Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Summary

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Abstract

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

Introduction

Peripheral arterial disease (PAD) is a common circulatory disorder in which the progression of atherosclerotic plaque formation leads to a narrowing of blood vessels in the limbs of the body. The recent increase in risk factors for atherosclerosis, including diabetes, obesity, and inactivity, has led to increasing prevalence of vascular disease¹. Currently, it is estimated that 12%–20% of the general population over 60 years old has peripheral arterial disease². A major consequence of peripheral arterial disease is the development of peripheral ischemia, most commonly found in the lower limbs. In severe cases, pati....

Protocol

Studies involving animals were performed with the approval of the University of Texas at Austin and the UTHealth Science Center at Houston Institutional Animal Care and Use Committee (IACUC), the Animal Care and Use Review Office (ACURO) of The United States Army Medical Research and Materiel Command Office of Research Protections, and in accordance with NIH guidelines for animal care.

1. Induction of diabetes and hyperlipidemia

1. Transition the New Zealand rabbits (4–6 months .......

Discussion

We have presented a preclinical model for inducing hind limb ischemia in rabbits with diabetes and hyperlipidemia. In many studies, there is ambiguity to the technique used to create hind limb ischemia in rabbits. In mice, the severity and recovery from hind limb ischemia is highly dependent on the location the ligation and technique used to induce ischemia. The significance of the technique presented in this work is that it allows for the consistent induction of ischemia that does not fully recover after 8 weeks in diab.......

Materials

Name	Company	Catalog Number	Comments
0.9% Sodium Chloride	Henry Schein Medical	1537468 / 1531434	250 mL bag / 1000 mL irrigation btl
1 mL Syringe	VWR	BD309628
10 mL Syringe	VWR	BD309695
10% Formalin	Fisher-Scientific	23-245684
18G Needle	VWR	89219-294
20G Needle	VWR	89219-340
25G Needle	VWR	89219-290
27G Needle	VWR	89219-288
5 mL Syringe	VWR	BD309646
5% Dextrose	Patterson Veterinary	07-800-9689
Acepromazine	Patterson Veterinary	VEDC207
Alfaxalone	Patterson Veterinary	07-891-6051
Alginate	Sigma-Aldrich	PHR1471-1G
Alloxan Monohydrate	Sigma-Aldrich	A7413
Angiography Equipment	Toshiba	Infinix-i
Angiography Injector	Medrad
Anti-Mouse Ab Alexa 594	Thermo Fisher Scientific	A-11032	Secondary Antibody for IHC
Anti-Rabbit Ab Alexa 488	Thermo Fisher Scientific	A-11008	Secondary Antibody for IHC
a-SMA Antibody	Abcam	ab5694	Primary Antibody for IHC
Baytril	Bayer Animal Health	724089904201	Enrofloxacin
Blood Chemistry Panel	IDEXX	2616	Rabbit Panel
Blood Pressure Cuff	WelchAllyn		Flexiport Disposable BP Cuff-infant size 7
Blood Pressure Monitor	Vmed Technology		Vmed Vet-Dop2
Bupivacaine	Henry Schein Medical	6023287
Buprenorphine	Patterson Veterinary	42023017905
Buprenorphine SR	ZooPharm
Calcium Sulfate	CB Minerals		Food and Pharmaceutical Grade USP and FCC
Chlorhexidine Scrub	Patterson Veterinary	07-888-4598
Chloroform	Fisher-Scientific	C298-4
Cholesterol	Sigma-Aldrich	C8503
DAPI	Thermo Fisher Scientific	62248
Ear Vein Catheter	Patterson Veterinary	SR-OX165	Surflo IV catheters
Endotracheal tube	Patterson Veterinary		Sheridan Brand, Depends on Rabbit Size
Glucometer	Amazon	B001A67WH2	Accu-Chek Aviva
Glucometer Test Strips	McKesson Medical-Surgical	788222	Accu-Chek Aviva Plus
Guidewire	Boston Scientific	39122-01
Hair Clippers	Amazon	B000CQZI3Q	Oster #40 blade
Heating Pad	Cincinnati Subzero	273
Heating Pad Pump	Gaymar		Gaymar T/Pump
Hemostat	Fine Science Tools	13009-12	Curved Mosquito Hemostat
Heparin	Patterson Veterinary
Insertion Tool	Merit Medical Systems	MAP550	metal wire insertion tool
Insulin	HPB Pharmacy		Novalin R & Novalin N
Insulin Syringes	McKesson Medical-Surgical	942674
Introducer	Cook Medical	G28954	3F Check Flo Performer Introducer
Isoflurane	Henry Schein Medical	1100734
Ketamine	Patterson Veterinary	856440301
Lactated Ringers	McKesson Medical-Surgical	186662
Lidocaine	McKesson Medical-Surgical	239936
Lidocaine/Prilocaine cream	McKesson Medical-Surgical	761240
Ligaloop	V. Mueller	CH117 / CH116	White Mini / Yellow Mini
Mazola Corn Oil	Amazon	B0049IIVCI
Medrad Syringe	McKesson Medical-Surgical	346920	150 mL
Meloxicam	Patterson Veterinary
Metal ball sutures	Ethicon-Johnson & Johnson	K891H	4-0 silk C-1 30"
Metzenbaum Scissors	Fine Science Tools	14019-13
Midazolam	Henry Schein Medical	1215470
Nitroglycerin	McKesson Medical-Surgical	927528
PECAM Antibody	Novus Biologicals	NB600-562	Primary Antibody for IHC
Perfusion Pump	Masterflex
Pigtail Catheter	Merit Medical Systems	1310-21-0053	3F pigtail
Polydioxanone (PDS II) suture	McKesson Medical-Surgical	129271	4-0 taper RB-1 (needle comes on suture)
Polydioxanone (PDS II) suture	McKesson Medical-Surgical	129031	4-0 reverse cutting FS-2
Polyglactin 910 (Vicryl) suture	Butler	7233-41	3-0 taper RB-1
Polyglactin 910 (Vicryl) suture	McKesson	104373	4-0 reverse cutting FS-2
Rabbit Chow (Alfalfa)	LabDiet	5321
Rabbit Restrainer	VWR	10718-000
Rib Cutters	V. Mueller
Scalpel	Fine Science Tools	10003-12
Scalpel Blade	Fine Science Tools	10015-00	#15 blade
Silk Sutures	Ethicon-Johnson & Johnson	A183H	4-0 silk ties 18"
Stainless Steel Ball	McMaster-Carr	1598K23	3-mm diameter
Surgical Drapes	Gepco	8204S
Syringe Pump	DRE Veterinary		Versaflow VF-300
Visipaque contrast media	McKesson Medical-Surgical	509055
Weitlaner Retractor	Fine Science Tools	17012-13