade, integridade e força.
Por terem me dado a oportunidade de escolher estudar e de chegar até aqui, mesmo
com muitas dificuldades financeiras. Ao meu pai, que sem qualquer estudo, entendia
a importância de meus estudos e que se enchia de orgulho para falar, com toda a sua
simplicidade, que eu sou cientista. À minha mãe que com muito sacrifício comprou
meu primeiro jaleco, que eu uso até hoje; pela força que tem para sustentar nossa
família. Ao meu irmão Rafhael, pelos sábios conselhos. Ao meu amigo e namorado,
Arthur, que a 200km de distância continua sendo a luz dos meus dias. Às minhas
amigas Fernanda, Sabrina, Raíssa e Cellini, que mesmo longe estão comigo, me
apoiam e me corrigem quando preciso.
Aos professores e alunos do CPNFC. Aos meus colegas de laboratório que

sempre me ajudam em todas as dificuldades pessoais e profissionais e sem os quais
este trabalho não seria possível. Por todas as vezes que aguentaram meu choro e
que sorriram comigo. Agradeço em especial aos meus colegas Aryanne e Paulo que
não mediram esforços para me ajudar sempre que precisei.
SUMÁRIO
LISTA DE ABREVIATURAS E SÍMBOLOS .................................................................. i
LISTA DE FIGURAS .................................................................................................. iii
RESUMO................................................................................................................... vii
ABSTRACT ................................................................................................................ ix
INTRODUÇÃO ............................................................................................................ 1
Regulação do equilíbrio hidroeletrolítico e hipertensão arterial ................................... 2
Dieta ocidental e síndrome metabólica ....................................................................... 8
OBJETIVOS .............................................................................................................. 13
MATERIAIS E MÉTODOS......................................................................................... 13
1. Modelos animais utilizados...........................................................................13
2. Protocolos de sobrecarga de sal e de sacarose...........................................14
3. Ingestão de ração, fluido e peso corporal.....................................................14
4. Canulação crônica.........................................................................................15
5. Registro de pressão arterial pulsátil (PAP) e frequência cardíaca (FC) nos
animais não anestesiados...................................................................................15
6. Avaliação dos reflexos barorreceptor e quimiorreceptor...............................15
7. Escala de sensibilidade ao sal......................................................................16
8. Pletismografia de cauda................................................................................16
9. Histologia cardíaca........................................................................................17
10. Avaliação da sensibilidade da região Rostro Ventrolateral do Bulbo
(RVLM).................................................................................................................18
11. Registro do Fluxo Sanguíneo Renal (FSR) e Aórtico (FSA).........................19
12. Confirmação histológica dos sítios de nanoinjeção na região RVLM...........20
13. Análise estatística .........................................................................................20
RESULTADOS .......................................................................................................... 21
Alterações cardiovasculares induzidas por sobrecarga de sal .................................. 21
1. Ingestão de fluido e ração e peso corporal...................................................21
2. Parâmetros cardiovasculares e reflexo barorreceptor..................................23
3. Ingestão de fluido e ração durante a escala de sensibilidade ao sal............30
4. Parâmetros cardiovasculares durante a escala de sensibilidade ao sal.......32
5. Análise da histologia cardíaca.......................................................................34
6. Alterações cardiovasculares induzidas pelas nanoinjeções de glutamato e
muscimol na região RVLM...................................................................................38
alterações cardiovasculares induzidas por sobrecarga de sacarose ........................ 46
1. Ingestão de fluido e ração e peso corporal...................................................46
2. Parâmetros cardiovasculares e reflexos barorreceptor e quimiorreceptor....47
DISCUSSÃO ............................................................................................................. 52
CONCLUSÕES E PERSPECTIVAS ......................................................................... 64
REFERÊNCIAS BIBLIOGRÁFICAS .......................................................................... 65
APÊNDICE I ............................................................................................................. 90
APÊNDICE II ............................................................................................................ 91
ANEXO I .................................................................................................................... 92
ANEXO II ................................................................................................................... 93
LISTA DE ABREVIATURAS E SÍMBOLOS
Ang II – Angiotensina II
ANP – Peptídeo Natriurético Atrial
ANS – Atividade Nervosa Simpática
AP – Área Postrema
AST – Área de secção transversa
AV3V – Região Antero Ventral do Terceiro Ventrículo
CVA – Condutância vascular aórtica
CVLM – Região Caudoventrolateral do Bulbo
CVR – Condutância vascular renal
DCV – Doenças cardiovasculares
ECA – Enzima conversora de angiotensina
ECA 2 – Enzima conversora de angiotensina 2
EPM – Erro padrão da média
FC – Frequência cardíaca
FSA – Fluxo sanguíneo aórtico
FSR – Fluxo sanguíneo renal
GABA – Ácido Gama-amino butírico
HDL – Lipoproteína de alta densidade
IB – Índice de barorreflexo
IML – Coluna intermédio-lateral
LDL – Lipoproteína de baixa densidade
MnPO – Núcleo Pré-óptico Mediano
Na+ - íon sódio
NaCl – cloreto de sódio
NTS – Núcleo do Trato Solitário
SNC – Sistema Nervoso Central
OMS – Organização Mundial da Saúde
OVLT – Órgão Vasculoso da lâmina Terminal
PA – Pressão arterial
PAM – Pressão arterial média
i
PAP – Pressão arterial pulsátil
PAS – Pressão arterial sistólica
PVN – Núcleo Paraventricular do Hipotálamo
RAA – Reflexo adiposo aferente
RVLM – Região Rostroventrolateral do Bulbo
RVMM – Região Rostroventromedial do Bulbo
SFO – Órgão Subfornical
SH – Salina hipertônica
SHR – Ratos espontaneamente hipertensos
SM – Síndrome metabólica
SRA – Sistema Renina-Angiotensina
TA – Tecido adiposo
VE – Ventrículo esquerdo
VLDL – Lipoproteína de muito baixa densidade
ii
LISTA DE FIGURAS
Figura 1: Média ± EPM da ingestão diária média de fluido (A), ração (B) e do
peso corporal (C) dos animais controle (n=5) e submetidos a sobrecarga de sal por 30
dias após o desmame (n=9) ao final dos períodos de tratamento e recuperação. *
diferente do grupo controle; p<0,05 ...........................................................................21
Figura 2: Média ± EPM da ingestão diária média de fluido (A) e ração (B) e do
peso corporal (C) dos animais controle (n= 6) e submetidos a sobrecarga de sal por
60 dias após o desmame (n=8) ao final dos períodos de tratamento e recuperação. *
diferente do grupo controle; p<0,05. .......................................................................... 22
Figura 3: Média ± EPM da pressão arterial média (PAM; A) e da frequência
cardíaca (FC; B) dos animais controle (n=5) e submetidos a sobrecarga de sal por 30
dias após o desmame (n=8) ao final dos protocolos experimentais.
.................................................................................................................................. 23
Figura 4: Média ± EPM da pressão arterial média (PAM; A) e da frequência
cardíaca (FC; B) dos animais controle (n=6) e submetidos a sobrecarga de sal por 60
dias após o desmame (n=6) ao final dos protocolos experimentais.
................................................................................................................................... 23
Figura 5: Traçados representativos ilustrando as alterações cardiovasculares
induzidas pela infusão de fenilefrina nos animais controle (A; n=5) e tratado por 30
dias após o desmame (B; n=8) e média ± EPM do índice de barorreflexo (IB) destes
animais ao final dos protocolos experimentais. *diferente do grupo controle; p<0,05.
................................................................................................................................... 25
induzidas pela infusão de nitroprussiato de sódio nos animais controle (A; n=5) e
tratado por 30 dias após o desmame (B; n=8) e média ± EPM do índice de barorreflexo
(IB) destes animais ao final dos protocolos experimentais. *diferente do grupo controle;
p<0,05........................................................................................................................ 26
induzidas pela infusão de fenilefrina nos animais controle (A; n=6) e tratado por 30
dias após o desmame (B; n=6) e média ± EPM do índice de barorreflexo (IB) destes
iii
................................................................................................................................... 27
tratado por 30 dias após o desmame (B; n=6) e média ± EPM do índice de barorreflexo
(IB) destes animais ao final dos protocolos experimentais. *diferente do grupo controle;
p<0,05. ...................................................................................................................... 28
Figura 9: Média ± EPM da ingestão diária média de fluido (A; ml/100g) e ração
(B; g/100g) dos animais controle (n=5) e submetidos a sobrecarga de sal por 30 dias
após o desmame (n=8) ao longo da escala de sensibilidade ao sal. * diferente do grupo
controle; p<0,05......................................................................................................... 29
Figura 10: Média ± EPM da ingestão diária média de fluido (A; ml/100g) e
ração (B; g/100g) dos animais controle (n=6) e submetidos a sobrecarga de sal por 60
dias após o desmame (n=9) ao longo da escala de sensibilidade ao sal. * diferente do
grupo controle; T diferente da 10ª semana;
p<0,05........................................................................................................................ 30
Figura 11: Média ± EPM da pressão arterial sistólica (A; PAS, mmHg) e da
frequência cardíaca (B; FC, mmHg) semanais dos animais controle (n=5) e
submetidos a sobrecarga de sal por 30 dias (n=8) em cada uma das etapas do
protocolo experimental. *diferente do grupo controle;
p<0,05........................................................................................................................ 32
Figura 12: Média ± EPM da pressão arterial sistólica (A; PAS, mmHg) e da
frequência cardíaca (B; FC, bpm) semanais dos animais controle (n=6) e submetidos
a sobrecarga de sal por 60 dias (n=8) em cada uma das etapas do protocolo
experimental. *diferente do grupo controle; # diferente da 8ª semana; T diferente da
10ª semana; p<0,05................................................................................................... 33
Figura 13: Fotomicrografias representativas dos corações corados pela técnica
de Reticulina de Gömori. (A) Controles 30 dias, n=4; (B) Tratados por 30 dias após o
desmame, n=4; (C) Controles 60 dias, n=4 e (D) tratados por 60 dias após o desmame,
n=4. Aumento de 40x. As setas brancas representam fibras colágenas do tipo I
iv
enquanto que as setas pretas representam as fibras do tipo
III................................................................................................................................ 34
Figura 14: Média ± EPM da área de secção transversa (AST) de cardiomiócitos
dos animais submetidos a sobrecarga de sal por 30 dias (A) e por 60 dias (B) e seus
respectivos grupos controle....................................................................................... 35
Figura 15: Média ± EPM da frequência relativa dos componentes do tecido
cardíaco dos animais submetidos à sobrecarga de sal por 30 dias e de seu grupo
controle. (A) cardiomiócitos; (B) tecido intersticial não fibrilar; (C) fibras colágenas tipo
I e (D) fibras colágenas tipo III................................................................................... 36
Figura 16: Média ± EPM da frequência relativa dos componentes do tecido
cardíaco dos animais submetidos à sobrecarga de sal por 60 dias e de seu grupo
controle. (A) cardiomiócitos; (B) tecido intersticial não fibrilar; (C) fibras colágenas tipo
I e (D) fibras colágenas tipo III. * diferente do grupo controle; p<0,05........................ 37
Figura 17: Fotomicrografia representativa de um corte coronal de bulbo de um
animal que recebeu nanoinjeções de azul de Evans em região correspondente ao
RVLM (setas). Os círculos indicam o Núcleo Ambiguos............................................ 38
Figura 18: Traçados representativos das alterações cardiovasculares
induzidas pelas nanoinjeções de soro e glutamato 10mM na região RVLM dos animais
controle (A) e submetidos a sobrecarga de sal por 60 dias após o desmame (B)
................................................................................................................................... 40
Figura 19: Média ± EPM das alterações de pressão arterial média (PAM,
mmHg, A), frequência cardíaca (FC, bpm, B), fluxos sanguíneos renal (FSR, % do
basal, C) e aórtico (FSA, % do basal, E) e condutâncias vasculares renal (CVR, % do
basal, D) e aórtica (CVA, % do basal, F) induzidas pelas pelas nanoinjeções de soro
fisiológico e glutamato 10mM na região RVLM de animais controle (n=4) e submetidos
a sobrecarga de sal por 60 dias após o desmame (n=4). *diferente das nanoinjeções
de soro fisiológico; T diferente do grupo controle, p<0,05.......................................... 41
Figura 20: Traçados representativos das alterações cardiovasculares
induzidas pelas nanoinjeções de soro e muscimol 2mM na região RVLM dos animais
controle (A) e submetidos a sobrecarga de sal por 60 dias após o desmame (B) .......43
v
Figura 21: Média ± EPM das alterações de pressão arterial média (PAM,
mmHg, A), frequência cardíaca (FC, bpm, B), fluxos sanguíneos renal (FSR, % do
basal, C) e aórtico (FSA, % do basal, E) e condutâncias vasculares renal (CVR, % do
basal, D) e aórtica (CVA, % do basal, F) induzidas pelas pelas nanoinjeções de soro
fisiológico e muscimol 2mM na região RVLM de animais controle (n=4) e submetidos
a sobrecarga de sal por 60 dias após o desmame (n=4). *diferente das nanoinjeções
de soro fisiológico, p<0,05.......................................................................................... 44
Figura 22: Média ± EPM da ingestão diária média de fluido (A; ml/100g) e de
ração (B; g/100g) padronizadas pelo peso corporal, da ingestão calórica (C, cal) e do
peso corporal (D, g) dos animais submetidos à sobrecarga de sacarose por 30 dias
na fase adulta (n=10) e do seu grupo controle (n=5). * diferente do grupo controle;
p<0,05........................................................................................................................ 46
Figura 23: Média ± EPM da pressão arterial média (PAM, mmHg) e da
frequência cardíaca (FC, bpm) dos animais controle (n=4) e submetidos a sobrecarga
de sacarose por 30 dias - síndrome metabólica (n=10) - ao final do tratamento.
*diferente do grupo controle; p<0,05.......................................................................... 47
induzidas pela infusão de fenilefrina nos animais controle (A; n=5) e submetidos a
sobrecarga de sacarose por 30 dias – síndrome metabólica (B; n=8) e média ± EPM
do índice de barorreflexo (IB; C) destes animais ao final dos protocolos experimentais.
*diferente do grupo controle; p<0,05. ................................................ 48
submetidos a sobrecarga de sacarose por 30 dias – síndrome metabólica (B; n=8) e
média ± EPM do índice de barorreflexo (IB; C) destes animais ao final dos protocolos
experimentais. *diferente do grupo controle; p<0,05................................................. 49
Figura 26: Traçados representativos ilustrando as alterações induzidas pela
infusão de cianeto de potássio nos animais controle (A; n=4) e submetidos a
sobrecarga de sacarose por 30 dias – síndrome metabólica (B; n=7) e média ± EPM
das respostas pressórica (C) e bradicárdica (D) destes animais ao final dos protocolos
experimentais. *diferente do grupo controle; p<0,05. ................................................ 50
vi
RESUMO
Atualmente as doenças cardiovasculares e suas complicações são as maiores causas

de morte em todo o mundo. Dentre estas doenças, a hipertensão arterial sistêmica
destaca-se por sua grande prevalência e por sua característica multifatorial. Diversos
estudos demonstram que, entre outros fatores, a alta ingestão de alimentos
industrializados, ricos em sal e em açúcar, contribui para a patofisiologia da
hipertensão arterial sal-dependente e relacionada à obesidade. Entretanto, as
alterações induzidas pela sobrecarga de sacarose sobre o sistema cardiovascular e a
regulação reflexa da pressão arterial ainda precisam ser esclarecidas. A partir do
exposto, objetivamos avaliar os efeitos da sobrecarga de sal de diferentes durações
durante o período pós-natal e da sobrecarga de sacarose na fase adulta sobre
parâmetros cardiovasculares de animais adultos. Avaliamos a pressão arterial média
(PAM), a frequência cardíaca (FC) e a sensibilidade barorreflexa dos animais
submetidos à sobrecarga de sal após o desmame e à sobrecarga de sacarose na fase
adulta. Nos animais submetidos à sobrecarga de NaCl, foram avaliadas também a
morfologia cardíaca e a sensibilidade da região Rostro Ventrolateral do Bulbo (RVLM).
Machos Wistar com 21 dias de vida receberam solução salina hipertônica (NaCl 0,3M)
durante 30 dias ou 60 dias (grupos experimentais). Os grupos controle foram mantidos
com água durante período equivalente. Após o tratamento, todos os grupos foram
mantidos com água e ração por um período de 15 dias (recuperação). Um grupo
distinto de animais adultos foi submetido à sobrecarga de sacarose por 30 dias. Ao
final dos protocolos experimentais, os animais foram submetidos ao procedimento de
canulação crônica. 24h após a cirurgia, os parâmetros cardiovasculares basais e suas
alterações induzidas por estimulação barorreflexa e/ou quimiorreflexa foram
registrados. O índice de barorreflexo (IB) foi calculado como a razão entre as
variações de FC e de PAM induzidas por cada infusão de fenilefrina e nitroprussiato
de sódio. Não foram observadas diferenças significativas entre os valores basais de
PAM e FC dos animais submetidos à sobrecarga de sal durante 30 dias em
comparação com os animais controle de mesma idade (PAM: cont.: 118,2 ± 3,7 mmHg
vs. exp.: 112,3 ± 4,0 mmHg. FC: cont.: 404,0 ± 10,6 bpm vs. exp.: 374,7 ± 9,1 bpm,
respectivamente). Entretanto, estes animais apresentaram diminuição da
vii
sensibilidade barorreflexa, em comparação com o grupo controle (IB: cont.: -2,441 ±
0,359 bpm/mmHg vs. exp.: -1,434 ± 0,086 bpm/mmHg, p<0,05). Os animais
submetidos a sobrecarga de sal por 60 dias apresentaram elevação de PAM (cont.
98,6 ± 2,6 mmHg vs. exp. 117,7 ± 4,2 mmHg; p<0,05) e de FC (cont. 365,4 ± 12,2 bpm
vs. exp. 392,5 ± 10,3 bpm; p<0,05) em comparação com seu grupo controle. Além
disso, a sensibilidade barorreflexa induzida por hipertensão se mostrou diminuída
nestes animais (cont. -1,83 ± 0,04 bpm/mmHg vs. exp. -1,24 ± 0,19 bpm/mmHg;
p<0,05). A sobrecarga de sal por 60 dias aumentou a frequência relativa de colágeno
tipo I nos corações dos animais adultos (cont.: 15,74 ± 0,61% vs. exp.: 19,79 ± 1,26%;
p<0,05), mas não promoveu hipertrofia de cardiomiócitos. Além disso, a resposta
pressora às nanoinjeções de glutamato na região RVLM se mostrou aumentada nos
animais tratados por 60 dias, em comparação com o grupo controle (cont.: Δ15,47 ±
2,56 mmHg vs. exp.: Δ34,31 ± 4,65 mmHg; p<0,05). Os animais tratados com
sacarose apresentaram elevação de PAM e FC em comparação com o grupo controle
(cont.: 102,5 ± 1,4 mmHg vs. exp.: 111,3 ± 0,9 mmHg; cont.: 334,7 ± 7,3 bpm vs. exp.:
371,6 ± 4,7 bpm; p<0.05). Além disso, apresentaram diminuição da sensibilidade
barorreflexa induzida por hipotensão (cont.: 5,0 ± 0,1 bpm/mmHg vs. exp.: 4,0 ± 0,1
bpm/mmHg; p<0,05) e aumento da resposta pressora após estimulação
quimiorreflexa (cont.: 14,9 ± 1,9 mmHg vs. exp.: 29,2 ± 5,5 mmHg; p<0,05). Os
resultados nos permitem concluir que as alterações na modulação reflexa da pressão
arterial induzidas pela sobrecarga de sal após o desmame precedem os efeitos
cardiovasculares induzidos por tal protocolo, indicando que modificações na
sensibilidade barorreflexa podem ser independentes de alterações permanentes de
pressão arterial. Além disso, a sobrecarga de sal após o desmame promove elevação
da sensibilidade da região RVLM nos animais adultos. Por sua vez, a sobrecarga de
sacarose promove elevação de PAM, FC e sensibilidade quimiorreflexa, enquanto que
a sensibilidade barorreflexa diminui. Em conjunto, estes resultados nos permitem
concluir que modificações da ingestão de sal ou sacarose produzem alterações
permanentes no controle da pressão arterial, sendo estas, em partes, desencadeadas
por alterações na regulação neural da circulação sanguínea.
viii
Palavras-chave: doenças cardiovasculares; dieta hipersódica; RVLM; síndrome
metabólica; quimiorreflexo.
ABSTRACT
Cardiovascular diseases (CVD) and their complications are the main causes of
death around the world nowadays. Among these diseases, the hypertension stands
out because of its great prevalence and multifactorial characteristic. Several studies
demonstrate that, among other factors, the increased intake of industrialized food (rich
in both salt and sugar) contribute to the pathophysiology of salt-dependent
hypertension and obesity-related hypertension. However, the high sucrose intake-
induced cardiovascular modifications still need to be clarified. Based on these
information, we sought to evaluate the effects of high salt intake of different durations
during the post-natal period and of high sucrose intake in adulthood on cardiovascular
parameters in adult animals. The present study sought to determine the cardiovascular
and autonomic effects of salt and sucrose overload in adult animals. We evaluated the
mean arterial pressure (MAP), the heart rate (HR) and the baroreflex sensitivity of the
animals submitted to salt overload after weaning and to sucrose overload in adulthood
and the cardiac morphology and Rostral Ventrolateral Medulla (RVLM) sensitivity after
salt overload. 21 days old male Wistar rats received hypertonic saline solution (NaCl
0,3M) for 30 or 60 days (experimental groups). The control groups were maintained
with tap water for equivalent period. After the treatment, the groups were maintained
with tap water and food for 15 days (recovery period). A distinct group of adult animals
was submitted to sucrose overload for 30 days. At the end of the experimental
protocols, the animals were chronically cannulated. 24h after the surgery, the basal
cardiovascular parameters and its modifications induced by baroreflex/chemoreflex
stimulation were recorded. The baroreflex index was calculated as the ratio between
HR and MAP changes after each infusion of phenylephyne and sodium nitroprusside.
There were no differences between baseline MAP and HR of the animals treated
during 30 days compared to the age-matched control animals (cont.: 118.2 ± 3.7
mmHg vs. exp.: 112.3 ± 4.0 mmHg; cont.: 404.0 ± 10.6 bpm vs. exp.: 374.7 ± 9.1 bpm).
ix
However, these animals presented diminished baroreflex sensitivity compared to the
control group (BI: cont.; -2.441 ± 0.359 bpm/mmHg vs. exp.: -1.434 ± 0.086
bpm/mmHg, p<0.05). The animals submitted to high salt intake for 60 days after
weaning presented increased MAP (cont. 98.6 ± 2.6 mmHg vs. exp. 117.7 ± 4.2 mmHg;
p<0.05) and HR (cont. 365.4 ± 12.2 bpm vs. exp. 392.5 ± 10.3 bpm; p<0.05) compared
to their control group. Moreover, the baroreflex sensitivity was diminished in those
animals (cont. -1.83 ± 0.04 bpm/mmHg vs. exp. -1.24 ± 0.19 bpm/mmHg; p<0.05). The
type I collagen relative frequency increased in the hearts of adult animals after 60 days
after salt overload (cont.: 15.74 ± 0.61% vs. exp.: 19.79 ± 1.26%; p<0.05) but no
differences were observed in the cardiomyocyte cross-sectional area. Furthermore,
the pressure response to glutamate nanoinjections into the RVLM was increased in
the animals treated for 60 days compared to the control group (cont.: Δ15.47 ± 2.56
mmHg vs. exp.: Δ34.31 ± 4.65 mmHg; p<0.05). The sucrose-treated animals
presented increased MAP and HR compared to the control group (cont.: 102.5 ± 1.4
mmHg vs. exp.: 111.3 ± 0.9 mmHg; cont.: 334.7 ± 7.3 bpm vs. exp.: 371.6 ± 4.7 bpm;
p<0.05). Furthermore, they presented diminished hypotension-induced baroreflex
sensitivity (cont.: 5.0 ± 0.1 bpm/mmHg vs. exp.: 4.0 ± 0.1 bpm/mmHg; p<0.05) and
increased pressure response after chemoreflex stimulation (cont.: 14.9 ± 1.9 mmHg
vs. exp.: 29.2 ± 5.5 mmHg; p<0.05). Our results allow us to conclude that changes in
the reflex regulation of blood pressure induced by salt overload after weaning precede
the cardiovascular effects induced by such protocol, indicating that baroreflex
sensitivity modifications can occur independently on permanent blood pressure
changes. Moreover, salt overload after weaning promotes increases in RVLM
sensitivity in adult animals. Furthermore, sucrose overload promotes MAP, HR and
chemoreflex sensitivity elevation, whereas it promotes diminish of baroreflex sensitivity
in adult rats. Taken together, our results allow us to conclude that changes in salt or
sucrose intake produce permanent modifications in the arterial pressure control and
such modifications are, in part, induced by changes in neuronal regulation of blood
circulation.
Key-words: cardiovascular diseases; salt overload; RVLM; metabolic syndrome,
chemoreflex sensitivity.
x
INTRODUÇÃO
A prevalência de doenças cardiovasculares (DCV) tem aumentado

significativamente desde a revolução industrial, especialmente no ocidente (1). De
fato, o advento de novas formas de produção industrial – em grande escala e
altamente automatizada – e a popularização de diversos tipos de meios de transporte
contribuem para o sedentarismo e o aumento do número de casos de DCV e diabetes.
Além disso, diversos estudos demonstram que o consumo exacerbado de
produtos industrializados e alimentos ricos em sal e açúcar desencadeiam efeitos
maléficos à saúde, como a resistência à insulina (2–4) e o aumento da pressão arterial
(PA) (2,5–7). A elevação sustentada da PA é um dos principais fatores de risco para
o desenvolvimento de DCV, sendo, juntamente com suas complicações, a maior
causa de mortes mundialmente (8). Outros fatores de risco para o desenvolvimento
destas doenças incluem hipercolesterolemia, diabetes, predisposição genética,
obesidade, sedentarismo e tabagismo (8,9). Enquanto que o sedentarismo e o
tabagismo podem ser evitados, os outros fatores de risco, além de não serem
evitáveis, tendem a ocorrer simultaneamente e caracterizam um quadro clínico
denominado Síndrome Metabólica (SM) (10).
Embora a patogênese da hipertensão arterial não seja completamente
conhecida, sabe-se que a alta ingestão de sal e ainda distúrbios da homeostase
hidromineral estão relacionados com esta patologia (6,7,11–15). A longo prazo, a
hipertensão arterial pode afetar órgãos como rins e coração (16–18).
Atualmente o consumo de alimentos altamente industrializados se inicia logo
na primeira infância, sendo que diversos produtos ricos em sal e em açúcar (como os
exemplificados no apêndice I) são direcionados, tanto em sua apresentação quanto
em sua divulgação, para as crianças. Além disso, no geral, produtos industrializados
apresentam menor custo e maior distribuição que seus equivalentes orgânicos,
fazendo com que sejam consumidos pelas diversas classes sociais(19). Diversos
estudos demonstram que a elevação no consumo destes produtos está diretamente
relacionada com a crescente incidência no número de casos de DCV observada nos
últimos anos (20–26), de modo que estudar os efeitos destas substâncias no
organismo se tornou de grande importância.
1
REGULAÇÃO DO EQUILÍBRIO HIDROELETROLÍTICO E HIPERTENSÃO
ARTERIAL
A manutenção do meio interno estável é o principal objetivo de todos os

processos fisiológicos (27) e está diretamente relacionada com a manutenção das
concentrações iônicas dos compartimentos intra e extracelulares. Assim, alterações
da osmolaridade do compartimento extracelular são percebidas por todos os tecidos
perfundidos e podem alterar o volume, o metabolismo e a função celular (28). Desta
forma, a regulação precisa do volume e da osmolaridade do compartimento
extracelular é fundamental para sobrevivência.
Dentre os diferentes tipos de sais inorgânicos presentes nos líquidos corporais,
o cloreto de sódio (NaCl) é o mais consumido e a concentração do íon sódio (Na+)
está diretamente relacionada à manutenção da homeostase dos fluidos corporais.
Assim, variações da concentração deste íon podem provocar fluxo osmótico entre os
compartimentos intra e extracelular modificando a concentração de todos os demais
componentes destes compartimentos. Grandes alterações na osmolaridade do
compartimento extracelular podem afetar a integridade da célula, por causar
alterações do volume e da concentração iônica intracelular, resultando em
convulsões, paralisia, coma e, em condições extremas, morte (29). Desta forma, não
é surpreendente observarmos a existência de diversos mecanismos homeostáticos
que visam à manutenção da concentração plasmática de sódio dentro de uma faixa
de variação restrita (30).
A detecção das variações de osmolaridade é realizada pelos osmorreceptores
centrais e periféricos, que enviam essas informações para diversas regiões do sistema
nervoso central (SNC), como o núcleo pré-óptico mediano (MnPO) e o núcleo
paraventricular do hipotálamo (PVN), modulando, assim, as respostas
comportamentais e vegetativas (29,31). Os osmorreceptores centrais estão
localizados em regiões próximas aos ventrículos cerebrais, e por isso, recebem a
denominação de órgãos circunventriculares. Dentre estes, destacam-se os neurônios
localizados no órgão sub-fornical (SFO) e no órgão vasculoso da lâmina terminal
(OVLT) (29,30,32). Os osmorreceptores periféricos estão localizados nos vasos
renais, intestinais e principalmente hepáticos (veia porta) e realizam a sua primeira
2
sinapse no SNC no Núcleo do Trato solitário (NTS) (33,34). Posteriormente, tais
informações são enviadas para regiões hipotalâmicas onde são processadas e
resultam em respostas autonômicas, principalmente através da ativação de neurônios
pré-motores simpáticos localizados na região Rostroventrolateral do bulbo (RVLM)
(35,36).
Estudos têm demonstrado que, quando expostos à hiperosmolaridade, os
osmorreceptores reduzem seu o volume intracelular devido à perda osmótica de água
(37,38). Ademais, evidências experimentais têm relacionado a diminuição do volume
celular com o aumento da excitabilidade e deflagração de potenciais de ação por
essas células (38,39). Uma vez detectadas as alterações da osmolaridade do fluido
extracelular, desencadeiam-se ajustes comportamentais e vegetativos que visam
reestabelecer o volume e/ou composição do compartimento extracelular (30,40).
Dentre os ajustes vegetativos, várias evidências experimentais têm
demonstrado que aumentos agudos da concentração plasmática de sódio
desencadeiam uma série de respostas autonômicas, cardiovasculares e hormonais.
Dentre essas respostas destacamos: a redução da atividade nervosa simpática (ANS)
renal (41–44), secreção do peptídeo natriurético atrial (ANP), ocitocina e vasopressina
(45–50), aumento da PA (50–54) e vasodilatação renal (51–56). Admite-se que estes
ajustes desencadeados pela hipernatremia, fazem parte de um conjunto de respostas
integradas que visam promover a natriurese, restabelecendo, assim, as condições
volêmicas normais.
Estudos de Weiss (1996) (42) demonstraram que a infusão intravenosa de
solução salina hipertônica (SH; NaCl 2,5M) induz aumento transitório de PA e que tal
aumento está relacionado com a secreção de vasopressina e com o aumento da ANS
lombar. Além disso, estudo realizado em nosso laboratório demonstrou que a infusão
de SH promove a diminuição da ANS renal e consequente vasodilatação deste
território (57). Em conjunto, estas respostas visam o aumento da pressão de perfusão
e do fluxo sanguíneo para o território renal, promovendo a natriurese.
De fato, a vasodilatação do território renal é um fator importante na excreção
de água e sódio, sendo um dos fatores cruciais a serem regulados após alterações
agudas da osmolaridade do compartimento extracelular (53). Diversos estudos
3
demonstram que a geração e a manutenção da resposta vasodilatadora do território
renal parecem depender de mecanismos neurais e hormonais (58–63). Os
mecanismos neurais parecem envolver a diminuição específica da ANS para o
território renal, uma vez que a condutância vascular renal aumenta, enquanto que não
ocorrem alterações significativas na condutância vascular para outros leitos, como o
mesentérico e para membros posteriores (41,52,62,63).
Diversas áreas do sistema nervoso central e aferentes periféricos (baro e
quimiorreceptores) participam do controle da homeostase hidromineral e da regulação
cardiovascular e estão envolvidos nas respostas simpatoinibitórias e vasodilatadoras
do território renal induzidas por hipernatremia (52,54,65). Estudos de Pedrino et al.
(52,53,64) demonstraram que lesões da região Antero-Ventral do Terceiro Ventrículo
(AV3V) e dos grupamentos noradrenérgicos A1 e A2 (nas regiões Caudoventrolateral
do bulbo – CVLM - e no NTS) abolem a vasodilatação e a simpatoinibição renal
induzidas por infusão de SH. Além disso, recentemente, estudo de Silva et al. (2015)
(66) demonstrou que a vasodilatação e a simpatoinibição renal após infusão de SH
são dependentes da integridade dos aferentes sinoaórticos, uma vez que a
desnervação destes aferentes abole tais respostas.
Diferentemente do que ocorre no território renal, estudos experimentais
demonstraram que a hiperosmolaridade plasmática promove aumento da ANS em
outros territórios (67). Holbein e Toney (2015) (68) demonstraram aumentos
específicos da ANS torácica, lombar e esplâncnica após infusão intravenosa de SH.
Estas alterações específicas de ANS para diferentes territórios alteram as funções
cardiovasculares em resposta às necessidades fisiológicas. Além disso, este aumento
de ANS em diversos leitos, exceto o renal, pode estar relacionado com a elevação de
PA observada após desafios osmóticos agudos.
É bem descrito na literatura que alterações da osmolaridade plasmática
desencadeiam respostas comportamentais e vegetativas que agem de modo a
restaurar as condições fisiológicas (29,30,69,70). Entretanto, quando tais alterações
de osmolaridade, especialmente a hipernatremia, são prolongadas, as respostas do
organismo, que são essenciais para a sobrevivência, podem desencadear efeitos
maléficos ao sistema cardiovascular.
4
Evidências experimentais e epidemiológicas demonstram que uma dieta rica
em sódio é um importante fator de risco para o desenvolvimento da hipertensão
arterial (39,71). De fato, aumentos da PA têm sido descritos em populações com altos
consumos de sódio em suas dietas (para revisão ver Horan et al. (1985) (72). Outros
estudos em modelos experimentais e ensaios clínicos fornecem provas convincentes
a respeito do efeito nocivo da ingestão de sódio na PA, tanto entre hipertensos quanto
entre normotensos (14,73). Além de seus efeitos sobre a PA, o excesso de consumo
de sódio na dieta tem sido associado diretamente com doença cardíaca coronariana,
acidente vascular cerebral e doenças não cardiovasculares (73).
Estudos pioneiros de Barker (1997) (74) sugerem que doenças desenvolvidas
na fase adulta estão relacionadas com determinadas condições a que o indivíduo foi
exposto nos estágios iniciais de vida, incluindo a fase pré-natal. Bao et al. (1995) (75)
demonstram que o risco de se desenvolver hipertensão arterial na fase adulta está
relacionado com os níveis de PA nas fases iniciais da vida. Consistente com estes
resultados, Li et al. (1995) (76) demostraram que valores altos de PA durante a
infância estiveram correlacionados positivamente com os altos valores da PA sistólica
e diastólica anos mais tarde. Neste sentido, Vidonho et al. (2004) (2) observaram
maior PA na prole adulta de mães que foram alimentadas com dieta hipersódica
(0,65M ou 1,3 M de NaCl) durante a gestação e a lactação.
Estudo recente realizado em nosso laboratório demonstrou que a sobrecarga
de sódio durante o período pós-natal e pós-desmame promove elevação da pressão
arterial média (PAM) e da frequência cardíaca (FC) na fase adulta (7, apêndice IIa).
Estes resultados estendem estudos anteriores, indicando que, não apenas as fases
pré-natais, mas também o período pós-natal e pós-desmame possui papel importante
no aumento da PA observado na fase adulta. Além disso, recentemente,
demonstramos que a sobrecarga de sal após o desmame, apesar de não alterar a
função cardíaca em condições basais, compromete o relaxamento cardíaco de
animais adultos após episódio de hipóxia, além de apresentar características
marcadamente arritmogênicas nestas situações (M.C.S.M., C.H.C. e G.R.P.,
observações não publicadas, apêndice IIb). Este estudo reforça o fato de que
5
alterações da homeostase hidromineral nas primeiras fases do período pós-natal
promovem alterações cardiovasculares persistentes até a fase adulta.
A ontogênese da hipertensão arterial sistêmica ainda não é completamente
conhecida, entretanto estudos em humanos e em modelos animais indicam que o
aumento da ingestão de sódio está associado a esta patologia (14,77–80). Além disso,
diversos estudos têm demonstrado a participação do SNC no desenvolvimento de
hipertensão arterial em modelos experimentais e em humanos (12,81).
O mecanismo pelo qual o SNC participa do desenvolvimento da hipertensão
depende das causas específicas do estado hipertensivo (82–84). Evidências
substanciais sugerem que a elevação da atividade simpática participe da patogênese
da hipertensão arterial, já que a simpatectomia e, ainda, agentes anti-hipertensivos
que atuam bloqueando a transmissão simpática são capazes de prevenir o
desenvolvimento ou a manutenção da hipertensão experimental (85–88). Além disso,
aumento do tônus simpático foi observado em ratos espontaneamente hipertensos
(SHR) (89).
A participação do SNC no controle da PA se dá principalmente através de sua
divisão simpática que inerva coração, rins, vasos e glândula adrenal, chamadas de
eferências barosensíveis (81). Tais eferências são caracterizadas por possuírem
atividade tônica modulada pelo barorreflexo. Os neurônios pré-ganglionares
simpáticos, localizados na coluna intermédio-lateral (IML) da medula espinhal
recebem um número reduzido de aferências de regiões supraespinhais, como o PVN,
o núcleo caudal da rafe, região rostroventromedial do bulbo (RVMM) e região
rostroventrolateral do bulbo (RVLM) (90). Dentre estas regiões, a região RVLM se
destaca como a principal região responsável pelo controle tônico da ANS.
Diversas evidências experimentais têm demonstrado que a integridade da
região AV3V é importante para a manutenção do equilíbrio hidroeletrolítico e para o
desenvolvimento e manutenção da hipertensão arterial em diversos modelos
experimentais (91–93). Estudos demonstram, por exemplo, que a ablação da região
AV3V compromete a emissão de comportamentos de sede e apetite ao sódio (94–96).
Além disso, Haywood et al. (1983) (92) demonstraram que a ablação desta região
6
previne o desenvolvimento da hipertensão arterial induzida por estenose parcial da
artéria renal (Goldblatt).
Um dos componentes da região AV3V, o MnPO é um importante centro de
integração do SNC para o controle hidroeletrolítico e da PA (97,98). De fato, estudos
recentes realizados em nosso laboratório demonstram a participação do MnPO na
manutenção da hipertensão arterial observada em SHR (99,100) e em animais
submetidos ao modelo de hipertensão Goldblatt (A.A.M., M.C.S.M. e G.R.P.,
observações não publicadas), além da sua participação na recuperação
cardiovascular induzida por SH em animais hemorrágicos (101). O MnPO recebe
informações de osmoreceptores centrais e se projeta para o PVN e este, por sua vez,
para o RVLM (90,102,103). Os neurônios da região RVLM que se projetam para os
neurônios pré-ganglionares simpáticos na IML são tonicamente ativos e respondem a
uma variedade de estímulos que alteram a PA (104). A região RVLM é considerada a
principal geradora do tônus simpático por ser, dentre as regiões que se projetam
diretamente para a IML, a única a apresentar atividade tônica essencial para a
manutenção do tônus vasomotor (105). Esta região participa do controle do equilíbrio
hidroeletrolítico, visto que recebe informações de osmorreceptores centrais, através
de regiões como o PVN, e promove, assim, alterações da ANS, a fim de restaurar a
osmolaridade plasmática (81).
Estudos têm demonstrado que uma dieta rica em sódio promove aumento
resposta pressora à injeção de glutamato na região RVLM, independente da forma
como o consumo de sódio é realizado (se pela água, com SH como única fonte hídrica,
ou pela ração) (12,106). Mais recentemente, Adams et al. (2007) (107) demonstraram
que este aumento da resposta pressora a injeções de glutamato ou GABA na RVLM
em animais submetidos a sobrecarga de sódio é causado por um aumento da
sensibilidade neuronal desta região, e não por mecanismos periféricos de controle da
PA. Neste trabalho, os autores sugerem, ainda, que tal alteração se deve a
mecanismos de plasticidade neuronal reversíveis e de desenvolvimento lento.
Entretanto, é importante ressaltar que nestes estudos, a dieta hipersódica foi ofertada
a animais adultos. Deste modo, não existem evidências experimentais a respeito de
7
alterações da sensibilidade e atividade neuronal na fase adulta induzidas por
alterações do equilíbrio hidroeletrolítico durante o período pós-natal.
DIETA OCIDENTAL E SÍNDROME METABÓLICA
A ocorrência concomitante de diversas alterações metabólicas que podem levar

ao desenvolvimento de DCV e diabetes é chamada de Síndrome Metabólica (SM) e
tem sido ostensivamente estudada na última década (3,108,109). A SM é
caracterizada pela ocorrências simultânea de pelos menos 3 dos principais fatores de
risco para o desenvolvimento de DCV, que são: obesidade, dislipidemia, resistência à
insulina e hipertensão arterial.
Uma alimentação desequilibrada, rica em gordura e açúcar, associada a um
estilo de vida sedentário promove um armazenamento excessivo de energia na forma
de lipídios, uma vez que a quantidade de caloria ingeridas é maior que a quantidade
gasta em atividade física. As moléculas de lipídio são armazenadas no tecido adiposo,
que pode ser visceral ou subcutâneo. De fato, sabe-se que o excesso de gordura
visceral (também chamada de central ou abdominal) está associado com
hiperglicemia e hiperinsulinemia e ainda a diminuição do HDL colesterol, fatores estes
associados com a resistência à insulina e aterosclerose (110). Diversos estudos têm
demonstrado que a obesidade apresenta forte correlação com o desenvolvimento de
DCV (3,111,112).
A glicose proveniente dos alimentos é absorvida para o interior das células pela
ação da insulina, um hormônio produzido pelas células beta do pâncreas (109,113).
A resistência à insulina ocorre quando as células do organismo se tornam menos
sensíveis aos efeitos deste hormônio (109,114). Uma vez que a glicose não é
absorvida pelas células, permanece na corrente sanguínea, desencadeando
novamente a produção de insulina. Esta condição geralmente superestimula as
células beta e diminui a sua capacidade de produzir insulina, levando à hiperglicemia
e ao diabetes tipo 2 (113,115). Além disso, a resistência à insulina promove danos
teciduais em diversos órgãos, como o fígado e os rins. De fato, o reflexo de
hiperinsulinemia aumenta a liberação de triglicerídeos pelo fígado para a corrente
sanguínea e, consequentemente, diminui os níveis de lipoproteína de alta densidade
8
(HDL) e aumenta o nível de lipoproteína de baixa densidade (LDL) (10,116,117). Tal
desbalanço nos níveis séricos de HDL e LDL colesterol é chamada de dislipidemia e
promove perturbações na estrutura, metabolismo e atividades biológicas das
moléculas aterogênicas (LDL) e antiaterogênicas (HDL) (118–122). O reflexo de
hiperinsulinemia pode também contribuir para a patofisiologia da hipertensão arterial,
através do aumento da reabsorção renal de água e da ANS (116,117). Por seu
conjunto de efeitos, a resistência à insulina é o principal fator de risco para o
desenvolvimento de diabetes tipo 2 e ainda de doença das artérias coronárias,
acidente vascular cerebral e doença arterial periférica (117,123,124).
A Organização Mundial da Saúde (OMS) estima que a quantidade de indivíduos
obesos duplicou desde 1980 até os dias atuais (22). Em 2014, cerca de 39% dos
indivíduos adultos (acima de 18 anos) apresentavam sobrepeso, enquanto que,
destes, 13% eram considerados obesos (22). Entre as crianças, a OMS estima que,
em 2013, cerca de 42 milhões de crianças menores de 5 anos de idade apresentavam
sobrepeso, sendo que destas, 31 milhões viviam em países em desenvolvimento.
Diversos são os estudos que relacionam o alto consumo de açúcares livres com
o ganho de peso em diversas faixas etárias (125,126). De fato, segundo a OMS, a
diminuição do consumo destes açúcares tem sido eficaz na diminuição do peso
corporal e, consequentemente, do risco de desenvolvimento de DCV (22,24,126–129)
Além do claro envolvimento de uma dieta rica em açúcares com o ganho de
peso corporal, o alto consumo destas substâncias também tem sido ostensivamente
relacionado com o desenvolvimento de diabetes tipo II (115,130–132). No geral,
alimentos industrializados apresentam grandes quantidades de frutose, que, quando
consumida em excesso, é responsável por diversas alterações metabólicas (133–
137).
Apesar de apresentar menor índice glicêmico em comparação com a glicose, a
frutose tem sido relacionada com o aumento das concentrações plasmáticas de
triglicerídeos e da taxa de síntese de lipídeos pelo fígado (131,138,139). Tais
triglicerídeos associados a ácidos graxos ou outros lipídeos estão associados com a
síntese de lipoproteína de densidade muito baixa (VLDL), substância importante no
desenvolvimento de doença hepática gordurosa não alcoólica (131,139). Nesta
9
situação, enquanto novos lipídeos são sintetizados no fígado, ácidos graxos são re-
esterificados e a oxidação de lipídeos no fígado é diminuída; deste modo o balanço
entre lipídeos produzidos e degradados no fígado fica comprometido, levando ao
acúmulo dessa substância no órgão (138,139). Distúrbios no metabolismo de lipídeos,
como os desencadeados por uma dieta rica em frutose, estão relacionados ao
desenvolvimento de resistência à insulina uma vez que podem levar a alterações da
fosforilação do receptor de insulina, reduzindo a sinalização celular por esta
substância (114,139).
Estudos de Saad et al. (2016) (136) demonstram que a alta ingestão de frutose
durante a gestação promove a programação fetal a diversos fatores de risco para
doenças na fase adulta, como obesidade, hipertensão e disfunções metabólicas,
sendo que estes efeitos são mais pronunciados em fêmeas. Deste modo, os autores
sugerem que limitar o acesso de gestantes a alimentos ricos em frutose pode ter
impacto significativo na saúde da prole a longo prazo.
Tem sido descrito na literatura que os efeitos metabólicos da ingestão de
frutose diferem daqueles evocados pela ingestão de glicose (137,140). Teff et al.
(2004) (137) demonstraram que o consumo de alimentos ricos em frutose resulta em
menores níveis de leptina e insulina circulantes e, ainda, maiores níveis de grelina e
triglicerídeos em comparação com o consumo de alimentos ricos em glicose. Uma vez
que insulina, grelina e leptina sinalizam o sistema nervoso central (SNC) acerca da
regulação do balanço energético a longo prazo (141), os autores sugerem que o
consumo de uma dieta rica em frutose possa elevar a ingestão calórica e contribuir
para o ganho de peso e a obesidade, além do desenvolvimento de aterosclerose e
doenças cardiovasculares associadas ao aumento de triglicerídeos plasmáticos. De
acordo com estes achados, Page et al. (2013) (140) observaram que a ingestão de
glicose reduz a ativação de regiões cerebrais relacionadas ao apetite, motivação e
processamento de recompensa (hipotálamo e ínsula, entre outras), além de aumentar
a saciedade em comparação com a ingestão de frutose. Neste estudo, os autores
relacionam a menor saciedade induzida pela ingestão de frutose aos reduzidos níveis
sistêmicos de insulina, que atua como hormônio sinalizador de saciedade.
10
Em outra abordagem, Rendeiro et al. (2015) (135) avaliaram os efeitos de uma
dieta rica em frutose desde a infância sobre parâmetros metabólicos e cognitivos na
fase adulta. Os autores observaram que sob sobrecarga de frutose, ocorre diminuição
da ingesta de ração, de modo que a ingestão calórica global permanece inalterada.
Assim, o estudo sugere que a frutose por si, mesmo na ausência de ingestão calórica
excessiva, promove elevação de peso corporal e de deposição de gordura,
potencialmente através da redução da atividade física, sem alterações das funções
cognitivas.
A sacarose, outra substância amplamente utilizada como adoçante tanto em
produtos industrializados como em alimentos preparados em casa, é um dissacarídeo
formado por uma molécula de glicose associada a uma molécula de frutose. De
maneira semelhante ao que ocorre com a frutose, a ingestão de sacarose tem
aumentado exponencialmente nos últimos anos de modo que determinar os efeitos
dessa substância no organismo se tornou foco de diversos grupos de pesquisa
(127,142–144).
Diversos são os estudos que demonstram os efeitos metabólicos de uma dieta
rica em sacarose (128,143,145-147). Coelho et al. (2010) (143) demonstraram que a
sobrecarga de sacarose em animais adultos promove elevação da PA, do tecido
adiposo (TA) epididimal e dos níveis circulantes de triacilgliceróis, insulina e leptina,
além da renina e da angiotensina II (Ang II) plasmáticas. No tecido adiposo, animais
submetidos a sobrecarga de sacarose 20% apresentam elevação de angiotensina 1-
7, receptores AT1 e AT2, enzima conversora de angiotensina 2 (ECA2) e
angiotensinogênio, enquanto que o conteúdo de angiotensina I e II e a atividade da
enzima conversora de angiotensina (ECA) estão diminuídos (143). Os autores
sugerem que as alterações dos componentes do sistema renina-angiotensina (SRA)
são específicas de alguns tecidos e podem estar relacionadas com a síntese
aumentada de ácidos graxos, a massa de TA e a hipertensão observada nestes
animais.
Outros estudos se dedicaram a avaliar os efeitos de uma dieta rica em sacarose
durante a gestação sobre diversos parâmetros metabólicos da prole. Kendig et al.
(2015) (147) observaram que a oferta de sacarose 10% durante a gestação promove
11
intolerância à glicose nas mães enquanto que não altera o tamanho da prole, o peso
ao nascer ou outros parâmetros metabólicos da prole em condições basais.
Parâmetros como glicose de jejum e sensibilidade a insulina apenas são alterados na
prole quando da alteração da dieta ou da prática de exercício físico (147). Além disso,
Choi et al. (2015) (142) demonstraram que a sobrecarga de sacarose durante a
gestação não altera o peso corporal da prole adulta, entretanto promove aumento da
atividade locomotora destes animais e ainda aumento da expressão do transportador
de dopamina e diminuição de diversos subtipos de receptores dopaminérgicos. Assim,
o estudo de Choi et al. (2015) (142) sugere que a exposição pré-natal a sacarose pode
ser um fator de risco para o desenvolvimento de transtorno de hiperatividade do déficit
de atenção.
Os efeitos da alta ingestão de sacarose durante a infância sobre parâmetros
comportamentais e metabólicos também tem sido estudados (127,128,135). Frazier
et al. (2008) (128) demonstraram que a sobrecarga de sacarose após o desmame
reduz a motivação para a busca de alimentos palatáveis, entretanto promove maior
ganho de peso quando da exposição a dieta hipercalórica. De fato, segundo os
autores, é possível que a exposição precoce a uma dieta rica em açúcares possa
modificar a expressão do chamado ‘fenótipo poupador’ e alterar a resposta dos
animais adultos ao seu ambiente.
Castellanos et al. (2015) (127) demonstraram que animais adultos que foram
submetidos a alta ingestão de sacarose desde a infância são normoglicêmicos, mas
apresentam TA retroperitoneal mais abundante, além de triglicerídeos, adiponectina e
leptina elevados, enquanto que insulina e PA tendem a aumentar. Os autores sugerem
que uma dieta rica em sacarose administrada cronicamente resulta em redistribuição
do tecido adiposo para a cavidade abdominal, contribuindo para elevar o risco destes
animais de desenvolver DCV e metabólicas; entretanto, o mecanismo pelo qual tal
redistribuição ocorre permanece desconhecido.
Os diversos estudos demonstram que a sobrecarga de sacarose, em qualquer
fase da vida, promove alterações comportamentais e metabólicas importantes e que
podem contribuir para o desenvolvimento e/ou agravamento de DCV. Entretanto, as
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alterações sobre o sistema cardiovascular e a regulação reflexa da PA induzidas pela
sobrecarga de sacarose ainda precisam ser esclarecidas.
OBJETIVOS
A partir do exposto, objetivamos avaliar os efeitos da sobrecarga de sal de

diferentes durações durante o período pós-natal e da sobrecarga de sacarose na fase
adulta sobre parâmetros cardiovasculares de animais adultos.
Em ambos os protocolos, avaliamos especificamente:
- a pressão arterial média (PAM) e a frequência cardíaca (FC);
- os reflexos barorreceptor e/ou quimiorreceptor.
Além disso, no que se refere à sobrecarga de sal após o desmame, buscamos
avaliar, mais especificamente:
- os efeitos cardiovasculares e o padrão de ingestão de fluidos e ração mediante
a ingestão de diferentes concentrações de NaCl na fase adulta;
- a morfologia cardíaca na fase adulta;
- as respostas cardiovasculares induzidas pela estimulação e pela inibição
farmacológicas da Região Rostroventrolateral do Bulbo (RVLM) na fase adulta.
MATERIAIS E MÉTODOS
1. Modelos animais utilizados
Todos os experimentos foram realizados com ratos da linhagem Wistar. A

sobrecarga de sal foi administrada a animais com 21 dias de vida, enquanto que a
sobrecarga de sacarose foi administrada a animais adultos (65 dias de vida; 250-
350g) (fornecidos pelo Biotério Central da Universidade Federal de Goiás (UFG)).
Os animais foram mantidos em salas climatizadas (temperatura 22-24ºC) com
acesso ad libitum à ração (0,4% ou 0,067M NaCl; AIN-93M; 148). Todos os
protocolos utilizados foram submetidos a aprovação da Comissão de Ética no Uso
de Animais (CEUA) da UFG, sob número de protocolo 023/2015 (Anexo I).
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2. Protocolos de sobrecarga de sal e de sacarose
O incremento na quantidade sódio (dieta hipersódica) ingerida pelos animais

dos grupos experimentais foi realizado através da adição de cloreto de sódio (0,3 M;
Synth, Diadema, SP, Brasil) na água ofertada a estes animais (grupos experimentais).
Aos grupos controle foi ofertada água. A administração da dieta hipersódica se iniciou
no vigésimo primeiro dia de vida e foi finalizada após 30 dias ou 60 dias de tratamento.
Após este período, estabeleceu-se um período de recuperação de 15 dias, em que foi
ofertada dieta normossódica (água e ração) para os animais de todos os grupos.
21º 51º ou 81º 66º ou 96º
Tratamento Recuperação
Água ou NaCl 0,3M Água
PAM e FC acordado
Escala de sensibilidade ao sódio
Histologia cardíaca
Sensibilidade do RVLM
O incremento na quantidade sacarose ingerida pelos animais foi realizado

através da adição de sacarose (20% ou 0,58M; Synth, Diadema, SP, Brasil) na água
ofertada a estes animais (grupo experimental). Ao grupo controle foi ofertada água. A
administração da dieta rica em sacarose teve duração de 30 dias.
3. Ingestão de ração, fluido e peso corporal
Os animais foram mantidos em gaiolas individuais e a ingestão diária de fluido

(de acordo com cada tratamento), de ração e o peso corporal dos animais foram
mensurados ao longo de todos os protocolos experimentais. A quantidade diária de
fluido e de ração ingerida foi determinada pela subtração da quantidade fornecida pela
quantidade remanescente na gaiola.
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4. Canulação crônica
Ao final dos protocolos experimentais, os animais foram anestesiados com

halotano 2% em O2 a 100%. Cateteres de polietileno (PE 10 soldado a PE 50),
preenchidos com solução de heparina (Hepamax, Blau Farmacêutica, Cotia, SP,
Brasil) acrescida de penicilina (Benzilpenicilina potássica, Eurofarma, Itapevi, SP,
Brasil), foram inseridos na aorta abdominal através artéria femoral e na veia cava
inferior através da veia femoral para o registro da pressão arterial pulsátil (PAP) e a
infusão de drogas, respectivamente. Os cateteres foram exteriorizados no dorso dos
animais e a sua extremidade oposta foi vedada com pinos de metal.
5. Registro de pressão arterial pulsátil (PAP) e frequência cardíaca

(FC) nos animais não anestesiados
Vinte e quatro horas após o procedimento cirúrgico, foi realizado o registro dos
parâmetros cardiovasculares dos animais não anestesiados e com livre
movimentação. O sinal da PAP foi obtido pela conexão do cateter da artéria a um
transdutor de pressão (MLT0699, ADInstruments, Bella Vista, Austrália) acoplado a
um amplificador (Bridge Amp, FE221, ADInstruments, Bella Vista, Austrália). Os dados
foram digitalizados em uma frequência de 2000 amostras.s -1 utilizando um conversor
analógico digital (PowerLab 4/25, ML845, ADInstruments, Bella Vista, Austrália). A
pressão arterial média (PAM) foi calculada a partir do sinal da PAP (LabChart 7 v7.3.7;
ADInstruments, Bella Vista, Austrália). A frequência cardíaca (FC) foi obtida através
dos sinais de pulso da PAP.
6. Avaliação dos reflexos barorreceptor e quimiorreceptor
Os animais tratados com sal e com sacarose foram submetidos à avaliação

do reflexo barorreceptor. Após o registro basal da PAP e da FC, os animais foram
submetidos a infusões sucessivas de diferentes concentrações de fenilefrina (1 µg/ml,
1,5 µg/ml e 2 µg/ml; 0,1 ml. Sigma-Aldrich, St. Louis, MO, EUA) e nitroprussiato de
sódio (10 µg/ml, 15 µg/ml e 20 µg/ml; 0,1 ml. Vetec, Duque de Caxias, RJ, Brasil) para
a avaliação da sensibilidade barorreflexa. A sensibilidade barorreflexa foi avaliada
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através da determinação do índice de barorreflexo (IB), dado pela razão entre as
alterações de máximas de FC e as alterações máximas de PAM (ΔFC/ΔPAM)
induzidas por cada uma das infusões de fenilefrina e nitroprussiato de sódio. O IB de
cada animal foi dado como a média dos IB’s para cada uma das soluções.
Além a avaliação barorreflexa, os animais submetidos à sobrecarga de
sacarose também foram submetidos à avaliação da sensibilidade quimiorreflexa. Para
isso, realizou-se a infusão de cianeto de potássio (KCN; Sigma-Aldrich, St. Louis, MO,
EUA. 400 µg/ml) e as respostas pressórica e bradicárdica foram avaliadas.
7. Escala de sensibilidade ao sal
Grupos distintos de animais submetidos a sobrecarga de sal por 30 dias ou

60 dias após o desmame foram submetidos à escala de sensibilidade ao sal. Após o
período de recuperação, os animais de todos os grupos (experimentais 30 dias,
experimentais 60 dias e seus respectivos grupos controle) foram submetidos a
ingestão espontânea de soluções salinas (NaCl) de concentrações crescentes (0,9%;
1,2%; 1,5% e 1,8%, com duração de 7 dias cada concentração) seguida por oferta de
água por mais 7 dias. A ingestão diária de ração e fluido e o peso corporal dos animais
foram mensurados desde os 15 últimos dias de tratamento até o final da escala de
sensibilidade.
8. Pletismografia de cauda
Os animais submetidos a sobrecarga de sal após o desmame tiveram as

variáveis cardiovasculares pressão arterial sistólica (PAS) e a frequência cardíaca
(FC) registradas através de pletismografia de cauda (3 registros por semana) nos
últimos 15 dias de tratamento e durante os períodos de recuperação e de escala de
sensibilidade ao sal. Para isso, inicialmente, os animais foram acomodados em uma
caixa para aquecimento (para promover a dilatação da artéria caudal).
Posteriormente, foram posicionados em um contensor de madeira com a cauda
exposta e um manguito de borracha, acoplado a um esfigmomanômetro, foi
posicionado na porção proximal da cauda. Um transdutor de pulso foi posicionado
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próximo ao manguito e os sinais de pulso da artéria caudal foram amplificados e
digitalizados (PowerLab; ADInstruments, Bella Vista, Austrália). Para a determinação
da PAS, o manguito foi insuflado e desinsuflado em intervalos regulares. Com a
insuflação do manguito, os sinais de pulso são silenciados, assim, a PAS é
considerada como o primeiro sinal de pulso a ser registrado após a desinsuflação do
manguito. A FC foi obtida através dos sinais da pressão de pulso e foi considerada
como a média de períodos de 10 s entre as insuflações do manguito. A PAS e a FC
diárias de cada animal foram consideradas como a média aritmética de pelo menos 3
valores dessas variáveis durante um mesmo registro.
A fim de diminuir o estresse causado pela manipulação e pela contensão, os
animais foram submetidos a 2 semanas de registros de adaptação, que não foram
utilizados para as análises.
9. Histologia cardíaca
Ao final do protocolo experimental de sobrecarga de sal, os animais de todos

os grupos foram eutanasiados e os corações coletados para análises histológicas. Os
segmentos de ventrículo esquerdo (VE) foram fixados em metacarn por 4 horas,
posteriormente desidratados em séries de soluções crescentes de etanol (Neon®, São
Paulo, SP, Brasil), xilol (Synth®, Diadema, SP, Brasil) e incluídos em paraplast
(Sigma-Aldrich, St. Louis, MO, EUA) (Ver Anexo II). Com o auxílio de um micrótomo
(Leica RM2155, Nussloch, Germany), as amostras foram seccionadas em cortes de 5
µm de espessura com intervalo de 150µm entre os cortes. Após secção, o material foi
fixado em lâminas (Exacta, 26x76x1x1.2mm), reidratado e submetido a citoquímica
em Reticulina de Gömori (Anexo II). Após a coloração de interesse, os cortes foram
desidratados e montados. Todas as imagens foram obtidas no microscópio Zeiss
Scope A1 (Oberkochen, Alemanha) sob luz convencional (40x de magnitude) e, em
seguida, foram digitalizadas no software Zen Lite 2012 e analisadas no software
Image Pro-Plus v6.1 para Windows (Media Cybernetics Inc., Silver Sprong, MD, USA).
a) Morfometria do cardiomiócito - Os cardiomiócitos foram mensurados a fim

de determinar a hipertrofia celular. Os cortes transversais do ventrículo esquerdo
foram microfotografados, e a área de secção transversa (AST) de um total de 200
17
cardiomiócitos por grupo foi mensurada. Somente as células cardíacas dispostas
longitudinalmente, com núcleo e limites celulares visíveis foram consideradas. A AST
de cada cardiomiócito foi mensurada na região correspondente ao núcleo.
b) Estereologia – As análises estereológicas foram utilizadas para quantificar

a frequência relativa (%) de cada um dos componentes do tecido cardíaco
(cardiomiócitos, fibras de colágeno dos tipos I e III e tecido conjuntivo não fibrilar).
Para isso foram capturados 30 campos fotomicrográficos aleatórios de cada grupo
experimental, a partir de cortes histológicos corados por Reticulina de Gömori. Estes
campos foram inseridos no sistema-teste multipontos com 130 pontos e 10 linhas,
proposto por Weibel (1963)(149), e os valores relativos foram determinados pela
contagem dos pontos coincidentes sobre cada tipo de componente em estudo,
seguido da divisão destes pelo número total de pontos do sistema-teste.
10. Avaliação da sensibilidade da região Rostro Ventrolateral do Bulbo

(RVLM)
Um grupo distinto de animais submetidos à sobrecarga de sal por 60 dias após

o desmame foi submetido à avaliação da sensibilidade da região RVLM. Após o
período de recuperação, os animais foram anestesiados com uretana (1,2 g ∙ kg -1 de
massa corpórea, i.v.; Sigma-Aldrich, St. Louis, MO, EUA), após indução anestésica
com halotana 2% em O2 a 100%. Cateteres de polietileno (PE 10 soldado a PE 50)
foram inseridos na aorta abdominal através artéria femoral e na veia cava inferior
através da veia femoral para o registro da PAP e a infusão do anestésico,
respectivamente. Após a canulação vascular, foi realizada traqueostomia, a fim de
reduzir a resistência das vias aéreas.
Após os procedimentos cirúrgicos gerais, os animais foram posicionados em
decúbito ventral em aparelho estereotáxico com bocal posicionado 11mm abaixo da
linha interaural. Nanoinjeções de 50 nL de soro fisiológico (NaCl 0,15M, Synth,
Diadema, SP, Brasil), L-glutamato (10 mM; Sigma-Aldrich, St. Louis, MO, EUA) ou
muscimol (agonista GABAa, 2mM; Sigma-Aldrich, St. Louis, MO, EUA) foram
realizadas na região RVLM. As coordenadas estereotáxicas utilizadas foram obtidas
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e modificadas a partir do Atlas Paxinos & Watson (150). As alterações
cardiovasculares evocadas por cada uma das nanoinjeções foram registradas. Após
os registros, foram realizadas nanoinjeções do corante Azul de Evans (Dinâmica,
Diadema, SP, Brasil), nas mesmas regiões em que foram nanoinjetados L-glutamato
e muscimol, para confirmação histológica. Foram utilizadas as seguintes coordenadas
estereotáxicas:
Antero-posterior +2,5 mm ao calamus scriptorios
Médio-lateral -2,0 e +2,0 mm ao calamus scriptorios
Dorso-ventral -2,5 mm ao calamus scriptorios
11. Registro do Fluxo Sanguíneo Renal (FSR) e Aórtico (FSA)
O fluxos sanguíneos (FS) renal (FSR) e aórtico (FSA) foram registrados por
fluxometria por tempo de trânsito. Sondas em miniatura (probe 1RB, Transonic
Systems, Inc., Ithaca, NY, EUA) foram implantadas ao redor da artéria renal esquerda
e da aorta e conectadas a um fluxômetro T206 (Transonic Systems, Inc., Ithaca, NY,
EUA), que permite determinar o fluxo em valores absolutos (ml ∙ min -1). Os sinais
obtidos foram enviados ao sistema de aquisição e análise de dados (PowerLab
System; ADInstruments, Bella Vista, Austrália). Os dados foram digitalizados em uma
frequência de 1000 amostras ∙ s -1. As variações do FSR e FSA foram calculadas como
a razão percentual em relação ao valor basal (%FSR/%FSA), de acordo com a
fórmula:
As condutâncias vasculares (CV) renal (CVR) e aórtica (CVA) foram obtidas

pela razão entre os respectivos fluxos sanguíneos e a PAM. As variações das CV
foram calculadas como variação percentual em relação ao basal (%CVR/%CVA),
utilizando-se a seguinte fórmula:
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12. Confirmação histológica dos sítios de nanoinjeção na região RVLM
Ao final dos experimentos, os animais foram perfundidos transcardiacamente

com soro fisiológico (NaCl 0,15M, Synth, Diadema, SP, Brasil) e solução de
formaldeído 10% (Synth, Diadema, SP, Brasil). Posteriormente, os bulbos foram
removidos e armazenados em solução de formaldeído 10% até a criotomia. Os bulbos
foram seccionados em cortes coronais de 40 µm de espessura utilizando um criostato
(CM 1860, Leica, Wetzlar, Alemanha). Posteriormente, os cortes foram posicionados
em lâminas de vidro e corados pela técnica de vermelho neutro. Após a secagem, os
cortes foram recobertos com lamínulas para posterior análise dos sítios de
nanoinjeção. Apenas os animais cujas nanoinjeções se localizaram nas regiões
correspondentes à região RVLM foram utilizados para as análises.
13. Análise estatística
Os dados experimentais obtidos foram expressos como média ± EPM (erro

padrão da média). Os valores basais das variáveis cardiovasculares, os valores de
ingestão e peso corporal, o IB e as alterações de PAM e de FC após estimulação
quimiorreflexa foram analisados através de teste t não pareado. Para as análises de
barorreflexo e quimiorreflexo foram utilizadas as respostas máximas das variáveis
cardiovasculares (PAM e FC) induzidas por cada uma das substâncias. Os valores de
ingestão e dos parâmetros cardiovasculares durante a escala de sensibilidade ao sal
foram analisados usando two way ANOVA seguida pelo teste LSD de Fisher. Para
análise das respostas cardiovasculares induzidas pelas nanoinjeções de L-glutamato
e muscimol na RVLM foi utilizada ANOVA one way. Foi assumida significância quando
p < 0,05. As análises estatísticas e a confecção dos gráficos foram realizadas usando
o GraphPad Prism v.06 para Windows.
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RESULTADOS
ALTERAÇÕES CARDIOVASCULARES INDUZIDAS POR SOBRECARGA

DE SAL
1. Ingestão de fluido e ração e peso corporal
A ingestão diária média de fluido e ração e o peso corporal dos animais

adultos que foram submetidos a sobrecarga de sal por 30 dias após o desmame (n=9)
e dos animais submetidos a sobrecarga de sal por 60 (n=11) dos seus respectivos
grupos controle (n=5 e n=12, respectivamente) e ao final dos períodos de tratamento
e recuperação estão expressos nas Figuras 1 e 2.
Os animais experimentais 30 dias apresentaram maior ingestão de salina
hipertônica NaCl em comparação com a ingestão de água do grupo controle durante
o período de tratamento (cont.: 18,8 ± 1,2 ml/100g de peso corporal vs. exp.: 61,2 ±
8,4 ml/100g de peso corporal, p<0,05), entretanto, não houve diferenças significativas
na ingestão de água de ambos os grupos no período de recuperação (cont.: 16,9 ±
0,8 ml/100g de peso corporal vs. exp.: 18,5 ± 1,1 ml/100g de peso corporal). Os
animais tratados apresentaram, também, maior ingestão média de ração/100g de
peso corporal em comparação com o grupo controle tanto no período de tratamento
(cont.: 10,7 ± 0,6 g/100g vs. exp.: 12,3 ± 0,4 g/100g; p<0,05) quanto no período de
recuperação (cont.: 8,7 ± 0,2 g/100g vs. exp.: 11,1 ± 0,4 g/100g; p<0,05). Além disso,
os animais experimentais apresentaram menor peso corporal em comparação com o
grupo controle, tanto no período de tratamento (cont.: 206,6 ± 5,9 g vs. exp.: 133,1 ±
7,9 g, p<0,05) quanto no período de recuperação (cont.: 266,7 ± 6,9 g vs. exp.: 205,10
± 9,7 g, p<0,05).
21
Figura 1: Média ± EPM da ingestão diária média de fluido (A), ração (B) e do peso corporal
(C) dos animais controle (n=5) e submetidos a sobrecarga de sal por 30 dias após o desmame (n=9)
ao final dos períodos de tratamento e recuperação. * diferente do grupo controle; p<0,05.
Similar ao que ocorreu com os animais tratados por 30 dias, os animais

tratados por 60 dias após o desmame apresentaram maior ingestão de salina
hipertônica NaCl em comparação como grupo controle no período de tratamento (cont.
15,50 ± 0,50 ml/100g de peso corporal vs. exp. 36,00 ± 6,80 ml/100g de peso corporal;
p<0,05) enquanto que a ingestão de água no período de recuperação não diferiu entre
os grupos (cont. 13,80 ± 0,90 ml/100g de peso corporal vs. exp. 12,30 ± 0,90 ml/100g
de peso corporal). Além disso, não houve diferenças significativas na ingestão de
ração entre os animais experimentais 60 dias e o grupo controle em ambos os
períodos (TRAT.: cont. 8,60 ± 0,30 g/100g de peso corporal vs. exp. 8,40 ± 0,40
g/100g de peso corporal. REC.: cont. 7,30 ± 0,10 g/100g de peso corporal vs. exp.
8,00 ± 0,30 g/100g de peso corporal; p<0,05). Entretanto, o peso corporal dos animais
tratados se mostrou diminuído em comparação com o grupo controle ao longo de todo
o protocolo experimental (TRAT.: cont. 313,64 ± 6.90 g vs. exp. 193,84 ± 18,79 g.
REC.: cont. 348,70 ± 10,90 g vs. exp. 280,45 ± 8,41 g; p<0,05) (Figura 2).
22
Figura 2: Média ± EPM da ingestão diária média de fluido (A) e ração (B) e do peso corporal
(C) dos animais controle (n= 6) e submetidos a sobrecarga de sal por 60 dias após o desmame (n=8)
ao final dos períodos de tratamento e recuperação. * diferente do grupo controle; p<0,05.
2. Parâmetros cardiovasculares e reflexo barorreceptor
Os parâmetros cardiovasculares basais (PAM e FC) dos animais adultos que

foram submetidos a sobrecarga de sal por 30 dias e dos animais controle ao final do
período de recuperação estão expressos na figura 3. Não foram observadas
diferenças significativas na PAM e na FC do animais experimentais em comparação
com os animais do grupo controle (cont. 118,2 ± 3,7 mmHg vs. exp. 112,3 ± 4,0 mmHg;
cont. 404,0 ± 10,6 bpm vs.exp. 374,7 ± 9,1 bpm).
23
Figura 3: Média ± EPM da pressão arterial média (PAM; A) e da frequência cardíaca (FC; B)
dos animais controle (n=5) e submetidos a sobrecarga de sal por 30 dias após o desmame (n=8) ao
final dos protocolos experimentais.
A Figura 4 apresenta os parâmetros cardiovasculares basais dos animais

tratados por 60 dias após o desmame e de seu grupo controle. Observamos que os
animais experimentais 60 dias apresentaram elevação de PAM (cont. 98,6 ± 2,6
mmHg vs. exp. 117,7 ± 4,2 mmHg; p<0,05) e de FC (cont. 365,4 ± 12,2 bpm vs. exp.
392,5 ± 10,3 bpm; p<0,05) em comparação com o grupo controle ao final do período
de recuperação.
Figura 4: Média ± EPM da pressão arterial média (PAM; A) e da frequência cardíaca (FC; B)
final dos protocolos experimentais. *diferente do grupo controle; p<0,05.
24
As Figuras 5 e 6 apresentam os traçados representativos e o índice de
barorreflexo (IB) dos animais tratados por 30 dias após o desmame e de seu grupo
controle mediante as infusões de fenilefrina e nitroprussiato de sódio,
respectivamente. Não houve diferenças significativas no IB dos animais tratados (n=8)
em comparação com os controle (n=4) mediante as infusões de nitroprussiato de sódio
(cont. 3,271 ± 0,3530 bpm/mmHg vs. exp. 3,336 ± 0,1350 bpm/mmHg), entretanto, os
animais tratados apresentaram menor IB mediante as infusões de fenilefrina, quando
comparados com os animais controle (cont. -2,441 ± 0,3590 bpm/mmHg vs. exp. -
1,434 ± 0,0860 bpm/mmHg; p<0,05).
25
Figura 5: Traçados representativos ilustrando as alterações cardiovasculares induzidas pela
infusão de fenilefrina nos animais controle (A; n=5) e tratado por 30 dias após o desmame (B; n=8) e
média ± EPM do índice de barorreflexo (IB) destes animais ao final dos protocolos experimentais.
*diferente do grupo controle; p<0,05.
26
infusão de nitroprussiato de sódio nos animais controle (A; n=5) e tratado por 30 dias após o desmame
(B; n=8) e média ± EPM do índice de barorreflexo (IB) destes animais ao final dos protocolos
experimentais. *diferente do grupo controle; p<0,05.
As figuras 7 e 8 apresentam os traçados representativos e o índice de

barorreflexo (IB) dos animais tratados por 60 dias após o desmame e de seu grupo
controle mediante as infusões de fenilefrina e nitroprussiato de sódio,
respectivamente. O IB dos animais tratados por 60 dias após o desmame se mostrou
27
diminuído em comparação com o grupo controle tanto após as infusões de fenilefrina
(cont. -1,83 ± 0,04 bpm/mmHg vs. exp. -1,24 ± 0,19 bpm/mmHg; p<0,05; Figura 7 C),
quanto após as infusões de nitroprussiato de sódio (cont. 2,12 ± 0,20 bpm/mmHg vs.
exp. 0,85 ± 0,22 bpm/mmHg; p<0,05; Figura 8 C).

infusão de fenilefrina nos animais controle (A; n=6) e tratado por 30 dias após o desmame (B; n=6) e
média ± EPM do índice de barorreflexo (IB) destes animais ao final dos protocolos experimentais.
*diferente do grupo controle; p<0,05.
28
infusão de nitroprussiato de sódio nos animais controle (A; n=6) e tratado por 30 dias após o desmame
(B; n=6) e média ± EPM do índice de barorreflexo (IB) destes animais ao final dos protocolos
29
3. Ingestão de fluido e ração durante a escala de sensibilidade ao sal
A Figura 9 apresenta os valores médios de ingestão de fluido e de ração dos

animais adultos submetidos a sobrecarga de sal por 30 dias no período pós-natal,
padronizados pelo peso corporal, em cada um dos períodos do protocolo
experimental. Não foram observadas diferenças significativas na ingestão de
fluido/100g de peso corporal entre os grupos tratado e controle durante a escala de
sensibilidade ao sal (NaCl 0,9%: cont.: 22,3 ± 0,1 ml/100g vs. exp.: 20,9 ± 1,9 ml/100g;
NaCl 1,2%: cont.: 20,1 ± 0,1 ml/100g vs. exp.: 25,7 ± 1,6 ml/100g; NaCl 1,5%: cont.:
28,8 ± 6,2 ml/100g vs. exp.: 29,6 ± 2,4 ml/100g; NaCl 1,8%: cont.: 44.6 ± 6,2 ml/100g
vs. exp.: 34,3 ± 2,7 ml/100g; Água: cont.: 13,6 ± 1,3 ml/100g vs. exp.: 16,1 ± 1,5
ml/100g). De forma similar, não houve diferenças significativas na ingestão diária
média de ração/100g de peso corporal entre os animais tratados e controle durante a
escala de sensibilidade ao sal (NaCl 0,9%: cont.: 7,8 ±0,1 g/100g vs. exp.: 8,6 ± 0,3
g/100g; NaCl 1,2%: cont.: 6,7 ± 0,1 g/100g vs. exp.: 7,1 ± 0,2 g/100g; NaCl 1,5%: cont.:
7,1 ± 0,4 g/100g vs. exp.: 7,6 ± 0,3 g/100g; NaCl 1,8%: cont.: 6,8 ± 0,2 g/100g vs. exp.:
7,0 ± 0,2 g/100g; Água: cont.: 8,7 ± 0,5 g/100g vs. exp.: 8,0 ± 0,4 g/100g).
Figura 9: Média ± EPM da ingestão diária média de fluido (A; ml/100g) e ração (B; g/100g)
longo da escala de sensibilidade ao sal. * diferente do grupo controle; p<0,05.
30
Os valores médios de ingestão de fluido e de ração dos animais adultos
submetidos a sobrecarga de sal por 60 dias no período pós-natal, padronizados pelo
peso corporal, em cada um dos períodos do protocolo experimental estão
apresentados na Figura 10. Podemos observar que os animais tratados apresentaram
maior ingestão de fluido em comparação com o respectivo grupo controle durante o
período de tratamento (cont.: 15,5 ± 0,5 ml/100g vs. exp.: 36,0 ± 6,8 ml/100g, na última
semana de tratamento, p<0,05). Entretanto, tais diferenças não foram observadas nos
outros períodos do protocolo (recuperação: cont.: 13,8 ± 0,9 ml/100g vs. exp.: 12,3 ±
0,9 ml/100g; NaCl 0,9%: cont.: 18,1 ± 2,8 ml/100g vs. exp.: 16,2 ± 0,9 ml/100g; NaCl
1,2%: cont.: 19,3 ± 3,7 ml/100g vs. exp.: 17,9 ± 1,7 ml/100g; NaCl 1,5%: cont.: 21,1 ±
3,3 ml/100g vs. exp.: 17,8 ± 0,8 ml/100g; NaCl 1,8%: cont.: 22,8 ± 2,5 ml/100g vs.
exp.: 23,2 ± 3,7 ml/100g; água: cont.: 11,4 ± 0,5 ml/100g vs. exp.: 10,3 ± 0,7 ml/100g).
Além disso, os animais Exp.60 apresentaram maior ingestão de ração por 100g de
peso corporal durante a primeira semana do período de recuperação (cont.: 7,8 ± 0,2
g/100g vs. exp.: 9,0 ± 0,7 g/100g; p<0,05); tal diferença não foi observada durante a
oferta de salina em suas diversas concentrações ou durante a segunda oferta de água.
Figura 10: Média ± EPM da ingestão diária média de fluido (A; ml/100g) e ração (B; g/100g)
longo da escala de sensibilidade ao sal. * diferente do grupo controle; T diferente da 10ª semana;
p<0,05.
31
4. Parâmetros cardiovasculares durante a escala de sensibilidade ao
sal
Os valores médios da pressão arterial sistólica (PAS) e da frequência cardíaca

(FC) dos animais submetidos a sobrecarga de sal por 30 dias e 60 dias são
apresentados nas Figuras 11 e 12.
Podemos observar que a PAS dos animais Exp.30 se mostrou elevada durante
o período de tratamento e na primeira semana do período recuperação (166,1 ± 3,9
mmHg e 154,9 ± 4,0 mmHg, respectivamente; p<0,05) em comparação com o grupo
controle (136,7 ± 2,5 mmHg e 136,8 ± 1,6 mmHg, respectivamente), entretanto esta
diferença não foi observada nos outros períodos do protocolo experimental (NaCl
0,9%: cont.: 128,6 ± 3,7 mmHg vs. exp.: 141,0 ± 0,1 mmHg; NaCl 1,2%: cont.: 135.6
± 5,4 mmHg vs. exp.: 137,8 ± 0,7 mmHg; NaCl 1,5%: cont.: 141.3 ± 1,3 mmHg vs.
exp.: 144.3 ± 3,6 mmHg; NaCl 1,8%: cont.: 143.6 ± 3,3 mmHg vs. exp.: 151,3 ± 0,7
mmHg; água: cont.: 141.1 ± 2,9 mmHg vs. exp.: 146.3 ± 5,8 mmHg). Não houve
diferenças significativas na frequência cardíaca dos grupos ao longo de todo o
protocolo experimental.
Figura 11: Média ± EPM da pressão arterial sistólica (A; PAS, mmHg) e da frequência cardíaca
(B; FC, mmHg) semanais dos animais controle (n=5) e submetidos a sobrecarga de sal por 30 dias
(n=8) em cada uma das etapas do protocolo experimental. *diferente do grupo controle; p<0,05.
32
Os animais Exp.60, como demonstrado na Figura 12 apresentaram maior PAS
em comparação com o grupo controle durante todos os períodos do protocolo
experimental (final do tratamento: cont.: 138,8 ± 4,4 mmHg vs. exp.: 167,6 ± 0,7
mmHg.; final da recuperação: cont.: 140,1 ± 2,2 mmHg vs. exp.: 157,6 ± 4,6 mmHg;
NaCl 0,9%: cont.: 130,8 ± 1,2 mmHg vs. exp.: 163,0 ± 1,1 mmHg; NaCl 1,2%: cont.:
132,6 ± 3,0 mmHg vs. exp.: 156.5 ± 3,8 mmHg; NaCl 1,5%: cont.: 140,5 ± 1,6 mmHg
vs. exp.: 159,4 ± 3,8 mmHg; NaCl 1,8%: cont.: 151,3 ± 1,4 mmHg vs. exp.: 164,6 ±
2,5 mmHg e água: cont.: 132,6 ± 3,5 mmHg vs. exp.: 160,4 ± 1,3 mmHg,
respectivamente; p<0,05). Observamos que a PAS dos animais tratados apresenta
queda significativa durante o período de recuperação, em comparação com o
tratamento, entretanto, permanece elevada em comparação com o grupo controle
neste período. A oferta de concentrações crescentes de NaCl eleva a PAS dos
animais tratados a valores semelhantes aos valores de PAS durante o tratamento.
Os animais do grupo controle apresentam aumento significativo de PAS
durante a ingestão de NaCl 1,8% em comparação com a PAS no período de
recuperação (151,3 ± 1,4 mmHg vs. 140,1 ± 2,2 mmHg; p<0,05), tal parâmetro retorna
a valores basais quando do retorno da oferta de água. A FC dos animais submetidos
a sobrecarga de sal por 60 dias após o desmame mostrou-se aumentada em
comparação com o grupo controle apenas ao final do tratamento e durante a segunda
oferta de salina hipertônica (cont.: 372,5 ± 8,5 bpm vs. exp.: 390,6 ± 4,0 bpm e cont.:
338,4 ± 5,9 bpm vs. exp.: 359,1 ± 1,9 bpm; p<0,05).
33
Figura 12: Média ± EPM da pressão arterial sistólica (A; PAS, mmHg) e da frequência cardíaca
(B; FC, bpm) semanais dos animais controle (n=6) e submetidos a sobrecarga de sal por 60 dias (n=8)
em cada uma das etapas do protocolo experimental. *diferente do grupo controle; # diferente da 8ª
semana; T diferente da 10ª semana; p<0,05.
5. Análise da histologia cardíaca
Fotomicrografias representativas dos corações de animais submetidos a

sobrecarga de sal por 30 dias ou por 60 dias após o desmame e de seus respectivos
grupos controle são apresentadas na figura 13.
34
Figura 13: Fotomicrografias representativas dos corações corados pela técnica de Reticulina
de Gömori. (A) Controles 30 dias, n=4; (B) Tratados por 30 dias após o desmame, n=4; (C) Controles
60 dias, n=4 e (D) tratados por 60 dias após o desmame, n=4. Aumento de 40x. As setas brancas
representam fibras colágenas do tipo I enquanto que as setas pretas representam as fibras do tipo III.
A figura 14 apresenta os valores médios da área de secção transversa (AST)

dos cardiomiócitos dos animais adultos dos grupos Exp.30 (Figura 14A) e Exp.60
(Figura 14B) em comparação com os seus respectivos grupos controle. Podemos
observar que a sobrecarga de sal após o desmame, independentemente de sua
duração, não promoveu hipertrofia de cardiomiócitos nos animais tratados, quando
35
comparados com seus respectivos grupos controle (30 dias: cont.: 14,68 ± 0,18 nm
vs. exp.: 14,38 ± 0,21 nm; 60 dias: cont.: 14,99 ± 0,20 nm vs.exp.: 14,63 ± 0,18 nm).
Figura 14: Média ± EPM da área de secção transversa (AST) de cardiomiócitos dos animais
submetidos a sobrecarga de sal por 30 dias (A) e por 60 dias (B) e seus respectivos grupos controle.
A frequência relativa (%) de cada um dos componentes do tecido cardíaco

(cardiomiócitos, fibras colágenas dos tipos I e III e tecido conjuntivo não fibrilar) dos
animais adultos dos grupos Exp.30 e Exp.60, em comparação com os seus grupos
controle, estão expressas nas Figuras 15 e 16, respectivamente. Não foram
observadas diferenças significativas na frequência relativa de cardiomiócitos (cont.:
41,82 ± 1,05% vs. exp.: 39,62 ± 1,04%), tecido conjuntivo não fibrilar (cont.: 1,44 ±
0,33 vs. exp.: 0,846 ± 0,286 %), colágeno tipo I (cont.: 20,72 ± 1,14% vs. exp.: 21,46
± 1,09%) ou colágeno tipo III (cont.: 36,03 ± 1,05% vs. exp.: 38,08 ± 1,60%) dos
animais tratados por 30 dias em comparação com seu grupo controle (Figura 15).
36
Figura 15: Média ± EPM da frequência relativa dos componentes do tecido cardíaco dos
animais submetidos à sobrecarga de sal por 30 dias e de seu grupo controle. (A) cardiomiócitos; (B)
tecido intersticial não fibrilar; (C) fibras colágenas tipo I e (D) fibras colágenas tipo III.
Já nos animais tratados por 60 dias, a frequência relativa de cardiomiócitos

(cont.: 48,31 ± 1,62% vs. exp.: 45,26 ± 1,44%) e de colágeno tipo III (cont.: 32,38 ±
1,04% vs. exp.: 34,03 ± 1,15%) não diferiu do grupo controle, enquanto que a
frequência relativa de tecido conjuntivo não fibrilar se mostrou diminuída (cont.: 2,46
± 0,50% vs. exp.: 0,92 ± 0,26%, p<0,05) e a frequência relativa de colágeno tipo I se
mostrou aumentada (cont.: 15,74 ± 0,61% vs. exp.: 19,79 ± 1,26%; p<0,05) em
comparação com o grupo controle (Figura 16).
37
Figura 16: Média ± EPM da frequência relativa dos componentes do tecido cardíaco dos
animais submetidos à sobrecarga de sal por 60 dias e de seu grupo controle. (A) cardiomiócitos; (B)
tecido intersticial não fibrilar; (C) fibras colágenas tipo I e (D) fibras colágenas tipo III. * diferente do
grupo controle; p<0,05.
6. Alterações cardiovasculares induzidas pelas nanoinjeções de

glutamato e muscimol na região RVLM
Uma vez que as alterações cardiovasculares (aumento de PAM e de FC e

diminuição da sensibilidade barorreflexa) foram mais pronunciadas nos animais
submetidos a sobrecarga de sal por 60 dias após o desmame, avaliamos se estas
alterações poderiam estar relacionadas com alterações de sensibilidade da região
38
RVLM. A Figura 17 apresenta um corte coronal de bulbo de um animal representativo,
que recebeu nanoinjeções de azul de Evans nas regiões correspondentes ao RVLM.
Figura 17: Fotomicrografia representativa de um corte coronal de bulbo de um animal que

recebeu nanoinjeções de azul de Evans em região correspondente ao RVLM (setas). Os círculos
indicam o Núcleo Ambiguos.
A figura 18 traz os traçados representativos das alterações cardiovasculares

induzidas pelas nanoinjeções de soro e glutamato na região RVLM dos animais
controle e submetidos à sobrecarga de sal por 60 dias. As alterações cardiovasculares
induzidas pelas nanoinjeções de glutamato e no RVLM dos animais controle (251,40
± 17,87 g; n=4) e submetidos a sobrecarga de sal por 60 dias após o desmame (221,00
± 5,80 g; n=4) estão expressas na Figura 19.
Como esperado, as nanoinjeções de soro fisiológico no RVLM não promoveram
alterações de PAM (Cont.: Δ1,03 ± 0,73mmHg; Exp.: Δ1,41 ± 0,83mmHg), FC (Cont.:
Δ0,19 ± 1,24 bpm; Exp.: Δ-0,46 ± 0,97 bpm) e de FSR (Cont.: Δ0,34 ± 0,30% do basal;
Exp.: Δ1,24 ± 0,62% do basal), CVR (Cont.: 99,33 ± 0,87 % do basal; Exp.: 100,17 ±
1,05% do basal) e FSA (Cont.: 101,33 ± 0,40% do basal; Exp.: 99,84 ± 0,77 % do
basal) e CVA (Cont.: 100,29 ± 0,36% do basal; Exp.: 98,78 ± 1,04 % do basal) em
nenhum dos grupos (Figuras 19 e 21).
39
Como demonstrado na Figura 19, as nanoinjeções de glutamato no RVLM
promoveram aumentos de PAM em todos os grupos; entretanto, as respostas
observadas nos animais experimentais foram significativamente maiores em
comparação com o grupo controle (Unilateral: cont.: Δ13,72 ± 1,69mmHg; exp.: Δ
25,31 ± 3,38 mmHg; p<0,05 em comparação com as nanoinjeções de soro. Bilateral:
cont.:Δ15,47 ± 2,56 mmHg vs. exp.: Δ 34,31 ± 4,65 mmHg; p<0,05 em comparação
com as nanoinjeções de soro e com o grupo controle. Figura 19A). Além disso, as
nanoinjeções de glutamato não alteraram a condutância vascular renal (cont.:
unilateral: -8,58 ± 2,57% do basal; bilateral: -9,35 ± 1,47% do basal. Figura 19D) ou
aórtica (cont.: unilateral: -4,51 ± 5,40% do basal; bilateral: -8,63 ± 2,39% do basal.
Figura 19F) dos animais do grupo controle enquanto que promoveram diminuição
significativa da CVR (exp.: unilateral: -13,38 ± 0,82% do basal, p<0,05 em comparação
com as nanoinjeções de soro; Bilateral: -21,15 ± 3,08% do basal, p<0,05 em
comparação com as nanoinjeções de soro e com o grupo controle. Figura 19D) e na
CVA (exp.: Unilateral: -16,64 ± 6,63% do basal. Bilateral: -29,80 ± 2,55% do basal,
p<0,05 em comparação com as nanoinjeções de soro e com o grupo controle. Figura
19F) dos animais tratados por 60 dias após o desmame. Não foram observadas
alterações significativas da FC (bilateral: cont.:1,48 ± 4,35 bpm; exp.: -7,37 ± 6,40
bpm. Figura 19B), do FSR (bilateral: Cont.: 2,64 ± 0,73% do basal; Exp. 1,13 ± 1,73%
do basal. Figura 19C) e do FSA (bilateral: cont.: -0,09 ± 3,15% do basal; exp.: -9,95 ±
1,27% do basal. Figura 19E) em nenhum dos grupos.
40
Figura 18: Traçados representativos das alterações cardiovasculares induzidas pelas
nanoinjeções de soro e glutamato 10mM na região RVLM dos animais controle (A) e submetidos a
sobrecarga de sal por 60 dias após o desmame (B).
41
Figura 19: Média ± EPM das alterações de pressão arterial média (PAM, mmHg, A),
frequência cardíaca (FC, bpm, B), fluxos sanguíneos renal (FSR, % do basal, C) e aórtico (FSA, % do
basal, E) e condutâncias vasculares renal (CVR, % do basal, D) e aórtica (CVA, % do basal, F)
induzidas pelas pelas nanoinjeções de soro fisiológico e glutamato 10mM na região RVLM de animais
controle (n=4) e submetidos a sobrecarga de sal por 60 dias após o desmame (n=4). *diferente das
nanoinjeções de soro fisiológico; T diferente do grupo controle, p<0,05.
42
Os traçados representativos das alterações cardiovasculares induzidas pelas
nanoinjeções de soro fisiológico e de muscimol 2mM nos animais controle e
submetidos à sobrecarga de sal por 60 dias estão expressos na figura 20. Como
demonstrado na figura 21, as nanoinjeções de muscimol no RVLM promoveram
diminuição da PAM nos animais controle e tratados em comparação com as
nanoinjeções de soro fisiológico (Bilateral: cont.: Δ-27,14 ± 8,24 mmHg; exp.: Δ-27,41
± 6,13 mmHg; p<0,05 em comparação com as nanoinjeções de soro. Figura 21A);
entretanto, este parâmetro não diferiu entre os grupos. Não foram observadas
alterações de FC, FSR, CVR, FSA e CVA após as nanoinjeções bilaterais de muscimol
nos grupos controle (Δ-12,05 ± 4,35 bpm, Figura 19B; -19,99 ± 7,83% do basal, Figura
19C; 19,64 ± 3,57% do basal, Figura 19D; -29,38 ± 11,05% do basal, Figura 19E; 4,06
± 8,25% do basal, Figura 19F) e experimental (Δ-16,49 ± 3,32 bpm, Figura 19B; -20,27
± 4,62% do basal, Figura 19C; 12,88 ± 8,85% do basal, Figura 19D; -28,57 ± 2,98%
do basal, Figura 19E; 0,68 ± 3,79% do basal, Figura 19F) em comparação com as
nanoinjeções de soro fisiológico.
43
Figura 20: Traçados representativos das alterações cardiovasculares induzidas pelas
nanoinjeções de soro e muscimol 2mM na região RVLM dos animais controle (A) e submetidos a
sobrecarga de sal por 60 dias após o desmame (B).
44
Figura 21: Média ± EPM das alterações de pressão arterial média (PAM, mmHg, A), frequência
cardíaca (FC, bpm, B), fluxos sanguíneos renal (FSR, % do basal, C) e aórtico (FSA, % do basal, E) e
condutâncias vasculares renal (CVR, % do basal, D) e aórtica (CVA, % do basal, F) induzidas pelas
pelas nanoinjeções de soro fisiológico e muscimol 2mM na região RVLM de animais controle (n=4) e
submetidos a sobrecarga de sal por 60 dias após o desmame (n=4). *diferente das nanoinjeções de
soro fisiológico, p<0,05.
45
ALTERAÇÕES CARDIOVASCULARES INDUZIDAS POR SOBRECARGA
DE SACAROSE
1. Ingestão de fluido e ração e peso corporal
Os valores médios da ingestão de fluido e ração dos animais submetidos a

ingestão de sacarose 20% por 30 dias na fase adulta (n=10) e dos animais controle
(n=5) são apresentados na figura 22. Os animais experimentais apresentaram maior
ingestão diária média de solução sacarose 20% em comparação com a ingestão de
água pelo grupo controle durante todo o tratamento (cont.: 10,05 ± 0,50 ml/100g/100g
vs. exp.: 19,00 ± 1,70 ml/100g; p<0,05). Além disso, apresentaram menor ingestão
diária média de ração (cont.: 6,80 ± 0,20 g/100g vs. exp.: 5,20 ± 0,20 g/100g; p<0,05)
e menor ingestão calórica em comparação com o grupo controle (cont.:142,50 ± 7,10
cal vs. exp.: 126,30 ± 3,50 cal; p<0,05). Não foram observadas diferenças
significativas no peso corporal dos animais experimentais em comparação com os
animais controle (cont. 351,2 ± 5,20g vs. exp.: 358,8 ± 13,30 g).
46
Figura 22: Média ± EPM da ingestão diária média de fluido (A; ml/100g) e de ração (B; g/100g)
padronizadas pelo peso corporal, da ingestão calórica total (C, cal) e do peso corporal (D, g) dos
animais submetidos à sobrecarga de sacarose por 30 dias na fase adulta (n=10) e do seu grupo controle
(n=5). * diferente do grupo controle; p<0,05.
2. Parâmetros cardiovasculares e reflexos barorreceptor e

quimiorreceptor
Os parâmetros cardiovasculares basais dos animais submetidos a sobrecarga

de sacarose por 30 dias na fase adulta e dos animais controle estão expressos na
Figura 23. Observamos que os animais experimentais apresentaram maior PAM
(cont.: 102,5 ± 1,4 mmHg vs. SM: 111,3 ± 0,9 mmHg; p<0.05) e maior FC (cont.: 334,7
± 7,3 bpm vs. SM: 371,6 ± 4,7 bpm; p<0.05) ao final do tratamento, quando
comparados com os animais controle
47
Figura 23: Média ± EPM da pressão arterial média (PAM, mmHg) e da frequência cardíaca
(FC, bpm) dos animais controle (n=4) e submetidos a sobrecarga de sacarose por 30 dias (n=10) - ao
final do tratamento. *diferente do grupo controle; p<0,05.
Os traçados representativos e os índices de barorreflexo (IB; ∆FC/∆PAM)

após as infusões intravenosas de fenilefrina (agonista α-adrenérgico) e nitroprussiato
de sódio (doador de NO) dos animais SM e controle são mostrados na Figura 24 e 25,
respectivamente. Não houve diferenças significativas entre o IB dos animais
experimentais em comparação com os animais controle após as infusões de fenilefrina
(cont.: -2,5 ± 0,1 bpm/mmHg vs. SM: -2,2 ± 0,2 bpm/mmHg; Figura 24 C). Entretanto,
os animais experimentais apresentaram menor IB em comparação com o grupo
controle para as infusões de nitroprussiato de sódio (cont.: 5,0 ± 0,1 bpm/mmHg vs.
SM: 4,0 ± 0,1 bpm/mmHg; p<0,05; Figura 25 C).
48
infusão de fenilefrina nos animais controle (A; n=5) e submetidos a sobrecarga de sacarose por 30 dias
– síndrome metabólica (B; n=8) e média ± EPM do índice de barorreflexo (IB; C) destes animais ao final
dos protocolos experimentais. *diferente do grupo controle; p<0,05.
49
infusão de nitroprussiato de sódio nos animais controle (A; n=5) e submetidos a sobrecarga de sacarose
por 30 dias – síndrome metabólica (B; n=8) e média ± EPM do índice de barorreflexo (IB; C) destes
As respostas pressóricas e bradicárdicas induzidas pela infusão de KCN e os

traçados representativos dos animais submetidos à sobrecarga de sacarose por 30
dias e do seu grupo controle são mostradas na Figura 26. Os animais experimentais
apresentaram maior resposta pressórica em comparação com os animais controle
50
(cont.: 14,9 ± 1,9 mmHg vs. exp.: 29,2 ± 5,5 mmHg; p<0,05; Figura 26 C), entretanto,
não houve diferenças significativas nas respostas bradicárdicas entre os grupos
(cont.: -191,2 ± 23,4 bpm vs. exp.: -197,4 ± 27,0 bpm; Figura 26 D).
Figura 26: Traçados representativos ilustrando as alterações induzidas pela infusão de cianeto
de potássio nos animais controle (A; n=4) e submetidos a sobrecarga de sacarose por 30 dias (B; n=7)
e média ± EPM das respostas pressórica (C) e bradicárdica (D) destes animais ao final dos protocolos
51
DISCUSSÃO
Nossos resultados demonstram que a sobrecarga de sal durante 30 dias após

o desmame não altera a PAM ou a FC na fase adulta, entretanto, promoveu diminuição
permanente da sensibilidade barorreflexa nos indivíduos adultos. Corroborando com
estudos anteriores (7), observamos ainda que os animais tratados por 60 dias
apresentaram elevação sustentada da PA sistólica ao longo de todo o experimento.
Ademais, observamos nestes animais aumento da frequência relativa de colágeno tipo
I no tecido cardíaco e aumento da resposta pressórica após nanoinjeções de
glutamato na região RVLM.
Estudos das últimas décadas demonstram que a oferta de soluções
hipertônicas como única fonte hídrica é um importante modelo de indução de anorexia,
denominada anorexia induzida por desidratação (151,152). Nesta situação, em que a
osmolaridade plasmática está elevada, a ingestão alimentar é diminuída visando a
redução da quantidade de água necessária à digestão e diminuindo a ingestão de
osmólitos pelos alimentos (151). Em nosso estudo, os animais submetidos a
sobrecarga de sal por 30 ou por 60 dias não apresentaram diminuição da ingestão
alimentar, entretanto apresentaram diminuição do peso corporal, em comparação com
os animais controle, mesmo após a retirada da dieta hipersódica. É possível que a
exposição prolongada ao estímulo osmótico modifique as respostas neuronais que
resultam na anorexia por desidratação, de modo que a ingestão alimentar não se
altere. Além disso, alterações hormonais ou de demanda metabólica podem estar
associadas ao baixo ganho de peso observado nos animais submetidos à dieta rica
em sal.
Diversos estudos indicam que a elevação da ANS é um importante fator para o
desenvolvimento e a manutenção do estado hipertensivo em diversos modelos de
hipertensão (153–157). De fato, a hipertensão sal-dependente está fortemente
relacionada à crônica excitação do sistema nervoso simpático (69,80,86,158–160).
Além disso, o desenvolvimento deste tipo de hipertensão em diversos modelos
experimentais é prevenido por lesões de regiões hipotalâmicas relacionadas com o
controle cardiovascular e do equilíbrio hidroeletrolítico, como a região AV3V, o SFO e
o PVN (85). Estudos de Adams et al. (2007) demonstraram que o aumento do
52
consumo de sal aumenta a atividade simpática e eleva a PA através da estimulação
de neurotransmissões glutamatérgicas no RVLM, resultando em aumento do tônus
vasomotor e de PA. Neste contexto, a sobrecarga crônica de sódio altera propriedades
neuronais intrínsecas e, consequentemente, eleva a excitação de neurônios pré-
motores simpáticos localizados na região RVLM. Baseado nessas informações, é lícito
supor que regiões hipotalâmicas e/ou neurônios pré-motores simpáticos possam estar
hiperativados após sobrecarga de sal durante o período pós-natal. De fato,
demonstramos que a ativação da neurotransmissão glutamatérgica na região RVLM
promoveu maior resposta pressora nos animais submetidos à dieta rica em sal,
indicando aumento da sensibilidade glutamatérgica desta região, o que acarretaria em
aumento do tônus simpático e nos consequentes aumentos de PA e FC observados
no presente estudo.
A fim de manter a correta perfusão e oferta de nutrientes e oxigênio aos
tecidos, a PA é estritamente controlada por diversos fatores que atuam em curto e
longo prazo, como os reflexos barorreceptor e quimiorreceptor e o sistema renina-
angiotensina (SRA). O SRA é um dos mais conhecidos sistemas de controle da PA e
as alterações de expressão de seus componentes podem ser outro fator contribuinte
para a elevação pressórica observada em situações de alta ingestão de sal. Estudos
recentes demonstram a existência de componentes do SRA necessários à produção
local de Ang II em diversos tecidos (161,162), como o cérebro (163), tecido adiposo
(164) e pâncreas (165). Diversos estudos demonstram que a sobrecarga de sal altera
a expressão de componentes-chave do SRA cerebral, especialmente de enzima
conversora de angiotensina (ECA), elevando a produção local de Ang II (166–169).
Assim, a Ang II cerebral atuando sobre receptores AT1 no Órgão Sub-fornical (SFO),
no PVN e no RVLM pode aumentar a produção de espécies reativas de oxigênio
nestas regiões, contribuindo para o aumento do tônus simpático característico de
modelos de hipertensão sal-dependente (170–173). Como mencionado
anteriormente, tal aumento do tônus simpático para diversos leitos está diretamente
relacionado ao aumento da PA, que é crucial para a excreção de sódio e para o
reestabelecimento das condições fisiológicas. Em nosso estudo, observamos que a
sobrecarga de sal promove elevação da PA, que, em nosso modelo, é sustentada
53
mesmo após a retirada da dieta rica em sal, indicando uma alteração permanente da
regulação da PA nestes animais.
Além do SRA, a regulação em curto prazo da PA ocorre especialmente pela
ativação do reflexo barorreceptor. Os barorreceptores arteriais são
mecanorreceptores, localizados no arco aórtico e no seio carotídeo, capazes de
detectar alterações da PA através do estiramento dos vasos em que encontram-se. O
aumento da pressão arterial promove estiramento celular e consequente aumento da
frequência de disparo de potenciais de ação destes neurônios, enviando informações
ao SNC, que promove uma série de ajustes a fim de restabelecer os valores basais
de PA. Em situações de estimulação contínua, como na hipertensão arterial, os
barorreceptores podem se adaptar e diminuir a sua sensibilidade a novos estímulos.
Além de alterar os parâmetros cardiovasculares basais, nossos resultados
demonstram que a sobrecarga de sal em fases do período pós-natal promove
diminuição da sensibilidade do reflexo barorreceptor. Embora diversos estudos
demonstrem que a sensibilidade barorreflexa está diminuída na hipertensão arterial
(7,174–176), existe intenso debate sobre qual condição se estabelece primeiro: a
hipertensão ou o comprometimento do reflexo barorreceptor. Em nosso estudo, os
animais submetidos à sobrecarga de sal por 30 dias apresentam apenas diminuição
da sensibilidade barorreflexa sem elevação da PA, enquanto que os animais tratados
por 60 dias apresentam elevação permanente de PA, em adição à diminuição da
sensibilidade barorreflexa. Em conjunto, estes resultados nos permitem inferir que, em
nosso modelo de sobrecarga de sal após o desmame, a diminuição da sensibilidade
barorreflexa se estabeleça anteriormente à elevação permanente da PA e da FC.
Estudos recentes realizados em nosso laboratório demonstram que animais
adultos submetidos a sobrecarga de sal por 60 dias após o desmame apresentam
diminuição da ingestão espontânea de sódio em situações em que ocorre a oferta
simultânea de água e de sódio (ABSM e AHFO, observações não publicadas).
Entretanto em nosso estudo, não observamos alterações das respostas
comportamentais à ingestão de diferentes concentrações de solução salina nos
animais adultos submetidos à sobrecarga de sal após o desmame. Tais divergências
podem ser explicadas pelo fato de que no presente estudo as soluções de NaCl foram
54
ofertadas como única fonte hídrica. Recentemente demonstramos que os animais
tratados por 60 dias apresentam menor ingestão de sódio após episódio de depleção
de sódio na fase adulta (7). Corroborando com estes achados, observações recentes
de nosso laboratório demonstram que estes animais apresentam diminuição, retardo
ou inibição na ingestão de solução hipertônica após a indução de diferentes tipos de
desidratação (ABSM e AHFO, observações não publicadas). Estes resultados
sugerem que a sobrecarga de sal após o desmame altera as respostas
comportamentais induzidas por alterações da concentração plasmática de sódio,
indicando possíveis alterações de sensibilidade de regiões hipotalâmicas, como a
região AV3V e órgãos circunventriculares, envolvidas no controle da sede e do apetite
ao sódio.
Nossos resultados indicam que o tratamento por 30 dias após o desmame não
altera a PAS ou a FC dos animais adultos submetidos a nova ingestão de soluções
salinas em concentrações crescentes (escala de sensibilidade). É possível que o
tratamento por 30 dias não seja capaz de alterar permanentemente vias centrais
envolvidas na respostas cardiovasculares à ingestão de sal e que outros mecanismos
de manutenção da osmolaridade (como a liberação de ANP e a adequada
vasodilatação do território renal) também estejam preservados nestes animais após a
retirada da dieta hipersódica. Diferentemente, os animais tratados por 60 dias
mantiveram a PAS elevada ao longo da escala de sensibilidade ao sal enquanto que
a elevação deste parâmetro ocorre apenas durante a oferta de salina NaCl 1,8% nos
animais controle, retornando aos valores basais quando do retorno da oferta de água.
Neste contexto, a sobrecarga de sal por 60 dias após o desmame pode ter alterado
definitivamente vias centrais envolvidas na regulação cardiovascular às alterações de
osmolaridade. No presente estudo, demonstramos que a região RVLM está mais
sensível a neurotransmissões excitatórias após o tratamento por 60 dias. Além disso,
anteriormente observamos que animais submetidos à sobrecarga de sal por 60 dias
apresentaram comprometimento da vasodilatação renal induzida por infusão de salina
hipertônica e que, nessa situação, a resposta pressórica é potencializada (7).
Estudo recente realizado em nosso laboratório demonstrou que os corações
isolados de animais adultos submetidos à sobrecarga de sal por 60 dias após o
55
desmame apresentam função cardíaca normal em condições basais. Entretanto, após
evento isquêmico e reperfusão tecidual, tais corações apresentam relaxamento
ventricular comprometido e ainda maior duração e severidade de arritmias de
reperfusão (MCSM, CHC e GRP, observações não publicadas, apêndice IIb). Alguns
estudos tem relacionado o alto consumo de sal com a disfunção diastólica do
ventrículo esquerdo em indivíduos hipertensos e normotensos (177–180). De fato,
Tzemos et al. (2008)(181) demonstraram que o alto consumo de sódio é capaz de
promover disfunção diastólica mesmo em animais normotensos e com o sistema
renina-angiotensina íntegro. Diversos estudos demonstram que a hipertrofia do
músculo cardíaco é um dos fatores associados ao comprometimento do relaxamento
do coração (182,183). Entretanto, estudos de Dupont et al. (2012) (184)
demonstraram que disfunções de relaxamento estão associadas com distúrbios da
homeostase intracelular de cálcio e sugerem, ainda, que as disfunção diastólicas
podem se desenvolver independentemente da hipertrofia cardíaca em SHR. De fato,
nossos resultados demonstram que a sobrecarga de sal após o desmame,
independente de sua duração, não promove hipertrofia de cardiomiócitos. Tzemos et
al. (2008) (181) sugerem que o excesso de sódio pode aumentar os níveis
intracelulares de cálcio ou ainda alterar os níveis de receptação de cálcio durante o
relaxamento muscular, o que comprometeria o relaxamento cardíaco. Assim, novos
estudos precisam ser realizados para analisarmos as possíveis alterações na
homeostase de cálcio causadas pela sobrecarga de sal após o desmame.
A fibrose do tecido cardíaco é outro fator importante no desenvolvimento de
disfunções diastólicas. As fibras colágenas são continuamente sintetizadas e
degradadas e as alterações da síntese das fibras colágenas cardíacas é crucial para
adaptar o órgão às necessidades do organismo (coração mais ou menos resistente
ao estiramento, por exemplo); por outro lado, a síntese exacerbada de apenas um tipo
de fibra pode, em longo prazo, afetar a função cardíaca (185–187). De fato, estudos
anteriores demonstraram que o aumento da deposição de fibras colágenas do tipo I -
um elemento inelástico - aumenta substancialmente a capacidade do coração de
resistir ao estiramento (187,188). Estudos de Yu et al. (1998) (189) demonstraram que
a alta ingestão de sal causa fibrose do miocárdio em SHR e em animais normotensos.
56
Neste estudo, os autores demonstram ainda que o sódio promove não apenas
hipertrofia ventricular esquerda, mas também fibrose intramiocardial, que pode levar
a disfunções sistólicas e diastólicas por aumentar a rigidez do tecido cardíaco. Além
disso, diferentes estudos demonstraram que tanto a sobrecarga pressórica, quanto a
alta ingestão de sal podem, separadamente, causar fibrose cardíaca (189–195). Os
mecanismos pelos quais a sobrecarga de sal promove fibrose do tecido cardíaco
permanecem por ser esclarecidos, entretanto, estudos de Lal et al. (2003)(195)
indicam que a aldosterona cardíaca pode participar deste efeito, uma vez que o
antagonismo de seus receptores previne tanto a hipertrofia quanto a fibrose cardíaca
induzidas por dieta rica em sal. Consistente com estes achados, Takeda et al. (2000)
(196) demonstram que a produção cardíaca de aldosterona está aumentada em
animais submetidos a dieta hipersódica. Além disso, estudos in vitro indicam que os
fibroblastos cardíacos elevam sua síntese de fibras colágenas na presença de
aldosterona (197). Estas evidências suportam a hipótese de que a fibrose do tecido
cardíaco após sobrecarga de sal ocorra por ação da aldosterona nos fibroblastos do
coração. Em nosso modelo, os animais foram submetidos a dieta rica em sódio e
apresentaram PA elevada, fatores contribuintes para o desenvolvimento de fibrose
cardíaca.
Além de comprometer o relaxamento cardíaco, a fibrose deste tecido pode
contribuir para o desenvolvimento e agravamento de eventos arrítmicos. O
acoplamento elétrico entre as células cardíacas é crucial para a sua contração
sincronizada e o batimento cardíaco efetivo e a conexão entre os citoplasmas de
células adjacentes é mediado pelas proteínas conexinas (198). Tais proteínas
participam do fluxo de iônico entre os citoplasmas e são um fator chave para a
propagação do impulso elétrico por todo o coração.
O aumento da deposição de colágeno no tecido cardíaco pode comprometer o
acoplamento elétrico entre os cardiomiócitos, comprometendo a propagação do
impulso elétrico e gerando contrações anormais e dessincronizadas das células
musculares cardíacas, o que caracteriza as arritmias. Neste sentido, observamos que
a sobrecarga de sal por 60 dias após o desmame promove aumento da frequência
relativa de colágeno tipo I na matriz extracelular do músculo cardíaco, o que,
57
provavelmente, contribui para a disfunção diastólica e para o agravamento das
arritmias observadas após evento isquêmico nos corações isolados de animais
adultos submetidos ao mesmo protocolo de dieta hipersódica utilizado no presente
estudo (MCSM, CHC, GRP observações não publicadas).
Dentre as regiões do SNC que se projetam para a IML e, assim, apresentam
controle direto da alça simpática do sistema nervoso autônomo, a região RVLM é a
única a apresentar atividade intrínseca tônica, sendo assim essencial para a
manutenção do tônus vasomotor simpático (104,199–201). Estudos demonstram que,
em animais adultos, uma dieta rica em sódio aumenta a resposta pressora a
neurotransmissores excitatórios e inibitórios na região RVLM (12,106) e estas
alterações podem estar relacionadas a alterações de sensibilidade neuronal (107). A
excitabilidade neuronal e suas alterações dependem do potencial de membrana das
células e de sua proximidade ao potencial limiar (potencial de membrana necessário
para a despolarização); deste modo, quanto maior o potencial de membrana, menor
o estímulo necessário para a despolarização e mais sensível é o neurônio. Assim, a
presença de substâncias que atuam sobre o neurônio alterando seu potencial de
membrana (chamadas de neuromoduladores, advindas da micróglia, por exemplo),
podem exercer papel importante no aumento de sensibilidade neuronal. Neste
sentido, diversos autores indicam que a ingestão de sal altera a sinalização
angiotensinérgica e promove aumento da produção de espécies reativas de oxigênio
no SNC, fatores estes que podem estar relacionados com as alterações de
sensibilidade neuronal da região RVLM (169,202–204).
Além das alterações de sensibilidade dos neurônios do RVLM, a ativação ou
inibição de vias neuronais que atinjam este núcleo podem ser um fator contribuinte
para as respostas observadas por Adams et al. (2007)(107). Neste sentido, estudos
de Li et al. (2005)(205) demonstram que animais hipertensos apresentam diminuição
da modulação GABAérgica sobre os neurônios pré-simpáticos do PVN, o que pode
contribuir para a excitação simpática observada na hipertensão arterial. Além disso,
Kim et al. (2011)(206) observaram que o estresse osmótico crônico inverte as
respostas dos neurônios secretores magnocelulares do PVN à neurotransmissão
GABAérgica para respostas excitatórias. Entretanto, a hipótese de que esta inversão
58
também ocorra em neurônios parvocelulares do PVN (que se projetam para a região
RVLM) precisa ser investigada.
Nosso resultados demonstram que os animais adultos que foram submetidos
a sobrecarga de sal após o desmame apresentam elevação das respostas pressora e
vasoconstritora após nanoinjeções de glutamato na região RVLM. Até o momento,
nenhum outro estudo na literatura demonstrou alteração da sensibilidade da região
RVLM induzida por alterações da homeostase hidromineral após o desmame. Estes
resultados corroboram com nossos resultados anteriores (7) demonstrando,
novamente, que as alterações provocadas pela sobrecarga de sal após o desmame
são persistentes até a fase adulta, mesmo após a retirada da dieta hipersódica.
Ademais, nossos achados estendem os achados de Adams et al. (2007)(107)
demonstrando que as alterações de sensibilidade da região RVLM induzidas por dieta
hipersódica são irreversíveis quando a dieta é ofertada após o desmame. Novos
experimentos são necessários para confirmar se tal alteração de sensibilidade
observada na neurotransmissão glutamatérgica também pode ocorrer em outras
neurotransmissões na região RVLM.
Em conjunto, nossos resultados demonstram que a sobrecarga de sal após o
desmame promove elevação da PA, diminuição da sensibilidade barorreflexa, que se
estabelece precocemente, e, ainda, fibrose miocárdica em animais adultos. Além
disso, tal sobrecarga altera as respostas cardiovasculares induzidas por dieta rica em
sal na fase adulta. Estas alterações podem ser resultado do aumento da sensibilidade
da região RVLM a neurotransmissores excitatórios observado nos animais adultos,
mesmo após a retirada da dieta hipersódica.
Em nosso estudo, observamos ainda que as respostas cardiovasculares
induzidas por sobrecarga de sal são bastante pronunciadas após o tratamento por 60
dias enquanto que não são observadas após o tratamento por 30 dias. Desta forma,
não podemos determinar se o estabelecimento destas alterações ocorre nos 30
últimos dias de tratamento (do 51º ao 81º dia de vida, fase que corresponde à
adolescência e início da fase adulta) (207) ou se são resultado da exposição
prolongada ao sal (desde a infância até a fase adulta). Assim, a padronização de um
protocolo que se estenda da adolescência à fase adulta (do 51º ao 81º dia de vida)
59
pode auxiliar na compreensão da participação das diversas fases da vida no
desenvolvimento de doenças cardiovasculares induzido por ingestão excessiva de sal.
Em relação aos animais tratados com solução de sacarose 20% por 30 dias
na fase adulta observamos maior ingestão de solução de sacarose e menor ingestão
de ração em comparação com o grupo controle. Observamos também que a
sobrecarga de sacarose promove elevação de PAM e da FC nos animais adultos,
além de diminuir a sensibilidade barorreflexa. Estes animais apresentam ainda
elevação da resposta pressórica induzida pela estimulação de quimiorreceptores
periféricos.
Estudos de Sheludiakova et al. (2012)(145) demonstram que uma dieta rica
em sacarose promove uma série de alterações metabólicas que elevam o risco de
desenvolver diabetes, doença hepática gordurosa não alcoólica e DCV mesmo com a
manutenção do peso corporal. Corroborando com estudos anteriores (135), no
presente estudo, os animais tratados apresentaram diminuição da ingesta de ração,
de modo que a ingestão calórica total também se mostrou diminuída em comparação
com o grupo controle. Assim, os animais submetidos a sobrecarga de sacarose não
apresentaram elevação de peso corporal em comparação com o grupo controle,
entretanto apresentaram elevação de PA e alterações de sensibilidade nos reflexos
baro e quimiorreceptor, fatores contribuintes para o desenvolvimento de DCV.
Estudos demonstram que a sobrecarga de sacarose induz aumento do tecido
adiposo visceral (127,143), que está diretamente relacionado com o aumento da ANS
(208–212). O tônus vasomotor simpático é um importante determinante da resistência
vascular periférica que, por sua vez, tem influência direta sobre os valores de PA (213–
216). Assim, a elevação da ANS contribui para a elevação da PA observada em
modelos de obesidade. Neste sentido, diversos estudos demonstram a existência de
um reflexo simpatoexcitatório originado no tecido adiposo, o reflexo adiposo aferente
(RAA) (159,161,163), desencadeado especialmente por citocinas (capsaicina,
bradicinina, adenosina ou leptina) neste tecido (159,161). A utilização de marcadores
anterógrados demonstrou que as fibras aferentes que partem do tecido adiposo
alcançam o SNC em regiões como o NTS, o PVN e o RVLM (217). Acredita-se que as
neurotransmissões glutamatérgica e gabaérgica no PVN participem do RAA, uma vez
60
que o bloqueio da neurotransmissão glutamatérgica abole o reflexo (218) enquanto
que o bloqueio da neurotransmissão gabaérgica intensifica o reflexo (219). Neste
contexto, o aumento da ANS observada em modelos de obesidade pode estar
diretamente relacionado com o aumento do tecido adiposo e com a intensidade do
RAA. Em nosso estudo, observamos elevação de PA, que pode estar relacionada com
o aumento da adiposidade induzida pela sobrecarga de sacarose, como demonstrado
por Coelho et al. (2010)(143).
Como mencionado anteriormente, o SRA é um importante mecanismo de
controle da PA e alterações de expressão de seus componentes podem estar
relacionados à patofisiologia da hipertensão arterial. Estudos de Boustany et al. (2004)
(220) demonstram que animais obesos apresentam aumento da atividade do SRA
sistêmico e do tecido adiposo, sendo que este aumento contribui para a hipertensão
arterial observada nestes animais. Segundo Coelho et al. (2010)(143) animais adultos
submetidos a protocolo similar ao conduzido no presente estudo apresentam elevação
de triacilgliceróis, insulina, leptina, renina e Ang II plasmáticos e ainda alterações na
expressão e na atividade de outros componentes do SRA, que ocorrem especialmente
no tecido adiposo e nos rins.
Diversos autores sugerem a hipótese de que o aumento da Ang II plasmática
esteja diretamente envolvido na elevação da ANS observada em diversos modelos de
obesidade e síndrome metabólica (162,170,221). A Ang II sistêmica pode atuar em
órgãos circunventriculares, promovendo a produção de espécies reativas de oxigênio,
que contribuem para a excitação simpática, atuando no SFO e OVLT (220), e para a
diminuição da sensibilidade barorreflexa, atuando na Área Postrema (AP) (221–223)
na obesidade. Ademais, a elevação de ANS induzida pela Ang II promoveria efeitos
órgão-específicos que associados aos efeitos da Ang II em cada órgão, são
característicos da síndrome metabólica, tais como elevação da produção de insulina
pelo pâncreas, da gliconeogênese hepática, da lipólise e da produção de leptina pelo
tecido adiposo, além dos conhecidos efeitos vasoconstritores deste peptídeo. Em
conjunto, estes fatores podem contribuir para o aumento de PA e a diminuição
barorreflexa observados em nosso modelo de sobrecarga de sacarose.
61
A leptina é uma citocina produzida pelo tecido adiposo e que tem sido
apontada como o elo entre a obesidade e o desenvolvimento de DCV (226–230). Sua
função principal está relacionada ao metabolismo de glicose e à regulação do
metabolismo em geral, uma vez que este hormônio informa o SNC acerca das
reservas energéticas periféricas (231–234). Diversos estudos relacionam o aumento
da leptina plasmática com a elevação da ANS observada em modelos de obesidade
(235,236). Estudos indicam que infusões sistêmicas de leptina elevam a ANS (237) e,
ainda, infusões centrais deste peptídeo aumentam a PA (238). Além disso, Lim et al.
(2013) (239) demonstraram que tal antagonismo promoveu diminuição de PA e a ANS
renal em coelhos obesos. Tais evidências nos permitem sugerir que a elevação da
leptina plasmática pode contribuir para a hipertensão arterial observada em nosso
modelo sobrecarga de sacarose.
Outro componente importante no aumento da ANS observado na obesidade
é o processo inflamatório bastante expressivo observado nesta condição (240–243).
Os adipócitos, em condições normais, produzem citocinas pró-inflamatórias (leptina,
IL-6, IL-12, IL-18, TNFα, IL-8 e CCL2/MCP-1, entre outras) e anti-inflamatórias
(adiponectina, proteínas C1q/TNF-relacionadas (CTRPs), omentina, entre outras)
importantes, que atuam no controle da PA, da homeostase energética, das funções
reprodutivas e ainda das respostas imunes, entre outras funções fisiológicas (241).
Em indivíduos com função metabólica normal existe equilíbrio entre a produção de
citocinas pró- e anti-inflamatórias. Á medida que o tecido adiposo aumenta durante o
desenvolvimento da obesidade, os adipócitos hipertrofiam, de modo que a produção
de citocinas pró-inflamatórias aumente, enquanto que a produção de citocinas anti-
inflamatórias diminua (241,244,245). Nesta situação, a infiltração de macrófagos no
tecido adiposo, juntamente com a proliferação e ativação de células do sistema imune
residentes neste tecido, promove o estabelecimento de um quadro inflamatório do
tecido adiposo (246). Além disso, as citocinas pró-inflamatórias alteram o padrão de
fosforilação do receptor de insulina após a ligação com o peptídeo, de modo que todas
as reações intracelulares decorrentes desta ligação ficam comprometidas, gerando
resistência à insulina no tecido adiposo (114,243,247). As citocinas pró-inflamatórias
produzidas pelo adipócito são transportadas pela corrente sanguínea por todo o corpo,
62
promovendo um estado global de inflamação em indivíduos obesos. O estado
inflamatório, especialmente no SNC e no fígado, promove também a resistência à
insulina nestes órgãos (118,243,248–250).
Além disso, a inflamação associada com o aumento do estresse oxidativo de
regiões do SNC, como o NTS e o RVLM, entre outras regiões, pode estar relacionado
com a elevação da ANS, contribuindo para a elevação de PA observada em modelos
de obesidade (170,173,251–254). De fato, estudos de Wei et al. (2015) (255)
demonstram que a injeção de TNFα ou IL-1β no SFO promove elevação de PA,
frequência cardíaca e ANS renal, sugerindo que as citocinas pró-inflamatórias
sistêmicas podem agir neste núcleo elevando mediadores inflamatórios e excitatórios
e culminando em excitação simpática. De fato, estudos de Beilharz et al. (2016) (256)
demonstram que uma dieta suplementada com açúcar, mesmo por curta duração,
pode induzir a produção de marcadores de inflamação central e periférica, com
elevação de citocinas pró-inflamatórias (TNF e IL-1) no hipocampo e no tecido
adiposo, o que pode ativar diversas vias de sinalização inflamatória. Uma vez que as
respostas barorreflexas são dependentes do sistema nervoso autônomo, as
alterações do componente simpático induzidas pela inflamação podem estar
associada com disfunções baroreflexas, como a observada em nosso estudo. Novas
análises do perfil inflamatório dos animais submetidos a sobrecarga de sacarose em
nosso estudo são necessários para avaliar a influência do processo inflamatório no
quadro cardiovascular e autonômico do modelo animal utilizado.
Em conjunto, alterações do RAA, da Ang II plasmática, da leptina e das
citocinas inflamatórias circulantes podem contribuir para a elevação da ANS na
obesidade, culminando na elevação da PA e da FC observadas em nosso modelo de
sobrecarga de sacarose.
Skrapari et al. (2007) (257) demonstraram que a sensibilidade barorreflexa
está comprometida em mulheres obesas. Neste estudo, os autores sugerem que o
índice de massa corporal (IMC, importante indicativo de obesidade) e a idade são,
nesse caso, determinantes da diminuição da sensibilidade barorreflexa (257).
Entretanto, Lazarova et al. (2007)(258) demonstram que a sensibilidade do reflexo
barorreceptor está diminuída em crianças e adolescentes obesos normotensos,
63
indicando que a adiposidade pode ser um fator mais importante na disfunção
barorreflexa. De fato, estudo de Alvarez et al. (2005) (259) demonstrou que a perda
de peso aumenta a sensibilidade barorreceptora em homens sobrepesados e obesos
e sugere ainda que o desbalanço simpato-vagal presente na obesidade contribua para
o comprometimento deste reflexo. Em nosso estudo, os animais submetidos a
sobrecarga de sacarose apresentam elevação da PA e, provavelmente, aumento da
adiposidade visceral e da Ang II plasmática, como demonstrado por Coelho et al.
(2010) (143), fatores estes que são determinantes para a diminuição da sensibilidade
do reflexo barorreceptor observada em nosso estudo.
Estudos de Porzionato et al. (2011) (260) demonstram que diversas isoformas
de leptina e receptores de leptina são expressos no corpúsculo carotídeo de ratos e
humanos. Assim, seria lícito supor que em modelos de obesidade e síndrome
metabólica, que apresentam níveis elevados de leptina plasmática, a sensibilidade
dos quimiorreceptores periféricos esteja aumentada pela ação desta substância no
corpúsculo carotídeo. Neste sentido, estudos de Paleczny et al. (2016)(261)
demonstraram que, em homens saudáveis, o sobrepeso ou a obesidade estão
acompanhados de um aumento da resposta pressora após estimulação
quimiorreflexa, enquanto que as respostas ventilatórias e de frequência cardíaca não
se alteram. Entretanto, os autores demonstraram que a hiperinsulinemia e a
resistência à insulina estão relacionadas à potencialização desta resposta, enquanto
que os níveis de leptina plasmática parecem não ter influência nos parâmetros
analisados. De modo similar, no presente estudo, observamos que os animais
submetidos a sobrecarga de sacarose apresentam elevação da resposta pressora
induzida pela estimulação de quimiorreceptores periféricos, enquanto que a reposta
bradicárdica não é alterada. Estudos anteriores demonstraram que a sobrecarga de
sacarose promove elevação de insulina e de leptina plasmática (143), fatores estes
que podem estar relacionado ás alterações de resposta pressora quimiorreflexa
observadas em nosso estudo.
CONCLUSÕES E PERSPECTIVAS
Nossos resultados demonstram que uma dieta rica em sal ou em açúcar

promove efeitos marcadamente nocivos ao aparato cardiovascular. Mais
64
especificamente, os resultados permitem concluir que modificações da ingestão de sal
ou de sacarose produzem alterações permanentes no controle da pressão arterial,
sendo estas alterações, pelo menos em partes, desencadeadas por modificações na
regulação neural da circulação sanguínea.
Uma vez que o consumo de alimentos industrializados, ricos nas duas
substâncias, tem aumentado exponencialmente, especialmente nas sociedades
ocidentais, entender seus efeitos sobre o organismo e alertar a população acerta
destes se torna de grande importância. Além disso, a ingestão destes alimentos se
inicia logo na primeira infância, de modo que o desenvolvimento de um protocolo de
sobrecarga de sacarose especificamente durante o período pós-natal pode contribuir
para a compreensão da participação desta fase da vida no desenvolvimento de
diabetes tipo II, obesidade e hipertensão arterial na fase adulta. A padronização de tal
protocolo pode contribuir também para a realização de um paralelo entre os efeitos da
sobrecarga de sal, já demonstrados anteriormente, e os efeitos da sobrecarga de
açúcar após o desmame sobre parâmetros cardiovasculares, autonômicos e
metabólicos na fase adulta.
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89
APÊNDICE I
Tabela de conteúdo de sódio (mg/porção) e de açúcares (g/porção) em

diversos produtos industrializados.
Sódio (mg/porção) Açúcares (g/porção)
Refrigerante tipo cola 10 21,1

(200ml)
Refrigerante tipo cola 28 -
zero açúcar (200ml)
Macarrão instantâneo 1607 -
(85g)
Chips de batata 129 -
ondulada (25g)
Achocolatado (20g) 28 18
Salsicha (50g) 625 -
Sorvete sabor flocos 39 16

(50g)
Chocolate ao leite 22 14
(25g)
Creme de avelã (15g) 20 8,5
Cheeseburger 726 -
(136,5g)
90
APÊNDICE II
Artigos publicados, aceitos ou submetidos para publicação. Os trabalhos aqui

apresentados são produto da pesquisa desenvolvida nesta tese.
91
British Journal of Nutrition (2014), 112, 1923–1932 doi:10.1017/S0007114514002918
q The Authors 2014
High sodium intake during postnatal phases induces an increase in arterial

blood pressure in adult rats
M. C. S. Moreira1, E. F. da Silva1, L. L. Silveira2, Y. B. de Paiva1, C. H. de Castro1, A. H. Freiria-Oliveira1,

D. A. Rosa1, P. M. Ferreira1, C. H. Xavier1, E. Colombari3 and Gustavo R. Pedrino1*
1
Department of Physiological Sciences, Center for Neuroscience and Cardiovascular Physiology,
Universidade Federal de Goiás, Goiânia, Goiás, Brazil
2
Department of Physiology, School of Medicine of Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto,
São Paulo, Brazil
3
Department of Physiology and Pathology, School of Dentistry, Universidade Estadual Paulista, Araraquara,
São Paulo, Brazil
(Submitted 12 March 2014 – Final revision received 30 July 2014 – Accepted 19 August 2014 – First published online 27 October 2014)
British Journal of Nutrition
Abstract
Epigenetic studies suggest that diseases that develop in adulthood are related to certain conditions to which the individual is exposed during
the initial stages of life. Experimental evidence has demonstrated that offspring born to mothers maintained on high-Na diets during preg-
nancy have higher mean arterial pressure (MAP) in adulthood. Although these studies have demonstrated the importance of prenatal phases
to hypertension development, no evidence regarding the role of high Na intake during postnatal phases in the development of this pathology
has been reported. Therefore, in the present study, the effects of Na overload during childhood on induced water and Na intakes and on
cardiovascular parameters in adulthood were evaluated. Experiments were carried out in two groups of 21-d-old rats: experimental
group, maintained on hypertonic saline (0·3 M- NaCl) solution and food for 60 d, and control group, maintained on tap water and food.
Later, both groups were given water and food for 15 d (recovery period). After the recovery period, chronic cannulation of the right femoral
artery was performed in unanaesthetised rats to record baseline MAP and heart rate (HR). The experimental group was found to have
increased basal MAP (98·6 (SEM 2·6) v. 118·3 (SEM 2·7) mmHg, P, 0·05) and HR (365·4 (SEM 12·2) v. 398·2 (SEM 7·5) beats per min,
P, 0·05). There was a decrease in the baroreflex index in the experimental group when compared with that in the control group. A water
and Na intake test was performed using furosemide. Na depletion was found to induce an increase in Na intake in both the control and
experimental groups (12·1 (SEM 0·6) ml and 7·8 (SEM 1·1), respectively, P , 0·05); however, this increase was of lower magnitude in the
experimental group. These results demonstrate that postnatal Na overload alters behavioural and cardiovascular regulation in adulthood.
Key words: Hypertension: Water intake: Sodium intake: Postnatal period
The maintenance of a stable internal environment is the main The regulation of blood pressure (BP) involves com-
target of all physiological processes(1,2), which is positively plex mechanisms, including local, hormonal, neuronal and
correlated with the regulation of ionic concentrations in renal regulation, that, working together, are responsible for
the intracellular and extracellular compartments. Among the the redistribution of blood through changes in peripheral
different types of inorganic salts present in the body fluids, vascular resistance and cardiac output. Experimental evidence
NaCl is the most predominantly consumed salt and Na con- has demonstrated that acute increases in plasma Na concen-
centration is directly related to the maintenance of body fluid trations trigger a set of autonomic(3 – 6), humoral(7 – 12) and
homeostasis(1,2). Changes in Na concentrations result in an renal responses. These adjustments comprise a set of inte-
osmotic flux between the intracellular and extracellular com- grated responses aimed at promoting increases in BP(13 – 15),
partments. Na influx or efflux affects the concentrations of all the renal vasodilation(13,14,16,17) and consequently natriuresis,
other components in these compartments. Therefore, it is not re-establishing normal volaemic conditions.
surprising that many homeostatic mechanisms exist to main- Dysfunctions in the control of plasma Na concentrations can
tain plasma Na concentrations with a limited rate of variation. lead to pathologies, among which hypertension predominates.
Abbreviations: BI, baroreflex index; BP, blood pressure; bpm, beats per min; FURO, furosemide; HR, heart rate; HS, hypertonic saline; MAP, mean arterial
pressure; RBF, renal blood flow; RVC, renal vascular conductance.
* Corresponding author: G. R. Pedrino, fax þ55 62 3521 1774, email pedrino@pq.cnpq.br, gpedrino@gmail.com
1924 M. C. S. Moreira et al.
In fact, epidemiological and experimental evidence has Experimental design

shown that a high-Na diet is a significant risk factor for the
The experimental group (high-Na diet) was provided hyper-
development of arterial hypertension(18,19).
tonic saline (HS; 0·3 M -NaCl; Sigma-Aldrich) for drinking
Recently, a group of researchers have described an
instead of water, while the control group (normal-Na diet)
experimental model for inducing hypertension, combining
was provided tap water. Both groups were given food regu-
increases in angiotensin II concentrations and Na circulation
larly throughout the experiments. High-Na diet feeding was
(angiotensin II salt hypertension) (‘Neurogenic Cardio-
started on the 21st day of life and was stopped after 60 d.
vascular Diseases Consortium’). In this model, normotensive
After this treatment, tap water was provided to both groups
animals are maintained on high-NaCl diets (2 % NaCl) and
for 15 d (recovery period).
subjected to chronic subcutaneous angiotensin II adminis-
tration to induce an increase in BP levels in 3 d(20,21). These
same researchers further demonstrated that subpressor doses Determination of plasma and urinary sodium
of angiotensin II do not lead to significant changes in concentrations and plasma osmolarity
BP, thus requiring the combination of a high-Na diet to
induce hypertension. Consistent with these findings, experi- During the last week of the treatment and recovery periods, rats
mental studies(18,19) have demonstrated a positive correlation were individually acclimatised in metabolic cages with free
between salt consumption and BP elevation. access to their respective drinking supplies to enable urine
Some epigenetic studies suggest that diseases developed collection and urinary volume measurement. The average
in adulthood are related to certain conditions to which the values of the 24 h urinary volume of the control and experi-
mental groups were calculated and recorded during the last
individual is exposed during the initial stages of life, including

prenatal phases. Bao et al.(22) demonstrated that the risk of 7 d of the treatment and recovery periods. Blood and urine
developing hypertension during adulthood is related to BP samples (0·5 ml each) were collected from control and experi-
levels during the initial stages of life. Li et al.(23) showed that mental rats at the end of the treatment and recovery periods.
high BP levels during childhood are positively associated To determine the haematocrit, blood samples were collected
with systolic and diastolic BP levels in later life. In addition, with the aid of a glass capillary and centrifuged. Blood samples
Vidonho et al.(24) found offspring born to mothers maintained were centrifuged for 5 min at 6000 g. Plasma was separated
on high-Na diets during pregnancy and lactation to have and stored at 2 208C. Plasma and urinary Na concentrations
higher BP levels in adulthood. were measured using a flame photometer (model 910M;
Although several studies have demonstrated the importance Analyser). Plasma osmolality was measured as freezing point
of prenatal phases to hypertension development, no study depression (Micro-Osmette 5004; Precision Systems).
has been carried out to evaluate the contribution of postnatal
phases to the development of this pathology. Therefore, Water and sodium intake test
the aim of the present study was to evaluate the effects
of increased Na consumption in postnatal phases on mean Adult rats from the experimental and control groups were sub-
arterial pressure (MAP) and heart rate (HR), barosensitivity, mitted to a water and Na intake test using furosemide (FURO).
changes in water and Na intakes induced by Na depletion, To carry out this test, after the last week of the recovery
and haemodynamic changes induced by hypernatraemia period, rats were maintained in metabolic cages over a 24 h
in adult rats. period, with free access to water and 0·3 M -NaCl in graduated
burettes. After this period, rats were administered a sub-
cutaneous dose of FURO (10 mg/kg body weight; Lasixw,
Sanofi Aventis). Then, food was removed from the cages
Materials and methods and rats were given access to water to prevent dehydration.
After 24 h of FURO administration, HS and water were pro-
Animals vided in graduated burettes and their intakes were measured
Experiments were carried out in 21-d-old male Wistar rats, every 30 min over the course of 2 h. Rats were not given
obtained from the Central Animal House of the Universidade access to food throughout the test period.
Federal de Goiás. In total, thirty rats were used as control
animals and twenty-eight rats from four litters were used as
Mean arterial pressure and heart rate recordings in
experimental animals. Rats were randomly selected from
unanaesthetised animals
each litter in the control and experimental groups. Rats were
housed in an acclimatised room (22– 248C) and given free After the recovery period, rats were anaesthetised with keta-
access to food and water as described previously (0·4 % mine (116 mg/kg body weight, intraperitoneal; Sespo) and
NaCl; AIN-93M(25)). All experimental and surgical procedures xylazine (20 mg/kg body weight, intraperitoneal; Rhobifarma)
were approved by the Institutional Animal Care and Use and submitted to a surgical procedure to implant a polyethy-
Committee of the Universidade Federal de Goiás (process lene (PE) tube (PE-10 connected to a PE-50) in the abdominal
no. 051/2010) and were performed in strict accordance with aorta through the right femoral artery for recording BP. The
the National Institutes of Health Guide for the Care and Use right femoral vein was catheterised for drug administration
of Laboratory Animals. (phenylephrine and sodium nitroprusside), and the cannulas
High sodium intake and hypertension in rats 1925
were led subcutaneously to exit between the scapulae. A pro- retroperitoneal incisions, miniature probes (Transonic Sys-
phylactic antibiotic was administered (penicillin, 60·000 IU/kg tems, Inc.) were implanted around the renal artery to record
body weight, intramuscular; Sigma-Aldrich), and rats were renal blood flow (RBF).
kept in individual cages. Later, 24 h after the surgery (recovery RBF was recorded by transit-time flowmetry. A miniature
period described as a standard procedure by several probe (1RB; Transonic Systems, Inc.) was implanted around
authors(26 – 30)), the pulsatile arterial pressure was obtained the left renal artery and attached to a flowmetry T206 device
by connecting the arterial cannula to a pressure transducer (Transonic Systems, Inc.) to determine the flow in absolute
attached to an amplifier (ETH-200; CB Sciences). Pulsatile values (ml/min). The signals obtained were sent to the data
pressure was recorded continuously with a PowerLab acquisition and analysis PowerLab System (ADInstruments).
System device (ADInstruments). MAP and HR were deter- Data were digitised at 1000 samples/s. Changes in the RBF
mined from the pulsatile signal using Chart software (version were calculated as a percentage of the baseline value
5.5.6; ADInstruments). The MAP and HR recordings were (%RBF). Renal vascular conductance (RVC) was determined
performed in unanaesthetised animals, with free movement as the ratio of RBF:MAP. RVC changes were calculated as a
within their cages, for 60 min. At the end of the baseline percentage of the baseline value (%RVC).
recordings of MAP and HR, the baroreflex test was performed. Na overload was induced through HS infusion (3 M -NaCl;
Sigma-Aldrich(9)). The infusion was realised by a cannula
implanted into the right atrium through the right jugular vein.
Baroreflex test
Administration was performed for 60 s, at a dose of 1·8 ml/kg
After the recovery period, to evaluate baroreflex integrity, body weight. Changes in MAP, HR, RBF and RVC were recorded
intravenous infusion (0·1 ml) of phenylephrine (10, 15 and for 10 min before and 60 min after the HS infusion.
20 mg/ml; adrenergic agonist; Sigma-Aldrich) and sodium
nitroprusside (100, 150 and 200 mg/ml; nitric oxide donor;
Data analysis
Sigma-Aldrich) was performed after the baseline recordings
of MAP and HR. The baroreflex index (BI) was evaluated as a The GraphPad Prism 5 software was used for performing the
mean index, calculated as the ratio of changes in HR:changes data analysis and drawing the graphs. In all the experiments,
in MAP induced by phenylephrine and sodium nitroprusside the independent variable was the treatment and the depen-
infusions. The mean index is expressed as beats per min dent variable was the measured response. The results are
(bpm) per mm of mercury, as described previously(20,31). expressed as means with their standard errors. Daily water,
HS and food intakes, plasma and urinary Na concentrations,
urinary flow, Na excretion rate and osmolality were analysed
Determination of haemodynamic changes induced by
using a one-way ANOVA. The baseline values of MAP, HR,
acute hypernatraemia
BI and haemodynamic parameters were analysed using the
After anaesthetic induction with halothane (2 %; Cristália Ltda) unpaired t test. The weekly average values of water/saline/
in 100 % O2, polyethylene tubes were inserted into the food intakes and body weight, induced water and Na intakes,
femoral artery and vein for MAP recordings and venous and variations in MAP, HR, %RBF and %RVC induced by HS
infusions, respectively, as described above. The anaesthesia infusion were analysed using a two-way repeated-measures
was maintained with urethane (1·2 g/kg body weight, intra- ANOVA followed by Fisher’s least significant difference
venous; Sigma-Aldrich). After vascular cannulation, tracheo- post hoc test. A P value ,0·05 was considered to denote
stomy was performed to reduce airway resistance. Through a significant difference.
Table 1. Daily water or hypertonic saline intake, food intake, body weight, urinary volume, urinary sodium concentration, urinary flow,
sodium excretion rate, plasma sodium concentration, plasma osmolarity and haematocrit of control and experimental rats during the
treatment and recovery periods
(Mean values with their standard errors)
Treatment period Recovery period
Control Experimental Control Experimental
Mean SEM Mean SEM Mean SEM Mean SEM
Water/hypertonic saline intake (ml) 36·6 1·6 116·7* 26·2 41·0 2·7 39·0 2·2
Food intake (g) 23·3 0·8 22·4 1·9 23·0 0·6 23·0 0·9
Body weight (g) 303·6 6·9 193·8* 18·8 335·0 8·6 244·0* 12·5
Urinary volume (ml) 7·5 4·1 98·7* 36·6 8·6 4·5 9·3 2·6
Urinary Na concentration (mM ) 45·0 5·5 153·6* 13·1 37·5 3·1 52·5 9·1
Urinary flow (ml/h) 0·31 0·17 5·43* 1·16 0·36 0·18 0·39 0·11
Na excretion rate (mmol/h) 0·015 0·005 0·85* 0·21 0·015 0·006 0·017 0·003
Plasma Na concentration (mM ) 135·7 11·3 141·7 4·6 148·5 9·2 144·8 15·2
Plasma osmolarity (mOsm) 302·8 4·7 303·4 7·6 302·7 4·0 301·0 4·5
Haematocrit (%) 43·1 0·8 41·4 1·3 44·2 1·3 45·9 0·7
* Mean value was significantly different from that of the control group in the respective period (P, 0·05).
Results during the recovery period (experimental: 52·5 (SEM 9·1) mM ;

control: 37·5 (SEM 3·1) mM ; Table 1).
Daily food, water and hypertonic saline intakes of the
control and experimental groups during the treatment
and recovery periods Effects of high sodium intake during postnatal phases
on plasma sodium concentrations, plasma osmolarity
At the end of the treatment period, during which HS solu-
and haematocrit
tion was provided to the experimental group and tap water
was provided to the control group, the daily fluid intake of As can be seen from Table 1, there were no differences
experimental rats (n 11; 116·7 (SEM 26·2) ml, P,0·05) was sig- in plasma Na concentrations between the control and
nificantly higher than that of control rats (n 12; 36·6 (SEM 1·6); experimental groups, either during the treatment period
Table 1). At the end of the recovery period, during which (135·7 (SEM 11·29) mM (n 7) v. 141·7 (SEM 4·61) mM (n 6),
tap water was provided to both groups, no difference was respectively) or during the recovery period (148·5 (SEM
observed in daily water intake between experimental rats 9·19) mM (n 7) v. 144·8 (SEM 15·23) mM (n 6), respectively).
(39·0 (SEM 2·2) ml) and control rats (41·0 (SEM 2·7); Table 1). Similarly, the plasma osmolarity values of control and experi-
At the end of the treatment and recovery periods, the daily mental rats were similar both during the treatment period
food intake values of both groups were similar (Table 1).
However, the body weight of experimental rats was lower (a) 150 *
than that of control rats at the end of the treatment period
*
Daily intake (ml)

(193·8 (SEM 18·8) v. 303·6 (SEM 6·9) g, respectively, P, 0·05;
* *
Table 1) and at the end of the recovery period (244·0 (SEM 100
12·5) v. 335·0 (SEM 8·6) g, respectively, P, 0·05; Table 1). * * *
Consistent with the results given in Table 1, Fig. 1 demon- *
strates that the weekly HS intake of experimental rats was 50
higher than the weekly water intake of control rats during
the treatment period. However, during the recovery period,
0
the absolute values of water intake of both groups were 0 1 2 3 4 5 6 7 8 9 10
similar. There was a decrease in the weekly food intake of
the experimental group over the treatment period when com- Treatment Recovery
pared with that of the control group. This was not observed Time (weeks)
during the recovery period. The average values of the (b) 25
weekly body weight of experimental rats were lower than
those of the weekly body weight of control rats during the 20
Daily intake (g)
treatment and recovery periods.

15
The values of water/HS intake, food intake, urinary volume, *
urinary flow and Na excretion rate per 100 g body weight 10 * * *
*
of the control and experimental groups during the treatment *
and recovery periods are given in Table 2. The absolute 5
*
values of Na excretion rate and daily water and food intakes
0
of experimental rats were similar to those of control rats 0 1 2 3 4 5 6 7 8 9 10
during the recovery period (Table 1). Experimental rats exhi-
Treatment Recovery
bited increases in water and food intakes and Na excretion Time (weeks)
rate per 100 g body weight when compared with control rats (c) 350
during the recovery period.
300
Body weight (g)
250
Effects of high sodium intake during postnatal phases on
200 *
urinary volume and sodium concentrations *
150
* *
Significant differences were observed in diuresis between 100 * *
control (n 6) and experimental (n 6) rats during the treat- *
50 *
ment period (7·5 (SEM 4·1) v. 98·7 (SEM 36·6) ml, respectively,
P, 0·05; Table 1). However, these differences were not 0
0 1 2 3 4 5 6 7 8 9 10
observed during the recovery period (control: 8·6 (SEM 4·5) ml
v. experimental: 9·3 (SEM 2·6) ml, respectively; Table 1). Treatment Recovery
The urinary Na concentrations of experimental rats were Time (weeks)
significantly higher than those of control rats during the Fig. 1. Average values of weekly water or hypertonic saline intake (a) and
food intake (b) and body weight (c) during the treatment and recovery
treatment period (153·6 (SEM 13·1) v. 45·0 (SEM 5·5) mM ,
periods. Values are means, with their standard errors represented by vertical
respectively, P,0·05; Table 1). These differences in urinary bars. * Mean value was significantly different from that of the control (X)
Na concentrations between the groups were not observed group (P, 0·05). W, Experimental group.
Table 2. Values of water/hypertonic saline intake, food intake, urinary volume, urinary flow and sodium excretion rate per 100 g body weight
of control and experimental rats
Treatment period Recovery period
Control Experimental Control Experimental
Mean SEM Mean SEM Mean SEM Mean SEM
Water/hypertonic saline intake (ml/100 g) 11·68 0·50 78·48* 21·61 12·23 0·87 16·93* 1·85
Food intake (g/100 g) 7·44 0·25 12·56* 1·35 6·93 0·13 9·88* 0·77
Urinary volume (ml/100 g) 2·55 1·35 119·5* 23·84 2·60 1·40 3·62 0·99
Urinary flow (ml/h per 100 g) 0·10 0·06 3·76* 0·93 0·11 0·058 0·15 0·04
Na excretion rate (mmol/h per 100 g) 0·0047 0·0017 0·59* 0·17 0·0045 0·002 0·0078* 0·001
* Mean value was significantly different from that of the control group in the respective period (P, 0·05).
(302·8 (SEM 4·67) mOsm (n 6) v. 303·4 (SEM 7·63) mOsm (n 6), Phenylephrine infusion resulted in a BI of 21·20 (SEM
respectively) and during the recovery period (302·7 (SEM 0·2) bpm/mmHg in experimental rats and a BI of 21·83 (SEM
4·14) mOsm (n 6) v. 301·0 (SEM 4·53) mOsm (n 6), respect- 0·04) bpm/mmHg in control rats (P,0·05; Fig. 4(a)). Sodium
ively). No differences were observed in the haematocrit nitroprusside infusion resulted in a BI of 0·85 (SEM
between the groups either during the treatment period (con- 0·22) bpm/mmHg in experimental rats and a BI of 2·12 (SEM
trol: 43·1 (SEM 0·8) % (n 8) v. experimental: 41·4 (SEM 1·3) % 0·2) bpm/mmHg in control rats (P,0·05; Fig. 4(b)).
(n 9)) or during the recovery period (control: 44·2 (SEM
1·3) % (n 8) v. experimental: 45·9 (SEM 0·7) % (n 9)).
Effects of high sodium intake during postnatal phases on
cardiovascular adjustments induced by acute sodium
Effects of high sodium intake during postnatal phases on overload
changes in water and sodium intakes induced by sodium
The baseline values of cardiovascular parameters of anaesthe-
depletion
tised control and experimental rats are given in Table 3.
The FURO treatment induced changes in water intake in The values of MAP, RBF and RVC were similar in both
both groups (Fig. 2(a)). However, the experimental group
(n 9) exhibited greater water intake than the control group
(n 9) (10 (SEM 1·2) v. 8·4 (SEM 0·8) ml after 120 min, respec- (a) 15
tively, P, 0·05; F ¼ 0·5172). As expected, Na depletion
Cumulative water
*†
induced an increase in Na intake in both the control group *† *†
intake (ml)
10
(12·1 (SEM 0·6) ml after 120 min; Fig. 2(b)) and the experimen-
*†
tal group (7·8 (SEM 1·1) ml after 120 min, P,0·05; F ¼ 4·016;
* *
Fig. 2(b)). However, the increase in Na intake induced by 5 *
Na depletion was of lower magnitude in the experimental
*
group (Fig. 2(b)).
0
0 30 60 90 120
Effects of high sodium intake during postnatal phases on Time (min)
the baseline values of mean arterial pressure and heart rate
in unanaesthetised animals (b) 15
NaCl 0·3 M intake (ml)
*
Unanaesthetised experimental rats (n 6) had higher MAP * *
*
Cumulative
(121·7 (SEM 7·3) mmHg, P,0·05; Fig. 3(a)) and HR (408·1 10

(SEM 7·5) bpm, P,0·05; Fig. 3(b)) levels when compared with
unanaesthetised control rats (n 6; MAP: 98·6 (SEM 3·0) mmHg
5 *† *†
and HR: 372·7 (SEM 12·4) bpm; Fig. 3). These results clearly
*† *†
indicate that high Na intake during postnatal phases induces
a chronic increase in MAP in unanaesthetised rats. 0
0 30 60 90 120
Time (min)
Effects of high sodium intake during postnatal phases
Fig. 2. Cumulative water intake (a) and 0·3 M -sodium intake (b) in response
on baroreflex index to subcutaneous administration of furosemide (10 mg/kg body weight) in con-
trol (X) and experimental (W) rats. Values are means, with their standard
Both phenylephrine and sodium nitroprusside infusions
errors represented by vertical bars. * Mean value was significantly different
induced a decrease in the BI of experimental rats (n 8) from that recorded at time 0 (P, 0·05). † Mean value was significantly differ-
when compared with that of control rats (n 9) (Fig. 4). ent from that of the control group (P, 0·05).
(a) 150 while decreasing baroreflex sensitivity; (2) an increase in Na

* intake during the postnatal period causes hypersensitivity to
MAP (mmHg)
Na appetite induced by Na depletion as well as a lesser
100
renal vasodilation induced by hypernatraemia; (3) high Na
intake promotes an increase in diuresis, natriuresis and fluid
50 intake during treatment; (4) high Na intake during postnatal
phases promotes an increase in natriuresis and water and
food intakes in need-free conditions in adult male rats.
0
Salt-dependent hypertension is strongly dependent on sym-
pathoexcitation(32). Moreover, lesions in hypothalamic regions
(b) 500
related to cardiovascular and hydroelectrolytic regulation,
* such as the anteroventral region of the third ventricle, subfor-
400
nical organ and paraventricular nucleus of the hypothalamus,
HR (bpm)
300
prevent or reduce increases in arterial pressure observed in
200 salt-dependent hypertension models(33). Another study has
demonstrated that an increase in salt consumption potentiates
100
sympathetic excitation and elevates arterial pressure by
0 stimulating glutamatergic input to the rostral ventrolateral
Fig. 3. Mean arterial pressure (MAP) (a) and heart rate (HR) (b) in unanaes- medulla(34). The constant stimulation of these regions by Na
thetised rats from the control (B) and experimental (A) groups. Values are overload leads to the stimulation of the rostral ventrolateral
means, with their standard errors represented by vertical bars. * Mean value
was significantly different from that of the control group (P, 0·05). bpm,
medulla, resulting in increased sympathetically mediated vaso-
Beats per min. motion and in pressor responses similar to those observed in
the present study. Thus, chronic Na overload could change
groups, while the HR value was significantly higher in the intrinsic neuronal properties and, consequently, increase
experimental group. the excitation of preganglionic sympathetic neurons(34). It is
Na overload induced a transient pressor response, with a not unreasonable to propose that hypothalamic and medullary
peak at 10 min (D 11·5 (SEM 3·0) mmHg), in the control group
cardiac presympathetic neurons may also be recruited during
(n 8), whereas it induced a greater response in the experimental
Na overload in the early stages of life, which would explain
group (n 5) (D 18·7 (SEM 1·7) mmHg, P, 0·05; F ¼ 1·213;
the positive chronotropy observed in the experimental
Fig. 5(a)). In the control group, RBF promptly increased after
group. Another plausible hypothesis is that the animals fed
Na overload (D 37·3 (SEM 6·0) % 10 min after infusion;
the high-Na diet became hypervolaemic, with increased
Fig. 5(c)) and remained elevated until 60 min after the infusion
blood volume being detected by stretch receptors located
(D 50·8 (SEM 4·1) %; Fig. 5(c)); furthermore, this increase
in the atria. Thus, the HR is increased to prevent congestive
was greater than that observed in the experimental group
heart failure (Bainbridge reflex)(35). However, we did not
(RBF: D 30·8 (SEM 11·0) % 60 min after infusion, P, 0·05;
F ¼ 1·984; Fig. 5(c)). In addition, HS infusion was also able to
induce a greater increase in RVC in the control group than in (a) 0·0
the experimental group during all the experimental periods
BI (bpm/mmHg)
(control: D 149 (SEM 4·0) % v. experimental: D 27·9 (SEM –0·5

12·3) % 60 min after the infusion; P,0·05; F ¼ 1·375; Fig. 5(d)).
–1·0
HS infusion resulted in a decreased HR in both groups
(control: D 2 18·9 (SEM 7·9) bpm v. experimental: D 216·0 –1·5
(SEM 4·2) bpm, 10 min after the infusion; Fig. 5(b)); how- *
ever, the HR of experimental rats remained lower than normal –2·0
levels 60 min after HS infusion (D 2 25·0 (SEM 10·2) bpm 60 min
after the infusion, P, 0·05; F ¼ 0·8036; Fig. 5(b)). (b)
2·5
BI (bpm/mmHg)
2·0
Discussion
1·5 *
Studies carried out in animals(24) and human subjects(22,23)
suggest that diseases that develop in adulthood are related 1·0
to certain conditions to which the individual is exposed 0·5
during the initial stages of life. Despite abundant evidence
highlighting the importance of prenatal phases to hyperten- 0·0
sion development, the relevance of postnatal phases to this Fig. 4. Baroreflex index (BI) of control (B) and experimental (A) rats induced
by phenylephrine (a) and sodium nitroprusside (b) infusions. Values are
condition is unclear. A number of interesting findings were
means, with their standard errors represented by vertical bars. * Mean value
recorded in the present study: (1) high Na intake during was significantly different from that of the control group (P, 0·05). bpm,
postnatal phases chronically increases arterial BP and HR Beats per min.
Table 3. Baseline values of mean arterial pressure, heart rate, renal differences when compared with the above-mentioned study.
blood flow and renal vascular conductance of anaesthetised control and Taken together, the results of the present study indicate that
experimental rats
high Na intake during the postnatal period can be more
harmful than high Na intake during the perinatal period, as
Control Experimental it promotes increases in spontaneous Na intake in adult-
hood, which is a risk factor for the development of several
Mean Mean
SEM SEM
diseases(26,27,37,38), such as hypertension(24,28,29).
MAP (mmHg) 122·8 5·3 118·8 7·4 FURO is a diuretic/natriuretic that reduces renal Na and
HR (bpm) 388·5 15·9 438·2* 8·7 water reabsorption, causing hypovolaemia. This hypovolae-
RBF (ml/min) 3·4 0·4 3·9 0·2
RVC (ml/min per mmHg) 0·0288 0·0044 0·0336 0·0012
mia activates the neuroendocrine and autonomic components
to maintain arterial pressure(30). In this condition, the acti-
MAP, mean arterial pressure; HR, heart rate; bpm, beats per min; RBF, renal blood vation of the sympathetic nervous system is related to the
flow; RVC, renal vascular conductance.
* Mean value was significantly different from that of the control group. increase in vascular tone, venous return, heart rate and con-
tractility and, moreover, to the increase in renal water and
observe any differences in the haematocrit of both groups, Na reabsorption(35). However, Na-seeking and drinking beha-
either during the treatment period or during the recovery viours, in addition to thirst, are required to induce Na appetite
period (Table 1). and to reset the physiological levels of water and extracellular
During the recovery period, experimental rats exhibited a solutes after a deficit(39). In the present study, rats fed the
higher Na excretion rate and higher daily water and food high-Na diet during early postnatal phases exhibited a Na
intakes per body weight when compared with control rats appetite induced by FURO that was significantly lower than
(Table 2). The results of the present study indicate that post- that induced in control rats. This disparity is probably due to
natal Na overload permanently alters the excretion patterns an increase in Na sensitivity in these animals: even on ingest-
and spontaneous intake patterns in adulthood. These results ing the same quantity of Na, the experimental group achieved
differ from those reported by Macchione et al. (36), who satiation before the control group and exhibited a decrease in
found that offspring born to mothers given free access to a Na intake. In fact, Macchione et al.(36) showed that central
hypertonic NaCl solution during the perinatal period exhibited structures involved in osmoreception are more activated in
decreased water intake in need-free conditions in adulthood. FURO-Na-depleted rats, suggesting a possible sensitisation of
It is worth noting that HS solution was provided to the rats osmosentitive circuits due to perinatal manipulations, a find-
after weaning in the present study, which would explain the ing that parallels the results of the present study.
(a) 25 (b) 40
*†
∆MAP (mmHg)
20
∆HR (bpm)
15
0
* * *
5 *
–20
*
* * * * † *†
–5 –40
–10 0 10 20 30 40 50 60 –10 0 10 20 30 40 50 60
Time (min) Time (min)
(c) 70 (d)
* * 70
*
* * * * *
55 *
∆RVC (% baseline)
∆RBF (% baseline)
55 *
*
40 40
*
25 25
*† *† *† *† *† *†
10 * *† 10
*† *† *†
–5 –5
†
–20 –20
–10 0 10 20 30 40 50 60 –10 0 10 20 30 40 50 60
Time (min) Time (min)
Fig. 5. (a) Mean arterial pressure (MAP), (b) heart rate (HR), (c) renal blood flow (RBF) and (d) renal vascular conductance (RVC) in anaesthetised rats from the
control (X) and experimental (W) groups exposed to hypertonic NaCl conditions (3 M , 1·8 ml/kg body weight during 60 s). Values are means, with their standard
errors represented by vertical bars. * Mean value was significantly different from that recorded at time 0 (P, 0·05). † Mean value was significantly different from
that of the control group (P, 0·05).
Some studies suggest that diseases that develop in adult- renal sympathoinhibition(14), which decreases peripheral
hood are related to specific conditions in the early stages of resistance and Na reabsorption, resulting in natriuresis. In
life(22,39,40). Nicolaidis et al.(41) revealed that pregnant rats sub- case this mechanism is unable to induce adequate plasma
mitted to extracellular dehydration generated offspring with Na concentrations, a widespread increase in sympathetic acti-
an increased Na appetite. Together, these results demonstrate vity promotes pressor responses, causing increases in renal
that Na sensitivity in adulthood can be determined before perfusion pressure, glomerular filtration rate and, conseque-
birth through different maternal –fetal influences, including ntly, natriuresis(49). In this regard, the increase in hypertensive
changes in hydromineral homeostasis. However, Nicolaidis responses to hypernatraemia observed in animals exposed
et al.(41) did not consider the possible role of postnatal to high-Na conditions 10 min after HS infusion could be
phases in the determination of Na sensitivity in adulthood. explained by a failure of the primary peptidergic and sym-
Our findings corroborate and extend these findings reported pathoinhibitory mechanisms, resulting in a widespread
by Nicolaidis et al.(41), as the increase in Na intake induced increase in sympathetic activity and therefore in sustained
in postnatal phases promoted the decrease of Na appetite arterial hypertension.
induced by Na depletion in adult rats. Although ontogenesis of essential hypertension is not
The results of the present study demonstrate that high Na completely clear, studies in both human and animal models
intake during postnatal phases chronically increases arterial indicate that an increase in Na intake is associated with this
BP in adulthood. In contrast to these results, Ufnal et al.(42) disease(23,50). Despite being a controversial subject, several
found that the ingestion of a solid diet during early postnatal studies have demonstrated that an increase in daily Na
phases does not change the Na intake pattern or the MAP in intake itself does not promote an increase in arterial pressure
adult animals. This might be because these authors used the in control animals. However, it is worth noting that high-Na
food as a source of Na intake elevation and gave the animals diets were fed to adult animals in these studies(23,50), which
free access to water, thus following a protocol different from differs from the protocol used in the present study. Another
that used in the present study. As the experimental animals study has demonstrated that mothers who have suffered epi-
were given free access to water, the ingested ions might sodes of vomiting and dehydration during the first 3 months
have been diluted in spite of these animals ingesting greater of pregnancy give birth to children who have increased systo-
amounts of Na. Due to this, it is likely that Na intake was lic pressure during adolescence(51). Vidonho et al.(24) found off-
not enough to significantly change the plasma osmolarity of spring born to mothers fed a high-Na diet (4 or 8 % NaCl) during
the experimental animals in terms of cardiovascular or beha- pregnancy and lactation to have higher arterial pressure
vioural parameters. In the present study, by contrast, the in adulthood. In addition, in the present study, the increase in
increase in Na intake occurred through drinking, thus prevent- Na intake (0·3 M- NaCl) during the postnatal period was found
ing the dilution of the ingested Na in the animals. to promote increases in MAP in unanaesthetised adult rats.
As can be seen from Fig. 4, rats fed the high-Na diet during The results of the present study show that not only the
the postnatal period exhibited a BI that was significantly lower uterine phases but also the postnatal phases determine arterial
than that of control rats, indicating a lower sensitivity of baror- pressure in adulthood. Interestingly, the arterial hypertension
eceptors. This might be due to a greater stimulation during in these animals was not reversed after removal of Na intake,
early periods, leading to neuronal adaptation. It has been indicating that the increase in Na consumption in postnatal
reported that during continuous or repetitive stimulation phases promotes permanent changes in pressoric levels. To
(constant within a pattern), the sensitivity of a neural cell is the best of our knowledge, no other study has shown that
redefined through adaptation(43). This continuous stimulation high Na intake during postnatal phases induces chronic
leads to an increase in the depolarisation threshold, but increases in arterial BP in adult male rats. Finally, it should be
does not trigger new action potentials(44). In this case, the emphasised that the present study was performed only in
main hypothesis is that baroreceptors would be unable to male animals to avoid possible hormonal influences
detect and correct changes in BP, which, in the long term, on cardiovascular, renal and behavioural parameters analysed,
would result in arterial hypertension. This hypothesis is sup- as food intake and the motivation for food can be modified
ported by previous studies that have shown impairment of by the phases of the oestrous cycle(47,52 – 56).
baroreflex function in some models of hypertension(44,45), Taken together, the results of the present study demon-
similar to the pressor changes observed in the present study. strate that changes in hydromineral homeostasis during the
It has previously been stated that transient pressor initial stages of postnatal life can change the behavioural
responses to Na overload may depend on increases in and cardiovascular adjustments induced by Na depletion and
lumbar sympathetic activity and vasopressin secretion(46,47). HS infusion. However, further studies are required to elucidate
In the present study, the pressor response to Na overload the mechanisms whereby such changes occur.
was found to be greater in rats exposed to high-Na conditions
(0·3 M- NaCl), but not in control rats. This is in good agreement
Acknowledgements
with the results of another study showing an increase in the
magnitude and duration of hypertensive responses to hyper- The present study was supported by Coordenadoria de Aper-
natraemia under renal vasodilation blockade(48). Moreover, it feiçoamento em Pesquisa (CAPES; http://www.capes.gov.br;
has been proposed that hypernatraemia initially induces a to M. C. S. M.), Conselho Nacional de Desenvolvimento
mechanism comprising secretion of vasoactive peptides and Cientı́fico e Tecnológico (CNPq; no. 477832/2010-5; 483411/
2012-4; http://www.cnpq.br; to G. R. P.) and Fundação expansion in anesthetized rats. Am J Physiol Regul Integr
de Amparo a Pesquisa do Estado de Goiás (no. 200910267 Comp Physiol 279, R884 –R890.
000352; http://www.fapeg.go.gov.br; to G. R. P.). The funders 14. Pedrino GR, Monaco LR & Cravo SL (2009) Renal vasodila-
had no role in study design, data collection and analysis, tion induced by hypernatraemia: role of alpha-adrenocep-
tors in the median preoptic nucleus. Clin Exp Pharmacol
decision to publish, or manuscript preparation.
Physiol 36, e83– e89.
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D. A. R., E. C., C. H. X. and G. R. P. conceived and designed of CNS sympathetic drive: implications for cardiovascular
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performed the experiments; M. C. S. M., A. H. F.-O., D. A. R., 16. Fujita T, Matsuda Y, Shibamoto T, et al. (1991) Effect of
P. M. F., C. H. d. C. and G. R. P. analysed the data; D. A. R., hypertonic saline infusion on renal vascular resistance in
E. C., C. H. X., P. M. F. and G. R. P. contributed the anesthetized dogs. Jpn J Physiol 41, 653– 663.
17. Colombari DS & Cravo SL (1999) Effects of acute AV3V
reagents/materials/analysis tools; M. C. S. M., A. H. F.-O.,
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D. A. R., P. M. F., C. H. d. C., E. C., C. H. X. and G. R. P. volume expansion. Hypertension 34, 762– 767.
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None of the authors has any conflicts of interest to declare. report on research challenges in nutrition and hypertension.
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1 Word count: Abstract (228); Text (3644)
2 References: 32; Figures and tables: 5
3 Sodium overload during post-natal phases impairs diastolic function and

4 exacerbates reperfusion arrhythmias in adult rats
5 MCS Moreira1, ADC Nunes2, PR Lopes1, CC Silva2, FCA Santos3, CH Castro2, GR

6 Pedrino1.
7 1Center for Neuroscience and Cardiovascular Research, Universidade Federal

8 de Goiás, Goiânia, Goiás, Brazil.
9 2Laboratory of Cardiovascular and Neurological Physiopathology, Department of

10 Physiological Sciences, Universidade Federal de Goiás, Goiânia, Goiás, Brazil.
11 3Department of Histology, Embryology and Cell Biology, Universidade Federal

12 de Goiás, Goiânia, Goiás, Brazil.
13
14
15 Running Tittle: Salt overload during childhood impairs the cardiac function
16
17 Corresponding author: Gustavo Rodrigues Pedrino
18 Department of Physiological Science
19 Universidade Federal de Goiás
20 Estrada do Campus, s/n. Zip code: 74001-970. PO: 131. Goiânia – GO - Brazil
21 Phone/Fax: +55-62-3521-1774
22 e-mail: pedrino@pq.cnpq.br / gpedrino@gmail.com
23 Disclosure: The authors declare no conflicts of interests.
24 Key words: hypertension, post-natal period, cardiac function, ischemia/reperfusion,

25 arrhythmia, diastolic dysfunction.
26 ABSTRACT
27 In recent study, we have demonstrated that rats submitted to sodium overload during
28 childhood present diminished baroreflex sensitivity, changes in sodium intake and
29 increased arterial blood pressure and heart rate in adulthood, even after the
30 withdrawal of high salt diet (HSD). However, no evidences regarding the effects of
31 HSD during post-natal phases in the cardiac function have been reported. Therefore,
32 the present study sought to determine the effects of HSD during the childhood on
33 isolated heart function and ischemia/reperfusion responses in adulthood. Experiments
34 were performed in two groups of 21-day-old males Wistar rats: experimental group,
35 maintained on hypertonic saline (0.3M-NaCl) solution and food for 60 days, and
36 control group, maintained on tap water and food. Later, both groups were given water
37 and food for 15 days. Posteriorly, the isolated rat hearts were perfused according the
38 Langendorff technique. After a basal period, the hearts were submitted 20min of
39 anoxia followed by 20min of reperfusion. HSD did not change the contractile function
40 of the adult rat hearts in baseline conditions. However, during the reperfusion period,
41 the experimental rat hearts exhibited an increase of left ventricular end-diastolic
42 pressure (23.10±5.152mmHg vs. 11.61±1.414mmHg; p<0.05), and increased
43 duration of reperfusion arrhythmias (208.8±32.87s vs. 75.00±7.849s; p<0.05)
44 compared to control group. These results indicate that sodium overload in post-natal
45 phases alters the isolated heart responses to the ischemia/reperfusion process,
46 indicating the possible arrhytmogenic effect of HSD.
47
48
49
50
51 INTRODUCTION
52 The long-term increase of blood pressure often affects several target organs, such as,
53 vessels, kidneys, and heart. Hypertension is a key factor to the development of
54 cardiac hypertrophy, which may lead to heart failure1,2.
55 The harmful effects of a high sodium diet (HSD) on blood pressure and cardiac
56 function in humans and animal models have been demonstrated by several studies
57 3,4. The spontaneously hypertensive rats (SHR) present increase of left hypertrophy,
58 fibrosis, and increased chamber stiffness with the aging, which allow to predict the
59 diastolic dysfunction observed in this hypertension model 5,6. In addition, it was
60 observed that HSD induced the shortening the time between the compensated and
61 the decompensated LV hypertrophy (Gao et al. (2011)3).
62 Several researchers have related the salt consumption with LV diastolic dysfunction
63 in hypertensive and normotensive condition 4,7. Recently, it was demonstrated that
64 HSD increases the diastolic dysfunction and the cardiac remodeling in transgenic
65 Ren2 adults female rats 4. In addition, Tzemos et al. (2008) demonstrated that sodium
66 consumption can also cause diastolic dysfunction in normotensive rats. In this study,
67 the authors have suggested that the salt excess would increases the intracellular
68 calcium (Ca2+) concentration, which would compromise the myocardial relaxation.
69 Diverse hypertension models present cardiac remodeling after the stabilization of the
70 hypertensive state5,9–11. Furthermore, another body of evidences indicates that the
71 salt loading induces and/or aggravates the cardiac remodeling3,7,9,12. This remodeling,
72 often characterized as an increase in collagen deposition on the cardiac tissue, is an
73 important contributor to both diastolic dysfunction and arrhythmias occurrence 13.
74 Recently, our group have demonstrated that the post-natal period has a key role in
75 the development of the hypertension14. We have observed that animals submitted to

76 sodium overload during childhood presented diminished baroreflex index, modified
77 sodium intake pattern, diminished hypernatremia-induced renal vasorelaxation and
78 increased mean arterial pressure and heart rate in adulthood, even with the
79 withdrawal of the HSD. However, it is unknown whether the HSD during childhood
80 affect the cardiac function in the adulthood. Thus, our study sought to evaluate the
81 effects of HSD during childhood on left ventricular contractile function of adult
82 animals.
83
84 MATERIALS AND METHODS
85 1. Animals
86 Twenty-one-days-old male Wistar rats provided by the animal facilities of the Federal
87 University of Goiás were submitted to high (experimental group) or normal sodium
88 diet (control group). The control and experimental animals were randomly selected in
89 each litter. All animals were kept in temperature-controlled rooms with 12 hour
90 light/dark cycles and the animals had free access to food and water (0.4% NaCl; AIN-
91 93M 15) or hypertonic saline. All the experimental procedures were approved by the
92 Institutional Animal Care and Use Committee of the Federal University of Goiás
93 (process number 051/2010) and were performed in strict accordance with the
94 National Institutes of Health Guide for the Care and Use of Laboratory Animals.
95
96 2. High sodium diet protocol
97 The experimental group (HSD) received 0.3M NaCl (Sigma-Aldrich, St Louis, MO,
98 USA) hypertonic saline as drinking supply, while control rats (normal sodium diet)
99 received tap water. Both groups received food regularly throughout the treatment.
100 The administration of HSD started on the 21st day of life and finished after 60 days of
101 treatment. After this period, tap water was offered to both groups for 15 days
102 (recovery period).
103
104 3. Isolated rat hearts
105 At the end of the recovery period, the rats were decapitated 10 to 15 minutes after an
106 intraperitoneal injection of 200 IU heparin. The hearts were carefully dissected and
107 perfused according to Langendorff technique. Briefly, the hearts were perfused
108 through the aortic stump with Krebs-Ringer solution (KRS) containing (in mmol/L)
109 NaCl (118.4), KCl (4.7), KH2PO4 (1.2), MgSO4•7 H2O (1.2), CaCl2•2 H2O (1.25),
110 glucose (11.7), and NaHCO3 (26.5). The perfusion flow was maintained at a constant
111 rate (10 mL/min) at 37°C and with constant oxygenation (5% CO2 and 95% O2). A
112 balloon was inserted into the left ventricle through the left atrium for isovolumetric
113 recordings of left ventricular pressures. Coronary perfusion pressure was measured
114 with a pressure transducer that was connected to the aortic cannula and coupled to a
115 data acquisition system (Dataq Instruments, Akron, OH, USA). After a basal period
116 (30 to 40 minutes), the hearts were submitted to 20 minutes of anoxia followed by 20
117 minutes of reperfusion. The isquemia/reperfusion Langendorff tecnique is a widely
118 used model to mimic situations of acute myocardial infarction and cardiac arrest.
119 Reperfusion arrhythmias were defined as the presence of ventricular fibrillation and/or
120 ventricular tachycardia in the reperfusion period. To get a quantitative measurement,
121 the arrhythmias were classified arbitrarily by their duration, in a modified scale from
122 the study of Ferreira et al. (2001)16. The occurrence of cardiac arrhythmias for up to 1
123 minute, was attributed the factor 0; 1 to 4 minutes was attributed the factor 2; 4 to 8
124 minutes was attributed the factor 4; 8 to 12 minutes was attributed the factor 6; 12 to
125 16 minutes was attributed the factor 8 and 16 to 20 minutes was attributed the factor
126 10. A value from 0 to 10 was obtained in each experiment and is indicated as
127 arrhythmia severity index (ASI). The VE hypertrophy was evaluated by the ratio VE
128 weight: tibia length.
129
130 4. Histological analysis
131 After the recovery period, the rats were decapitated and the intermediate segment of
132 the left ventricles were collected for histological analysis. The segments were fixed in
133 metacarn solution (see Table 1, Annex I) during 4 hours. After fixation, the tissues
134 were dehydrated in ethanol series (Neon®), cleared in xylene (Synth®) and
135 embedded in paraffin (Sigma®; see Table 2, Annex I). After the inclusion, three 5µm
136 thickness sections (with 150 µm of interval) were obtained from each animal using a
137 microtome (Leica RM2155, Nussloch, Germany). After the cutting, the sections were
138 fixed in laminas (Exacta, 26x76x1x1.2mm), rehydrated (see Table 3, Annex I) and
139 submitted to the cytochemistry technique using Gömori’s reticulin (see Table 4,
140 Annex I). After the staining, the sections were dehydrated (see Table 4, Annex I) and
141 covered with coverslips.
142
143 4.1 Morphometry of cardiomyocytes and stereology
144 The cross section area of cardiomyocytes were measured in order to determine the
145 degree of cellular hypertrophy. The transversal sections were photographed and,
146 later, the cross-sectional area (CSA) of 200 cardiomyocytes for each group was
147 measured. Only the cells arranged longitudinally, with visible nuclei and cellular limits
148 were considered. The CSA (µm) of each cardiomyocyte was measured in the region
149 of the nucleus.

150 The stereology was used in order to quantify and specify the relative frequency (%) of
151 each component of the cardiac tissue, such as cardiomyocites, collagen fibers (type I
152 and III) and non-fibrous connective tissue (which includes cells, ground substance,
153 blood vessels and nerves). To this, 30 random photomicrographic fields from each
154 experimental group were captured from the histological sections stained with
155 Gömori’s Reticulin. The fields were inserted in the multipoints test with 130 points and
156 10 lines, proposed by Weibel (1963)17 and the relative values were determined by
157 counting the coincident points on each component in study; the relative frequency
158 was calculated by the ratio between the counting points and the total points in the test
159 system. The stereological analysis was performed in the image analyzing system, in
160 the Image Pro-Plus v 6.1 program for Windows (Media Cybernetics Inc., Silver
161 Spring, MD, USA). All the photographs were obtained in the Zeiss Scope A1
162 microscope under conventional light. The stereological analysis and the
163 morphometric analysis were performed using 40x magnification photographs.
164
165 5. Statistical analysis
166 The statistical analysis and the graph confection were performed in the GraphPad
167 Prisma v.6. All the data were expressed as mean ± standard error of the mean
168 (SEM). The cardiac function data were analyzed using variance analysis (ANOVA)
169 followed by Newman Keuls post-test. The morphometric and stereological data were
170 analyzed using unpaired Student t-test.
171
172 RESULTS
173 1. Isolated rat hearts

174 As observed in the figure 1, in the basal condition, the left ventricular end-systolic
175 pressure (LVESP; Fig. 1A; 95.60 ± 11.68 vs. 96.87 ± 15.46 mmHg), left ventricular
176 end-diastolic pressure (LVEDP; Fig. 1B; 10.40 ± 0.7293 vs. 11.96 ± 1.458 mmHg),
177 maximal rate of left ventricular pressure rise (+dP/dt; Fig. 1C; 2296 ± 291.2 vs. 2360
178 ± 398.7 mmHg/s), maximal rate of left ventricular pressure decline (-dP/dt; Fig. 1D
179 1630 ± 253.3 vs. 1551 ± 283.5 mmHg/s), heart rate (HR, Fig. 1E; 233.8 ± 19.36 bpm
180 vs. 246.8 ± 16.78 bpm), left ventricular developed pressure (calculated as the
181 difference between LVESP and LVEDP) (LVDevP; Fig. 1F; 85.20 ± 11.85 mmHg vs.
182 84.91 ± 14.80 mmHg) and perfusion pressure (Fig. 1G; 89.74 ± 11.52 mmHg vs.
183 78.81 ± 12.31 mmHg) was not different between the control (n=9) and the
184 experimental rat hearts (n=8). Similarly, during the reperfusion period, there were no
185 differences between the control animals and the experimental animals in the LVESP
186 (Fig. 1A; 86.14 ± 8.393 vs. 83.22 ± 9.169 mmHg), +dP/dt (Fig. 1C; 2033 ± 181.8
187 mmHg/s vs. 1607 ± 353.0 mmHg/s) -dP/dt (Fig. 1D; 1247 ± 161.1 vs. 924.6 ± 160.3
188 mmHg/s), HR (Fig. 1E; 238.4 ± 21.66 bpm vs. 235.5 ± 13.92 bpm), LVDevP (Fig. 1G;
189 74.53 ± 8.745 mmHg vs. 60.13 ± 11.19 mmHg) or perfusion pressure (Fig. 1F; 75.08
190 ± 9.383 mmHg vs. 78.76 ± 8.985 mmHg). However, the experimental hearts
191 presented a significant increase in the LVEDP during the reperfusion period
192 compared to the control hearts (11.61 ± 1.414 mmHg vs. 23.10 ± 5.152 mmHg;
193 p<0.05) (Figure 1B).
194 Furthermore, the experimental hearts rats presented an increased duration of
195 arrhythmias (Figure 2A; 208.8 ± 32.87 s vs. 75.00 ± 7.849 s; p<0.05) and increased
196 arrhythmia severity index (ASI; Figure 2B; 3.0 ± 0.378 arbitrary units vs. 1.0 ± 0.378
197 arbitrary units; p<0.05) compared to the control animals.
198
199 2. Histological analysis
200 As observed in the Figure 3, no differences were observed in the cross-sectional area
201 of the cardiomyocytes of the animals submitted to sodium overload (14.99 ± 0.20 µm;
202 n=200) when compared to the control group (14.63 ± 0.18 µm; n=200). The figure 4
203 shows representative photomicrographs of cross sections of the left ventricle of both
204 control (A) and experimental (B) animals, at 40x magnification, stained with Gömori’s
205 reticulin. There was no differences in the relative frequencies of the cardiomyocites
206 between the groups (Figure 5A; control group: 48.31 ± 1.62%, n=30, vs. experimental
207 group: 45.26 ± 1.44%, n=30; p<0.05), however the relative frequencies of the non-
208 fibrous connective tissue were significant smaller in the experimental group when
209 compared to the control group (Figure 5B; control group: 2.46 ± 0.50%, n=30 vs.
210 experimental group: 0.92 ± 0.26%, n=30; p<0.05). A significant increase was
211 observed in the collagen type I relative frequency (Figure 5C) in the hearts of the
212 experimental animals (19.79 ± 1.26%, n=30; p<0.05) when compared to the control
213 group (15.74 ± 0.61%, n=30). However, no differences were observed in the collagen
214 type III relative frequency (Figure 5D) in the hearts of the experimental animals,
215 compared to the control group (control group: 32.38 ± 1.04%, n=30 vs. experimental
216 group: 34.03 ± 1.15%, n=30).
217
218 DISCUSSION
219 Our results indicate that sodium overload during childhood did not change the cardiac
220 function of isolated hearts in basal conditions. However, these hearts presented an
221 important impairment of the diastolic function and increase of the arrhythmias severity
222 after ischemia-reperfusion.

223 Several factors can contribute to the development of the diastolic disturbances 18. The
224 diastolic dysfunction can be developed by the progress of the hypertensive state 19.
225 Using different ages of normotensive and SHR animals, LeGrice et al., (2012) 19 have
226 demonstrated, by morphologic and hemodynamic data, four stages of hypertensive
227 progress: systemic hypertension, diastolic dysfunction, early systolic failure and
228 decompensated cardiac failure.
229 Some researchers have related the salt consumption with LV diastolic dysfunction in
230 hypertensive and normotensive individuals 7,8. Tzemos et al. (2008)8 demonstrated
231 that sodium consumption can cause diastolic dysfunction also in normotensive rats,
232 with normal renin-angiotensin system activity. In that study, the authors suggest that
233 the salt excess would increases the intracellular Ca2+ concentration, which would
234 compromise the myocardial relaxation 8.
235 In our study, it was not observed cardiomyocyte hypertrophy in animals submitted to
236 HSD. Thus, it is possible that the diastolic function compromising observed during
237 reperfusion could be caused by the increased intracellular Ca 2+ concentration or by
238 dysfunctions of Ca2+ reuptake during the muscular relaxation. In fact, Dupont et al.
239 (2012) have demonstrated that relaxation dysfunction is associated with abnormal
240 intracellular calcium homeostasis, suggesting that diastolic dysfunction may be
241 revealed independently of LV hypertrophy in SHR.
242 Besides the increased intracelular Ca2+ concentration, the cardiac tissue fibrosis is
243 another key factor that can cause diastolic dysfunction. Regarding this factor, Yu et
244 al. (1998)9 have demonstrated that HSD causes myocardial fibrosis both in SHR and
245 normotensive animals. In addition, this study indicates that sodium promotes not only
246 LVH (left ventricular hypertrophy) but also intramyocardial fibrosis that, participating in
247 the abnormal myocardial stiffness, leads to systolic and diastolic dysfunctions 9.
248 Different research groups have shown that myocardial fibrosis can be caused by
249 pressure overload or high salt intake, separately 5,9. In this sense, recent study of
250 Dadson et al. (2016)21 have demonstrated that pressure overload (induced by aortic
251 constriction) induces increase in the myocardial collagen content and such increase is
252 reverted after unload. Thereby, it is not surprising to suggest that the animals
253 submitted to salt overload during childhood, whose are expose to both pressure
254 overload and HSD, could develop myocardial fibrosis and consequent diastolic
255 dysfunction. In fact, our results demonstrate that animals submitted to sodium
256 overload in post-natal phase present increase in collagen type I relative frequency in
257 adulthood. The collagen fibers are continuously synthesized and degraded and the
258 shift of the equilibrium of collagen fibers synthesis to one or another fiber type is
259 crucial to adapt the heart to the organism requirements (more resistance or more
260 resilience, for example); on the other hand, it can, long term, affect the cardiac
261 function22–24. In fact, previous studies have demonstrated that increases in the
262 deposition of type I collagen fibers (an inelastic element) substantially increases the
263 cardiac ability to resist stretch 24,25. This factor can contribute to the diastolic
264 dysfunction observed in these animals after an anoxia event.
265 Our data also demonstrate that the HSD in childhood promotes increased time and
266 severity of arrhythmias during ischemia/reperfusion process in adulthood. The
267 ischemia/reperfusion of the myocardium induces several types of ventricular
268 arrhythmias in humans and animal models, such as ventricular premature beats,
269 ventricular tachycardia and ventricular fibrillation 26. The arrhythmias are the major
270 cause of death in ischemia/reperfusion process and can be caused by both cardiac
271 fibrosis13,27,28 and unbalance in calcium (Ca2+) homeostasis 29.

272 The electrical coupling between the cardiac cells is crucial to their synchronized
273 contraction and the effective heart beating. The protein connexin, present in the
274 cellular membrane, allows the connection between the cytoplasm of two adjacent
275 cardiac cells13. Such proteins participates of the current flow between the cytoplasms
276 and are a key factor to the electrical impulse propagation throughout the cardiac
277 muscle cells. The increase in the collagen deposition in the heart tissue can
278 compromise the electrical coupling between the cardiomyocytes, impairing the
279 propagation of the electrical impulse and generating abnormal and desynchronized
280 contraction of the cardiac muscle cells, which characterizes the arrhythmias. In fact,
281 the hearts of the animals submitted to sodium overload presented increase in the
282 collagen type I deposition, which could contributes to the increased reperfusion
283 arrhythmias observed in those animals.
284 As mentioned before, changes in Ca2+ homeostasis is another factor that can
285 contribute to the prolongation of reperfusion arrhythmias. During the myocardial
286 ischemia, the intracellular pH decreases considerably due to ATP decreasing and
287 anaerobic glycolysis 29. The increase of intracellular concentration of H+ stimulates the
288 Na+H+ and the Na+Ca2+ exchanges, which results in an increase of the cytosolic Ca2+
289 29. In the absence of arrhythmias, the intracellular Ca 2+ concentration returns to the
290 normal levels in the myocytes that survive 29. However, the return of Ca2+
291 concentration to normal levels depends on myocyte ATP and Na + levels and the
292 integrity of Ca2+ handling proteins 29. During the reperfusion period, the high cytosolic
293 Ca2+ levels promote negative effects on the cardiomyocyte, such as hypercontracture,
294 electrical instability, and contractile dysfunction30.
295 Baldo et al. (2012)31 have demonstrated that high salt diet is a determining factor of
296 the arrhythmias levels in normotensive infarcted animals, once the ventricular
297 premature beats was increased in the salt-loaded rats. Furthermore, Marketou et al.
298 (2013)32 have observed that a higher Na+ excretion rate, which revealed a HSD, is
299 positively related to the premature ventricular contractions in patients with essential
300 hypertension.
301 Our results demonstrate that isolated hearts of rats submitted to sodium overload
302 during childhood exhibit prolonged arrhythmia during the reperfusion process, when
303 compared to the control animals. Thus, we can hypothesize that the sodium overload
304 could compromise the Ca2+ homeostasis, leading the return of the Ca2+ to the
305 baseline values to last longer in the experimental animals.
306 Summarizing, our results demonstrate that a high salt diet compromises the cardiac
307 function after anoxia event, diminishing the ventricular relaxation, and demonstrating
308 a possible arrhythmogenic effect, once the duration and severity of arrhythmias were
309 increased. Furthermore, we have demonstrated that the increased salt intake in
310 childhood also promotes increase in the collagen relative frequency in the heart of the
311 adult animals. To the best of our knowledge, no other study has shown that high salt
312 diet during childhood increase the chance of death, caused by severe arrhythmia
313 after an anoxia event, such as infarction and cardiac arrest, in adulthood. However,
314 other experiments are required to clarify the mechanisms thereby the cardiac
315 commitment occurs.
316
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417 Figure legends
418 Figure 1: Effects of sodium overload during post-natal phases on isolated rat
419 hearts. Left ventricular end-systolic pressure (A); left ventricular end-diastolic
420 pressure (B); dP/dt maximum (C); dP/dt minimum (D; mmHg/s); heart rate (E; bpm);
421 perfusion pressure (F); left ventricular developed pressure. Values are mean ± SEM.
422 *different from control group, p<0.05.
423 Figure 2: Effects of sodium overload during post-natal phases on reperfusion
424 arrhythmias in isolated hearts. (A) time of arrhythmia (s); (B) arrhythmia severity index
425 (ASI). Values are mean ± SEM. *different from control group, p<0.05.
426 Figure 3: Cross-sectional area (µm) of the cardiomyocytes of animals submitted
427 to sodium overload during post-natal phases and control animals. Values are mean ±
428 SEM.
429 Figure 4: Representative photomicrographs of the cross sections of the left
430 ventricle of both control animals (A) and animals submitted to sodium overload during
431 post-natal phases (B) stained with Gömori’s reticulin. (40x magnification). Type I
432 collagen fibers are indicated by the white arrows; type III collagen fibers are indicated
433 by the black arrows.
434 Figure 5: Relative frequency of each component of the left ventricular cardiac
435 tissue. Percentage of tissue surface correspondent to cardiomyocytes (A), non-
436 fibrous connective tissue (B), collagen type I (C) and collagen type III (D) in the left
437 ventricle of animals submitted to sodium overload during post-natal phases and
438 control animals. Values are mean ± SEM. *different from control group, p<0.05.
ANNEX I
Table 1. Description of the fixer Metacarn

Reagent Volume (ml) Temperature (°C)
Methanol PA 60 Room temperature
Chloroform PA 30 Room temperature
Acetic acid PA 10 Room temperature
Table 2. Description of the tissue processing in paraplast

Deidratation
Reagent Time (min) Procedure Temperature (°C)
Ethanol PA 80% 60 immersion Room temperature
Diafanization
Xylol PA 60 immersion Room temperature
Infiltration
Paraplast I 60 immersion 58º- 60ºC
Paraplast II 60 immersion 58º- 60ºC
Inclusion Paraplast Until the sectioning Embedment 58º- 60ºC
Table 3. Rehydration to cytochemistry

Distilled water 5 immersion Room temperature
Table 4. Cytochemistry using Gömori reticulin
Potassium permanganate 2 Dripping Room temperature
Oxalic acid 1 Dripping Room temperature
Alum ferric 2 Dripping Room temperature
Ammoniacal silver 2 Dripping Room temperature
Formol 10% 3 Dripping Room temperature
Golden chloride 0,2 Imersion Room temperature
Sodium hyposulphite 2 Dripping Ambient
Room temperatureRoo
Picric acid 2 Room temperature
Dehyidration and cover with coverslips Room temperature
Reagent Time (min) Procedure m temperature(°C)
Temperature e
REVIEW
published: 25 August 2015
doi: 10.3389/fphys.2015.00234
Does the sympathetic nervous

system contribute to the
pathophysiology of metabolic
syndrome?
Marina C. dos Santos Moreira 1 , Izabella S. de Jesus Pinto 1 , Aline A. Mourão 1 ,
James O. Fajemiroye 2 , Eduardo Colombari 3 , Ângela A. da Silva Reis 4 ,
André H. Freiria-Oliveira 1 , Marcos L. Ferreira-Neto 5 and Gustavo R. Pedrino 1*
1
Department of Physiological Sciences, Center for Neuroscience and Cardiovascular Research, Federal University of Goiás,
Goiânia, Brazil, 2 Laboratory of Pharmacology of Natural Products, Federal University of Goiás, Goiânia, Brazil, 3 Department
of Physiology and Pathology, School of Dentistry, Universidade Estadual Paulista, Araraquara, Brazil, 4 Department of
Biochemistry and Molecular Biology, Federal University of Goiás, Goiânia, Brazil, 5 Laboratory of Experimental Physiology,
Faculty of Physical Education, Federal University of Uberlândia, Uberlândia, Brazil
Edited by: The metabolic syndrome (MS), formally known as syndrome X, is a clustering of several
Valdir Andrade Braga,
Federal University of Paraiba, Brazil
risk factors such as obesity, hypertension, insulin resistance, and dislypidemia which
Reviewed by:
could lead to the development of diabetes and cardiovascular diseases (CVD). The
J. Thomas Cunningham, frequent changes in the definition and diagnostic criteria of MS are indications of the
University of North Texas Health
controversy and the challenges surrounding the understanding of this syndrome among
Science Center, USA
Marli Cardoso Martins-Pinge, researchers. Obesity and insulin resistance are leading risk factors of MS. Moreover,
State University of Londrina, Brazil obesity and hypertension are closely associated to the increase and aggravation of
*Correspondence: oxidative stress. The recommended treatment of MS frequently involves change of
Gustavo R. Pedrino,
Department of Physiological Science,
lifestyles to prevent weight gain. MS is not only an important screening tool for the
Federal University of Goiás, Estrada identification of individuals at high risk of CVD and diabetes but also an indicator
do Campus, s/n, PO Box 131,
of suitable treatment. As sympathetic disturbances and oxidative stress are often
74001-970 Goiânia, Brazil
pedrino@pq.cnpq.br; associated with obesity and hypertension, the present review summarizes the role of
gpedrino@gmail.com sympathetic nervous system and oxidative stress in the MS.
Specialty section: Keywords: obesity, insulin resistance, hypertension, cardiovascular diseases, central nervous system
This article was submitted to
Integrative Physiology,
a section of the journal Introduction
Frontiers in Physiology
Received: 25 June 2015 Cardiovascular diseases (CVD) are major causes of morbidity and mortality worldwide (WHO,
Accepted: 05 August 2015 2013). The risk factors of CVD include hypertension, hypercholesterolemia, diabetes, genetic
Published: 25 August 2015 predisposition, obesity, sedentary lifestyle, and smoking (WHO, 2013). Unlike other risk factors,
Citation: smoking and sedentary lifestyle are preventable. The risk factors that cannot be prevented often
Moreira MCS, Pinto ISJ, Mourão AA, occur together. The clustering of these risk factors has been studied extensively (Kagota et al., 2010;
Fajemiroye JO, Colombari E, Reis Salazar et al., 2011; Gyawali et al., 2015). Reaven (1988) observed that insulin-resistant subjects
ÂAS, Freiria-Oliveira AH, Ferreira-Neto often manifest obesity, high level of low-density lipoprotein (LDL)-cholesterol, low level of high-
ML and Pedrino GR (2015) Does the
density lipoprotein (HDL)-cholesterol, fasting hyperglycemia and increased arterial blood pressure.
sympathetic nervous system
contribute to the pathophysiology of
These manifestations with multiple risk factors to the development of CVD were called Syndrome
metabolic syndrome? X (Reaven, 1988; Grundy et al., 2004). Later, the Syndrome X was renamed as Metabolic Syndrome
Front. Physiol. 6:234. (MS). In other words, MS is a clustering of several metabolic changes that affect the organism and
doi: 10.3389/fphys.2015.00234 often result in CVD.
Frontiers in Physiology | www.frontiersin.org 1 August 2015 | Volume 6 | Article 234

Moreira et al. Sympathetic nervous system and metabolic syndrome
The prevalence of CVD has increased since the advent of Major Components of Metabolic Syndrome
industrial revolution especially in western societies (Gottlieb
et al., 2008). The less active lifestyle being promoted by modern Abdominal Obesity
transportation system, exclusion of manual jobs with high scale Obesity is defined by WHO as an abnormal or excessive fat
mechanized production and widespread consumption of fat rich accumulation that causes health problems (Mendis et al., 2015).
food has contributed greatly to the increasing cases of obesity The body mass index (BMI) is a parameter being are used to
and MS. The consumption of industrial products which are classify overweight and obesity in adults. BMI is calculated using
also rich in sugar and salt elicits harmful effects on insulin the formula weight/height2 (kg/m2 ). WHO defines a BMI greater
resistance (Vidonho et al., 2004; Salazar et al., 2011) and blood than or equal to 30 kg/m2 as obesity. Although the prevalence
pressure (Vidonho et al., 2004; He et al., 2008; Moreira et al., of obesity has been doubled worldwide since 1980, it is still a
2014). According to Stoian and Stoica (2014), MS patients exhibit preventable disease. Nowadays, obesity kills more people than
lower concentrations of serum phosphate and magnesium as underweight globally. In 2014, it was reported that 39% of the
compared to subjects who did not meet the criteria for the adults (≥18 years) were overweight and 13% obese. About 42
diagnosis of this syndrome. Phosphate and magnesium are million of people under 5 years of age were overweight in 2013
crucial to carbohydrate metabolism, thus, it is not surprising to (Mendis et al., 2015).
suggest that lower level of these ions in MS patients can lead Basically, obesity is caused by the imbalance between
to a decrease in peripheral utilization of glucose as well as the the calories consumed and calories expended. The higher
development or aggravation of insulin resistance (Stoian and consumption of calories as compared to the calories expended
Stoica, 2014). has being associated with weight gain. The accumulation of
Some studies have demonstrated that oxidative and adipose tissue in the abdominal region causes abdominal obesity.
inflammatory stress play an important role in the initiation Several studies have demonstrated that abdominal obesity which
and progression of CVD (Toshima et al., 2000; Libby et al., 2002). can be measured by the waist circumference has a strong
It has been established that obesity could lead to an increase in correlation with the development of CVD (Ferreira and Moisés,
the circulating markers of oxidative stress and inflammation 2000; Salazar et al., 2011; Traissac et al., 2015).
(Festa et al., 2000; Keaney et al., 2003). As obesity is an important Considering the strong relationship among obesity, MS
risk factor of MS, the establishment of a definitive relationship and cardiovascular risk factors, it is important to unravel
between inflammation and oxidative stress has attracted a lot of the autonomic disturbances in obesity. Though the autonomic
interest. However, there are still dearth of information in this imbalance is not homogeneous in obesity, some studies
regard. demonstrated sympathetic hyperactivity in most part of obese
In order to unravel the pathophysiology of MS, several individuals (Lopes and Egan, 2006). In the obese individuals, the
definitions and diagnostic parameters have been proposed. The sympathetic tone is increased in target organs such as kidney,
World Health Organization (WHO) defined MS as a group of risk skeletal muscle and peripheral vessels to elicit hypertension (Esler
factors of CVD and diabetes (Alberti and Zimmet, 1998; Grundy et al., 2001; Lopes and Egan, 2006).
et al., 2004). WHO considered insulin resistance as the primary
cause of MS (Grundy et al., 2004). This diagnostic consideration Hypertension
predicts diabetes in MS. However, the need of a specific test for Hypertension is, by definition, a sustained increase in the
insulin resistance makes MS diagnostic criteria by WHO less arterial blood pressure (WHO, 2013). Nowadays, hypertension
accessible (Grundy et al., 2004). According to WHO, the insulin and its complications is one of the major cause of death
resistance can be identified through one of the following factors: worldwide. Factors such as increase in the sympathetic activity
(i) type 2 diabetes; (ii) impaired fasting glucose; or (iii) impaired and dysregulation in sodium homeostasis could cause an
glucose intolerance. Besides the insulin resistance, at least two increase in the arterial blood pressure. Although the pathogenesis
other risk factors are required to diagnose the syndrome (Alberti of hypertension is still unclear, salt consumption has been
and Zimmet, 1998; Grundy et al., 2004). Some of the diagnostic implicated as one of the important causes of this disease (da Costa
criteria for MS are shown in Table 1. Lima et al., 1997; Ito et al., 1999; Contreras et al., 2000; Sacks
et al., 2001; Rodriguez-Iturbe and Vaziri, 2007; He et al., 2008;
Moreira et al., 2014). In addition, sedentary lifestyle, obesity and
TABLE 1 | WHO—Diagnostic criteria for metabolic syndrome. smoking are other factors that contribute to increase in blood
pressure (Filho et al., 2008). The long-term increase in arterial
Factor Limit levels blood pressure often affects some organs, like heart and kidneys.
The higher the blood pressure, the greater is the resistance needed
High blood pressure ≥140/≥90 mmHg or antihypertensive medication
by heart to function. A higher blood pressure could lead to
Plasma triglycerides ≥150 mg/dL (1.7 mM)
an increase in the frequency and contractile force. In the long-
HDL cholesterol
term, this changes in blood pressure could compromise cardiac
Men <35 mg/dL (0.9 mM)
function (Zile, 2002; Cingolani et al., 2003; Chen-Izu et al., 2007).
Women <39 mg/dL (1.0 mM)
According to WHO, hypertension is a systolic blood pressure
Body mass index >30 kg/m2
equal to or above 140 mmHg and/or diastolic blood pressure
Urinary albumin excretion rate ≥20 µg/min
equal to or above 90 mmHg (WHO, 2013).

Endothelial dysfunction has been identified in hypertension. (ONOO–). This reaction could lead to a significant reduction in
Endothelium is a cellular layer on the vascular wall that the bioavailability of endothelial NO.
controls the traffic of small and large molecules, and maintains Nitric oxide are highly relevant in endothelial dysfunction
the integrity of vascular wall (Carvalho et al., 2001). The because in this pathology, production may be reduced due
endothelium controls the expansion and contraction of blood to changes in NOS3 (Nitro oxide synthase type 3) protein as
vessels by producing local mediators that are involved in observed in animal models of cardiovascular disease such as SHR,
vasodilation (endothelium-derived relaxing factors–EDRFs) DOCA, diabetic rats (Hink et al., 2001; Sullivan et al., 2002). The
or vasoconstrictor (endothelium-derived constriction factors– reduction in the bioavailability of NO is considered as part of the
EDCFs) in response to changes in blood flow or vasoactive mechanism for endothelial dysfunction in oxidative stress. NO
agents (Carvalho et al., 2001). The endothelium has multiple could reacts with O2 to cause vasoconstriction, vascular damage
and important roles in physiological events: (i) it acts as and lipid peroxidation (Virdis et al., 2002).
hemodynamic sensor by receiving and transmitting signals from In summary, endothelial dysfunction is characterized by
the extracellular matrix and cells; (ii) it produces mediators that an imbalance in the release of vasoconstrictors and the
interfere with growth, activity, migration and cell death; and (iii) endothelium-dependent relaxants. Hence, increases in EDCFs
it preserves the adaptive changes to circulatory requirements are common in pathophysiological condition like hypertension.
(Carvalho et al., 2001).
The principal EDRFs are nitric oxide (NO), the endothelium- Insulin Resistance
derived hyperpolarizing factor (EDHF) and prostacyclin (PGI2). The insulin-resistance occurs when the body cells become less
Angiotensin II (Ang II) and superoxide or reactive oxygen sensitive and resistant to insulin. Insulin, an hormone which
species (ROS) are among the main EDCFs (Kang, 2014). In facilitate glucose absorption, is produced in the beta cells of
pathophysiological situations, such as hypertension, an increase the pancreas (The IDF consensus worldwide definition of the
of EDCFs occurs. In this condition, study has shown an MS: IDF, 2006). Once the glucose cannot be absorbed by the
increase in the release of endothelial derived contractile factors cells, it remains in the blood and subsequently triggers off the
cyclooxygenase (e.g., PGH 2) in response to acetylcholine production of more insulin (hyperinsulinaemia reflex). The over
and angiotensin II in aorta and mesenteric arteries of SHR production of insulin often wears off beta cells and diminishes
(Côrtes et al., 1996). This result suggests endothelial dysfunction its capacity to produce insulin. This condition generally leads to
in hypertension. Under physiological conditions, there is a hyperglycaemia and type 2 diabetes (WHO and IDF, 2006; The
balance between the release, and production of the most IDF consensus worldwide definition of the MS: IDF, 2006). The
important relaxing and contractile factors. This balance could be insulin-resistance promotes damage to several insulin-sensitive
changed by attenuation of vasodilatory effect of endothelium. An organs such as liver and kidneys. The hyperinsulinaemia reflex
apparent decrease in vascular endothelial-dependent relaxation increases the release of triglycerides by liver into the bloodstream
is called endothelial dysfunction (Carvalho et al., 2001). The and subsequent decrease in the level of HDL cholesterol, increase
mechanisms involved in endothelial dysfunction as found in in the level of small and dense particles of LDL cholesterol
hypertension is multifactorial. Preclinical studies have shown (Reaven, 1988, 2003; Yoon et al., 2014). The hyperinsulinaemia
an increase in the basal activity of nitric oxide synthase reflex can contribute to the pathophysiology of the essential
(NOS) and a decrease in the expression and activity of hypertension through an increase in renal water absorption
soluble guanylyl cyclase in smooth muscle of spontaneously and/or increase in the sympathetic activity (Reaven, 2003; Yoon
hypertensive rats (Bauersachs et al., 1998; Kojda et al., 1998). et al., 2014). The insulin resistance is a major risk factor of type
On the other hand, in Dahl rats with salt-sensitive hypertension, 2 diabetes and atherosclerotic complications such as coronary
there is a decrease in the responsiveness of vascular smooth artery disease, stroke and peripheral arterial disease (Yoon et al.,
muscle cells due to a decrease in the eNOS activity and 2014).
NO production (Luscher and Vanhoutte, 1986; Hayakawa
and Raij, 1998). EDHF has been described as one of the Dislypidemia
principal mediators of endothelium-dependent vasorelaxation The dislypidemia is considered as an unbalance serum level
in normotensive animals. The contribution of EDHF-mediated of LDL (which increases) and HDL cholesterol particles
relaxation appears significantly greater in small resistance vessels (which decreases) (Reaven, 1988). As defined by Kaur (2014),
than in large conduit vessels (Félétou and Vanhoutte, 2006). dyslipidemia is characterized by spectrum of qualitative lipid
A reduction in the release of EDHF may lead to endothelial abnormalities that reflect perturbations in the structure,
dysfunction and subsequently arterial hypertension (Fujii et al., metabolism, and biological activities of both atherogenic
1992). lipoproteins and antiatherogenic HDL cholesterol. Dislypidemia
The oxygen and oxidative reactions in the body are vital for is frequently associated to insulin resistance. As mentioned
energy supply and defense against invaders. Under physiological above, the hyperinsulinaemia reflex, caused by the insulin
conditions, the enzyme superoxide dismutase is responsible for resistance, promotes increase in the hepatic release of
preventing the formation of reactive species of oxygen such as triglycerides to the blood. This release could decrease the
peroxynitrite (ONOO-) which has a detrimental effects in the level of HDL cholesterol and increase the level of small and
body (McIntyre et al., 1999). However, under oxidative stress denser particles of LDL cholesterol (Reaven, 1988, 2003; Yoon
condition as in MS, a large amount of O2 reacts with NO to form et al., 2014).

The increase in serum level of small and denser LDL (Francisco et al., 2006). IL-18 is a proinflammatory cytokine
cholesterol particles could lead to the accumulation of with pleiotropic action which induces the secretion of the
triglyceride in the vessels and development of atherosclerosis cytokines IL-6, TNF-α, IL-1β and endothelial adhesion molecules
among other cardiovascular complications (Reaven, 1988, 2003). (Francisco et al., 2006). This cytokine exerts chemotaxis
of human T cells to promote the recruitment into the
Inflammation and Oxidative Stress in Metabolic atherosclerotic plaque. It has been proposed that the IL-18
Syndrome induces the expression of several matrix metalloproteinases,
The increase in oxidative stress and inflammatory state are which may weaken the fibrous cap of injury atherosclerotic
known to play an important role in the initiation and progression (Hung et al., 2005).
of CVD (Toshima et al., 2000; Libby et al., 2002). Obesity and The cytokines CD40 and CD40L are expressed by
MS have been associated with increased circulating markers of macrophages, lymphocytes T, platelets, endothelial cells and
oxidative stress and inflammation (Festa et al., 2000; Keaney muscle cells flat (Hung et al., 2005). The system CD40/CD40L
et al., 2003). However, there are still few information on the exerts various pro-inflammatory and pro-thrombotic effects by:
relationship between MS and inflammation/oxidative stress. The (i) stimulating the production of endothelial cells free radicals
possible link between MS and inflammation is resistance to to antagonize nitric oxide production; (ii) inducing expression
insulin (RI) (Volp et al., 2008). A defective insulin action in of endothelial cells and smooth muscle adhesion molecules; (iii)
target tissues (muscle, liver, and adipose tissue) could increase stimulating the expression of proinflammatory cytokines and
chronic inflammation (Dandona et al., 2007). T cells elaborate chemokines; (iv) inducing tissue factor expression on endothelial
inflammatory and anti-inflammatory properties of cytokines by and smooth muscle cells to promote an increase in potential
stimulating macrophages, endothelial cells and smooth muscle thrombogenic plate; and (v) participating in the activation of
cells (Volp et al., 2008). The key inflammatory cytokines platelet (Angelico et al., 2006). Data has shown that CD40 which
markers include interleukin-6 (IL-6), tumor necrosis factor- is expressed on the surface of platelets could activate platelet to
α (TNF-α), interleukin-8 (IL-8), interleukin-1β (IL-1β), CD40, promote thrombus formation (Angelico et al., 2006).
CD40L, and C-reactive protein (Kon et al., 2005; Wu and C-Reactive Protein (CRP) is synthesized by the liver and
Wu, 2006). Since the adipocytes cells are the main producers regulated by cytokines, predominantly IL-6, TNF-α, and IL-1
of cytokines proinflammatory, there is a strong relationship (Abdellaoui and Al-Khaffaf, 2007). Its levels are increased
between increased secretion and higher levels of cytokines in during acute inflammatory process. Mild increase in the level
obese people. This phenomenon increases the risk of developing of CRP also occurs in chronic inflammatory condition such as
MS (Vanhala et al., 2006). atherosclerosis. The levels of this protein almost triple in the
IL-6 is a pro-inflammatory cytokine involved in the presence of peripheral vascular disease risk (Abdellaoui and Al-
development of hyperinsulinemia and MS. This cytokine Khaffaf, 2007). It has been shown that MS patients have CRP
increases lipolysis to release free fatty acids and glycerol while serum levels significantly higher than people without MS (Bahia
reducing the expression of insulin receptor substrate-1 (IRS-1) et al., 2006).
and GLUT4 in muscle and liver tissues (Francisco et al., Macrophages are a heterogeneous population of immune cells
2006). Though IL-6 is mainly secreted by adipocytes, it is that have a range of roles in both the induction and resolution
produced by smooth muscle cells, endothelial cells, monocytes of inflammation (Dey et al., 2015). The pleiotropic responses are
and macrophages and may contribute to the development of coordinated through distinct programs of macrophage activation
atherosclerotic lesions through its paracrine, autocrine, and classified as classical (or M1) and alternative (or M2) activation
endocrine effect. Studies have correlated the values of serum IL-6 (Gordon, 2003; Martinez et al., 2008).
with the waist circumference (Rexrode et al., 2003). People with It has been shown that the immune response that is activated
central obesity are at increased risk to develop MS. during inflammation and obesity-induced insulin resistance has
The TNF-α is a cytokine with autocrine, paracrine and the same M1 mechanism (Takeda et al., 2003). In classical
endocrine action (Ruan and Lodish, 2003). In obese humans, activation of macrophages, lipids derived from bacteria bind
there is an inverse correlation between TNF-α and glucose to Toll-like receptor 4 (TLR4) and activate signaling pathways
metabolism (Winkler et al., 2003). Insulin signaling suppression that induce, for example, the release of NF-kB inflammatory
by the TNF-α reduces the translocation of glucose transporter molecules (TNF, IL-6 and 2) (Takeda et al., 2003). In obesity
(GLUT-4) to the membrane and consequently promotes a and inflammation, saturated fatty acids activate TLR4 to promote
decrease in insulin-mediated glucose uptake by cells. It is also inflammatory responses (Shi et al., 2006; Kim et al., 2007; Nguyen
known that the expression of mRNA and TNF-α secretion are et al., 2007). In fact, acute infusion of lipids into mice potentiates
higher in obese person (Hsueh and Law, 2003). the insulin resistance in both adipose tissue and skeletal muscle in
IL-1β is responsible for increase in the expression of a TLR4-dependent manner (Kim et al., 2007). In addition, other
endothelial adhesion molecules which facilitate the aggregation studies have shown that white adipose tissue of obese rats have
of other inflammatory cells in the activated endothelium mainly macrophages with classically activated—M1 phenotype
(Francisco et al., 2006). IL-1β, together with TNF-α, stimulates (Bouloumié et al., 2005; Ferrante, 2007). Alternatively, during
IL-6 production by smooth muscle cells and increases the the resolution of inflammation, the balance of macrophage
expression of macrophages. This process is associated with activation toward an M2 phenotype occurs in order to promote
the progression of inflammatory and atherosclerosis processes clearance of debris and inhibit the production of inflammatory

mediators to restore tissue homeostasis (Mills et al., 2014). et al. (2000) demonstrated that chronic subclinical inflammation
M2 macrophages produce anti-inflammatory cytokines and is a key feature of the MS and components of MS (dyslipidemia,
express endocytic receptors. These cells promote the clearance abdominal obesity, and hypertension) increase in parallel to the
of apoptotic cells, proliferation and wound healing (Mills et al., increasing levels of plasma CRP in non-diabetic individuals. It
2014). Together, these data suggest a model in which increased is possible that chronic inflammation triggers MS (Festa et al.,
flux of saturated fatty acids, as seen in obese states stimulates 2000), overnutrition, cytokine hypersecretion, insulin resistance,
classical macrophage activation, tissue inflammation, and insulin and diabetes in predisposed individuals (Festa et al., 2000). Festa
resistance. et al. (2000) also suggested that the decrease in insulin sensitivity
Although the number of classically activated adipose tissue may lead to increase in CRP expression by counteracting the
macrophages increases with obesity, adipose tissue of lean physiological effect of insulin on hepatic protein synthesis
animals contains a moderate number of macrophages. The (Campos and Baumann, 1992; Festa et al., 2000).
adipose tissue macrophage of lean mice under non-inflammatory Although there are many studies focusing on oxidative
conditions express high levels of substances encoded by genes stress, inflammation and MS (Campos and Baumann, 1992;
of alternatively activated M2 macrophages that promotes tissue Roebuck, 1999; Festa et al., 2000; Furukawa et al., 2004;
homeostasis and repair (Hung et al., 2005; Vanhala et al., 2006). Tangvarasittichai, 2015), there is still no clear consensus about
As suggested by Palaniappan et al. (2003), obesity results in a the causal relationship among the triad oxidative stress—
change in the activation pathway of macrophages. inflammation—MS.
Van Guilder et al. (2006) evaluated a possible synergistic effect
of MS and obesity on the circulating markers of oxidative stress
and inflammation. In that study, the authors observed that MS The Sympathetic Nervous System and the
heightens oxidative stress and inflammatory burden in obese Metabolic Syndrome
adults, thereby suggesting that MS and obesity have a synergistic
effect on oxidative stress and inflammation markers (Van Guilder The sympathetic nervous system (SNS) is an arm of the
et al., 2006). Furthermore, the authors suggested that the autonomic nervous system which plays vital role in the regulatory
increased oxidative and inflammatory stress may contribute to mechanisms of blood pressure, sodium balance and maintenance
risk of coronary heart disease and cerebrovascular disease in of homeostatic state. The SNS is fundamental in the control
obese adults with MS (Van Guilder et al., 2006). of daily energy expenditure through the regulation of resting
Several studies have related the adipose tissue to the elevated metabolic rate and thermogenesis in response to physiological
markers of oxidative stress and inflammation once the expression stimuli, changing energy states, food intake, carbohydrate
and secretion of such markers increase in proportion to adiposity consumption and hyperinsulinemia (Thorp and Schlaich, 2015).
(Mohamed-Ali et al., 1998; Bertin et al., 2000; Kern et al., 2001; Furthermore, the activation of sympathetic nerves in target
Van Guilder et al., 2006). However, as observed by Van Guilder organs like liver, pancreas, skeletal muscle, and adipose tissue can
et al. (2006), the adiposity alone do not seems to be the primary elicit acute catabolic responses (i.e., glycogenolysis and lipolysis)
cause of oxidative stress and inflammation once the markers are (Thorp and Schlaich, 2015).
increased only in the obese individuals diagnosed with MS. Over activation of SNS is strongly associated with two
In addition, an increase in glucose metabolism has been components of the MS, i.e., obesity and hypertension
implicated in oxidative stress—MS, relation (Furukawa et al., (Tentolouris et al., 2006). In fact, enhanced SNS activation
2004; Tangvarasittichai, 2015). During the metabolism of glucose exerts unfavorable effects like cardiac hypertrophy, arterial
(glycolysis and tricarboxylic acid—TCA cycle), the electron remodeling, and endothelial dysfunction on the cardiovascular
donors NADH (nicotinamide adenine dinucleotide) and FADH2 system (Grassi and Seravalle, 2006). Increase in sympathetic
(flavin adenine dinucleotide) are generated (Tangvarasittichai, activity enhances systemic and regional norepinephrine spillover
2015). In the case of over nutrition or obesity, a large amount and elevate resting heart rate. This condition has been linked
of glucose is oxidized in such a way that an increase in the to hypertension, obesity, and insulin resistance (Mancia et al.,
generation of NADH and FADH2 in the mitocondrial electron 2007). Furthermore, it has been shown that high levels of fasting
transport chain subsequently result in an increase in superoxide insulin, an index of insulin resistance, were positively associated
generation (Tangvarasittichai, 2015). Furthermore, the increase with the low-to-high frequency (LF/HF) ratio of the heart rate
in free fatty acid and acetyl coenzyme A (CoA) oxidation (due variability (HRV)—an index of the sympathovagal balance at the
to the excessive of free fatty acids) in TCA cycle generate heart level (Emdin et al., 2001).
more molecules of NADH and FADH2 to be oxidized, and In view of the strong relationship among obesity, MS and
overproduction ROS (Furukawa et al., 2004; Tangvarasittichai, the development of cardiovascular risk factors, it is important to
2015). Moreover, NADPH oxidase is involved in fatty acids— elucidate autonomic disturbances that occur in obese individuals.
ROS generation in adipocytes once the treatment with NADPH As cited by Hall et al. (2000), there are two lines of evidences
oxidase inhibitor blocks the ROS generation (Furukawa et al., about the central nervous system involvement in obesity-induced
2004; Tangvarasittichai, 2015). hypertension: (i) increase in sympathetic activity in obese as
Like oxidative stress, inflammatory state is also an important compared to lean subjects; and (ii) attenuation of obesity-related
feature of MS. Inflammation is one the manifestations of hypertension through pharmacological blockade of adrenergic
oxidative stress (Roebuck, 1999; Tangvarasittichai, 2015). Festa activity (Landsberg and Krieger, 1989; Grassi et al., 1995;

Hall et al., 2000). Though the autonomic disorders are not In patients with type 2 diabetes mellitus (T2DM), MS has
homogeneous in obesity, some studies have demonstrated that approximately 70% of prevalence rate (Monami et al., 2007;
most individuals exhibit sympathetic hyperactivity (Lopes and Bianchi et al., 2008). The basic mechanism involved in the
Egan, 2006). Both baroreflex sensitivity (BRS) and impaired pathogenesis of T2DM is the insulin resistance. The insulin
HRV in obese women (Skrapari et al., 2007). Esler et al. (2001) resistance, in turn, is strongly associated with sympathovagal
demonstrated that the sympathetic tone in obese individuals imbalance. Furthermore, many data suggest the involvement
is increase in some target organs like kidney, skeletal muscle of increased SNS activity in insulin resistance (Mancia et al.,
and vessels. Sympathetic hyperactivity in obesity indicates that 2007). Epidemiological studies have found a correlation between
obesity impairs renal-pressure natriuresis, increases renal tubular insulin resistance and hypertension (Modan and Halkin, 1991;
sodium reabsorption and causes hypertension (Kassab et al., Skyler et al., 1995). In patients with type 1 diabetes mellitus
1995; Hall et al., 2000). (T1DM), the hypertension is usually developed after the onset
It is known that sympathetic disturbances are directly of nephropathy and it is associated with rennin-angiotensin
related to increase in arterial blood pressure (Tan et al., 2010; induced SNS activation (Perin et al., 2001). In contrary, the
Oliveira-Sales et al., 2014). In humans, several features confirm prevalence of hypertension in patients with T2DM is extremely
typical increase in sympathetic tone as observed in obesity. common (Perin et al., 2001). Thus, it can be assumed that Insulin
Various studies have demonstrated increased blood pressure and resistance and hypertension as observed in the MS are closely
serum catecholamine levels in obese individuals. The loss of linked with sympathetic overactivation (Frontoni et al., 2005).
weight is associated to the decrease in plasma concentration The SNS activity plays a crucial role in the regulation
norepinephrine (Tuck, 1992; Lopes and Egan, 2006). Obese of circulation and blood pressure (Fisher and Paton, 2011;
hypertensive children show increase in sympathetic nerve activity Zubcevic et al., 2011). Sympathetic vasomotor and cardiac
(Rocchini et al., 1989; Lopes and Egan, 2006). However, in neural activities are induced by the sympathetic preganglionic
these patients, a low salt diet (or hyposodic diet) is capable of neurons in the spinal cord. These neurons receive tonic excitatory
promoting a decrease in arterial pressure (Rocchini et al., 1989; drive from pre-sympathetic networks within the brainstem and
Lopes and Egan, 2006). These studies point out the fact that hypothalamus (Dampney, 1994; Guyenet et al., 1996; Dampney
sympathetic hyperactivity is related to sodium retention and et al., 2002; Madden and Sved, 2003). Increased in SNS activity
increase of arterial blood pressure in obese children (Lopes and has been linked to the pathogenesis of hypertension in humans
Egan, 2006). Furthermore, muscular sympathetic nerve activity with essential hypertension (Abboud, 1984; Mancia et al., 1999;
(MSNA) is increased in obese (normotensive and hypertensive) Esler et al., 2001; Guyenet, 2006). Sympathetic overload is
as compared to non-obese normotensive individuals (Grassi implicated in the pathogenesis and/or deterioration of essential
et al., 2000). Moreover, it has been shown that the MSNA and the hypertension through the modification of heart rate, cardiac
plasmatic norepinephrine are reduced and the BRS is increased output, peripheral vascular resistance and renal sodium retention
after weight loss in normotensive obese individuals (Grassi et al., (Grassi and Seravalle, 2006). The study of sympathetic nerve
1998; Lopes and Egan, 2006). The SNS response is blunted in firing rate in hypertensive patients has shown sympathetic
obese subjects despite the fact that plasma insulin levels were overactivity in young, middle-aged, and elderly hypertensive
almost 45% higher in the obese as compared to the lean patients (Grassi et al., 2000). Some studies with essential hypertensive
(Tentolouris et al., 2003). patients have plasmatic overflow of norepinephrine. This
The heart rate variations are a visible effect of the autonomic overflow indicates an increase in the activation of sympathetic
influences on the heart in cases of emotional stress. An outflow to the heart, kidneys and cerebrovascular circulation of
inability to sustain varying heart rate is an important risk these individuals (Esler et al., 1991; Mancia et al., 2007). These
factor to the development of CVD (Hemingway et al., 2001; observations are evidences that some target organs are negatively
Brunner et al., 2002). The study of Brunner et al. (2002) affected by increased blood pressure (Grassi et al., 2007).
demonstrated a relative sympathetic dominance and a lower Moreover, increase in heart rate has been observed in subjects
vagal tone to the heart in MS cases, thereby indicating unbalance with MS as compared to those without MS (Mancia et al., 2007).
sympathovagal in those individuals. Jamerson et al. (1993) In this way, the physical inactivity can be associated with obesity
demonstrated an inverse relationship between sympathetic as well as to the increase in cardiac sympathetic drive in the MS.
vascular tone and the insulin-mediated cellular consumption The SNS disturbances are closely related to all the main
of glucose. Thus, it is not surprising to speculate that the features of the MS. Although SNS participation in the
increased SNA as observed in obese individuals increases the pathophysiology of MS is clear, it is difficult to determine
vascular constriction and impairs the glucose transportation into whether the metabolic changes are responsible for sympathetic
the cells (Grassi et al., 2000; Lopes and Egan, 2006). Previous disturbances or vice versa.
studies with obese models have implicated vascular constriction
in insulin-resistance (Laakso et al., 1990; Lopes and Egan, 2006).
Specific alpha-adrenergic vasoconstriction seems to be more Role of Leptin in the Elevation of
malefic on glucose consumption than the angiotensin-induced Sympathetic Activity
vasoconstriction (Jamerson et al., 1993; Lopes and Egan, 2006).
This assumption suggests, once again, sympathetic influence on Obesity, an important risk factor for the development of MS, is
glucose metabolism. characterized by the increase in size and number of adipocytes

(cells that produce adipokines, Martínez-Martínez et al., 2014). (which could lead to the weight loss) could be a promising
Leptin is one of the adipokines that has been postulated as a link therapy as it decreases insulin resistance. Since there are direct
between obesity and cardiovascular damage (Martínez-Martínez relationships between sedentary lifestyle and cardiovascular
et al., 2014). Leptin is a peptide synthesized and secreted from risk factors, the benefits of physical exercises on the prognosis
white adipose tissue (Head et al., 2014) in addition to other of the MS are clear (Lakka et al., 2003; Rennie et al., 2003).
sources like placenta, stomach, and heart (Zeidan et al., 2011). In fact, translational studies demonstrate lower insulin levels
Leptin expression can be induced by obesity, insulin and TNF- and increased insulin sensitivity in athletes as compared to
α. This adipokine has been implicated in numerous physiological match-aged sedentary individuals (Nuutila et al., 1994; Roberts
functions such as immune response and reproduction (Frühbeck, et al., 2013). Eriksson et al. (1997) demonstrated that one
2002; Mark, 2013). However, its main action is related to glucose single session of exercise increase the glucose offer mediated by
homeostasis and regulation of appetite. This hormone provides insulin in healthy and insulin resistant obese and type 2 diabetes
feedback to the CNS on the status of peripheral energy reserves individuals. Chronic exercise improves insulin sensibility in
(Rahmouni, 2010). healthy, non-diabetic obese, type 1 and 2 diabetic individuals
Plasma leptin concentrations are significantly elevated in (Eriksson et al., 1997). Also, the use of drugs like metformin and
several rodent and human models of obesity in a proportional thiazolidinediones (insulin sensitizers) have also been prescribed
manner to adiposity (Magni et al., 2000). Although circulating for insulin resistance treatment (Einhorn et al., 2003; Grundy
levels of leptin rise in obesity, these individuals are thought to et al., 2004).
be leptin resistant due to lack of satiation (Magni et al., 2000). As previously mentioned, the serum phosphate and
It is known that systemic infusion of leptin increased renal magnesium is reduced in MS patients (Stoian and Stoica,
sympathetic nerve activity (RSNA) and supplies (Haynes et al., 2014). The compensatory hyperinsulinemia can promote
1997). Central infusion of this peptide increases blood pressure decrease of serum phosphate and magnesium, a vicious cycle
(Shek et al., 1998). Head et al. (2014) recently demonstrated that which contributes to the pathophysiology of MS (Stoian and
central infusion of leptin antagonist in obese rabbits is able to Stoica, 2014). Hence, a pharmacological treatment that restores
return blood pressure to their basal levels (Head et al., 2014). the level of these ions could be beneficial (Stoian and Stoica,
These findings may contribute to understand an obesity-induced 2014) to MS patients.
hypertension. Furthermore, recent studies have demonstrated
that antagonism of central leptin receptor, LepR, caused a Final Considerations
reduction in BP and HR in hypertensive mice (Tumer et al.,
2007). The cardiovascular effects of leptin administration are not The definition and diagnostic criteria of MS are still controversial
exclusively due to its central action. In fact, it has been shown that for obvious reasons. The divergent opinions on the diagnosis
systemically administration of leptin antibodies elicited similar of MS among health institutions constitute challenges to its
changes as compared to central administration (Tumer et al., treatment and identification of individuals at high risk of CVD
2007). and diabetes. The diagram presented in the Figure 1 below
In an animal model of non-obese animals with summarizes the complex relationship between some of the MS
hyperleptinemia, leptin increased systolic blood pressure and components.
promoted an increase in intrarenal and systemic oxidative stress A clinical diagnosis of MS is critical as it affects therapeutic
(Beltowski et al., 2004). In these model, the increase in ROS cause strategy. A multidisciplinary approach including lifestyle
inactivation of nitric oxide. This effect can explain the leptin- changes, pharmacological and surgical approaches could be
associated hypertension in this model (Beltowski et al., 2004; helpful toward the management of this syndrome. Physical
Martínez-Martínez et al., 2014). Renal sodium retention has inactivity, insulin resistance, advance age, hormonal factors
also been implicated in hyperleptinemia hypertension (Martin (androgens and corticosteroids), and diets rich in fats
et al., 2008). Beltowski et al. (2004) have demonstrated that which promote abdominal obesity or adiposity have been
hyperleptinemia decreases urinary sodium excretion to promote consistently identified as major risk factors of MS. Atherogenic
volume retention and consequent increase in arterial blood dyslipidemia, elevated blood pressure, smoking, elevated glucose,
pressure. Other harmful effects such as atherosclerosis (Gruen prothrombotic, and proinflammatory state are cardiovascular
et al., 2007), inflammation, thrombosis and cardiac myocyte risk factors that accompany MS. Change in lifestyle and anti-
hypertrophy (Northcott et al., 2012) have been associated obesity drugs among others could engender effective prevention
with leptin. Based on these observations, it is reasonable to or treatment of MS. Although, lifestyle changes remain first-line
suggest that pharmacological approaches targeting leptin’s effects therapy for the improvement of all the underlying metabolic
could represent a potentially useful therapeutic strategy for risk factors, cases of unsuccessful lifestyle modification therapy
the treatment of obesity-associated hypertension among other can be substituted with anti-obesity treatment. Lifestyle therapy
cardiovascular disease. could dampen MS progression at every stage. This kind of
therapy does not treat each risk factor in isolation but rather
Treatment of Metabolic Syndrome target multiple risk factors simultaneously. Although lifestyle
therapy may not modify any given risk factor as much as drug,
Since MS is a clustering of dysfunctions, different treatment its benefit lies in the fact that it produces moderate reduction
strategy has been adopted. WHO suggested treatments focusing in all metabolic risk factors. In most situations, drug therapies
on insulin resistance as first-line therapy. A more active lifestyle might be required in the case of worsening condition of MS.

FIGURE 1 | Hypothetical diagram summarizing the relationship between the components of the metabolic syndrome.
The effectiveness of drugs targeting individual risk components receptors blockers and peroxisome proliferator–activator
of MS separately is still uncertain. This approach could lead receptor-γ agonism could lower blood pressure, vascular
to aggressive use of medications at the expense of lifestyle and cardiac sympathetic tone as well as plasmatic glucose
therapy. The ineffectiveness of some available drugs for the simultaneously. Though new drug development programme
treatment of MS has been compounded by the impractical looks interesting, better understanding of MS, improved
approach of simultaneous prescription and administration diagnostic criteria and treatment strategy is key to future clinical
of all the drugs that could modify all of the risk factors. practice.
Current efforts being made to combine drugs into a single
capsule that targets multiple risk factors seems ingenious as Acknowledgments
it could reduce the burden of polypharmacy. A single drug
that can affect multiple metabolic risk factors simultaneously This work was supported by Fundação de Amparo a Pesquisa
and provide health benefit to MS patient seems promising. do Estado de Goiás (FAPEG) and by Conselho Nacional de
Drugs that act as angiotensin and adrenergic (alpha and beta) Desenvolvimento Científico e Tecnológico (CNPq).
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Neuronal circuits involved in osmotic challenges
Marina Conceição dos Santos Moreira1, Lara Marques Naves1, Stefanne Madalena
Marques1, Elaine Fernanda Silva1, Eduardo Colombari2, Gustavo Rodrigues Pedrino1.
1Center for Neuroscience and Cardiovascular Research, Universidade Federal de Goiás,

Goiânia - GO – Brazil.
2Department of Physiology and Pathology, Universidade Estadual Paulista Júlio de

Mesquita Filho, Araraquara - SP – Brazil.
Running title: CNS and osmotic challenges
Corresponding author: Gustavo Rodrigues Pedrino
Department of Physiological Science
Federal University of Goiás
Estrada do Campus, s/n. Zip code: 74001-970. PO: 131. Goiânia – GO - Brazil
Phone/Fax: +55-62-3521-1774 e-mail: pedrino@pq.cnpq.br / gpedrino@gmail.com
Key words: Body fluid homeostase, osmossensory areas, acute hyperosmotic challenge,
chronic hyperosmotic challenge.
Summary
The maintenance of plasma sodium concentration within a narrow limit is crucial to
life. When it differs from normal physiological patterns, several mechanisms are activated in
order to restore body fluid homeostasis. Such mechanisms may be vegetative and/or
behavioral, and several regions of the central nervous system (CNS) are involved in their
triggering. Some of these are responsible for sensory pathways that perceive a disturbance
of the body fluid homeostasis and transmit information to other regions. These regions, in
turn, initiate adequate adjustments in order to restore homeostasis. The main cardiovascular
and autonomic responses to a change in plasma sodium concentration are: i) changes in
arterial blood pressure and heart rate; ii) changes in sympathetic activity to the renal system
in order to ensure adequate renal sodium excretion/absorption, and iii) the secretion of
compounds involved in sodium ion homeostasis (ANP, Ang-II, and ADH, for example). Due
to their cardiovascular effects, hypertonic saline solutions have been used to promote
resuscitation in hemorrhagic patients, thereby increasing survival rates following trauma. In
the present review, we expose and discuss the role of several CNS regions involved in body
fluid homeostasis and the effects of acute and chronic hyperosmotic challenges.
Introduction
The maintenance of body homeostasis is crucial to life and is directly related to
hydromineral balance. The sodium ion (Na+) is, among all the inorganic ions of the organism,
the one most often consumed (through dietary salt, NaCl), and is also the most important
determinant of extracellular osmolarity. Variations in sodium plasma concentration cause
osmotic flow between the intracellular and extracellular compartments, which can affect all
perfused tissues and alter volume, metabolism, and cellular functioning. Left uncontrolled,
these osmotic changes can harm the organism and contribute to the development of
cardiovascular diseases and their related complications. The central nervous system (CNS)
detects variations in the volume, tonicity, and composition of the extracellular compartment
through peripheral and central receptors. Once detected, changes in the plasma sodium
concentration will trigger a set of responses to re-establish normal physiological conditions.
Studies have demonstrated that minimal elevations in plasma osmolarity and reductions in
circulating volume (dehydration) are strong stimuli in the development of thirst behavior
(Antunes-Rodrigues et al. 2004; Fitzsimons 1998). Regarding vegetative adjustments,
hypernatremia triggers responses that modulate the renal excretion of water and sodium.
Among these responses, we highlight changes in sympathetic nerve activity (Chen and
Toney, 2003; Pedrino et al., 2008; Shi, Stocker et al., 2007; Stocker et al., 2013); renal
vasodilation (Morita and Vatner 1985; Pedrino et al., 2005; Pedrino et al., 2006; Pedrino et
al., 2009); renin, atrial natriuretic peptide (ANP), and oxytocin secretion (Antunes-Rodrigues
et al. 1991, 1992; Godino et al., 2005; Haanwinckel et al., 1995; Huang et al. 2000; Huang
et al., 1995; Morris and Alexander, 1988; Rauch et al., 1990); and natriuresis and diuresis
(Bealer 1983; Bealer et al. 1983; Bie 1980; Bourque et al., 1994; Schoorlemmer et al., 2000;
da Silva et al. 2013).

Central circuitry involved in hyperosmotic activation
Central osmoreceptors localized in the forebrain lamina terminalis, the organum
vasculosum of the lamina terminalis (OVLT), and the subfornical organ (SFO) have been
described as participating in the autonomic adjustments activated by acute hyperosmotic
challenges (Johnson et al., 1996; Shi et al., 2007). OVLT and SFO neurons are known as
circumventricular organs (CVOs) because they lack a complete blood–brain barrier
(McKinley and Johnson 2004; Stocker et al. 2013; Toney et al. 2003). These neurons can
respond to blood-borne solutes that do not have free access to other regions of the brain
(McKinley & Johnson, 2004). In fact, studies have reported that the OVLT and SFO contain
neurons that exhibit intrinsic osmosensitivity (Bourque 2008; Ciura and Bourque 2006).
Experimental evidence has shown that either electrolytic lesion or inhibition of OVLT
neurons by muscimol (GABAa receptor agonist) significantly reduced both lumbar and renal
sympathoexcitatory responses promoted by the intracarotid infusion of hypertonic NaCl
solutions (Shi et al., 2007). Moreover, Antunes et al. (2006) showed that a transection that
disrupts the connections of the circumventricular organs (CVO) attenuated the
sympathoexcitation induced by hyperosmotic perfusate in the arterially perfused
decorticated in situ rat preparation. Indeed, raising the sodium concentration in the plasma
or cerebrospinal fluid increases Fos expression, a marker of neuronal activation in the OVLT
(Shi et al., 2008). Furthermore, SFO neurons contribute to the hypertension induced by
chronic Ang II administration in rats on a high-salt diet (Ang II–salt model) (Osborn et al.
2014). In addition to initiating autonomic adjustments in response to hypernatremia, SFO
and OVLT also participate in triggering behavioral adjustments to hyperosmotic stimuli.
Furthermore, combined electrolytic lesions of the OVLT and SFO attenuate the drinking of
water induced by plasma hypernatremia (McKinley et al. 1992). These findings emphasize
the role of the osmosensitive neurons of the lamina terminalis in the sympatoexcitatory and
behavioral responses to hyperosmotic challenges.
Lamina terminalis neurons do not project directly onto sympathetic preganglionic
neurons. Cerebrospinal fluid hypernatremia, capable of increasing renal sympathetic nerve
activity (RSNA) and blood pressure, also induces increased Fos expression in OVLT
neurons with axonal projections to the paraventricular nucleus of the hypothalamus (PVN)
(Shi et al., 2008). OVLT and SFO osmosensitive neurons are responsible for triggering
sympathetic reflex adjustments to hyperosmolality mainly through a descending pathway to
PVN (McKinley et al., 1992; Shi et al., 2008; Swanson and Sawchenko, 1983). In turn, PVN
neurons activate downstream sympathetic regulatory targets, including both presympathetic
neurons localized in the rostral ventrolateral medulla (RVLM) (Brooks et al., 2004) and
sympathetic preganglionic neurons of the spinal cord (Badoer and De Matteo, 2003).
The area postrema (AP) is another region known to participate in central
osmosensory mechanisms. A study by Carlson et al. (1997) demonstrated that intragastric
hyperosmolarity activates several regions of the CNS, such as the supraoptic nucleus
(SON), PVN, the nucleus of the solitary tract (NTS), AP, and lateral parabrachial nucleus
(LPBN), and that such activation is mediated by peripheral osmoreceptors throughout the
splanchnic and vagal afferent projections. Following these observations, Carlson et al.
(1998) demonstrated that PVN responses to intragastric hyperosmolarity are dependent on
the connections with the AP, whereas the responses of other regions (NTS, SON, and
LPBN) are independent of AP integrity. The authors have suggested, therefore, that
peripheral osmosensitive projections to both the NTS and AP modulate the activity of the
SON and PVN. Moreover, both nuclei are necessary for a complete response; however, the
NTS – but not the AP – may control hypothalamic function through projections to the LPBN.
Acute hyperosmotic challenges
In the past, the effects of acute changes in body fluid osmolality through hypertonic
NaCl infusion have been studied and is well established. Weiss et al. (1996) showed that an
intravenous infusion of hypertonic saline (HS) (2.5 M NaCl) produces a transient increase in
arterial blood pressure (ABP) and that this response is related to increases in lumbar
sympathetic activity and vasopressin release. By contrast, the authors reported that HS
infusion is able to induce long-lasting renal vasodilation and decreases in RSNA.
Renal vasodilation is a significant factor in the regulation of water and sodium
excretion, and it is one of the variables adjusted in response to acute variations in the
osmolality of extracellular fluid (Pedrino et al. 2008). The mechanisms involved in the
genesis and maintenance of renal vasodilation induced by increases in osmolality are
complex and appear to involve neural and hormonal mechanisms (Amaral et al., 2014;
Burnett et al., 1984; Haanwinckel et al. 1995; Huang et al. 1995; Shen et al., 1991; Wakitani
et al., 1985). Regarding the neural mechanisms, studies have related reductions in RSNA
to renal vasodilation and consequent natriuresis (Pedrino et al. 2008, 2012; da Silva et al.
2013; Weiss et al. 1996), showing that HS infusion causes a sustained increase in ABP and
renal vascular conductance (RVC) but no changes in mesenteric or hindquarter vascular
conductance. These findings indicate that vasodilation induced by HS infusion is specific to
the renal vascular region.
Central areas and peripheral afferents known to contribute to body fluid homeostasis
and cardiovascular regulation (Silva et al., 2013; Pedrino et al., 2009; Pedrino et al., 2005)
have been described by their involvement in renal vasodilation and by their
sympathoinhibition to acute hypernatremia.
Acute/chronic electrolytic lesions of the lamina terminalis region eliminated HS-
induced renal vasodilation (Pedrino et al., 2005). The chemical lesion of noradrenergic
neurons located in the caudal ventrolateral medulla (CVLM; A1) and NTS (A2) prevented
renal sympathoinhibition induced by intravenous HS infusion (Pedrino et al., 2012, 2008).
More recently, we have shown that sinoaortic denervation (SAD) abolished renal
vasodilation and sympathoinhibition induced by acute hypernatremia (Silva et al. 2015).
These findings suggest the involvement of central regions and peripheral receptors in the
renal hemodynamic and autonomic adjustments induced by acute NaCl overload.
In opposition to these results, experimental investigations have also demonstrated
that hyperosmotic stimulation increases SNA in other regions (Shi et al., 2007). Specifically,
acute intravascular infusion of hypertonic NaCl increases thoracic, lumbar, and splanchnic
SNA (Holbein and Toney 2015). These changes in the sympathetic outflow generated by
sodium overload have been described as altering cardiovascular functions and appear to
serve an important homeostatic role. Furthermore, such SNA increases in several regions
(excepting the renal region) seem to contribute to the increased BP observed after an acute
osmotic challenge.
Hypertonic solutions and hemorrhagic shock
Hemorrhagic shock is defined as the decrease of ABP due to drastic blood loss. Such
pathology is characterized by tissue hypoperfusion leading to decreases in nutrients and
oxygen supply to the body’s tissues (Krausz, 2006). Hemorrhagic shock can promote a
series of disturbances in the physiological state, leading to multiple organ failure and causing
high levels of mortality (Angele et al., 2008; Xu et al. 2013).
It has been shown that trauma is currently the main cause of death among younger
people (under 40 years old), and several studies have demonstrated that approximately
40% of such deaths result from uncontrolled blood loss (Curry and Davis, 2012). Therefore,
treating hemorrhagic shock and promoting the cardiovascular recovery of hypovolemic
patients are crucial to prevent death. Thus, the use of fluid resuscitation in patients in
hemorrhagic shock has been studied in the past to determine its cardiovascular effects and
therapeutic potential. Among several types of solutions used in hypovolemic patients, such
as blood substitute and colloid, crystalloid, isotonic, and hypertonic solutions, hypertonic
saline (HS) has stood out because it combines the cardiovascular benefits of the other
solutions even at lower volumes (Tremblay et al., 2001).
The treatment of hemorrhagic patients using hypertonic solutions has been practiced
for more than 85 years. Pioneering studies by Velasco et al. (1980) have shown that the
administration of HS (7.5% NaCl) in dogs submitted to hemorrhagic shock promoted the
restoration of blood pressure and cardiac output and resulted in the survival of all the dogs
in the study. The control group, which received the same volume of isotonic solution, did not
show hemodynamic improvement or survival. In the same sense, de Felippe et al. (1980)
demonstrated hemodynamic improvement in patients with refractory shock in intensive care
units following hypertonic saline infusion.
Various studies have sought to determine the cardiovascular effects of intravenous
HS infusions on hypovolemic hemorrhage (Lopes et al., 1981; Rocha-e-Silva et al., 1986;
Younes et al., 1985). These studies demonstrated that hypertonic saline infusion promoted
an instantaneous elevation in mean arterial blood pressure, increased pulse pressure, and
re-established cardiac output and mesenteric, renal, and hind-limb circulation.
Although the hemodynamic responses to the infusion of HS in animals subjected to
bleeding have already been established, the involvement of the CNS in these responses is
still under investigation. Recent studies have linked peripheral afferents to cardiovascular
recovery induced by HS. In 2009, de Almeida Costa et al. observed that, in the absence of
carotid afferents, cardiovascular recovery induced by HS after hemorrhagic shock is
compromised, indicating that such afferents are crucial to HS cardiovascular effects,
whereas it is independent of the integrity of baroreceptor afferents. In the same sense,
Pedrino et al. (2011) demonstrated the participation of peripheral chemoreceptors in the
detection of changes in plasma osmolarity. Such studies have demonstrated that the ligation
of the artery responsible for irrigating the carotid corpuscle and its consequent inactivation
interferes with the cardiovascular recovery induced by HS in hypovolemic rats. Taken

together, such results support the hypothesis that increasing plasma sodium concentration
activates several mechanisms that are dependent on peripheral afferents in order to restore
physiological conditions in hypovolemic animals. In the absence of chemoreceptor afferents,
such reflex mechanisms are compromised, indicating that these afferents play a key role in
body fluid homeostasis.
The peripheral chemoreceptor afferents execute their first synapses in the NTS; from
such nuclei, the information can be transmitted to various regions of the CNS, particularly to
hypothalamic structures such as the lamina terminalis. The lamina terminalis includes
structures such as the OVLT, the ventral portion of the median pre-optic nucleus (MnPO),
the preoptic periventricular nucleus, and the more medial aspects of the medial preoptic
nucleus (Andresen and Kunze, 1994; Bourque, 2008; Cunningham and Sawchenko, 1988;
Saphier, 1993; Tanaka et al. 1997, 2002).
In past years, the lamina terminalis has been the focus of several studies that
demonstrated that this region is an important integration center involved in the processing
and correcting osmolarity and volume changes of extracellular compartments.
Cardiovascular, endocrine, renal, and behavioral responses are recruited following the
lamina terminalis’s activation in an effort to restore plasma sodium concentration and blood
volume. The lamina terminalis establishes reciprocal connections with CNS structures that
are known to participate in cardiovascular regulation and hydroelectrolytic balance, such as
the vasomotor nuclei of the ventral surface of the medulla, RVLM and CVLM, SFO, and the
limbic system (Tanaka et al., 1992; Tucker et al., 1987; Zardetto-Smith and Johnson, 1995).
Interestingly, studies have demonstrated that the lamina terminalis is also involved
in cardiovascular recovery induced by hyperosmotic solution in rats submitted to
hypovolemia. In 1992, Barbosa et al. observed that the integrity of the lamina terminalis is
important to HS effects; lamina terminalis-lesioned rats submitted to hemorrhagic shock do
not show cardiovascular recovery following HS infusion.

Moreover, a recent study from our laboratory demonstrated that MnPO integrity is
crucial to cardiovascular recovery induced by HS infusion following hemorrhagic shock;
following the pharmacological blockade of such nuclei, recovery is abolished (Amaral et al.,
2014). Consistent with these results, unpublished observations from our laboratory
demonstrate that inhibition of this region impairs the recovery of ABP and RSNA induced by
HS in rats submitted to hemorrhagic shock (Figure 1; L.M.N and G.R.P., unpublished
observations). Taken together, these results indicate that MnPO integrity is required for the
development of cardiovascular and sympathetic responses induced by HS in hypovolemic
conditions.
The MnPO is an integrative nucleus corresponding with body fluid homeostasis. Such
nuclei receive projections from regions important to hydroelectrolytic balance, such as the
A1 and A2 noradrenergic neurons, the SFO, and OVLT. Furthermore, the MnPO projections
to the PVN promote the humoral and autonomic adjustments required to restore
homeostasis. Thus, it is not unreasonable to suggest that the cardiovascular recovery
induced by HS infusion after hemorrhagic shock could also depend on the integrity of others
of these regions, such as the A1 and A2 noradrenergic groups.
Recently, we studied the participation of the A1 neuronal group in HS cardiovascular
effects in hypovolemic rats. Our results demonstrate that A1-lesioned animals present
cardiovascular recovery induced by HS similar to sham rats after hemorrhagic shock (Figure
2; L.M.N. and G.R.P unpublished observations). Such observations allow us to conclude
that the A1 noradrenergic neurons alone are not crucial to cardiovascular recovery induced
by HS infusion in hypovolemic animals. However, the participation of the A2 noradrenergic
neurons in this process is still under investigation.
Chronic hyperosmotic challenges
It has been well established that high salt intake results in harmful effects to the
organism, especially to the cardiovascular system. He et al. (2008) investigated the

relationship between high salt intake and blood pressure levels in children and adolescents.
The results demonstrated that there was a significant association between high salt intake
and raised levels of systolic blood pressure and pulsatile pressure. The authors observed
that an increase of 1 g/day in salt intake was related to an increase of 0.4 mmHg in systolic
and 0.6 mmHg in pulsatile blood pressure. In experimental models, the addition of dietary
salt has been associated with the development of exacerbated arterial hypertension. Dahl
et al. (1963) reported that two-thirds of the rats fed a high salt diet presented with increased
blood pressure. In these animals, raised levels of ABP were maintained even after the re-
establishment of normal NaCl intake. It is suggested that this effect could be etiological,
indicating the existence of a group of salt-sensitive animals.
Several studies have demonstrated that salt intake enhances SNA and ABP, even in
normotensive salt-resistant animals (Appel et al., 2011; Stocker et al., 2010). In this sense,
Simmonds et al. (2014) found evidence that dietary salt intake modulates the
responsiveness of central autonomic circuits promoting functional changes in the regulation
of SNA and ABP. These results indicate that the level of dietary NaCl was directly correlated
to the magnitude of the SNA and ABP responses during activation of sciatic afferents,
stimulation of aortic depressor nerve, activation of vagal nerve afferents, volume expansion,
and increased NaCl concentration in the cerebrospinal fluid. Furthermore, consistent with a
sensitization to the central autonomic pathways, a high NaCl diet promoted increased blood
pressure variability (BPV) independent of changes in mean ABP (Simmonds et al., 2014).
Brooks et al. (2004) studied the hyperosmolality effects on the excitatory drive of the
RVLM. In this study, the authors observed that the excitatory amino acids (EAA) driving the
RVLM blockade, using kynurenic acid (KYN), did not promote changes in the cardiovascular
parameters of water-replete rats; however, such a blockade promotes a great decrease of
ABP in dehydrated animals, indicating that the EAA drive is increased in this case.
Furthermore, the EAA drive of the RVLM is partially influenced by plasma osmolality once
the depressor response of KYN into the RVLM is diminished after the re-establishment of
osmolality in previously hypovolemic rats. In normovolemic animals receiving HS, the KYN
did not promote immediate hypotension; however, such a response developed gradually
after three hours. These results demonstrate that acute and chronic increases in plasma
osmolality promote an increase of the EAA drive of the RVLM.
Adams et al. (2007) showed that increased dietary salt exaggerates pressure
responses induced by the RVLM. The study determined that the enhanced pressor
responses were directly attributable to a greater increase in SNA, whereas these enhanced
responses were balanced by a greater responsiveness of RVLM neurons to inhibitory input.
In this study, the authors demonstrated that pharmacological stimulation or inhibition of the
RVLM produced a significantly greater response (increase or decrease) in renal SNA,
splanchnic SNA, and ABP in rats drinking NaCl for 14 days versus in rats drinking water.
The enhanced SNA and ABP responses were not observed in rats drinking NaCl for 1 to 7
days but were present in animals under this treatment for 21 days, indicating its relationship
to chronic salt intake. The withdrawal of a high NaCl diet re-established the baseline levels
of these responses. These findings indicate that the potentiated ABP responses are
dependent on a slowly developing and reversible form of neuronal plasticity. In this sense,
Simmonds et al. (2014) demonstrated that, in animals submitted to different levels of dietary
salt, the magnitude of SNA and ABP responses to electrical stimulation of sciatic afferents
or intracerebroventricular infusion of NaCl were increased according to the level of salt
intake. The authors observed that there was a direct correlation between the level of dietary
salt intake and the sympathoinhibitory responses produced by acute volume expansion,
stimulation of the aortic depressor nerve, or cervical vagal afferents. By contrast,
sympathetic and blood pressure responses to chemoreflex activation produced by hypoxia
or hypercapnia were not affected by increased sodium intake.
In addition, the authors observed that the ability of dietary salt intake to modulate
cardiovascular effects is negatively affected by chronic lesions of the lamina terminalis,
indicating that the integrity of this area is crucial to salt-induced responses. In this study,
animals submitted to a high-salt diet presented with elevated BPV but normal levels of MAP.
These findings indicate that dietary salt intake works through the forebrain hypothalamus to
modulate various centrally mediated cardiovascular reflexes and increase BPV.
The neuronal plasticity induced by salt overload was also the focus of a study by Kim
et al. (2011), who observed that GABAa receptor-mediated transmission in the
magnocellular neurosecretory cells of the PVN is inhibitory in almost 75% of the neurons
under resting conditions. However, for animals submitted to NaCl 2% as a drinking supply
for 7 days, the GABAa, post-synaptic potentials (PSPs), and currents (PSCs) of these cells
were almost always excitatory. Such transmission becomes excitatory when there is a
sustained demand for AVP and oxytocin secretion (Kim et al., 2011; De Koninck, 2007).
Several studies in animal models (Brown et al., 2009; Cook et al., 2007; He and
MacGregor, 2009) demonstrated that, at present, high salt intake is directly related to
increased morbidity of and mortality resulting from cardiovascular events. This increased
salt intake promotes elevation of ABP, increasing the possibility of myocardial infarction,
stroke, coronary revascularization, or cardiovascular death. In an epidemiological study,
Costa et al. (2012) demonstrated that increased sodium intake promotes higher oxidative
stress, inflammatory response, myocardial stretching and remodeling, and higher total
mortality after myocardial infarction in human patients. Taken together, the studies reinforce
the understanding of the potentially harmful effects of a high-salt diet on cardiovascular
parameters.
As previously described, the negative cardiovascular effects of high salt intake in
adult animals are well established in the literature. Furthermore, it is known that the perinatal
period (pregnancy and lactation) is crucial to the organism’s development throughout
adulthood (Bao et al., 1995; Barker, 1997; Li et al., 1995; Vidonho et al., 2004). However,
little information exists regarding the post-natal period and its contributions to the organism’s
development. Recently, several researchers aimed to investigate the importance of
hydromineral imbalance during the post-weaning period in the development of the organism
to adulthood.
A study from Coelho et al. (2006) evaluated the effects of sodium restriction and
sodium overload after rats were weaned. The authors observed that a high-salt diet
promotes increased ABP; diminished body weight in adulthood; increased food intake, L-
thyroxine concentration, and energy expenditure; lower non-fasting leptin; decreased
plasma thyroid-stimulating hormone; and diminished plasma angiotensin II. In animals
submitted to a low-salt diet, an increase in brown adipose tissue Ang II content was
associated with decreased uncoupling protein 1 expression and energy expenditure. In this
group, a low angiotensin II content in white adipose tissue was also found. These results
were from rats that were still under a high-, normal-, or low-sodium intake regimen. The
authors concluded that chronic alteration in salt intake is associated with changes in body
weight, food intake, hormonal profile, energy expenditure, and tissue angiotensin II content.
A recent study in our laboratory evaluated the effects of sodium overload during
childhood on cardiovascular and behavioral parameters in adulthood. The study also sought
to evaluate whether the sodium-overload effects were present even after withdrawing the
high-sodium diet (Moreira et al. 2014). The authors observed that animals submitted to high
salt intake presented increased MAP and HR, diminished baroreflex sensitivity, lower body
weight, diminished sodium, increased water-induced intake, and compromised
cardiovascular responses induced by acute hypernatremia. The results indicated that
sodium-overload-induced modifications persist until adulthood, even after the withdrawal of
a high-sodium diet.
Taken as a group, the studies demonstrated that chronic hyperosmolality or salt
intake promotes increases in ABP and decreases in baroreflex sensitivity and alters the
cardiovascular and autonomic responses to acute hyperosmolality. Such modifications can
be transitory in adult animals but persist throughout adulthood when the hydromineral
disturbance occurs during the early stages of life. Furthermore, sodium loading alters the
sensitivity and/or activity of specific central nuclei involved in cardiovascular and autonomic
control. Such effects can be determinants in the development of cardiovascular diseases.
In fact, chronic high-salt intake has often been related to hypertension, as demonstrated by
several studies.
Osmotic transduction signals
As previously noted, several regions of the CNS are responsible for the detection of
changes in plasma osmolarity and angiotensin concentration. The central osmoreceptors
send this information to other regions of the CNS, especially to the MnPO and the PVN
(Saper and Levisohn, 1983; Saper et al.,1983; Tucker et al., 1987). Such regions promote
necessary adjustments to sympathetic nerve activity and vasopressin/oxytocin secretion in
order to restore the normal physiological state.
Several researchers have sought to determine how the peripheral and central
osmoreceptors perceive changes in osmolarity (Bourque et al., 1994; Ciura & Bourque,
2006). Previous studies demonstrated that neurons from the SFO and OVLT are depolarized
by increases in plasma sodium concentration and hyperpolarized by decreases in such
parameters (Bourque et al., 1994). Other studies have observed that variations in
hypothalamic neuronal activity are associated with the glutamatergic postsynaptic potentials
from the OVLT neurons (Bourque et al., 1994). In fact, studies by Shi et al. (2008)
demonstrated that the PVN-projecting OVLT neurons are activated following intracarotid
hypertonic saline infusion. Such observations indicate that increased osmolarity stimulates
both SFO and OVLT and that sending information to hypothalamic regions is dependent on
glutamatergic neurotransmission, especially from the OVLT.
It has been demonstrated that the SON, another hypothalamic region, is also
osmosensitive (Mason, 1980) and that hyperosmolarity of body fluids depolarizes the SON
neurons independently of synaptic connections (Bourque, 1989; Oliet & Bourque, 1992).
However, the mechanisms whereby such regions are stimulated are still under investigation.
In 1993, Oliet and Bourque determined that changes in body fluid osmolarity induce changes
in cell volume. Such volume change modulates the activity of membrane mechanosensitive
cation channels in an adequate way to modulate the membrane voltage and action potential
discharge. Therefore, the SON neurons are stimulated by deformations of their membranes,
with changes in permeability enabling the action potential generation.
More recently, Ciura and Bourque (2006) demonstrated that the murine neurons of
the OVLT are intrinsically sensitive to extracellular fluid osmolarity increases. The authors
observed that hypertonicity increases the membrane conductance to cations, resulting in
the depolarization of osmoreceptor potential and enhancing the action potential discharge.
Moreover, such a cascade of osmosensory transduction is dependent on the transient
receptor potential vanilloid 1 (TRPV1), since in the absence of this receptor or after its
blockage, the OVLT capacity to detect changes in osmolarity is lost. Thus, the OVLT
neurons may act as first-order osmoreceptors, and a product of the TRPV1 genes may be
crucial to the transduction of the osmosensory signals (Ciura & Bourque, 2006).
In summary, the central osmoreceptors are depolarized by changes in cell volume
caused by plasma hyperosmolarity. Before today, it was widely known that the TRPV1
receptors are involved in the osmosensory processes. However, such processes are not
completely understood.
Final considerations
The perception of changes in body fluid osmolarity and the adequate cardiovascular,
autonomic, and behavioral adjustments are crucial to the maintenance of life. The sodium
ion is one of the most important determinants of extracellular fluid osmolarity; therefore, the
balance between its consumption and excretion is crucial to body fluid homeostasis. With
its central importance to life, the osmosensory mechanism is a complex group of pathways
that cooperate to produce hydroelectrolytic balance. However, the malfunction of even one
of these mechanisms can be harmful to the organism and can trigger the development of
cardiovascular diseases with several complications that, left untreated, can lead to death.
Thus, understanding the functioning of the components involved in body fluid homeostasis
is important in order to understand its malfunction and, it is hoped, to treat consequent
disease processes and prevent death.
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Figures
Figure 1: Cardiovascular and autonomic effects of HS infusion in rats submitted to
hemorrhagic shock after saline or muscimol nanoinjections into the MnPO. Variations of
mean arterial pressure (MAP; A) and renal sympathetic nerve activity (RSNA; B) following
HS infusion in both control (saline) and experimental (muscimol) animals after hemorrhagic
shock. The grey bar represents hemorrhagic shock; the dashed line represents HS infusion;
the arrow represents MnPO nanoinjection of saline or muscimol. *Different from 0; †
Different from control group; p <0.05.

Figure 2: Cardiovascular effects of HS infusion in A1-lesioned rats and sham rats submitted
to hemorrhagic shock. A) Neuronal count of the A2 and C2 (NTS), A1 (CVLM) and C1
(RVLM) neuronal groupments after immunocytochemistry to tyrosine hydroxilase, indicating
a reduction in the neuronal number of the A1 groupments with no changes in the other
groupment. B) Variations of mean arterial pressure (MAP) and renal vascular conductance
(RVC) following HS infusion in both control and A1-lesioned animals after hemorrhagic
shock. The grey bar represents hemorrhagic shock; the dashed line represents HS infusion.
*Different from 0; † Different from control group; p <0.05.

ANEXO I
Parecer da Comissão de Ética no Uso de Animais (CEUA- UFG)
92
MINISTÉRIO DA EDUCAÇÃO
UNIVERSIDADE FEDERAL DE GOIÁS
PRÓ-REITORIA DE PESQUISA E INOVAÇÃO
COMISSÃO DE ÉTICA NO USO DE ANIMAIS/CEUA
Goiânia, 04 de maio de 2015.
PARECER CONSUBSTANCIADO REFERENTE AO ATENDIMENTO DE

PENDÊNCIA DO PROTOCOLO Nº. 023/15
I. IDENTIFICAÇÃO:
1. Título do projeto: Efeitos do aumento da ingestão de sódio durante o período pós-natal sobre o
controle hidroeletrolítico e sobre a sensibilidade e atividade dos neurônios da região rostroventral do
bulbo (RVLM) na fase adulta
2. Pesquisador Responsável: Andre Henrique Freiria Oliveira
3. Unidade/Órgão do pesquisador: ICB/UFG
4. Pesquisadores Participantes: Andre Henrique Freiria Oliveira, Gustavo Rodrigues Pedrino,
Aryanne Batiosta Soares de Melo, Marina Conceição dos Santos Moreira
5. Unidade onde será realizado: ICB/UFG
6. Data de apresentação do protocolo a CEUA: 06/04/15
7. Data de Atendimento das Pendências: 28/04/15
II - Parecer da CEUA:
Informamos que a Comissão de Ética no Uso de Animais/CEUA da Universidade Federal de Goiás, após
análise das adequações solicitadas APROVOU o pedido de emenda de prorrogação do prazo do projeto
acima referido, e o mesmo foi considerado em acordo com os princípios éticos vigentes.
Reiteramos a importância deste Parecer Consubstanciado, e lembramos que o(a) pesquisador(a)
responsável deverá encaminhar à CEUA-PRPI-UFG o Relatório Final baseado na conclusão do estudo e
na incidência de publicações decorrentes deste, de acordo com o disposto na Lei nº. 11.794 de
08/10/2008, e Resolução Normativa nº. 01, de 09/07/2010 do Conselho Nacional de Controle de
Experimentação Animal-CONCEA. O prazo para entrega do Relatório é de até 30 dias após o
encerramento da pesquisa, prevista para conclusão em 30/06/2019.
III - Data da reunião: 04/05/15
RENATA
MAZARO:12343522812
2015.05.05 16:49:25 -03'00'
Dra. Renata Mazaro e Costa
Coordenadora da CEUA/PRPI/UFG
Comissão de Ética no Uso de Animais/CEUA

Pró-Reitoria de Pesquisa e Inovação/PRPI-UFG, Caixa Postal: 131, Prédio da Reitoria, Piso 1, Campus Samambaia (Campus II) -
CEP:74001-970, Goiânia – Goiás, Fone: (55-62) 3521-1876.
Email: ceua.ufg@gmail.com
ANEXO II
Procedimentos para processamento histológico de corações:
Fixador tecidual Metacarn
Reagente Volume (ml) Temperatura (ºC)
Metanol PA 60 Ambiente
Clorofórmio PA 30 Ambiente
Ácido acético PA 10 Ambiente
Processamento do tecido em paraplast

Desidratação
Reagente Tempo (min) Procedimento Temperatura (ºC)

Etanol PA 80% 60 Imersão Ambiente
Diafinização
Xilol PA 60 Imersão Ambiente
Infiltração
Paraplast I 60 Imersão 58-60
Paraplast II 60 Imersão 58-60
Paraplast de Até secção Emblocamento 58-60
inclusão
Reidratação para citoquímica em geral

Água destilada 5 Imersão Ambiente
93
Citoquímica em reticulina de gomori
Permanganato de 2 Gotejamento Ambiente
postássio
Ácido oxálico 1 Gotejamento Ambiente
Alúmen férrico 2 Gotejamento Ambiente
Prata amoniacal 2 Gotejamento Ambiente
Formol 10% 3 Gotejamento Ambiente
Cloreto de ouro 0,2 Imersão Ambiente
Hipossulfito de 2 Gotejamento Ambiente
sódio
Ácido pícrico 2 Gotejamento
Desidratação e montagem
Desidratação para citoquímica em geral

A montagem consiste na adesão de uma lamínula (Precision®) com Entellan® sobre

o material processado.
94

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UNIVERSIDADE FEDERAL DE GOIÁS

INSTITUTO DE CIÊNCIAS BIOLÓGICAS

ALTERAÇÕES CARDIOVASCULARES INDUZIDAS PELO

MARINA CONCEIÇÃO DOS SANTOS MOREIRA

Na qualidade de titular dos direitos de autor, autorizo a Universidade Federal de

1. Identificação do material bibliográfico: [ ] Dissertação [X] Tese

2. Identificação da Tese ou Dissertação

Nome completo do autor: Marina Conceição dos Santos Moreira

3. Informações de acesso ao documento:

Concorda com a liberação total do documento [X] SIM [ ] NÃO1

________________________________________ Data: 06 /03 / 2017

ALTERAÇÕES CARDIOVASCULARES INDUZIDAS PELO

Tese apresentada ao Programa de

ALTERAÇÕES CARDIOVASCULARES INDUZIDAS PELO

Aprovada em: 17 / 01 / 2017.

Agradeço aos meus pais, Benedito e Maria Auxiliadora, pelo esforço e

Aos professores e alunos do CPNFC. Aos meus colegas de laboratório que

Atualmente as doenças cardiovasculares e suas complicações são as maiores causas

A prevalência de doenças cardiovasculares (DCV) tem aumentado

A manutenção do meio interno estável é o principal objetivo de todos os

DIETA OCIDENTAL E SÍNDROME METABÓLICA

A ocorrência concomitante de diversas alterações metabólicas que podem levar

A partir do exposto, objetivamos avaliar os efeitos da sobrecarga de sal de

1. Modelos animais utilizados

Todos os experimentos foram realizados com ratos da linhagem Wistar. A

O incremento na quantidade sódio (dieta hipersódica) ingerida pelos animais

21º 51º ou 81º 66º ou 96º

Água ou NaCl 0,3M Água

O incremento na quantidade sacarose ingerida pelos animais foi realizado

3. Ingestão de ração, fluido e peso corporal

Os animais foram mantidos em gaiolas individuais e a ingestão diária de fluido

Ao final dos protocolos experimentais, os animais foram anestesiados com

5. Registro de pressão arterial pulsátil (PAP) e frequência cardíaca

6. Avaliação dos reflexos barorreceptor e quimiorreceptor

Os animais tratados com sal e com sacarose foram submetidos à avaliação

7. Escala de sensibilidade ao sal

Grupos distintos de animais submetidos a sobrecarga de sal por 30 dias ou

Os animais submetidos a sobrecarga de sal após o desmame tiveram as

Ao final do protocolo experimental de sobrecarga de sal, os animais de todos

a) Morfometria do cardiomiócito - Os cardiomiócitos foram mensurados a fim

b) Estereologia – As análises estereológicas foram utilizadas para quantificar

10. Avaliação da sensibilidade da região Rostro Ventrolateral do Bulbo

Um grupo distinto de animais submetidos à sobrecarga de sal por 60 dias após

Antero-posterior +2,5 mm ao calamus scriptorios

Médio-lateral -2,0 e +2,0 mm ao calamus scriptorios

Dorso-ventral -2,5 mm ao calamus scriptorios

11. Registro do Fluxo Sanguíneo Renal (FSR) e Aórtico (FSA)

As condutâncias vasculares (CV) renal (CVR) e aórtica (CVA) foram obtidas

Ao final dos experimentos, os animais foram perfundidos transcardiacamente

13. Análise estatística

Os dados experimentais obtidos foram expressos como média ± EPM (erro

ALTERAÇÕES CARDIOVASCULARES INDUZIDAS POR SOBRECARGA

1. Ingestão de fluido e ração e peso corporal

A ingestão diária média de fluido e ração e o peso corporal dos animais

Similar ao que ocorreu com os animais tratados por 30 dias, os animais

2. Parâmetros cardiovasculares e reflexo barorreceptor

Os parâmetros cardiovasculares basais (PAM e FC) dos animais adultos que

A Figura 4 apresenta os parâmetros cardiovasculares basais dos animais

As figuras 7 e 8 apresentam os traçados representativos e o índice de

Figura 7: Traçados representativos ilustrando as alterações cardiovasculares induzidas pela

A Figura 9 apresenta os valores médios de ingestão de fluido e de ração dos

Os valores médios da pressão arterial sistólica (PAS) e da frequência cardíaca

5. Análise da histologia cardíaca

Fotomicrografias representativas dos corações de animais submetidos a