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 compliance with the Use of Laboratory Animals and Institutional Animal Care and Use Committee guidelines at Chengdu University of Traditional Chinese Medicine (Record number: 2019-DL-002). All animal research data have been documented following the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. Male Sprague Dawley (SD) rats weighing 250 g &#177; 20 g and aged 6-8 weeks were utilized for this study. The specifics regarding the animals, reagents, and equipment employed are listed in the Table of Materials.
1. Animal preparation
<ol>
	<li>Induce and maintain deep anesthesia in rats using Zoletil 50 (50mg/kg, IM) and Xylazine Hydrochloride (40mg/kg, IP).&#160;Ensure the body temperature is maintained at 37 &#177; 0.5 &#176;C using a rectal probe connected to a heating pad throughout the surgical procedure. Apply veterinary ointment to the rat&#39;s eyes to prevent them from drying out.</li>
	<li>Shave the hair from the head and neck of the rats. Use depilatory cream to remove fur from the head and neck, then wash off the cream with normal saline. Disinfect the skin at the surgical site by applying ethanol and povidone-iodine three times using sterile cotton balls.</li>
	<li>Make a 2 cm incision in the middle of the rat&#39;s head along the direction of the sagittal suture using a scalpel, and carefully remove the muscles covering the skull.</li>
	<li>Thin the skull on the ischemic side of the rat using a skull drill. Use normal saline to cool down and remove debris during the grinding process. Record the baseline blood flow of the rats using laser speckle contrast imaging (LSCI).</li>
	<li>Apply ethanol and povidone-iodine three times to the skin of the neck using sterile cotton balls. Make a 2 cm incision along the midline of the neck using a surgical blade. Use retractors to laterally pull back the skin and salivary glands, exposing the sternocleidomastoid and cervical muscles.</li>
	<li>Separate the sternocleidomastoid and cervical muscles to expose the carotid territory. Identify the vascular anatomy of the common carotid artery (CCA), the internal carotid artery (ICA), and the external carotid artery (ECA). Based on vascular anatomy, separate the CCA, as well as the CCA-derived ECA and ICA15. 
	NOTE: The anatomical structure of the rat indicates that the CCA region is mainly covered by sternocleidomastoid and cervical muscles, with the ICA and ECA derived from the CCA. After separation exposes the CCA, a Y-shaped fork can be seen along the CCA, representing the ICA and ECA. Be cautious not to damage the vagus nerve, which runs parallel to the CCA.</li>
</ol>
2. Occlusion of the MCA
<ol>
	<li>Tie an easily untangled knot over the external carotid artery (ECA) and common carotid artery (CCA) using a 3-0 silk thread to temporarily block blood flow. Position a vessel clip on the CCA approximately 0.5 cm from the first knot.
	<ol>
		<li>Dye the needle of a 5 mL syringe black. Create a small puncture in the CCA using the black-dyed needle and mark the pinholes in black.</li>
	</ol>
	</li>
	<li>Insert the monofilament nylon suture into the CCA through the black mark. Open the vascular clip and guide the nylon wire into the Internal Carotid Artery (ICA) until it halts with slight resistance. Tighten the second knot securely to keep the monofilament nylon suture in place inside the artery, preventing displacement from the blocking position.</li>
	<li>Note the time of ischemia at this point and measure the blood flow value on the ischemic side using LSCI. Administer drops of bupivacaine to the neck wound for analgesia and wound closure. Remove the anesthesia mask and allow the rat to recover. 
	NOTE: If the monofilament nylon suture encounters difficulty entering the ICA, retract it slightly and attempt insertion again.</li>
</ol>
3. Reperfusion of MCAO
<ol>
	<li>Following 90 min of ischemia, re-anesthetize the rats with isoflurane. Measure cerebral blood flow from the ischemic side using LSCI and ensure the monofilament nylon suture has not shifted.</li>
	<li>Untie the knots on the external carotid artery (ECA) to allow blood reperfusion. Release the knot on the common carotid artery (CCA), secure the monofilament suture, and withdraw the suture. Apply a vascular clip ahead of the fixed monofilament suture to prevent bleeding.</li>
	<li>Replace the first CCA knot with a vascular clip. Use tweezers to rotate the vessel incision sideways (Figure 1). Clamp the incision with tweezers and ligate it using 6-0 thread to repair the incision. Remove the vascular clip, check for leaks, and confirm complete reperfusion (Video 1). 
	NOTE: Perform ligation to repair the incision to avoid excessive vessel clamping, which could lead to CCA stenosis.</li>
	<li>Record cerebral cortical blood flow values using LSCI after incision ligation to confirm successful reperfusion. Close the neck incision with sutures and administer 1,00,000 units of penicillin and 2 mL of saline to prevent infection and dehydration.</li>
	<li>Maintain warmth until the rats regain consciousness. Offer soft food post-surgery and monitor the animals&#39; vital signs.</li>
</ol>
4. Evaluation of nerve function and cerebral ischemic injury
<ol>
	<li>Once the rats have fully awakened, their neural function will be assessed by researchers unaware of the animal grouping. Utilize the Longa score scale (Table 1) and the modified neurological severity score (mNSS) scale (Table 2) to evaluate the neurological function of all rats.</li>
	<li>After 24 h post-surgery, euthanize the rats under isoflurane-induced anesthesia (4% isoflurane at 4 L/min oxygen) via cervical dislocation16 (following institutionally approved protocols). Rinse the brain with ice water to remove any remaining blood.</li>
	<li>Divide the brain tissue into 5 sections and stain them with TTC. Capture images of both sides of the brain tissue slices, measure the ischemic area of the front and back sides using Image J software, and calculate the infarction rate using the following formula: 
	<img alt="Equation 1" src="/files/ftp_upload/67060/67060eq01.jpg" style="margin: auto;" /> 
	NOTE: S1 and S2 represent the infarct area of the proximal forebrain and proximal brainstem sides of the brain slices, respectively. The infarct area of each brain slice is corrected using Swanson&#39;s formula, where h represents the thickness of the brain slice. This formula, previously outlined by the author in prior research17, allows for a more accurate calculation of the infarct lesion size.</li>
	<li>Analyze the data using statistical and graphing software to assess the model&#39;s success rate and stability.</li>
	<li>Employ the immunofluorescence method to label cortical neurons in rats, further validating the neuronal damage caused by this method. After 24 h of ischemia-reperfusion in rats, cardiac perfusion using pre-cooled PBS was performed to obtain brain tissue.</li>
	<li>Embed and freeze the brain tissue into sections. Use neuronal antibodies such as the immature neuronal marker Doublecortin (DCX)18, neuronal nucleus marker Neuronal Nuclei (NeuN)18, and neuron dendritic marker Microtubule-associated protein-2 (MAP-2)19. Observe the expression of ischemic neurons using laser confocal microscopy.</li>
</ol>

Modified MCAO/R Model with Preserved ECA for Enhanced Reperfusion and Stability

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The middle cerebral artery occlusion (MCAO) model induced by a monofilament nylon suture is the most common method used for preparing MCAO models. This approach is widely adopted in preclinical studies and has gained recognition from many practitioners due to its simplicity, lack of need for craniotomy, minimal surgical trauma, and ability to achieve reperfusion.
There are two classical surgical techniques for intraluminal filament MCAO: the Koizumi method8 and the Longa method9. The main difference between these methods lies in how the suture is introduced to occlude the MCA. In the Koizumi method, the suture is inserted through the common carotid artery (CCA) incision into the internal carotid artery (ICA). The front end of the suture is coated with silicone to block the starting point of the MCA. Conversely, in the Longa method, the suture is inserted into the ICA through the severed external carotid artery (ECA). The front end of the suture is expanded into a spherical shape to block the starting point of the MCA (Figure 5). During reperfusion, the suture is gently withdrawn to restore blood flow.
The Longa method involves introducing the suture into the internal carotid artery (ICA) through the severed external carotid artery (ECA). This process requires the separation and disconnection of the ECA, as well as the separation and removal of small branches, which are then cut off using an electrocoagulation pen to prevent bleeding. Consequently, this method demands careful and precise operation. Moreover, the short residual end of the severed ECA can make it easy for the suture to slip off during insertion, further increasing the complexity of the procedure. This challenge is particularly pronounced when preparing a mouse model of cerebral ischemia, as it often necessitates the use of a microscope to ensure accuracy, thus adding to the overall complexity of the operation.
The primary concern with the Koizumi method lies in the ligation of the common carotid artery (CCA), which results in compensatory reperfusion of brain tissue on the ischemic side solely through the Circle of Willis. The CCA is a crucial branch of the aortic arch from the heart and serves as the main artery supplying cerebral blood flow perfusion. Therefore, the cerebral ischemia-reperfusion model created by the Koizumi method often experiences a state of hypoperfusion. This hypoperfusion is not limited to the middle cerebral artery region but extends to the entire ischemic hemisphere on the same side. Even after 35 days of modeling, the blood flow value does not return to baseline, suggesting that the Koizumi method should be regarded as an ischemic model with chronic hypoperfusion rather than an ischemia-reperfusion model10.
In contrast, the Longa method can achieve a blood flow value of 100% in MCAO/R animals. However, it is crucial to note that achieving complete recanalization does not necessarily mean that the Koizumi method should be abandoned. When considering clinical translation, the Koizumi method may be more suitable for patients with spontaneous slow recanalization after artery occlusion. However, this phenomenon is relatively rare in clinical practice. Only a small percentage of ischemic stroke patients meet the criteria for thrombolysis and receive effective treatment within the treatment time window20,21. Furthermore, some patients do not achieve complete revascularization even after thrombolytic therapy22. Hence, the Koizumi method can be utilized to simulate the conditions of such patients.
On the other hand, the Longa method allows vessels to rapidly recanalize after occlusion, making it more suitable for simulating patients who receive effective thrombolytic therapy or thrombectomy in clinical practice. For simulating patients who have not undergone thrombolytic therapy, the permanent cerebral ischemia model prepared by the Koizumi method may offer a more complete occlusion and a simpler surgical procedure.
The Longa method for inducing middle cerebral artery occlusion/reperfusion (MCAO/R) can lead to more significant reperfusion injury by preserving common carotid artery (CCA)-derived vascular recanalization23,24. However, it&#39;s important to note that the external carotid artery (ECA) needs to be cut off and permanently ligated during the Longa method, which disrupts the complete blood flow structure of the brain and is not consistent with clinical patients. Moreover, the lingual, maxillary, and occipital arteries are branches of the ECA13. Thus, cutting or permanently ligating the ECA can cause eating and drinking disorders, resulting in weight loss25. Previous studies have indicated that the deaths of mice following MCAO modeling are primarily due to inadequate food or water intake rather than cerebral ischemic injury14,26. Additionally, ligation of the ECA can reduce retinal blood flow and induce retinal damage in the form of cell death27.
In addition, the most important indicator for stroke research is infarct volume; however, the volume of infarcts induced by the suture method has been proven to have a significant standard deviation, even following strict surgical and data collection protocols. In previous reports, the infarct volume was associated with the degree of reperfusion28, and the reperfusion of the stroke model prepared by Koizumi&#39;s method depends on the Willis circulation29,30,31. However, it has been proven that the Willis cycle has high anatomical variability, especially in C57Bl/6 mice, where 90% of animals may lack one or two posterior communicating arteries32, which is the key determinant of high variability in ischemic injury33. Therefore, preserving the perfusion of CCA can reduce the variability of infarct volume34. It was reported that the average coefficient of variation in infarct size in stroke trials was 29.5%31, which contributed to the low statistical power of stroke trials. It also poses a huge challenge for potential drug discovery. This undoubtedly increases the cost of development and clinical translation and can lead to researchers missing out on potentially effective drugs. It also increases the number of animals to be included in the statistical analysis of the experiment, which further violates the 3R principle of animal ethics.
Therefore, in this study, modeling methods were modified to ensure the integrity of brain tissue blood flow structure and sufficient reperfusion after cerebral ischemia. The main modifications include two steps: (1) inserting the suture into CCA using a 5 mL syringe needle instead of making a small incision; this step reduces the incision of the CCA and is more conducive to ligating the incision; (2) withdrawing the suture and use finer silk threads to ligate the needle opening of the CCA, without affecting the blood flow perfusion of the CCA. Of course, this also requires careful surgical operation, but is different from the introduction of suture via ECA; this method preserves the integrity of the cerebral blood flow structure while allowing blood flow to be fully reperfused through CCA, making the operating procedure simpler and reducing the variability of infarct volume. Some researchers have used similar research methods to repair incisions with tissue glue, but this has the potential for thrombosis, which will bring uncontrollable effects. And it takes longer to stabilize the incision25.
In conclusion, this study provides a modified approach to the preparation of the cerebral ischemia-reperfusion model that allows for adequate blood perfusion after filament withdrawal and can result in significantly reproducible infarct volumes and neurological damage. All of the above results demonstrate that the modified method is feasible and reproducible and may provide a reference for practitioners to prepare models to facilitate the effective clinical translation of cerebral ischemic drugs.

According to the World Health Organization, stroke has remained the second leading cause of death worldwide for the past decade, with a high incidence rate, high mortality, and high disability rate1,2. As the global population ages, the incidence of stroke is expected to increase in developing countries, potentially becoming the leading cause of premature death and disability in adults. Additionally, there is a trend for strokes to occur at a younger age3. The loss of the labor force after a stroke also places a heavy burden on families and society4. Therefore, the development of safe and effective treatments poses a major challenge in stroke research.
Animal models serve as crucial tools for studying the prevention and treatment of human diseases. The successful translation of stroke treatment strategies relies on the reproducibility and reliability of stroke animal models5,6. The middle cerebral artery (MCA) is a common site for clinical stroke, making the MCAO model the closest model to human ischemic stroke. The MCAO model, prepared using the suture method, has been favored by researchers due to advantages such as no craniotomy and easy control of ischemic time. It has been utilized in over 40% of neuroprotective experiments7. However, despite its numerous advantages, the operational details of this model remain a controversial topic for many researchers.
For the suture-induced middle cerebral artery occlusion (MCAO) model, reperfusion occurs by withdrawing the suture. Currently, two main methods are used for suture insertion: Koizumi's method8 and Longa's method9. In Koizumi's method, the suture enters the internal carotid artery (ICA) mainly through the common carotid artery (CCA) incision, while in Longa's method, it passes through the severed external carotid artery (ECA) into the ICA. During reperfusion, the Koizumi method requires permanently ligating the CCA incision and relies on the circle of Willis for reperfusion10. However, some studies suggest that effective reperfusion cannot be achieved solely through the compensatory supply of the circle of Willis after losing the CCA supply. Moreover, the circle of Willis exhibits high anatomical variability, especially in C57Bl/6 mice, increasing infarction variability and reducing experimental data reliability. Consequently, this method has been increasingly questioned by researchers11.
Longa's method involves inserting a suture through the severed ECA and then permanently ligating the internal carotid artery (ICA) once the suture is withdrawn. This preserves CCA patency, allowing blood perfusion up to 100% of baseline values. However, this method necessitates separating the external carotid artery and small arterial branches, cutting them off, or electrocoagulating them, making the procedure challenging. It also disrupts the brain's complete blood flow structure, which differs from the clinical patient's state12. Importantly, studies indicate that cutting or ligating the ECA can cause ischemic lesions in the muscles controlling chewing and swallowing, affecting animal diet and leading to postoperative animal death and severe sensory and motor damage in rats13,14.
Hence, a modified model preparation method is urgently needed to address these issues. This study introduces a modified MCAO modeling method that repairs the CCA insertion incision and achieves effective reperfusion. The procedure is simple, practical, and feasible, inducing significant neurological damage and replicable infarct lesions and providing valuable guidance for stroke researchers.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Animal anesthesia system</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>R500IE</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal temperature maintainer</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>69020</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3 secondary antibody</td>
			<td>Wuhan Saiweier Biotechnology Co., Ltd</td>
			<td>GB21303</td>
			<td></td>
		</tr>
		<tr>
			<td>DAP1 antibody</td>
			<td>Wuhan Saiweier Biotechnology Co., Ltd</td>
			<td>G1012</td>
			<td></td>
		</tr>
		<tr>
			<td>DCX antibody</td>
			<td>Wuhan Saiweier Biotechnology Co., Ltd</td>
			<td>GB13434</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Beyotime Biotechnology Co., LTD</td>
			<td>C0265</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad Software</td>
			<td>GraphPad Prism 8.0</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td>ImageJ software</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluran</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>R510-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser speckle blood flow imaging system</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>PeriCam PSI NR</td>
			<td></td>
		</tr>
		<tr>
			<td>MAP-2 antibody</td>
			<td>Wuhan Saiweier Biotechnology Co., Ltd</td>
			<td>GB11128</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniature hand-held skull drill</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>87001</td>
			<td></td>
		</tr>
		<tr>
			<td>monofilament suture</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>250-280g</td>
			<td></td>
		</tr>
		<tr>
			<td>NeuN antibody</td>
			<td>Wuhan Saiweier Biotechnology Co., Ltd</td>
			<td>GB11138</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT embedding agent</td>
			<td>BIOSHARP</td>
			<td>BL557A</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin sodium</td>
			<td>Chengdu Kelong Chemical Co., Ltd.</td>
			<td>17121709-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick Antigen Retrieval Solution for Frozen Sections</td>
			<td>Beyotime Biotechnology Co., LTD</td>
			<td>P0090</td>
			<td></td>
		</tr>
		<tr>
			<td>SD rats</td>
			<td>SPF ( Beijing ) Biotechnology Co.,Ltd.</td>
			<td>250-280g</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Beyotime Biotechnology Co., LTD</td>
			<td>ST795</td>
			<td></td>
		</tr>
		<tr>
			<td>TTC</td>
			<td>Chengdu Kelong Chemical Co., Ltd.</td>
			<td>2019030101</td>
			<td></td>
		</tr>
	</tbody>
</table>

a modified model preparation for middle cerebral artery occlusion reperfusion

The middle cerebral artery occlusion reperfusion (MCAO/R) model is crucial for understanding the pathological mechanisms of stroke and for drug development.However, among the commonly used modeling methods, the Koizumi method often faces scrutiny due to its ligation of the common carotid artery (CCA) and its inability to achieve adequate reperfusion. Similarly, the Longa method has been criticized for disconnecting and ligating the external carotid artery (ECA). This study aims to introduce a modified model preparation method that preserves the integrity of the ECA, involves inserting a monofilament nylon suture through the CCA, repairing the ligated CCA incision, and maintaining reperfusion from the CCA. Reperfusion of blood flow was confirmed using laser speckle flow imaging. Evaluation methods such as the Longa scale, Modified Neurological Severity Score, triphenyltetrazolium chloride (TTC) staining, and immunofluorescence labeling of neurons demonstrated that this approach could induce stable ischemic nerve damage. This modified MCAO/R model protocol is simple and stable, providing valuable guidance for practitioners in the field of cerebral ischemia.

Laser speckle flow imaging demonstrated that prior to the occlusion of the monofilament nylon suture, there was abundant blood flow in the middle cerebral artery (MCA) area, and the baseline blood flow values of the rats were recorded. Following the occlusion of the MCA, the blood flow value on the ischemic side of the brain rapidly decreased. Before withdrawing the suture, the blood flow values on the ischemic side were rechecked to confirm whether the suture was occluding the MCA. The results indicated only a slight change in blood flow. Upon withdrawing the suture, the blood flow perfusion swiftly recovered (Figure 2). Video 1 also shows the robust filling pulse of the common carotid artery, suggesting that this method can achieve sufficient blood flow perfusion post-cerebral ischemia.
The results of the neurological function scores (Figure 3) revealed that compared to the sham surgery group, the Longa score of the model group was 1.80 &#177; 0.27 (p &#60; 0.05), and the mNSS score was 7.4 &#177; 0.89 (p &#60; 0.05). These scores indicate that the middle cerebral artery occlusion/reperfusion (MCAO/R) model prepared using this method led to significant neurological damage. Video 2 demonstrates the characteristic behavior of model group rats rotating while crawling, a hallmark of stroke-induced injury, further confirming the validity of this method.
TTC staining demonstrated that the infarct rate in the model group rats was 5.58 &#177; 1.79 (p &#60; 0.05), with the infarct area primarily affecting the striatum and cortex, and the infarct size remained relatively stable. Immunofluorescence staining illustrated the loss of cortical neurons following ischemia-reperfusion (Figure 4). These findings collectively indicate that this method can establish a stable MCAO/R model suitable for evaluating preclinical drug efficacy.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67060/67060fig01v5.jpg" /> 
Figure 1: Schematic diagram of CCA&#39;s connector ligation. The yellow circle denotes the insertion point and ligation site of the wire plug, while the ligation position (yellow arrow) indicates where the common carotid artery (CCA) was ligated with the wire. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67060/67060fig01large.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/67060/67060fig02v5.jpg" /> 
Figure 2: Laser speckle blood flow images at the times of rats on the ischemic side pre-, post-occlusion, and pre, and post-reperfusion. (A) Cerebral blood flow images of rats, where blue areas indicate low blood flow values and red areas indicate high blood flow values. Scale bars: 200 mm. (B) Changes in cerebral ischemia-reperfusion in rats, presented as mean &#177; standard deviation, with a sample size of n = 5. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67060/67060fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67060/67060fig03v5.jpg" /> 
Figure 3: Results of infarct area and neurological function scores in the sham group and model group. (A) TTC staining depicted the infarcted areas of brain tissue in both the sham group and the model group, with white areas indicating infarcted tissue and red areas indicating normal tissue. (B) Quantitative results of the Longa and mNSS scores, as well as the infarction rate, are presented. Statistical significance compared to the sham group is denoted by *p &#60; 0.05, with a sample size of n = 5. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67060/67060fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67060/67060fig04v5.jpg" /> 
Figure 4: Results of cortical neuron injury in sham group and model group. Expression of NeuN (A), DCX (B), and MAP-2 (C) in cortical neurons. Scale bars: 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67060/67060fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67060/67060fig05v5.jpg" /> 
Figure 5: Diagram of MCA occlusion and reperfusion by Koizumi, Longa, and the modified method. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67060/67060fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Motor Tests/strong&#62;</td>
			<td>Points</td>
		</tr>
		<tr>
			<td>Raising rat by the tail</td>
			<td>3</td>
		</tr>
		<tr>
			<td colspan="2">1 Flexion of forelimb</td>
		</tr>
		<tr>
			<td colspan="2">1 Flexion of hindlimb</td>
		</tr>
		<tr>
			<td colspan="2">1 Head moved &#62; 10&#176; to vertical axis within 30 s</td>
		</tr>
		<tr>
			<td>Placing rat on the floor</td>
			<td>3</td>
		</tr>
		<tr>
			<td colspan="2">0 Normal walk</td>
		</tr>
		<tr>
			<td colspan="2">1 Inability to walk straight</td>
		</tr>
		<tr>
			<td colspan="2">2 Circling toward the paretic side</td>
		</tr>
		<tr>
			<td colspan="2">3 Fall down to the paretic side</td>
		</tr>
		<tr>
			<td>Sensory tests</td>
			<td>2</td>
		</tr>
		<tr>
			<td colspan="2">1 Placing test (visual and tactile test)</td>
		</tr>
		<tr>
			<td colspan="2">2 Proprioceptive test (deep sensation, pushing the paw against the table edge to&#160;</td>
		</tr>
		<tr>
			<td colspan="2">stimulate limb muscles)</td>
		</tr>
		<tr>
			<td>Beam balance tests</td>
			<td>6</td>
		</tr>
		<tr>
			<td colspan="2">0 Balances with steady posture</td>
		</tr>
		<tr>
			<td colspan="2">1 Grasps side of beam</td>
		</tr>
		<tr>
			<td colspan="2">2 Hugs the beam and one limb fall down from the beam</td>
		</tr>
		<tr>
			<td colspan="2">3 Hugs the beam and two limbs fall down from the beam, or spins on beam (&#62;60 s)</td>
		</tr>
		<tr>
			<td colspan="2">4 Attempts to balance on the beam but falls off (&#62;40 s)</td>
		</tr>
		<tr>
			<td colspan="2">5 Attempts to balance on the beam but falls off (&#62;20 s)</td>
		</tr>
		<tr>
			<td colspan="2">6 Falls off: No attempt to balance or hang on to the beam (&#60;20 s)</td>
		</tr>
		<tr>
			<td>Reflexes absent and abnormal movements</td>
			<td>4</td>
		</tr>
		<tr>
			<td colspan="2">1 Pinna reflex (head shake when touching the auditory meatus)</td>
		</tr>
		<tr>
			<td colspan="2">1 Corneal reflex (eye blink when lightly touching the cornea with cotton)</td>
		</tr>
		<tr>
			<td colspan="2">1 Startle reflex (motor response to a brief noise from snapping a clipboard paper</td>
		</tr>
		<tr>
			<td colspan="2">1 Seizures, myoclonus, myodystony</td>
		</tr>
		<tr>
			<td>Maximum points</td>
			<td>18</td>
		</tr>
	</tbody>
</table>
Table 1: Modified neurological severity score.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Zea-Longa scores</td>
		</tr>
		<tr>
			<td>Score</td>
			<td>Symptom</td>
		</tr>
		<tr>
			<td>0 points</td>
			<td>No neurological deficits</td>
		</tr>
		<tr>
			<td>1 point</td>
			<td>Mild: unable to completely extend the right forepaw when the tail is lifted</td>
		</tr>
		<tr>
			<td>2 points</td>
			<td>Moderate: circles to right/left when walking</td>
		</tr>
		<tr>
			<td>3 points</td>
			<td>Severe: tumbles to right/left when walking</td>
		</tr>
		<tr>
			<td>4 points</td>
			<td>Unable to walk spontaneously with loss of consciousness</td>
		</tr>
	</tbody>
</table>
Table 2: Zea-Longa scores.
Video 1: Key steps for ligature repair of CCA incision during reperfusion in rats. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67060/video 1.zip" target="_blank">Please click here to download this Video.</a>
Video 2: Behavioral videos of rats undergoing MCAO/R. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67060/video 2.zip" target="_blank">Please click here to download this Video.</a>

Author Spotlight: A Novel Approach to Cerebral Ischemia Modeling &#8211; Enhancing Reperfusion and Simplifying Procedure

This protocol describes the process of preparing for middle cerebral artery occlusion reperfusion via the common carotid artery.

A Modified Model Preparation for Middle Cerebral Artery Occlusion Reperfusion

The middle cerebral artery occlusion reperfusion (MCAO/R) model is crucial for understanding the pathological mechanisms of stroke and for drug ...

a-modified-model-preparation-for-middle-cerebral-artery-occlusion

Nanyang Technological University

Research

JoVE Journal

Medicine

803 Views.  Foshan University. This protocol describes the process of preparing for middle cerebral artery occlusion reperfusion via the common carotid artery.

Video: Author Spotlight: A Novel Approach to Cerebral Ischemia Modeling – Enhancing Reperfusion and Simplifying Procedure

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 ...

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 ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

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.