Cerebral Ischemic Coma Model Induced by Modified Four-Vessel Occlusion

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 limbs of the rats began twitching, 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 diseases^1,2, 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 development^6,7,8.

The purpose of this study is to introduce a model of inducing global ischemic coma through electrocoagulation of bilateral vertebral arteries (VA) and temporary ligation of bilateral common carotid arteries (CCA), 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 CCAs^9,10,11,12. 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 Table of Materials.

1. Implantation of EEG electrodes

1. Inject 0.05mg/kg of atropine subcutaneously 15 min before anesthesia to prevent respiratory obstruction and asphyxia caused by secretions. Administer an intramuscular injection of 20 mg/kg zoletil and 5 mg/kg xylazine to anesthetize the rats¹⁴. Use tweezers to clamp the toes of the rat to confirm deep anesthesia.
2. Remove hair from the rat's head with a hair shaver. Fix the rat's head on a brain stereotaxic device using non-penetrating ear bars. Use sterile cotton balls to apply ethanol and povidone iodine three times to the surgical site to disinfect the skin.
3. Cut the skin of the rat's head along the decapitated suture with a surgical blade. Remove the muscle covering the skull and completely expose the skull. Use sterile cotton swabs to stop bleeding throughout the process.
4. Blow dry the surface of the skull with an ear wash ball to help dental cement adhere tightly to the skull. Mark the installation position of the skull nail (Diameter 1.2 mm, Length 3 mm) with a black marker (Figure 1, step 1). The specific positions are the anterior fontanelle point and four other sites.
5. Use the needle of a 10 mL syringe to rotate and drill through four areas in sequence. Insert four skull nails into the skull in sequence, ensuring contact with the cerebral cortex (Figure 1, steps 2-3).
   NOTE: Use sterile cotton swabs to absorb blood in case of bleeding to prevent rusting of the bone nails.
6. Wrap the silver wire of the EEG electrode around the skull nail. Embed the electromyographic electrode into the muscle and fix it with a 6-0 suture.
7. Mix the denture base resin with self-setting denture powder and fix the electrode to the skull. Use an ear wash ball to blow air onto the surface of the dental cement to accelerate curing.
8. Inject 10,000 units of penicillin to prevent infection. House each rat in a separate cage to prevent mutual tearing and damage to the electrodes. Subcutaneously injected 0.2 mg/kg meloxicam for three consecutive days to alleviate postoperative pain. Allow 3 days for recovery of rat wounds and electrode fixation (Figure 1, step 4).

2. Surgical process of cerebral ischemic coma model

1. Three days later, re-anesthetize the rats and place them in a supine position. Use sterile cotton balls to disinfect the surgical site via three repeated applications of iodine followed by an alcohol rinse/wipe. Make an incision about 2-3 cm long with a scalpel from the upper margin of the sternum lengthwise along the middle of the neck (Figure 1, step 5).
2. Bluntly separate the subcutaneous tissue and sternohyoid muscle, fully exposing the trachea and the longus colli muscles on both sides of the trachea¹⁵.
   NOTE: Avoid stimulating the trachea throughout the entire procedure.
3. Bluntly separate the longus colli muscles from the level of the thyroid gland downward, exposing the first and second cervical vertebrae. Expand the neck area with a rat tissue dilator, fully exposing the surgical site.
4. Use fine forceps to carefully separate the muscles and tissues visible in the cervical intervertebral space, exposing the characteristic location where the vertebral artery enters the first cervical vertebra. It can be observed that the vertebral artery passes through the first cervical vertebra (Figure 1, step 6).
   NOTE: Placing a 1 mL syringe under the neck provides a clearer surgical manipulation space.
5. Preheat the electrocoagulation pen and insert it into the area for 3-5 s to ensure that the vertebral artery is electrocoagulated and severed. Separate the muscles and fascia along the inner edge of the sternocleidomastoid muscle, expose and free the bilateral CCAs, and tie a loose knot.
   NOTE: The electrocoagulation pen must be preheated; otherwise, it cannot quickly coagulate the vertebral artery, leading to bleeding. Both vertebral arteries are electrocoagulated and loose knots are tied around the CCAs.
6. Quickly tighten the first loose knot to block blood flow in the CCA when the rats regain consciousness and exhibit limb twitching, while the EEG detector detects active EEG and EMG signals. The rats will struggle for a few seconds and then gradually lose consciousness (Figure 1, step 7).
7. After releasing the fixation, observe that the limbs of the rat are stiff, the righting reflex disappears, but breathing is maintained. At this point, the electromyogram (EMG) presents a straight line, and the EEG is rapidly suppressed, indicating that the 4-VO induced cerebral ischemia model was successful¹⁶.
   NOTE: Respiratory depression occurs in some rats during bilateral CCA ligation. Rapid mechanical stimulation can restore spontaneous respiration in some rats.
8. According to the "needle control tie method¹⁶," bind the CCA with a 0.5 mm diameter syringe needle using 6-0 nylon thread about 1.5 cm away from the bifurcation of the CCA, ICA, and ECA. Carefully pull out the needle; this second knot will subsequently cause the carotid artery to narrow (Figure 1, step 8).
   NOTE: The ligature used for CCA stenosis needs to be made of nylon material, which is stable. Nylon thread is not affected by blood and does not thicken; otherwise, it can cause extreme stenosis of the CCA in rats and increase the mortality rate.
9. After 30 min of ischemia, untie the first knot, and the CCA will undergo reperfusion, but the second knot will result in CCA stenosis, inducing a sustained coma (Figure 1, step 9). Close the subcutaneous tissue with absorbable monofilament sutures and the skin with non-absorbable monofilament sutures.

3. Animal recovery

1. Place the rats on insulation pads and inject 10,000 units of penicillin to prevent infection. Subcutaneously inject 0.2 mg/kg meloxicam for three consecutive days to alleviate postoperative pain.
2. After 60 min of reperfusion, ensure the rats gradually recover.

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 hippocampus^17,18, which is the primary functional brain area responsible for cognitive memory^19,20...

Materials

Name	Company	Catalog Number	Comments
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