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).

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 &#177; 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
<ol>
	<li>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.</li>
	<li>Remove hair from the rat&#39;s head with a hair shaver. Fix the rat&#39;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.</li>
	<li>Cut the skin of the rat&#39;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
</ol>
2. Surgical process of cerebral ischemic coma model
<ol>
	<li>Three days later, re-anesthetize the rats and place them in a supine position. Use sterile cotton balls to&#160;disinfect the surgical site via three repeated applications of iodine followed by an&#160;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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>According to the &#34;needle control tie method16,&#34; 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.</li>
	<li>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.</li>
</ol>
3. Animal recovery
<ol>
	<li>Place the rats on insulation pads and inject 10,000 units of penicillin to prevent infection.&#160;Subcutaneously inject&#160;0.2 mg/kg meloxicam for three consecutive days to alleviate postoperative pain.</li>
	<li>After 60 min of reperfusion, ensure the rats gradually recover.</li>
</ol>

Single-Step Surgery for Inducing Cerebral Ischemic Coma in Rats

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The authors have nothing to disclose.

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 &#34;locked-in syndrome&#34; 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.

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 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 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16 channel microfiber photoelectrode array</td>
			<td>Jiangsu Yige Biotechnology Co., Ltd</td>
			<td>2605</td>
			<td></td>
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			<td>4-0 Surgical suture</td>
			<td>Nantong Holycon Medical Devices Co.,Ltd.</td>
			<td>B-104</td>
			<td></td>
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			<td>6-0 Surgical suture</td>
			<td>Ningbo MEDICAL Needle Co., Ltd.</td>
			<td>JM1216-742417</td>
			<td></td>
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			<td>EEG electrode</td>
			<td>Kedou Brain machine Technology Co., LTD</td>
			<td>KD-EEGEMG</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrocoagulation pen</td>
			<td>CONPUVON Company</td>
			<td>465</td>
			<td></td>
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			<td>Lunion Stage Automatic Sleep Staging System</td>
			<td>Shanghai Lulian Intelligent Technology Co., Ltd.</td>
			<td>1336</td>
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			<td>Miniature hand-held skull drill</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>87001</td>
			<td></td>
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			<td>Penicillin sodium</td>
			<td>Chengdu Kelong Chemical Co., Ltd.</td>
			<td>17121709-2</td>
			<td></td>
		</tr>
		<tr>
			<td>SD rats</td>
			<td>SPF ( Beijing ) Biotechnology Co.,Ltd.</td>
			<td>180-220g</td>
			<td></td>
		</tr>
		<tr>
			<td>Skull nail</td>
			<td>GLOBALEBIO,LTD</td>
			<td>/</td>
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			<td>Stereotaxic instrument</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>68801</td>
			<td></td>
		</tr>
		<tr>
			<td>Zoletil 50</td>
			<td>Vic Trading (Shanghai) Co., LTD</td>
			<td>BN 88SHA</td>
			<td></td>
		</tr>
	</tbody>
</table>

cerebral ischemic coma model induced by modified four-vessel occlusion

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.

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 rats exhibited limb twitching, while EEG and EMG signals were active and no longer in a straight line, 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67161/67161fig01.jpg" /> 
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&#39;s passage through the spine, providing a clearer indication of the electrocoagulated vertebral artery&#39;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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67161/67161fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67161/67161fig02.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67161/67161fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

Cerebral Ischemic Coma Model Induced by Modified Four-Vessel Occlusion

Coma caused by cerebral ischemia is the most serious complication of cerebral ischemia. Four-vessel occlusion can establish a cerebral ischemic coma model ...

cerebral-ischemic-coma-model-induced-by-modified-four-vessel-occlusion

Nanyang Technological University

Research

JoVE Journal

Medicine

410 Views.  South China University of Technology. This protocol describes the process for inducing a cerebral ischemic coma model using a modified four-vessel occlusion method.

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

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history have&#160;remained poorly understood. Recently, we developed a reliable, reproducible animal model of PION and tested the treatment effect of some neurotrophic factors in this model1. The purpose of this video is to demonstrate our photochemically induced model of posterior ischemic optic neuropathy, and to evaluate its effects with retrograde labeling of retinal ganglion cells. Following surgical exposure of the posterior optic nerve, a photosensitizing dye, erythrosin B, is intravenously injected and a laser beam is focused onto the optic nerve surface. Photochemical interaction of erythrosin B and the laser during irradiation damages the vascular endothelium, prompting microvascular occlusion mediated by platelet thrombosis and edematous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback, owing to a loss of axonal input - a remote, injury-induced and clinically relevant outcome. Thus, this model provides a novel platform to study the pathophysiologic course of PION,&#160;and can be further optimized for testing therapeutic approaches for optic neuropathies as well as other CNS ischemic diseases.

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.