EVALUATION OF THE ANTI-ATHEROSCLEROTIC EFFICACY OF HERBAL FORMULATIONS IN RABBIT MODEL OF ATHEROSCLEROSIS INDUCED BY HIGH-FAT DIET AND ILIAC ARTERY ATHEROMA
1.0
TEST SYSTEM DETAILS:
Species : Oryctolagus cuniculus (Rabbits)
Strain : New
Zealand White
Age : 3-5 months
Sex : Male
No. of animals : 8/Group
Total animals : 56
2.0
TEST ARTICLES DETAILS
Lagenaria siceraria-based herbal formulation.
3.0
VEHICLE DETAILS
The test articles will be formulated
by utilizing 0.5% methylcellulose as the vehicle.
4.0
ALLOCATION OF GROUPS:
|
Group No. |
Group
Description |
Disease
Induction procedure |
Treatment
administered |
Dose
Volume and Route |
|
G1 |
Sham Control |
Normal laboratory diet × 5 weeks and exposure with
manipulation of iliac artery |
0.5% MC, p.o., b.i.d. |
10 ml/kg, p.o. |
|
G2 |
Disease Control |
Atherogenic diet × 5 weeks + Balloon injury in the iliac artery |
0.5% MC, p.o., b.i.d. |
|
|
G3 |
Reference Control |
Simvastatin-5 mg/kg, p.o., q.d. (morning) + 0.5% MC,
p.o., q.d. (evening) |
||
|
G4 |
Treated with low dose |
LD: 3-10
mg/kg, b.i.d. |
||
|
G5 |
Treated with intermediate dose 1 |
ID-1: 10-30
mg/kg, b.i.d. |
||
|
G6 |
Treated with intermediate dose 2 |
ID-2: 30-100
mg/kg, b.i.d. |
||
|
G7 |
Treated with high dose |
HD: 100-300
mg/kg, b.i.d. |
Abbreviations: MC: Methylcellulose, p.o.-per os. q.d.: quaque
die; bid: bis in die.
5.0
METHOD:
·
Healthy
animals will be selected for the study, randomized based on body weight and will
be assigned to 7 groups consisting of 8 animals each.
·
Animals
of the Group G1 will be designated as sham-control and administered 0.5% MC, p.o., b.i.d., two weeks prior to disease
induction and continued till the end of the experiment.
·
Disease
control animals (assigned to group G2) will receive 0.5% MC, p.o., b.i.d.., two weeks prior to disease induction and continued
till the end of the experiment.
·
Animals
of group G3 will be treated with 0.5% MC, p.o., b.i.d.., two weeks prior to
disease induction and reference drug simvastatin at the dose of 5 mg/kg, p.o., q.d.
in the morning and 0.5% MC, p.o., q.d. in the evening, concurrent with the
disease induction protocol.
·
Animals
of group G4-G7 will be treated with drug, at different incremental dose
levels as outlined in Section 4.0 of Annexure-I, twice daily, two weeks prior to disease induction and continued
till the end of the experiment.
·
After
Male New Zealand rabbits will be fed an atherogenic diet consisting of 1%
cholesterol and 6% peanut oil for 5 weeks. One week after initiating the
high-cholesterol diet, iliac artery atheroma will be created. Briefly, surgery
site will be disinfected with povidone-iodine solution. Animals will be
anesthetized using ketamine (25 mg/kg) and xylazine (3 mg/kg). Hair will be
removed from the ventral area just below the knee joints using animal hair
clippers. Skin will be cleaned with suitable disinfectant. Saphenous artery
will be located and a small skin incision of about 1.5 cm in length will be
made using a scalpel. A small portion of saphenous artery will be exposed with
small, curved forceps without damaging the femoral vein and femoral nerve. Two
loose ligature loops (5-0 silk) will be placed beneath the saphenous artery and
one ligature loop towards the distal end of the artery will be tied off. A
microvascular clamp will be placed above the ligature to stop the blood flow
from the iliac artery. Saphenous artery will be lifted up with the help of the
tied ligature and small arteriotomy incision will be made using a 24-gauge
needle. Incision flaps will be elevated with fine forceps and a 2 French
Fogarty arterial embolectomy catheter will be inserted into the saphenous
artery. Microvascular clamps will be removed and the catheter will be advanced
till the sixth mark (20 - 25 cm) corresponding to a position roughly 2-5 cm
above the iliac bifurcation. Balloon will be inflated with 0.1 mL normal saline
using a 1 mL syringe. Balloon catheter will be pulled back by 6 cm through the
iliac artery towards the point of insertion, while rotating the catheter.
Balloon will be deflated by pulling back the plunger of the syringe. The steps
of inserting and pulling back catheter will be repeated three times to ensure
complete endothelial denudation. Catheter will be removed, and the ligature
loop will be immediately tied just above the arteriotomy site to stop bleeding.
Suitable antiseptic will be applied all around the periphery of the wound. Skin
incision will be closed with a 5-0 suture. Animals will be maintained on the
same atherogenic diet for an additional 4 weeks.
·
Animals
will be bled by ear vein at 0, 7, 14, 28 and 35 days for measurements of plasma
cholesterol, triglycerides, glucose, LDL and HDL.
·
At
the end of the treatment period, animals will be euthanized under an overdose
of thiopentone anesthesia. Subsequently, both uninjured and injured iliac
artery will be collected and divided in two portions. One portion will be fixed
in 4% buffered formaldehyde for further histological, immunohistochemical and
morphometric analysis. The other portion will be snap frozen in liquid nitrogen
and stored immediately at -80°C for molecular parameters,
6.0
PARAMETERS TO BE EVALUATED:
·
Body
weight: Twice a week.
·
Plasma
levels of Total cholesterol, HDL-C, LDL-C, Glucose
·
Gene expression
analysis of injured and uninjured iliac artery by Real Time PCR: Col1a1, MMP9,
MCP-1, VCAM, pro and anti-inflammatory genes.
·
Histological
analysis of arterial sections (Oil red O, Hematoxylin & Eosin, IHC-
macrophage, α-smooth muscle actin).
7.0
REFERENCE(S):
1. Jain, M.; Frobert,
A.; Valentin, J.; Cook, S.; Giraud, M.N. The Rabbit Model of Accelerated
Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon
Injury. J Vis Exp 2017, doi:10.3791/55295.
2. Khanna, V.; Jain, M.; Singh, V.; Kanshana, J.S.;
Prakash, P.; Barthwal, M.K.; Murthy, P.S.; Dikshit, M. Cholesterol diet
withdrawal leads to an initial plaque instability and subsequent regression of
accelerated iliac artery atherosclerosis in rabbits. PLoS One 2013, 8, e77037, doi:10.1371/journal.pone.0077037.
3. Kanshana, J.S.; Khanna, V.; Singh, V.; Jain,
M.; Misra, A.; Kumar, S.; Farooqui, M.; Barthwal, M.K.; Dikshit, M. Progression
and Characterization of the Accelerated Atherosclerosis in Iliac Artery of New
Zealand White Rabbits: Effect of Simvastatin. J Cardiovasc Pharmacol 2017,
69, 314-325,
doi:10.1097/FJC.0000000000000477.
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