Author Spotlight: Enhancing Cerebral Ischemia Research with a Simplified Rat Model

Rong Ma; Rui Li; Jianlong Liang; Hongyan Yang; Qian Xie; Xuxin Zeng; Jialiang Guo

doi:10.3791/67161

Medicine

Cerebral Ischemic Coma Model Induced by Modified Four-Vessel Occlusion

Published: July 5, 2024 doi: 10.3791/67161

Rong Ma*^1,2, Rui Li*², Jianlong Liang², Hongyan Yang², Qian Xie², Xuxin Zeng², Jialiang Guo²

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

* These authors contributed equally

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.

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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

Inject 0.1 mL 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.
Remove hair from the rat's head with a hair shaver. Fix the rat's head on a brain stereotaxic device. Use sterile cotton balls to apply ethanol and povidone iodine three times to the surgical site to disinfect the skin.
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.
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.
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.
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.
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.
Inject 100,000 units of penicillin to prevent infection. House each rat in a separate cage to prevent mutual tearing and damage to the electrodes. Allow 3 days for recovery of rat wounds and electrode fixation (Figure 1, step 4).

2. Surgical process of cerebral ischemic coma model

Three days later, re-anesthetize the rats and place them in a supine position. Use sterile cotton balls to apply iodine and disinfect the surgical site three times. 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).
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.
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.
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.
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.
Quickly tighten the first loose knot to block blood flow in the CCA when the rats regain consciousness and exhibit a righting response. The rats will struggle for a few seconds and then gradually lose consciousness (Figure 1, step 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.
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.
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). Stitch up the incision using 4-0 suture.

3. Animal recovery

Place the rats on insulation pads and inject 100,000 units of penicillin to prevent infection.
After 60 min of reperfusion, ensure the rats gradually recover.

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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 consciousness, and EEG and EMG activity increased. When the rat was about to complete the righting reaction, the CCA was quickly ligated. At this moment, all four blood vessels responsible for the blood supply to the brain were blocked, resulting in global cerebral ischemia. The EEG and EMG activities were rapidly inhibited, almost forming a straight line, and the rat no longer struggled, and the eyeballs appeared gray and white (Figure 2A). If the rats cannot be induced into a coma, it means that the model has failed.

After 30 min, the first ligation thread of the CCAs was released for reperfusion, but the second ligation thread placed in front of the CCAs kept the perfusion level low, inducing continuous coma in the rats for about 60 min. With the gradual compensation and blood perfusion of the body, the rats gradually woke up (Figure 2B). The EEG and EMG of the rats recovered almost completely after waking up.

Figure 1: Position diagram of the electrocoagulation vertebral artery. Steps 1-3 outline the protocol for implanting the electrodes, and step 4 tests whether the electrodes are functioning properly. Steps 5-6 detail the surgical incision procedure at the front of the neck and bilateral vertebral arteries (VAs) for electrocoagulation. Yellow arrows and arcs indicate the key sites for electrocoagulation. Silk threads are used to simulate the vertebral artery's passage through the spine, providing a clearer indication of the electrocoagulated vertebral artery's characteristic location. Steps 7-9 describe the protocols for ligation of the bilateral common carotid arteries (CCAs) to induce coma. All procedures adhere to the Animal Use guidelines. Please click here to view a larger version of this figure.

Figure 2: Electroencephalogram (EEG) and electromyogram (EMG) changes from coma to recovery. (A) Waveform of EEG, EMG, and activity levels in rats during cerebral ischemic coma. (B) Diagram of EEG, EMG, and activity levels in rats transitioning from coma to awakening. The entire process lasts approximately 90 min. Please click here to view a larger version of this figure.

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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^,²¹. Therefore, four-vessel occlusion can also be used to simulate a vascular dementia model²²^,²³. In summary, the four-vessel occlusion-induced global cerebral ischemia model has been widely used to simulate the aforementioned clinical diseases.

In 1979, Pulsinelli et al.¹⁰ achieved global cerebral ischemia and reperfusion by blocking the bilateral common carotid arteries and bilateral vertebral arteries. Since then, the model has gradually become an internationally recognized classic preparation method for global cerebral ischemia models²⁴^,²⁵^,²⁶. However, this method has some problems, such as individual differences in the transverse process and foramen of the first cervical vertebra, including small, irregular, and curved foramina, resulting in incomplete electrocoagulation. Additionally, long-term electrocoagulation or excessive electric current may pose a risk of damaging the brainstem and spinal cord. Insufficient electrocoagulation of the vertebral artery can lead to significant bleeding and other issues¹⁵^,¹⁶^,²⁷.

Therefore, scholars have continually made improvements to ensure the integrity of electrocoagulation of the vertebral artery and reduce the variability of the model²⁸^,²⁹. Due to the invisibility of electrocoagulating the vertebral artery through the vertebral foramen, Todd et al.³⁰ fully exposed the vertebral artery and burned it under direct vision by drilling through the atlas foramen, thereby increasing the accuracy of electrocoagulation. However, this surgical method is highly invasive and can damage the cervical spine. The thin wall of the pterygoid foramen can easily damage the vertebral artery, causing massive bleeding and resulting in a relatively high mortality rate. Sugio et al.¹² visualized the vertebral artery and permanently severed it by inserting an electric needle into the left and right intervertebral foramina of the second cervical vertebra. Toda et al.³¹ and Lu et al.²⁷ exposed the vertebral artery between the first and second transverse processes outside the atlas joint based on this anatomical structure and performed electrocoagulation. These researchers solved the problem of instability in electrocoagulation when the vertebral artery was not visible by exposing it behind the neck and performing electrocoagulation.

However, rats undergoing surgery on both the abdomen and back still had an increased risk of infection, so some scholars have attempted to operate from the front of the neck. Sun Wei et al.¹⁵ achieved global cerebral ischemia-reperfusion by separating the vertebral artery from the anterior neck, ligating or clipping the small arteries, and continuously or intermittently clipping the CCA. This step requires meticulous operation, as the vertebral artery is difficult to separate due to its subtle nature. Additionally, small arterial clamps are difficult to accurately clamp the vertebral artery. If the separation is not clear and clean enough, it will lead to incomplete clamping or ligation, which undoubtedly increases the instability of the model.

This study provides a simple and feasible method for vertebral artery electrocoagulation. The key step is not to separate the vertebral artery but to locate the transverse foramen where the vertebral artery crosses and enters the first cervical vertebra. This is the entrance to the vertebral artery, which has a curved feature, allowing novices to quickly locate it. After preheating the electrocoagulation pen, insert it into the curved position to quickly coagulate the vertebral artery, lasting for 3-5 s to ensure complete occlusion. Both vertebral artery electrocoagulation and CCA ligation were performed in one operation, reducing the risk of infection and the duration of the experiment.

We also found that the death of the model animal was mainly caused by the blockage of all four blood vessels responsible for cerebral blood supply and was not related to the number of surgical operations. However, completing all operations in one surgery did indeed shorten the experimental period. Additionally, the modified method can also be used to prepare the global cerebral ischemia-reperfusion model. The four-vessel occlusion-induced cerebral ischemia coma model can also simulate the "locked-in syndrome" model in traditional Chinese medicine³². Therefore, the model introduced in this study can also provide a reference for practitioners of traditional Chinese medicine.

In previous literature¹⁶^,³³^,³⁴, CCA stenosis ligation was performed 30 min after ischemia. However, after 30 min of ischemia, some rats showed slight consciousness due to compensation. Mechanical stimulation could cause these rats to wake up, leading to failure of the coma model. Therefore, in our study, CCA stenosis treatment was carried out immediately after the onset of coma. At this time, the coma depth was profound, making it difficult to stimulate the rats to wake up, ensuring the stability of the model. The CCA was in a clamped state during this treatment, and the stenosis treatment would not affect blood flow.

Additionally, in the neck incision of rats, it was observed that the vertebral arteries in three spinal intervals could be blocked by electrocautery or ligation. However, there is a characteristic sign of vertebral artery entry in the first spine, making it very suitable for electrocautery blockade. Due to the lack of characteristic markers between the second and third vertebrae, as well as between the third and fourth vertebrae, suture ligation is more suitable. However, there is a drawback of insufficient ligation. Therefore, in this study, electrocautery was chosen to cut off the vertebral artery between the first and second vertebrae.

Additionally, it should be noted that the success of this model is closely related to the weight of the rats. It has been reported that rats weighing between 180-200 g are more suitable for inducing coma. In this study, it was also found that rats weighing less than 180 g can hardly withstand the damage of four blood vessels simultaneously, leading to a high mortality rate. Conversely, if the weight is greater than 220 g, the rat will wake up quickly and not remain in coma long enough to meet the model standard.

Moreover, the diameter of the pinhole used for narrowing the CCA is another factor affecting the duration of coma in rats. Studies have shown that a pinhole diameter of 0.6 mm can induce continuous coma in rats for 6-8 h with a high survival rate. A pinhole diameter of 0.45 mm caused extreme arterial stenosis in rats, leading to failure of severely injured rats to recover, while a diameter of 0.7 mm resulted in coma lasting 1-3 h, but with significant individual differences¹⁶.

However, in the preliminary experiment, a pinhole diameter of 0.6 mm could not induce coma for more than 60 min, while a diameter of 0.5 mm could induce coma for 1-2 h. This differs from the study by Xiaobing Jia et al., which may be due to differences in anesthetics and modeling methods. Further study is needed in the future to elucidate these differences.

In conclusion, this study presents a simple, feasible, and beginner-friendly method for four-vessel occlusion. It serves as a valuable reference for practitioners studying cerebral ischemic coma or global cerebral ischemia.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

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

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

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