A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

Summary

This article demonstrates an efficient surgical approach to establish acute ischemia in mice with a small incision. This approach can be applied by most research groups without any laboratory upgrades.

Abstract

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most animal laboratories. Contrary to the conventional approach for double ligation of the femoral artery (DLFA), a smaller incision on the right inguinal region was made to expose the proximal femoral artery (FA) to perform DLFA. Then, using a 7-0 suture, the incision was dragged to the knee region to expose the distal FA. Magnetic resonance imaging (MRI) on bilateral hind limbs was used to detect FA occlusion after the surgery. At 0, 1, 3, 5, and 7 days after the surgery, functional recovery of the hind limbs was visually assessed and graded using the Tarlov scale. Histologic evaluation was performed after euthanizing the animals 7 days after DLFA. The procedures were successfully performed on the right leg in ten ApoE^-/- mice, and no mice died during subsequent observation. The incision sizes in all 10 mice were less than 5 mm (4.2 ± 0.63 mm). MRI results showed that FA blood flow in the ischemic side was clearly blocked. The Tarlov scale results demonstrated that hind limb function significantly decreased after the procedure and slowly recovered over the following 7 days. Histologic evaluation showed a significant inflammatory response on the ischemic side and reduced microvascular density in the ischemic hind limb. In conclusion, this study introduces a modified technique using a miniature incision to perform hind limb ischemia (HLI) using DLFA.

Introduction

There is an unmet need for preclinical animal models for research in vascular diseases such as peripheral artery disease (PAD). Despite the advanced developments in diagnosis and treatment, there were more than 200 million patients with PAD in 2018¹, and their number is constantly increasing. Although several novel therapeutic approaches^2,3,4,5,6,7 have been described, successful translation of these therapeutic modalities into clinical application ....

Protocol

NOTE: All experimental procedures were performed according to the EC guideline EC 2010/63/EU and have been approved by the local German legislation (35-9185.81/G[1]239/18). Ten male ApoE^-/- mice with the C57BL/6J background, weighing 29.6-38.0 g, were housed on a 12 h light/dark cycle and fed a western diet (1.25% cholesterol and 21% fat) and water ad libitum for 12 weeks from the age of 8 weeks. HLI was performed on 20-week-old mice as described below.

1. Induction of HLI in ApoE^-/- mice

1. Prepare the required equipment and tools for surgery (see the Table of Materials and

Results

Characteristics of ApoE^-/- mice
DLFA surgeries were successfully performed on 10 mice to establish the HLI model, and none of the mice died after the procedure. To follow changes in body weight, mice were weighed before the DLFA procedure (Pre-DLFA) and 7 days after the DLFA surgery (Post-DLFA). Pre-DLFA weights ranged from 29.6 to 38.0 g (mean 34.74 ± 2.47 g), and post-DLFA weights ranged from 26.5 to 34.1 g (mean 30.77 ± 2.15 g), which were significantly.......

Discussion

This study reports a modified, simplified, and surgically efficient approach to establish an HLI model in ApoE^-/- mice using double ligation in the proximal and distal regions of the FA through a 3-4 mm incision without any required laboratory upgrades. The main characteristic of this method is the smaller size of the incision compared to previously reported studies describing mouse HLI models^8,9,10,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10x Phosphate buffer saline	Roth	9143.1	Used for haematoxylin and eosin stain and immunohistochemistry stain
30% H2O2	Roth	9681.2	Used for immunohistochemistry stain
6-0 absorbable sutures	PROLENE	8776H	Used for stitching the skin
6-0 absroable suture	PROLENE	EP8706	Used in Surgery
7-0 absorbable sutures	PROLENE	EH8021E	Used for ligating the artery
7-0 absroable suture	PROLENE	EP8755	Used in Surgery
Acetic acid	Roth	6755.1	Used for haematoxylin and eosin stain
Albumin Fraktion V	Roth	8076.2	Used for immunohistochemistry stain
Autoclave	Systec GmbH	Systec VX-150	Used for the sterilisation of the surgical instruments
Axio vert A1 microscope	Carl Zeiss	ZEISS Axio Vert.A1	Used for viewing and taking the pictures from haematoxylin and eosin stain and immunohistochemistry stain
Bruker BioSpec 94/20 AVIII	Bruker Biospin MRI GmbH	N/A	Scan the femoral artery blockage
Buprenovet Sine 0,3mg/ml	Bayer AG	2542 (WDT)	Used in post operative pain-management. Dose - 0.1 mg/kg body weight every 8 hours for 48 h after operation
CD31 antibody	Abcam	ab28364	Used for immunohistochemistry stain
Eosin Y solution 0.5 % in water	Roth	X883.1	Used for haematoxylin and eosin stain
Epitope Retrieval Solution pH 6	Leica Biosystems	6046945	Used for immunohistochemistry stain
Ethanol ≥ 99,5 %	Roth	5054.1	Used for haematoxylin and eosin stain and immunohistochemistry stain
Fentanyl	Cayman Chemical	437-38-7	Used for anesthesia
Fine point forceps	Medixplus	93-4505S	Used for separating the artery from nerve and vein
Glass bead sterilisator	Simon Keller	Type 250	Used for sterilisation of the surgical instruments
Graefe iris forceps curved	VUBU	VUBU-02-72207	Used for blunt separation of skin and subcutaneous tissue
Hair Remover cream, Veet (with aloe vera)	Reckitt Benckiser	108972	Remove hair from mice hind limbs
Heating plate	STÖRK-TRONIC	7042092	Keep the satble temperature of mice
Hematoxylin	Roth	T865.2	Used for haematoxylin and eosin stain and immunohistochemistry stain
Leica surgical microscope	Leica	M651	Enlarge the field of view to facilitate the operation
Liquid DAB+Substrate Chromogen System	Dako	K3468	Used for immunohistochemistry stain
Male ApoE-/- mice	Charles River Laboratories	N/A	Used for establish the Peripheral artery disease mice model
Medetomidine	Cayman Chemical	128366-50-7	Used for anesthesia
Micro Needle Holder	Black & Black Surgical	B3B-18-8	Holding the needle
Micro suture tying forceps	Life Saver Surgical Industries	PS-MSF-145	Used to assist in knotting during surgery
Microtome	Biobase	Bk-Mt268m	Used for tissue sectioning
Midazolam	Ratiopharm	44856.01.00	Used for anesthesia
MR-compatible Small Animal Monitoring and Gating System Model 1025	SA Instruments	N/a	monitoring vital signs of animal during MRI scan
Octeniderm farblos	Schülke & Mayr GmbH	180212	used for disinfection of the skin
Ointment for the eyes and nose	Bayer AG	1578675	Keep the eyes wet under the anesthesia
Paraformaldehyde	Roth	0335.1	Used for fixation of the tissue
Pentobarbital	Nembutal	76-74-4	Used for anesthesia
Saline	DeltaSelect	1299.99.99	Used for anesthesia
Spring handle scissors with fine, sharp tips	Black & Black Surgical	B66167	Used for cutting the artery
SuperCut Scissors	Black & Black Surgical	B55992	Used for cutting the skin
Triton X-100	Roth	9002-93-1	Used for immunohistochemistry stain
Western diet, 1.25% Cholesterol	ssniff Spezialdiäten GmbH	E15723-34	Diet for the mice
Xylene	Roth	4436.3	Used for haematoxylin and eosin stain and immunohistochemistry stain