Cerebral Ischemic Coma Model Induced by Modified Four-Vessel Occlusion

Published: July 05, 2024

doi:

Rong Ma*^1,2, Rui Li*², Jianlong Liang, Hongyan Yang, Qian Xie, Xuxin Zeng, Jialiang Guo

¹School of Chemistry and Chemical Engineering,South China University of Technology, ²School of Medicine,Foshan University

Summary

This protocol describes the process for inducing a cerebral ischemic coma model using a modified four-vessel occlusion method.

Abstract

Coma caused by cerebral ischemia is the most serious complication of cerebral ischemia. Four-vessel occlusion can establish a cerebral ischemic coma model for disease research and drug development. However, the commonly used four-vessel occlusion method mainly involves inserting an electrocoagulation pen into the bilateral pterygoid foramen of the first cervical vertebra behind the neck to electrocoagulate the vertebral arteries. This process carries the risk of incomplete electrocoagulation, bleeding, and damage to the brainstem and spinal cord. Twenty-four hours after surgery, re-anesthetized rats undergo carotid artery ligation in front of the neck. Two surgeries expose the rats to a higher risk of infection and increase the experimental period. In this study, during a single surgical procedure, an anterior cervical incision was used to locate the key site where the vertebral artery penetrates the first cervical vertebra. The bilateral vertebral arteries were electrocauterized under visual conditions, while the bilateral common carotid arteries were separated to place loose knots. When the rats showed consciousness of the inversion reaction, the bilateral common carotid arteries were quickly ligated to induce ischemic coma. This method can avoid the risk of infection caused by two surgical operations and is easy to perform with a high success rate, providing a useful reference for relevant practitioners.

Introduction

Ischemic brain injury is the most common brain injury in clinical practice, accounting for approximately 75% of cerebrovascular disease cases. Ischemia can lead to severe secondary brain injuries and diseases¹^,², and coma is the most severe symptom caused by ischemic hypoxic brain injury. It is also the final pathway for many critical conditions³. Coma is a critical and severe illness in clinical practice that is difficult to manage⁴. The longer the coma lasts, the greater the potential danger. Prompt awakening is the primary goal in preventing the deterioration and progression of the condition. Although naloxone injection has a wide range of clinical applications in promoting wakefulness, it still has some side effects⁵. Therefore, the development of safe and effective wakefulness-promoting drugs is an urgent problem that needs to be addressed. Establishing a simple and easy-to-operate brain ischemic coma model is essential for elucidating the pathogenesis of ischemic coma and for drug development⁶^,⁷^,⁸.

The goal of this study is to introduce a model of global ischemic coma induced by electrocoagulation of the vertebral artery (VA) and temporary ligation of the common carotid artery (CCA) simultaneously, which is simple and user-friendly for novices. The previous protocol involved exposing the bilateral pterygoid foramen of the first posterior cervical vertebra during the first operation and electrically burning the pterygoid foramen to block the bilateral VAs. A second operation was performed 24 h later to induce total ischemic coma by ligation of the bilateral CCAs⁹^,¹⁰^,¹¹^,¹². However, due to invisibility, there is a risk of incomplete electrocoagulation, bleeding, brainstem, and spinal cord injury, as well as a prolonged experimental period. Therefore, it is necessary to address these issues.

Here, we present an improved method for modeling ischemic coma. The main procedure involves making a median anterior neck incision, performing electrical resection of the bilateral VAs under visual conditions, and briefly ligating the bilateral CCAs during a single operation to block the blood supply to the entire brain, causing rapid electroencephalogram (EEG) inhibition and leading to coma. This method also induces a brief continuous coma after reperfusion. This procedure is easy to perform, novice-friendly, and reduces the risk of secondary trauma infection in animals, thereby shortening the experimental period.

The protocol is suitable for the study of global ischemic coma caused by cardiac arrest. It is also ideal for the study of ischemic dementia, mainly because the hippocampal brain area is extremely sensitive to ischemia; thus, transient cerebral ischemia can lead to damage or even loss of hippocampal neurons¹³, resulting in cognitive dysfunction. Therefore, the protocol can provide a reference for practitioners studying cerebral ischemia, ischemic coma, and ischemic dementia.

Protocol

The experimental protocol was conducted in accordance with the requirements of the Use of Laboratory Animals and Institutional Animal Care and Use Committee at Foshan University (Record number: 2023-643656). Male Sprague Dawley (SD) rats (200 g ± 20 g, 6-8 weeks old) were used for this study. All animal research data have been written up in accordance with the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. The details of the reagents and equipment used in the study are listed in the Tabl…

Representative Results

Due to inflammation and other stimulation caused by the implantation of electrodes, the EEG may be unstable, so the rats need to recover for 3 days. Rats with normal EEG and EMG after 3 days could be included for coma model preparation. When the rats were anesthetized, EEG and EMG activity was slightly suppressed but proceeded smoothly. There was no significant change in EEG and EMG activity after electrocoagulation blocking the bilateral VAs. After about 30 min, the drug was metabolized, the rats gradually regained cons…

Discussion

Four-vessel occlusion induces global ischemic and hypoxic brain injury, which can simulate acute coma, cardiac arrest, asphyxia, shock, severe arrhythmia, and other critical clinical conditions caused by cerebral ischemia in clinical practice. Meanwhile, four-vessel occlusion can lead to damage mainly in the hippocampus¹⁷^,¹⁸, which is the primary functional brain area responsible for cognitive memory¹⁹^,²⁰…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (82173781 and 82373835), Postdoctoral research project (BKS212055), Science and Technology Innovation Project of Foshan Science and Technology Bureau (2320001007331), Guangdong Basic and Applied Basic Research Foundation (2019A1515010806), Key Field Projects (Intelligent Manufacturing) of General Universities in Guangdong Province (2020ZDZX2057), and the Scientific Research Projects (Characteristic Innovation) of General Universities in Guangdong Province (2019KTSCX195).

Materials

16 channel microfiber photoelectrode array	Jiangsu Yige Biotechnology Co., Ltd	2605
4-0 Surgical suture	Nantong Holycon Medical Devices Co.,Ltd.	B-104
6-0 Surgical suture	Ningbo MEDICAL Needle Co., Ltd.	JM1216-742417
EEG electrode	Kedou Brain machine Technology Co., LTD	KD-EEGEMG
Electrocoagulation pen	CONPUVON Company	465
Lunion Stage Automatic Sleep Staging System	Shanghai Lulian Intelligent Technology Co., Ltd.	1336
Miniature hand-held skull drill	Rayward Life Technology Co., Ltd	87001
Penicillin sodium	Chengdu Kelong Chemical Co., Ltd.	17121709-2
SD rats	SPF ( Beijing ) Biotechnology Co.,Ltd.	180-220g
Skull nail	GLOBALEBIO,LTD	/
Stereotaxic instrument	Rayward Life Technology Co., Ltd	68801
Zoletil 50	Vic Trading (Shanghai) Co., LTD	BN 88SHA

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