E-Mail:
marcovaz@esef.ufrgs.br
Pedido'051028-546
BRI9907443
Usuario:
Phys Ther
1986 66(6) pags. 946-53 / Greathouse DG, Nitz AJ:
Matulionis DH; Curtier DP / Effects of short-term
electrical stimulation on the ultrastructure of rat
skeletal muscles. ((iah) MEDLINE_1966-1992 pmia
3714812]
Fonte de referéncia:(iah) MEDLINE_1966-1992
pmid: 3714812,
Prof Dr. Marco Aurelio Vaz
Rua Mariz e Barros 392 Ap 501
'90690-390 - Porto Alegre - RS
BRASIL
E-Mail
marcovaz@esef.ufrgs.br
HH NNETLTINE
AT
| | antl
Phys Ther
1986 66(6) pags. 946-53 / Greathouse DG, Nitz AJ
Matulionis DH, Currier DP / Effects of short-term
electrical stimulation on the ultrastructure of rat
skeletal muscles. {(iah) MEDLINE_1966-1992 pmia:
3714812]
BRI9907443
pull)
Pedido 051028-546
Local: BR66.1
Opgées: BR66.1 /BR512.1/ BR1.1
Atendido / Paginas:
Rejeltado / Motivo:
65/80 Effects of Short-term Electrical Stimulation on the
Ultrastructure of Rat Skeletal Muscles
DAVID G. GREATHOUSE,
ARTHUR J. NITZ,
DANIEL H. MATULIONIS,
and DEAN P. CURRIER
‘The purpose of this study was to determine the effects of short-term, 2,500-Hz
ccarrier-wave frequency electrical stimulation on the ultrastructure of fast-twitch
rat skeletal muscles. Thirteen Spraque-Dawley male rats were divided into three
‘groups: daily treatment (Group 1), every-other-day treatment (Group 2), and
‘control (Group 3). The medial quadriceps femoris and hamstring (semitendinosus
‘and semimembranosus) muscles of the right thigh were treated with short-term
electrical stimulation. After treatment, the animals were sacrificed, and their
‘treated and sham quadriceps femoris and hamstring muscles were removed,
fixed by Immersion, and processed for electron microscopy. Morphometric meas-
‘urements were made on electron micrographs using a Videoplan computer
‘system, and the results were analyzed statistically. The results revealed that
‘mitochondria, triads, and glycogen content of the fast-twitch muscles changed
to resemble those of slow-twitch muscles. In view of these results, clinicians
‘should consider the possibility that similar changes ight occur in humat
‘subjects under clinical condition
Key Words: Electric stimulation, Muscles, Physical therapy.
Electrical stimulation is used as a
treatment modality in physical therapy.
Increased use of such treatment has
been prompted in light of the develop-
‘ment of a new form of stimulator, that
is, the Russian “new generation” type of
clectrial generator." This type of treat-
‘ment modality has been claimed to re-
Tieve pain in injured areas, increase local
blood flow, strengthen muscles, cause
‘muscle hypertrophy, and facilitate mus-
cle contraction," As impressive as these
observations are, no information is
Major Greathouse is Director, US Army-Baslor
LUntvestty Program in Physical Therapy. US Army
‘Acar of Heath Sciences, For Sim Houston
{TX Tazds (USA). Atthe ime otis stady be was
‘Uloctoral candidate i the Deparment of Anat
‘omy, Unisersy oF ‘Kentucky. Lexington, KY
ish
‘De Nit ig Asan Profesor, Department of
Physical Therapy University of Kentucky.
‘br, Mituionis Associate Pofeso2, Depart
ment of Anatomy. Unwersty of Kent
De. Cure is Profesor and Chairman, Depa
‘ment of Phys Therapy, University of Kentcky
“Fis study was sport bythe Deparment of
Anatomy and in pat by the Tobacco an Heath
‘esearch Insutue (Gram #44007), Univesity of
Kentucky.
The opinion or assertions contained herein are
the private sews ofthe authors and are aot 1 Be
onsrud atofal a reflectng the views oe
Deparment ofthe Army the Department
Deters
Phi ance was submited December 26, 1984
vs ith he hrs for reisionT8 weeks: and was
‘ceed November 11985,
946
available regarding the actual structural
changes that might occur with short
term (ie, less than four weeks) electrical
stimulation, Evidence in the literature
exists, however, that indicates that elec-
trical stimulation, using different wave-
forms and frequencies produced by
other generators, alters skeletal muscles
structurally. Reddanna et al showed that
short-term electrical stimulation of frog
skeletal muscle resulted in a decrease of
glycogen and a reduction in the total
‘muscle protein content.‘ Ogilvie and
Rhein, analyzing the combined effects
of exercise and short-term electrical
stimulation (prodding) on rat skeletal
muscle, noted a disruption of the A
band, I band, and Z disk.° Eriksson et
al assessed the effects of short-term, low
frequency electrical stimulation on the
human quadriceps femoris muscles and
found an acute depletion of phosphagen
and glycogen stores, but no changes in
muscle enzyme activities, fiber type
characteristics, and volume fraction and
‘number of mitochondria
The muscle alterations noted after
short-term stimulation are not in com-
plete accord with those observed after
long-term stimulation (je, more than
four weeks’ duration). Several investi-
gators stimulated fast-twitch muscle fi-
bers for extended periods and observed
fan increase in width of the Z disk but
no disruption."""" An increase in the
volume fraction and number of mito-
chondria after long-term clectrical stim
ulation was also observed.""* The T-
tubule systems of fast-twitch muscle
fiber type were reduced in extent to
that of slow-twitch muscle in 2 10 14
days” The T-tubule system was not
investigated in the short-term electrical
stimulation studies. Sree et al demon
strated that the transformation of fat
twitch muscle to slow-twitch muscle by
long-term electrical stimulation was
produced not only by continuous ele
trical stimulation by trains of impulses
ata low frequency but also by electrical
stimulation by trains of impulses at a
higher frequency." Their studies ind
cate that muscle fiber transformation
depends more on the total contractile
activity of muscle than on the pattem
of electrical stimulation."®
The literature regarding electrical
stimulation therapy is incomplete and
often conflicting, which clearly indicates
the need for further research in this area.
In view of this, our study was designed
to evaluate the short-term effects ofthe
Russian type of electrical stimulation on
the ultrastructure of normal rat skeletal
muscle. Particular attention was paid to
subcellular structures of muscle cell,
that is, mitochondria, glycogen, and
triads, which if altered could be related
PHYSICAL THERAPY
The
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PHYSIC fo. a change in characteristics of fast-
twitch to slow-twitch muscle fiber types.
MATERIALS AND METHODS
Thirteen healthy male Spraque-Daw-
ley rats weighing between 225 and
249 g were used in this study. The rats
‘were three months old at the onset of
the study. The animals were kept in
Bioctean cages, three in each cage. The
bottoms of the cages consisted of an
open wire mesh. The animals were ex-
posed to alternating periods of artificial
light and darkness with the Tight cycle
starting at 7 AM and terminating at 7
PM.
‘The 13 rats were divided randomly
into three groups: Group 1 (rats 1-5
experimental). Group 2 (rats 6-10, ex-
perimental), and Group 3 (rats 11-13,
control). The animals were anesthetized
daily before their morning treatment by
an intraperitoneal injection of sodium
pentobarbitol (20 mg/kg). The use of
anesthesia was necessary to pacify the
animals during the electrical stimulation
treatments and to make the treatments
painless, although anesthesia is not used
when this type of modality is used on
‘human subjects. The rats were anesthe-
tized for a total period of about 30 min-
utes during each treatment session.
Before the first treatment, the right
thighs and legs ofthe treatment animals
were shaved with electric shears. The
animals then were placed in a supine
position on the laboratory table and se-
cured to the tabletop with tape. After
the animals’ right thighs were prepared
with a conductive medium,* metal sur-
face disk electrodes 5 mm in diameter
were placed on the anterior-medial and
anterior-lateral right thighs 15 mm prox-
imal to the knee joints and secured 10
the thighs with Velero fasteners.” The
Electrostim 180-2 stimulator? was used
to produce the electrical current. The
‘ight thighs of the animals were sub-
Jeted 10 15-second trains of 50-Hz elec-
‘tical impulses followed by S0-second
rest periods. The carrier-wave frequency
was 2,500 Hz. This sequence was con-
ducted 10 times during each treatment
session, The intensity of the stimulus
was 6 mA, which was three times the
amount of current needed to elicit a
visible contraction of the quadriceps
femoris and hamstring muscles. This
“aquasnic 100, Parke Laboratories ne, 307
Washington St Orange, NI 17030.
SNM. Ine, 38 N Hammes Ave Joliet,
cons!
PHYSICAL THERAPY
stimulus produced a tetanic contraction.
After the electrical stimulation, the rats
‘were placed temporarily in a cage for
‘observation until they revived from the
anesthesia No adverse effects of the
daily anesthesia on the animals were
noted.
Group | rats were treated five times a
‘week (Monday—Friday) for three weeks
for a total of 15 treatments. Group 2
animals were treated every other day for
a total of 12 treatments. The control
‘group received no treatment. The left
thighs of the animals in both treatment
‘groups served as the sham thighs. The
‘medial quadriceps femoris and medi
hamstrings (semitendinosus and semi
‘membranosus) were the muscles chosen
to assess the alterations in muscle fiber
ultrastructure asa result of the electrical
stimulation,
Although every effort was made 10
use clinical methods of electrical stimu-
lation, we acknowledge that the proce-
dures we used in the animal model
could not totally imitate those used clin-
ically in human models. Pacification
(using anesthesia) and restraint of ani-
‘mals was necessary so that electrical
stimulation could be applied. In addi-
tion, the relative size of the electrodes
‘that we used in the animal model was
larger than those used to stimulate hu-
‘man muscles. Thus, more muscle mass
‘was stimulated in the animal model
than in human models. Furthermore,
the intensity of electrical current that we
used in our study was different in pro-
portion to muscle mass than that used
clinically. Finally, the muscle response
to electrical stimulation may differ be-
cause of diverse species differences. It
should also be recognized, however, that
human studies often fail to provide in-
formation because such investigations
cannot be manipulated sufficiently to
conform to the experimental design. For
this reason, animal research is appropri-
ate for experimental studies of the ef
fects of electrical stimulation,
After the final treatment, girth meas-
urements of the treated and sham thighs
and legs of those animals that received
electrical stimulation were obtained,
‘The animals were placed in a supine
position and secured tothe tabletop with
tape. The knees were flexed to approxi-
mately 90 degrees, and the legs were
fixed in this position with tape. The
girths of the thigh and leg were measured
‘5 mm proximal and distal to the knee
joint with a nonelastic tape measure.
RESEARCH
Before the autopsy of the muscles, the
animals were injected intraperitoneally
with a lethal dose (70 mg/kg) of sodium
pentobarbitol. Incsions were made in
the thigh, andthe relaxed medial quad-
riceps femoris and hamstring (semiten-
dinosus and semimembranosus) mus-
cles were removed.“ Longitudinal strips
of muscle were dissected from the belly
of each muscle and immersed within
‘wo minutes in a. cacodylate buffered
3.5% glutaraldehyde fixative solution at
4°C. The muscle strips were then cut
into 1-x 1-x 1 S-mm blocks. The tissue
remained immersed in the fixative so-
lution fora total of 1.5 hours.
‘fier inital fixation, the blocks of
muscle tissue were washed in a cacodyl-
ate bulfer containing 2% sucrose. The
tissues were postfixed by immersion for
one hour in a cacodylate buffered solu-
tion of 2% osmium tetroxide. The mus-
cle tissue was dehydrated in a graded
series of ethanol, passed through pro-
pylene oxide, and embedded in Epon
812.” Thre blocks from each biopsy of
muscle tissue were selected randomly
and sectioned longitudinally at 600 10
800 nm in thickness on an LKB 8880
Ultramicrotome The sections. were
stained with lead citrate. The tissue was
examined and photographed with a
Phillips EM 400 electron microscope-§
Four micrographs were made of the
centrally located muscle fiber from each
biock at an original magnification of
10,000 x, and then enlarged photo-
graphically to 27,000 x. The muscle
fiber chosen for study and photography
was located most centrally on the sup-
porting grid. This method, described
by Engel and Stonnington," ensured
random sampling of the muscle fibers.
The centrally located fibers were repre-
sentative of other fibers in the same
muscle. Twelve micrographs of muscle
tissue were obtained from each animal,
Yielding a total of 36 micrographs from
controls and 120 micrographs from
treated rats foreach muscle.
Various subcellular components per
unit volume of selected muscle cell ey-
toplasm were measured and quantified
(morphometrcally and algorithmically)
using the Zeiss Videoplan Image Analy-
sis System.|| We used area-perimeter
{LKB Produker AB, $16125 Bromma, Sock
hole, Sweden
{NV Philips Gloilumpentvishem, Eindoven,
‘The Netherlands
Tart Zs In, Thorood, NY 10598 TABLE 1
Girth Measurements of Treated Animals
‘Above Knee (thigh) Below Knee (eg)
Group Rat (erm (om)
Rightt Left DiMA L) Right” Left Dim — L)
1 1 8 8S 4 mR 65 7
1 a Tol ee 7 6 6t a
1 3 8486 8 er 8. 1
1 4 88 80 8 6 61 2
1 5 9 80 10 7 68 4
2 0 7 7 68 "
2 7 9 a7 8 7% 7 3
2 8 88S. 4 7 © 64 6
ely OF ae oie 08 6 2 88 4
2 0 98 a9 9 B70 3
* Denotes treated thigh org,
TaBLe 2
“Test Results for Girth
surements of Treated Animals
(Group _Dieronce” tp
1 Thigh 755-002
1 NS.
2 or
2 2?
Ditference
* Significant.
analysis to calculate volume fraction
(Vy) and surface volume (Sv) of the
mitochondria and the triads using the
formulae:
Wm A/AT w
Sv=4/xxU/AT — (2)
where A is the sum of the structure
areas, AT is the sum of the reference
area (cell cytoplasm), and U is the sum
of the structure section perimeters in
AT" Volume fraction of glycogen par-
ticles was determined using the point
counting procedure in which more than
528 independent test points (“hits”) on
a multipurpose grid that intercepted gly-
cogen particles were identified. The vol-
lume fractions of these struftures were
estimated using the equation:
Voi = Pri a
where Vvi is the volume fraction (%)
‘occupied by a structure and Ppi is equal
to the fraction of test points intercepted
by the structure.” The Wvi reported rep-
resents the percentage volume of the
lycogen particles per unit volume of
‘muscle tissue in the sampled region."”
‘The width of the Z disk ofthe sarcomere
located in each corner of a micrograph
‘was measured using the Zeiss Videoplan
948
Image Analysis System. Four Z disks
were measured for each micrograph, 10-
taling 48 disks for each muscle for each
animal.
Data Analysis
The thigh- and leg-girth measure
‘ments of the treated animals (Groups 1
and 2) were analyzed in the following
‘manner. The difference between treat-
‘ment and sham thigh girth in individual
animals was determined. Group means
for thigh and leg girth differences were
determined and analyzed statistically
using the paired f test. The volume frac-
tion; surface area per volume; and num-
ber of mitochondria, triads, and glyco-
gen particles were analyzed statistically
using a one-way analysis of variance 10
compare differences between groups
(daily treatment—Group 1, every-
other-day treatment—Group 2, and
control—Group 3). Scheffe's multiple
‘comparison procedure was used post hac
10 determine intergroup differences. All
statistical analyses were performed at
the .05 level of significance.
RESULTS:
General Observations
Throughout the electrical stimulation
period, the animals appeared to be in
‘00d health, No limp or gait defect im
the treated extremities after the electri-
cal stimulation was noted. Also, no ad-
verse effect of repeated anesthesia on the
rats was noted. Forty minutes after the
administration of anesthesia, the ani-
mals were normally active. The food
and water intake of the treated animals,
did not differ from that of the controls.
After two weeks of electrical stimula.
tion treatment, the thigh and leg size
increased notably. Girths of these re
sions by the end ofthe treatment period
(three weeks) were larger than those of
the sham extremities (Tab. 1). When
analyzed statistically, the difference in
the girth of the treated thighs of both
‘groups was significantly different (p =
002) when compared with sham values
(Tab. 2). A statistical difference (p =
03) between treated and sham leg girths,
however, was noted only in the animals
of Group 2 (Tab. 2).
Ultrastructural Assessment
Morphotogy—control animals. The
fine structure of the normal control me-
dial quadriceps femoris muscle was dif-
ferent from that of the hamstring muscle
(semitendinosus and semimembrano-
sus), Subcellular components vary nor-
mally in different muscles according 10
fiber type.'*" The medial quadriceps
femoris muscle was characterized by a
relatively sparse number of mitochon-
dria, a sizeable number of triads, and
clusters of glycogen particles located be-
tween the muscle myofibrils (Fig. 1a).
In contrast, the medial hamstring mus-
cle (Fig. 1b) appeared to have fewer
mitochondria than the medial quadri-
‘eps femoris muscle, but the number of
triads and the glycogen content of the
medial hamstring muscle appeared to
be greater than that seen in the medial
quadriceps femoris muscle, The ultra.
structure of quadriceps femoris and
hhamstring muscles from the sham ex-
tremities was similar to those of the
normal controls.
Morphology—treatment animals.
‘The number and size of triads in some
micrographs of the treated quadriceps
femoris and hamstring muscles (Figs. 2
and 3) appeared to be slightly decreased
‘when compared with those of the con-
trols. We acknowledge, however, that in
other micrographs these characteristics
of the triad appeared to be unaffected
by electrical stimulation treatment. The
‘mitochondria in the medial quadriceps
femoris muscle of a Group 1 animal
(Fig. 2) and the medial hamstring mus-
cle (Fig. 3) in both Groups | and 2
appeared 10 be more numerous and
Jager in size than these organelles in the
controls (Figs. 1a and Ib). There was no
apparent change in the number and size
of the mitochondria of the medial quad-
riceps femoris muscles of the Group 2
PHYSICAL THERAPY
Morp
TH
squat
rep
mals
207
cal st
the e
nifica
was 1
frat
reps
(oo
(Tab
triad
cers
aie
treatn
and?
with v
(oss
analy
tetve
the su
ofthe
tistical
and s
The
media
Signi
in Gr
am?) v
tes 0
both
‘muscle
surface
plasm
Volum animals, as compared with those of the
controls. A differential effect on the mi:
tochondria was observed after different
electrical stimulation treatments. In
zencral, the amount of glycogen seemed
fo be influenced by the electrical stim-
ulation treatments in a similar manner
to the differential effect on the mito-
chondria. When compared with the con-
trols, the number of glycogen particles
was decreased in the medial hamstring
muscles (Fig. 3) of both treatment
groups. There was no apparent decrease,
however, in the number of glycogen par-
ticles in the medial quadriceps femoris
muscles of either treatment group when
compared to the controls. Other fea-
tures, A bands, I bands, and Z disks,
were unaffected by electrical stimulation
treatment and resembled those features
of the control animals,
Morphometric Analysis
‘Triads. The mean triad number per
square micrometer of the medial quad-
Ticeps femoris muscle fibers of the ani-
mals in Group 1 (0.68/um’) and Group
2(0.71/um*) was unaltered after electri-
cal stimulation in contrast to those of
the controls (0.95/um’) (Tab. 3). A sig-
nificant decrease (p = .005), however,
was noted in the mean triad volume
fraction (% volume) in the medial quad-
riceps femoris muscle fibers of Group |
(0.92%) and Group 2 (0.94%) animals
(Tab. 3). Quantification of the mean
triad surface area in the medial quadri-
cxps femoris muscle cells demonstrated
a significant decrease (p = .03) after
treatment in Groups 1 (0.41 ym?/um?)
and 2 (0.48 um*/um?) when compared
With values noted in untreated animals
(0.63 wm'/um’) (Tab. 3). A post hoc
analysis did not reveal any differences
between cither the volume fraction or
the surface area per unit volume means
of the animals in Groups I and 2. Sta-
tistical analyses are shown in Tables 4
and 5.
‘The mean number of triads in the
medial hamstring muscle cells decreased
significantly (p = 0.004) after treatment
in Groups 1 (0.68/um*) and 2 (0.66/
um?) when compared with control val-
vues (0.98/um*) (Tab. 3). In addition,
both treatment procedures caused a sig-
nificant decrease (p = .0001) in the
‘mean volume fraction of triads in these
muscle fibers (Tab. 3). The mean triad
surface area per unit volume of sarco-
plasm in the medial hamstring muscle
Volume 66 / Number 6, June 1986
Fig. 1a. Medial quacticeps femoris muscle
‘of control animal. Mitochondria (M). ads
(double arrownead), glycogen particles (ar.
ownead) (x 22,000). 1b- Medal hamstring
‘muscle ofa contro arimal Few mitochoncria
(M) are present, but tiads (double arrow-
head) and glycogen particies (arrowhead) are
‘numerous (x 22,000).
fibers also decreased significantly (p<
0.0001) in Group 1 (0.39 um?/um’) and
Group 2 (0.39 um?/umm?) animals when
compared with the surface area in the
control animals (0.74 wm?/um') (Tab.
3). A past hoe analysis did. not reveal
any differences between the triad num-
ber, volume fraction, or surface area per
unit volume means of the medial ham-
string muscles of the animals in Groups
1 and 2, To summarize, the size and
number of triads in both the medial
quadriceps femoris and hamstring mus-
les of the animals in the treatment
groups decreased as a result of three
Fig. 2. Medial quacriceps femons muscle
(ofa Group 1 anmal. Note the many large
‘mitochondria (M). Triads (double arrowneac),
‘alycogen particles (artownoad) (x 22.000),
Fig. 3. _Mectal hamstring muscle of a Group
anal. Mitochondria (M) are numerous, but
‘nad (double arrowhead) ane glycogen par-
ticles (arrowhead) are rare (x 22,000),
‘weeks of electrical stimulation, as com-
pared with the controls.
Mitochondria, No difference was seen
in the mean number per square mi-
crometer of mitochondrial profiles in
the medial quadriceps femoris fibers of
either Group 1 (0.75/um:) or Group 2
(0.53/am') animals when compared
with that of the control animals (0.63/
um?) (Tab. 6). The mean mitochondrial
volume fraction (5.1%) in these muscle
cells of Group 1 animals increased sig-
nificantly (p = .02) over the volume
fraction (3.6%) of the control animals’
‘medial quadriceps muscles (Tab. 6). The
949 mean mitochondrial surface area per
unit volume of cytoplasm in the medial
quadriceps femoris muscle fibers of
Group 1 animals (0.99 ym"/um?) also
increased significantly (p = .03) when
‘compared with the mitochondrial sur-
face area per unit volume in these mus-
cle fibers of the control animals (0.75
um?/um?) (Tab. 6). No difference was
seen in mitochondrial volume fraction
and surface area per unit volume of
sarcoplasm in the medial quadriceps
femoris muscle fibers of Group 2 ani-
‘mals when compared with control ani-
mals (Tab. 6).
‘Quantification revealed a significant
increase (p = .04) in the mean number
of mitochondria per square micrometer
in the medial hamstring muscle cells of
both Group | (0.86/um") and Group 2
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