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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 .......

Representative Results

Following induction of diabetes and initiation of the 0.1% cholesterol diet, the total cholesterol for the rabbits with diabetes and cholesterol diet was 123.3 ± 35.1 mg/dL (n = 6 male rabbits) averaged overall time points and rabbits. The BGL level for these rabbits was 248.3 ± 50.4 mg/dL (n = 6 male rabbits). A time course for blood chemistries and leg blood pressure ratios in a typical rabbit is shown in Figure 3 in comparison to rabbits under a higher choles.......

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.......

Acknowledgements

The authors gratefully acknowledge funding through the Department of Defense Congressionally Directed Research Program (DOD CDMRP; W81XWH-16-1-0582) to ABB and RS. The authors also acknowledge funding through the American Heart Association (17IRG33410888), the DOD CDMRP (W81XWH-16-1-0580) and the National Institutes of Health (1R21EB023551-01; 1R21EB024147-01A1; 1R01HL141761-01) to ABB.

....

Materials

NameCompanyCatalog NumberComments
0.9% Sodium ChlorideHenry Schein Medical1537468 / 1531434250 mL bag / 1000 mL irrigation btl
1 mL SyringeVWRBD309628
10 mL SyringeVWRBD309695
10% FormalinFisher-Scientific23-245684
18G NeedleVWR89219-294
20G NeedleVWR89219-340
25G NeedleVWR89219-290
27G NeedleVWR89219-288
5 mL SyringeVWRBD309646
5% DextrosePatterson Veterinary07-800-9689
AcepromazinePatterson VeterinaryVEDC207
AlfaxalonePatterson Veterinary07-891-6051
AlginateSigma-AldrichPHR1471-1G
Alloxan MonohydrateSigma-AldrichA7413
Angiography EquipmentToshibaInfinix-i
Angiography InjectorMedrad
Anti-Mouse Ab Alexa 594Thermo Fisher ScientificA-11032Secondary Antibody for IHC
Anti-Rabbit Ab Alexa 488Thermo Fisher ScientificA-11008Secondary Antibody for IHC
a-SMA AntibodyAbcamab5694Primary Antibody for IHC
BaytrilBayer Animal Health724089904201Enrofloxacin
Blood Chemistry PanelIDEXX2616Rabbit Panel
Blood Pressure CuffWelchAllynFlexiport Disposable BP Cuff-infant size 7
Blood Pressure MonitorVmed TechnologyVmed Vet-Dop2
BupivacaineHenry Schein Medical6023287
BuprenorphinePatterson Veterinary42023017905
Buprenorphine SRZooPharm
Calcium SulfateCB MineralsFood and Pharmaceutical Grade USP and FCC
Chlorhexidine ScrubPatterson Veterinary07-888-4598
ChloroformFisher-ScientificC298-4
CholesterolSigma-AldrichC8503
DAPIThermo Fisher Scientific62248
Ear Vein CatheterPatterson VeterinarySR-OX165Surflo IV catheters
Endotracheal tubePatterson VeterinarySheridan Brand, Depends on Rabbit Size
GlucometerAmazonB001A67WH2Accu-Chek Aviva
Glucometer Test StripsMcKesson Medical-Surgical788222Accu-Chek Aviva Plus
GuidewireBoston Scientific39122-01
Hair ClippersAmazonB000CQZI3QOster #40 blade
Heating PadCincinnati Subzero273
Heating Pad PumpGaymarGaymar T/Pump
HemostatFine Science Tools13009-12Curved Mosquito Hemostat
HeparinPatterson Veterinary
Insertion ToolMerit Medical SystemsMAP550metal wire insertion tool
InsulinHPB PharmacyNovalin R & Novalin N
Insulin SyringesMcKesson Medical-Surgical942674
IntroducerCook MedicalG289543F Check Flo Performer Introducer
IsofluraneHenry Schein Medical1100734
KetaminePatterson Veterinary856440301
Lactated RingersMcKesson Medical-Surgical186662
LidocaineMcKesson Medical-Surgical239936
Lidocaine/Prilocaine creamMcKesson Medical-Surgical761240
LigaloopV. MuellerCH117 / CH116White Mini / Yellow Mini
Mazola Corn OilAmazonB0049IIVCI
Medrad SyringeMcKesson Medical-Surgical346920150 mL
MeloxicamPatterson Veterinary
Metal ball suturesEthicon-Johnson & JohnsonK891H4-0 silk C-1 30"
Metzenbaum ScissorsFine Science Tools14019-13
MidazolamHenry Schein Medical1215470
NitroglycerinMcKesson Medical-Surgical927528
PECAM AntibodyNovus BiologicalsNB600-562Primary Antibody for IHC
Perfusion PumpMasterflex
Pigtail CatheterMerit Medical Systems1310-21-00533F pigtail
Polydioxanone (PDS II) sutureMcKesson Medical-Surgical1292714-0 taper RB-1 (needle comes on suture)
Polydioxanone (PDS II) sutureMcKesson Medical-Surgical1290314-0 reverse cutting FS-2
Polyglactin 910 (Vicryl) sutureButler7233-413-0 taper RB-1
Polyglactin 910 (Vicryl) sutureMcKesson1043734-0 reverse cutting FS-2
Rabbit Chow (Alfalfa)LabDiet5321
Rabbit RestrainerVWR10718-000
Rib CuttersV. Mueller
ScalpelFine Science Tools10003-12
Scalpel BladeFine Science Tools10015-00#15 blade
Silk SuturesEthicon-Johnson & JohnsonA183H4-0 silk ties 18"
Stainless Steel BallMcMaster-Carr1598K233-mm diameter
Surgical DrapesGepco8204S
Syringe PumpDRE VeterinaryVersaflow VF-300
Visipaque contrast mediaMcKesson Medical-Surgical509055
Weitlaner RetractorFine Science Tools17012-13

References

  1. Mozaffarian, D., et al. Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation. 133 (4), e38-e360 (2016).
  2. Roger, V. L., et al.

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