High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Summary

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

Abstract

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

Introduction

Peripheral arterial disease (PAD), characterized by reduced blood flow to the extremities due to atherosclerosis, affects 6.5 million people in the United States and 200 million people worldwide¹. Patients with PAD experience reduced limb function and quality of life, and those with CLTI, the most severe form of PAD, are at increased risk for amputation and death with a 5-year mortality rate nearing 50%². In clinical practice, patients with ankle-brachial indices (ABI) <0.9 are considered to have PAD, and those with ABI <0.4 associated with either rest pain or tissue loss as having CLTI³. ....

Protocol

All animal experiments described in the protocol were approved by the University of Miami Institutional Animal Care and Use Committee (IACUC). FVB mice, both male and female, aged 8-12 weeks, were used for the study.

1. Preparation of L-NAME solution

1. Under sterile conditions in a laminar flow hood, prepare an L-NAME stock solution by dissolving 1g of L-NAME powder (see Table of Materials) with 20 mL of sterile water to make a 50 mg/mL of solution. Store the stock solution in 300-500 µL aliquots at -20 °C for up to 3 months.
2. To make a working L-NAME solution, thaw an aliquot o....

Results

This protocol details a reliable means of inducing ischemia and tissue loss in the murine footpad using a combination of femoral artery and vein coagulation with L-NAME administration, a nitric oxide synthase inhibitor, in susceptible FVB mice. Figure 1 details the anatomy of the murine hindlimb vasculature and indicates the sites of the femoral artery and vein coagulation (yellow X), just proximal to the lateral circumflex femoral artery (LCFA) and proximal to the saphenopopliteal junction........

Discussion

While mouse hindlimb ischemia is the most widely used preclinical model to study neovascularization in PAD and CLTI, there is significant variation in ischemia severity and recovery depending on the specific mouse strain used and the site, number, and method of arterial disruption. The combination of femoral artery ligation and IP administration of L-NAME can reliably induce hindlimb gangrene in FVB mice¹¹. The same treatment results in hindlimb ischemia without tissue loss in C57BL/6 mice, wherea.......

Materials

Name	Company	Catalog Number	Comments
Binder clips (small)	Office supply store
Buprenorphine (sustained-release)
Butterfly needle (25 G with Luer-Lok)	VWR	10148-584
Confocal laser scanning microscope	Leica	TCS SP5
DiI (1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)	Invitrogen	D282
Electrocautery device	Gemini Cautery System	5917
Ethanol (100%)	VWR	89370-084
Fiji (ImageJ) software	NIH		Used version 2.1.0. Free download, no license required.
Foam biopsy pads	Fisher Scientific	22-038-221
Formalin (neutral buffered, 10%)	VWR	89370-094
FVB mice	Jackson Laboratory	001800
Glucose	Sigma-Aldrich	G7528	Used version 2.1.0.
HCl (1 M)	Sigma-Aldrich	13-1700
Imaris software	Oxford Instruments		Used version 9.6.0.
Isoflurane	Pivetal	NDC 46066-755-04
KCl	Sigma-Aldrich	P9333
Ketamine
L-NAME (Nω-Nitro-L-arginine methyl ester hydrochloride)	Sigma-Aldrich	N5751
Laser Doppler perfusion imager	MoorLDI	moorLDI2-HIR	Used moorLDI V5 software.
Microscope slides (25 x 75 x 1 mm)	VWR	48311-703
Na2HPO4	Sigma-Aldrich	S7907
NaCl	Sigma-Aldrich	S7653
NaH2PO4	Sigma-Aldrich	S8282
NaOH	Sigma-Aldrich	S8263
Needles (27 G)	BD	305109
Povidone-iodine swabstick (10%)	Medline	MDS093901ZZ
Surgical instruments	Roboz Surgical		Fine forceps, needle driver, spring scissors, and hemostat are recommended.
Suture (5-0 absorbable)	DemeTECH	G275017B0P
Syringes (10 mL)	BD	305482
Three-way stopcocks	Cole-Parmer	19406-49
Vascular Analysis Plugin			Free download, no license required. See reference: Elfarnawany (2015).
Xylazine