TITLE: Procedure of trans-carotid iliac artery stenting in rabbit-a novel model to

evaluate vascular stent

AUTHORS: Patil P. B
1
., Patil D. B
2
., Parikh, P. V
2
., Rajapurkar, M
3
., Lele S. S
4
.,
Rajapurkar, M. M
5
. and Kelawala, N. H
6
.
1. Ph.D. Student, Laboratory for Transplantation and Regenerative Medicine,
G.U-, Sweden.
2. Professor, Department of Surgery, Anand Veterinary College, AAU, India.
3. Assistant Professor, Department of ENT, Karamsad Medical College, India.
4. Interventional Cardiologist, Bhailal Amin Cardiac hospital, India.
5. Medical Director and Head, Dept. of Nephrology, MPUH, Nadiad, India.
6. Professor and Principal, Department of Surgery, Navsari Veterinary College,
India.
CORRESPONDING AUTHOR Pradeep B. Patil vetdrpradip@yahoo.com
KEYWORDS: ante-grade, trans-carotid, iliac artery, stenting, rabbit
SHORT ABSTRACT: Use of rabbit as a compliant animal model with early vascular
lesions proved to be highly explored model for interventional research including
stents. However, here we present a simple trans-carotid iliac artery stenting
procedure, which has additional advantages over conventional retrograde method.
LONG ABSTRACT:
Stents as endoluminal splinting devices are increasingly used as an alternative to
surgery by providing inner mechanical support for internally stenosed or externally
compressed hollow structures e.g. arteries (aorta, coronary, periphery); glandular
ducts (bile, pancreatic); gastrointestinal tract (esophagus, colon); respiratory passages
(nasal cavity, trachea, bronchi); urinary passages (ureters, urethra) and veins in
human and animals. In this fast moving science world, competition for medical
products is huge. Sometimes in the stent world, it is referred to as stent war;
however to provide a piece of data in short period with distance prognosis, a
compliant model is always preferred. Rabbit is one such model, used in newly
developed vascular stent evaluation. In this video article, procedure for antegrade
approach of iliac artery stenting has been demonstrated. In the world of animal
ethics and cost, this model provides an option of implanting two vascular stents in
the same animal, which not only avoids individual variation but also allows following
3R of animal ethics (i, e. Replacement, Reduction, Refinement)
INTRODUCTION:
In the field of interventional therapeutics stents has emerged as one of the rampantly
growing area and used as a device to alleviate cardiovascular disease. Rapid
development of vascular lesions in experimental rabbit model similar to humans
potentiates that the model is uniquely suited for the study of cellular mechanism of
restenosis. The antegrade procedure of stenting in rabbit model
1
does not produce
vascular injury and conserves the flow domain near the site of an implanted stent as
compared with traditional retrograde (trans-femoral) stenting procedure in rabbit iliac
arteries
2
, which can have in vivo clot formation due to surgical maneuver by
catheterization
3
. Retrograde procedure of vascular stenting
4,5
led to an
unacceptably high rate of vascular compromise in the limbs secondary to vascular
injury. Further, this trans-femoral approach may render alterations in the localized
flow environment distal to the stent, subsequently influencing neointimal hyperplasia
6

7
within the stented region. Hence, to overcome these disadvantages and increase
the efficacy of the catheterization procedure, the normograde trans-carotid approach
for iliac artery stenting in rabbits was followed.
Protocol
Comparative evaluation of dual drug eluting (Heparin-Sirolimus) stents was
performed with FDA approved drug eluting stent (Sirolimus) in external iliac arteries
of rabbit model as below.
1. Ethical consideration: Experiment was performed under Institutional Animal
Ethics Committee (IAEC) of Veterinary College at Anand Agricultural
University, India
2. Animals
a. In the present protocol, 21 apparently healthy New Zealand White
(NZW) adult rabbits of either sex, >8 months age with average mean
body wt of 2.168 kg (range: 1.650 3.000 kg) were used.
b. Rabbits were tagged with threads of number plate tied around hind
legs and identity cards were kept on cage doors.
c. Rabbits were maintained under ideal and uniform environmental
conditions with ad lib access to feed and water.
d. Rabbits were separated and caged in-group of two or three one week
before the operative procedure as per need.
e. Each rabbit was provided with green grass, overnight soaked (Bengal
gram) (100 gm) in the morning and vegetables (cabbage and carrot)
and pellets (100 gm) in the evening with access to ad lib water in cage
throughout the day.
f. Single person throughout the research period handled the rabbits.
g. Rabbits were restrained by holding neck scruff with left hand while
supporting back of animal with palm to keep them dorsal recumbence.
3. Instrumentation
a. Image Intensifier Television
1
(IITV) was used for fluoroscopic imaging
(FI) and angiography during experimentation, which displays lives as
well as still X-ray images with direct storing capacity.
b. Incision (3-5cm) on neck skin was taken with the help of Blade No. 15
in B. P. handle No. 3.
c. Haemorrhages were controlled using thermo-cautery
2
.
d. Stent Delivery System Specification (SMT, 2006 company literature)
i. Shaft Diameter - 2.6 Fr (proximal), 1.9 Fr (distal)
ii. Nominal Pressure - 8 atm
iii. Rated Burst Pressure - 16 atm

1
Image Intensifier Television, Medico, Imaging Co, India.
2
Thermocautery- Jorgenssen Laboratories, Inc. Loveland, Co. Mfg. Lot: 0205-2
iv. Marker Bands - Encapsulated marker bands
v. Balloon Overhand - Minimal with concave balloon shoulders
vi. Usable length - 140 cms
vii. Guide wire (GW) - 0.014"
viii. Guiding Catheter - 5 Fr Compatible
ix. Tip - Tapered Soft Tip
x. Entry Profile - 0.017
e. Stents: (SMT, 2006 company literature)
i. SRL Eluting Stent (SES) [Supralimus
3
]
ii. Heparin-Sirolimus Eluting Stent (HSES) [Syncronium
4
]
f. Balloon for inflating stent: Balloon has two platinum markers for
accurate stent positioning made of ultra thin polyamide balloon material
with short soft tip used for inflation and deployment of stent in target
area.
g. Torque: Torque is used for directing tip of guide wire (GW) and
straightening the loop of GW in the heart. It is also used to divert the tip
of GW during passage through the arterial branching to specific artery.
h. Balloon dilator
5
: Balloon dilator is used for inflating balloon using
pressure for balloon angioplasty or inducing balloon injury or deploying
stent. The nominal pressure (NP) for inflation and deployment of SMT
made Supralimus and Synchronium stents is 8 atm and a rated burst
pressure is 16 atm. Units range is vacuum to 0 and 0 to 25 atm or
vacuum to 0 and 0 to 350 psi.
i. Guide Catheter (GC): GC having distal shaft diameter of 2.9 Fr,
proximal shaft diameter of 1.9 Fr was dorsally forwarded to posterior
aorta using 5 Fr introducer sheath.
j. Guide Wire (GW): Flexible metal ATW (FI marker) GW
6
of 0.014
diameter (0.36 mm) was used for the guidance and stent deployment
at the target site (i.e. just below to the quadrification of posterior aorta
at the level of last lumbar) in external iliac arteries of rabbits. The GW
has steerable GW with total length of 195 cm and Flexible tip of 3 cm.
Tip Flexibility is floppy and passed through mini GC of 1.9 Fr (0.64)
diameter and 140 cm length having wire entry/exit point at 25 cm far
from balloon with 2.6 Fr (1.02 mm) diameter.
k. Intracath 24 Fr
l. Introducer Sheath 5Fr
m. Jelco No. 16
n. Microtome
7
Microtome (RM 2125RT) and the Tungsten Carbide blade
(No. 22227) were used for sectioning the resin embedded blocks of
stented arteries with stents. The angle of knife was kept at 4
o
, which
enabled to cut at 5m sections.
4. Preoperative procedure

3
Supralimus : SES manufactured by Sahajanand Medical Technologies Pvt. Ltd., India
4
Synchronium Stent manufactured by SMT Pvt. Ltd., India
5
Balloon Dilator- Basix, Merit made in USA
6
[Cordis Corporation (Johnson & Johnson), Miami, Fl 33102-5700, USA]
7
Microtome Leica Company, Germany
a. A day prior to the experiment, rabbits was weighed and ventral neck
region were shaved and prepared for aseptic intervention, in supine
position, which provides uneventful restraining.
b. In the rabbits, needles of 20 G, 22 G and 26 G were used for injecting
medicine using subcutaneous (s.c.), intravenous (i.v.) and
intramuscular (i.m.) routes at scruff of neck / flank regions, marginal ear
vein and cranial aspect of quadriceps muscle respectively.
c. On the day of the trial after preoperative preparation, the Rabbits in all
the three groups were pre-medicated with Inj. Meloxicam
8
@ 0.05
mg/kg body wt i.m. 30 min. prior to general anaesthesia.
d. All rabbits were sufficiently hydrated one hour before stenting
procedure with warm Lactated Ringers solution given s.c. at standard
maintenance dose of 50-70 ml / kg body wt in divided doses on
multiple sites of either flank region and neck scruff (Rosenthal, 2001).
Inj. Enrofloxacin
9
@ 5 mg/kg body wt i.m. o.i.d. for 3 days.
5. Anesthesia
a. Rabbits were anaesthetized for surgical procedures and imaging studies.
Induction of general anaesthesia was done with a mixture of inj.
Ketamine HCl
10
(40 mg/kg body wt) inj. Diazepam
11
(0.5 mg/kg body
wt) and inj. Xylazine HCl
12
(2.3 mg/kg body wt) given i.m.
b. General anaesthesia was maintained by administration of 1/3
rd
of the
calculated induction dose as per the requirement.
c. Rabbits were operated under stage III of anaesthesia.
d. Rabbits were secured in dorsal recumbence with head under general
anesthesia and neck was fully extended to ensure a patent airway.
6. Anaesthetic monitoring
a. The onset and duration of sleep were also observed and recorded.
b. The depth of anaesthesia was monitored by the ear pinch and hind leg
withdrawal reflex (which must be present).
c. In absence of foreleg withdrawal reflex (disappears later), anaesthesia
was maintained lightly.
d. Chewing is sign of light anaesthesia and was considered as rapid awaking
sign.
e. Drugs like inj. Dexamethasone
13
and inj. Doxapram
14
were kept ready in
emergency kit.
7. Operative procedure
a. After blunt dissection and avoiding handling of vagus nerve, carotid
artery was isolated and separated from rest of the tissue.
b. Three 6-0 silk threads were put around the carotid, which helps in intra-
cath insertion.
c. The artery was cannulated using a 24 G intra-cath and GW advanced
under fluoroscopic imaging.

8
Meloxicam, Melonex, Neovet, INTAS, A bad, Gujarat, India.
9
Enrofloxacin, Bayer, India.
10
Ketamine HCl, KETMIN50, Themis medicare Ltd, Goregaon (W), Mumbai
11
Diazepam Injection, Anxol, Manish Pharmaceutical pharmaceutical Ltd., Mumbai, India -400043
12
Xylazine HCl, Xylaxin, Indian Immunological Ltd, Gallapadu, Guntur, Dist. A. P., India-522408
13
Dexamethazone Na PO
4
Injection, Karnataka Antibiotics & Pharmaceutical Ltd., India
14
Doxapram, Corapram, Cipla, Mumbai, India.
d. With steady hold on carotid artery, the introducer sheath (5 Fr) was
progressed into the carotid artery for angiogram
8,9
however few
authors used 4Fr
10,11
.
e. The GW looped into the ascending aorta, which guided gently into the
descending aorta under guidance of fluoroscopy.
f. The soft tip GW was oriented dorsally towards common iliac artery via
cranial, caudal aorta and aortic quadrification.
g. The left external iliac artery was wired first using torque.
h. A 3 mm diameter noncompliant coronary balloon angioplasty (BA)
catheter with radio-opaque balloon was advanced under FI to the
proximal common iliac artery, where it was inflated to 8 atm for 60
seconds to induce deep arterial injury.
i. Then, GW was advanced into the proximal portion of the right external
iliac artery for balloon injury.
j. Balloon catheter was carefully removed while the GW remained in
place.
k. A sterile distilled water soaked guage was used continuously to avoid
drying of tissue and reducing risk of tearing vessel due to friction from
GW and GC.
l. The stent on millennium matrix delivery system was advanced to the
distal portion of the iliac artery using the GW then; FDA approved stent
as control was deployed at the site of arterial injury.
m. The GW was taken out and repositioned into the left iliac artery for
deploying stent of interest.
n. Both stents were stented at same distance from iliac branching.
o. Balloon was slowly taken out under FI after deflating.
8. Post-stenting procedure
a. Successful deployment and patency of the vessels after stents
implantation was determined in rabbits intra-operatively and
postoperatively before stent retrieval with angiography using diluted
Urografin
15
.
b. Special needle catheter (Jelco No. 16)
16
was introduced into the carotid
artery. The quick spurting of the blood from the catheter indicated the
correct arterial placement. After the proper placement of the
angiography catheter, contrast angiography was ascertained by
fluoroscopy.
c. For proper visualization of angiogram of iliac arteries, animal was kept
in supine recumbency with stifle joints abducted. After contrast dye
injection, NS
17
@ 5 ml / kg was injected through same catheter. The
GW was removed.
d. Immediately after angiography, catheter was removed from the carotid
artery.
e. Left common carotid artery was ligated with Vicryl 3-0; neck
musculature was sutured using interrupted sutures pattern with Vicryl
3-0 and skin with silk 2-0.

1S
Urografin 76% Each ml contains Diatrizoic Acid dehydrate U.S.P. equivalent to Diatrizoic Acid
anhydrous U.S.P.597.30mg; Meglumine U.S.P. equivalent to Diatrozoate Sodium 100.00mg
Diatrozoate meglumine 660.00mg. German Remedies Ltd., Worli, Mumbai, India.
16
Jelco 16 G, Johnson & Johnson, Netherlands.
17
NS Merck, Gujarat, India.
f. Suture line was sealed with thin cotton layer soaked in diluted
Betadine.
g. The average experimental time was 90 min from induction until
emergence from general anesthesia.
9. Post operative care
a. Rabbits were allowed to recover after stent deployment and maintained
in warren for a period until stent was retrieved.
a. Immediately after surgery, rabbits were allowed to recover in warm
environment by keeping under heating lamp to maintain body
temperature.
b. Greens as feed and water was offered immediately after surgery in
recovery chamber.
c. Sutures were protected with light dressings. The rabbits recovered
from anaesthesia uneventfully.
h. Post operatively inj. Enrofloxacin @ 5 mg/kg i.m. s.i.d.
12
and inj.
Meloxicam @ 0.05 mg/kg i.m. s.i.d. was given for 3 days.
10. Post operative monitoring
a. The rabbits were monitored for 5 days for any adverse effects or
complications.
b. The postoperative rabbits were monitored for physical (grooming,
temperature, hopping) and behavioral activities (dullness, teeth
grinding, appetite and water intake).
c. It is advisable to check urine color and texture (hair, droppings).
11. Evaluation of stent in vivo and in vitro
a. None of the animal was treated with anticoagulation or antiplatelet
therapy after the operation.
b. On the day of stent retrieval, rabbits received same anesthetic
combination and dose rate as mentioned in stenting procedure.
c. Stents were retrieved from anesthetized rabbits (supine position) with
midline laparotomy incision to expose stented external iliac arteries.
d. The stented vessels were then carefully and bluntly dissected free of
the surrounding adventitial connective tissue.
e. The descending aorta was ligated after securing access distally to both
the femoral arteries.
f. The femoral vessels were then transected above after securely ligating
and the two-stented vessel specimens separated carefully for bench
dissection at the level of the aortic quadrification.
g. Rabbits were euthanized using Thiopentone-Sodium
18
@ 10 mg / kg
body wt i.v. through catheter fixed at marginal ear vein.
h. The specimens were labeled and fixed in formaldehyde for histological
examination.
i. Resin embedding technique was followed for sectioning tissue sample
along with stent struts.
j. Microtome
19
(RM 2125RT) and the Tungsten Carbide blade (No.
22227) were used for sectioning the resin embedded blocks of stented
arteries with stents.

18
Thiopentone NaThiopentone Injection, Thiosol, Neon Laboratory Ltd, Andheri, India-400093
19
Microtome Leica Company, Germany
k. The angle of knife was kept at 4
o
, which enabled to cut at 5m
sections.
l. We used Haematoxylin and Eosin (HE), Martius-Scarlet-Blue (MSB)
staining to visualize microscopic lesions however SEM and
immunohistochemistry can also be performed for further evaluation.
m. Evaluation was performed using histological grade points (neointimal
hyperplasia, apparent re-endothelization, non-obstructive peri-strut
fibrin, peristrut inflammatory response, lamina intima disruption, luminal
narrowing) and morphometric analysis.
12. Tips for hurdles and safety measures
a. Major cause of death was gross injury by impaired GW tip leading to
loss of blood in the form of haematoma or leakage during stenting
procedure. Use of new GW with soft tip for every rabbit and injecting
saline before operation will prevent possible death.
b. Death due to vagal stimulation and tearing of vessel can be avoiding by
less handling of vagus nerve and using papaverine-lignocaine drops
during procedure for vessel dilatation.
c. GW or GC tearing of vessel can be prevented using gentle handling,
securing entry points personally, keeping vessel wet using wet gauge
piece thought the procedure.
d. Allow animal to recover on warm chamber with full access to food
(especially green grass).
e. The transition from the common carotid artery to the aortic arch
presented an anatomical obstacle which can be overcome in
subsequent placement attempts by using torque with suitable
judgments and restoring or positioning of animal manually.
13. Advantage of this method
a. This procedure shows possibility of bilateral stenting in same animal,
which allows true comparison.
b. This model allows reducing number of animals used in experiment (3R
of animal ethics).
c. It allows easy monitoring.
d. Trans-carotid iliac artery stenting prevents handling of area of interest.
e. Among many animal models of cardiovascular diseases (CVD), rabbit
has been preferred more due to early vascular lesion development.
Also, due to ease in handling, monitoring and cost effectiveness.
14. Discussion
a. The angiographic images revealed dark column of vascular branches
because of the increased radio-density of the blood vessels, which may
be attributed to mixing of the iodinated contrast in the vascular system
with blood, which is further, carried diluted down stream. Despite the
considerable developments in the non-invasive vascular techniques,
stenting under Fluoroscopic Angiography (FA) was the easily
maneouverable, non-invasive, reliable, economical and a qualitative
technique for the investigation of vascular disease and has superseded
other techniques for e.g. Magnetic Resonance Imaging (MRI) or
Computerized Tomography (CT) guided stenting.
b. Variation in rabbit carotid anatomical location was not detected. The
introduction of stent delivery catheters through the left carotid artery
facilitated access to the distal iliac arteries where stent deployment was
performed.
c. In the present study, an antegrade trans-carotid balloon injured iliac
artery in vivo rabbit model for stenting was performed within the
interventional region in the absence of flow disturbances introduced by
the antegrade method of stent deployment. We found the present
technique of stent implantation as relatively easy to perform. The
model allowed detailed examination of the influence of stent
implantation on histological grade points. A trans-carotid approach to
the iliac vessels is mandatory to maintain the patency of the iliac
vessels distal to the stent deployment.
d. Absence of any vascular compromise in the hind limbs post stenting in
any of the rabbits studied reinforces this belief. The near non-invasive
vascular techniques for stenting, using trans-carotid/antegrade/normo-
grade model was efficient and easy to operate yielding consistent
results as compared to hind leg (femoral) approach. This technical
development should help researchers to use trans-carotid balloon
injured iliac artery rabbit model for vascular interventional research.
15. Conclusions
a. The semi-invasive vascular technique for stenting, using trans-carotid
antegrade model was effective and yields desired results using proper
instrumentation. It provides good access to the desired length of artery
and allows easy catheterization.
b. Is model of normograde trans-carotid iliac artery stenting is
reproducible, comfortable and reliable model, which can be used to
yield consistent and satisfactory results for various interventional
studies.
16. Disclosure
This procedure has been performed under IAEC at Anand Veterinary College
and performed at JPAC, Muljibhai Patel Urological Hospital. Sahajanand
Medical Stent Company and Muljibhai Patel Society for Research in Nephro-
Urology, Nadiad were sponsoring organisations of this study.
NB: Earlier version of protocol for stenting technique has been published as an
article in below reference:
Patil, P. B., et al. (2007). Catheterization of carotid artery for iliac stenting in rabbits.
IJVS, 28(2): 133-134
ROLE Of AUTHORS:
1. PPB: video, manuscript, experiment, data analysis and interpretation
2. PDB: manuscript, experiment, data analysis and interpretation
3. PPV: manuscript, experiment, data analysis and interpretation
4. RM:, experiment
5. LSS: experiment, data analysis and interpretation
6. RMM: experiment, data analysis and interpretation
7. KNH: experiment

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