A Rat Model of Middle Cerebral Artery Occlusion/Reperfusion without Damaging the Anatomical Structure of Cerebral Vessels

Published: May 17, 2024

doi:

Zhang Jiren, Jia Xue, Sun Yufei, Li Huiwen, Yang Jian, Qi Shilei, Zhao Dongman, Xie Lei, Jia Mingming, Wang Tian, Zhang Pinyuan

¹Department of Neurosurgery,The Third Hospital of Hebei Medical University, ²School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation, Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong,Yantai University

Summary

The current protocol describes a rat model of middle cerebral artery occlusion/reperfusion that preserves the anatomical structure of cerebral vessels without causing damage.

Abstract

Ischemic stroke stands as the primary cause of long-term disability and mortality among adults worldwide. Animal models of ischemic stroke have significantly contributed to our understanding of its pathological mechanisms and the development of potential treatments. Presently, there are two common methods involving filament (endovascular suture) techniques to induce animal models of cerebral ischemia. However, these methods have inherent limitations, such as reduced blood perfusion to the brain, damage to the external carotid artery system, impaired food and/or water intake, and sensory dysfunction of the face. This article introduces a new method for inducing a rat ischemic stroke model without compromising the cerebral vascular anatomy. In this study, the common carotid artery (CCA) of Sprague-Dawley rats was exposed, and an incision was made. A filament was then inserted through the incision into the internal carotid artery to occlude the middle cerebral artery. After 1.5 h of induced ischemia, the occluding filament was fully removed from both the internal carotid artery and the CCA. The incision in the CCA was subsequently sutured using 11-0 microsurgical sutures under a microscope (magnification 4x). Through the utilization of microsurgical techniques to repair the CCA, this study successfully developed a unique method to induce an ischemic stroke model in rats while preserving the anatomical integrity of cerebral blood vessels.

Introduction

Stroke, the leading cause of death and long-term neurological dysfunction in adults worldwide, encompass various types, with ischemic stroke accounting for approximately 81.9% of cases, while cerebral hemorrhage and subarachnoid hemorrhage account for 14.9% and 3.1%, respectively¹. Ischemic stroke often results from atherosclerosis or occlusion of the middle cerebral artery (MCA), leading to local cerebral blood flow reduction and subsequent brain damage. This reduction in nutrient supply to ischemic cells triggers energy depletion, causing membrane integrity loss, cell swelling, and eventual lysis. Additionally, factors like increased intracellular calcium, excitatory amino acid release, heightened free radical production, and inflammatory cell activation contribute to brain tissue injury post-stroke².

The treatment goals for ischemic stroke are multifaceted: reduce ongoing neurological damage and lower mortality and long-term disability; prevent complications arising from immobility and neurological dysfunction; and minimize the risk of stroke recurrence. Given the complexity of ischemic stroke pathophysiology, a robust stroke model is essential for the research and development of treatments targeting ischemic stroke.

Currently, middle cerebral artery occlusion (MCAo) is a common animal model of ischemic stroke³. MCAo is typically performed by inserting a silicon-tipped or flame-blunted monofilament through the cervical vessels until it reaches the origin of the middle cerebral artery (MCA). In 1986, Koizumi and colleagues developed a technique involving the insertion of a silicon-tipped monofilament through an incision in the common carotid artery (CCA), allowing it to reach the entrance of the MCA and block its blood flow⁴. In 1989, Longa’s group reported another MCAo method that involved introducing a monofilament through an incision in the external carotid artery (ECA). The monofilament then traverses the bifurcation of the internal carotid artery (ICA) and ECA before reaching the starting point of the MCA⁵.

The limitations of the two existing methods for inducing MCAo are evident. Koizumi’s method necessitates the permanent blockage of the ipsilateral CCA, leading to reduced MCA perfusion since reperfusion solely relies on the circle of Willis⁴. Conversely, Longa’s method involves cutting the ECA, resulting in damage to the ECA system and subsequent impairment of facial muscle function⁵. This damage can impact food and water intake, as well as cause sensory dysfunction of the face, affecting rehabilitation and the assessment of neurological function post-ischemic stroke⁶.

The current protocol seeks to address these limitations by developing a new method to induce an ischemic stroke model without damaging the anatomical structure of cerebral vessels. This novel approach utilizes microsurgical techniques to repair the CCA, thus avoiding the drawbacks associated with previous methods.

Protocol

The experimental procedure received approval from the Institutional Animal Care and Use Committee at Yantai University (Approval Number: YTDX20230410) and adhered to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats weighing between 300-350 g were utilized for this study. A comprehensive list of the animals, reagents, and equipment used is provided in the Table of Materials. 1. Animal preparation</p…

Representative Results

The process of microsuture The filament was withdrawn near the incision of the CCA until the white filament tip was visible (Figure 1A). Micro clamps were then placed on the CCA (Figure 1B). Under a microscope at 4x magnification, the incision of the CCA was sutured using microsurgical sutures in an interrupted pattern (Figure 1C). Subsequently, the microclamps were removed from the CCA, restoring the anatomic…

Discussion

MCAo is a widely used method to create animal models of stroke, effectively replicating the stroke process observed in patients by restoring blood flow after ischemia¹⁰^,¹¹^,¹². Our laboratory has developed an ischemic stroke model that enables reperfusion from the CCA without causing damage to the ECA. Following the induction of ischemia, microsurgical repair of the CCA is performed. Although direct measurement of CCA blood flow …

Declarações

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Hebei Province Introducing Foreign Intelligence Project in 2023. We thank Lisa Kreiner, PhD, from Liwen Bianji, (Edanz) (www.liwenbianji.cn), for editing the English text of a draft of this manuscript.

Materials

10% formalin	Beijing Labgic Technology Co.,Ltd.	BL913A
11-0 microsurgical sutures	Ningbo Medical Suture Needle Co., Ltd.	3/8 1×5
2,3,5-triphenyltetrazolium chloride (TTC)	SIGMA	T8877
3-0 silk suture	Ningbo Medical Suture Needle Co., Ltd.	3-0
4% isoflurane	Tianjin Ringpu Bio-Technology Co., Ltd.	20221102
6-0 silk suture	Ningbo Medical Suture Needle Co., Ltd.	6-0
70% ethanol	Shandong Lierkang Medical Technology Co., Ltd.	20230408
Filament	Xinong Technologies Co Ltd.	2838-A5
Ketamine	Jiangsu Hengrui Pharmaceutical Co., Ltd.	230613BL
Male Sprague Dawley rats	Jinan Pengyue Experimental Animal Breeding Co., Ltd.	SCXK (Lu) 20190003
Microclips	Shanghai Jinzhong Medical Device Factory	W40150
Microscope	ZEISS	S100 OPMI pico
Nursing box	Beijing Fuyi Electrical Appliance Co., Ltd.	FYL-YS-100L
Xylazine	SIGMA	PHR3263

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