This study was supported by the Grants from the Nature Science Foundation of Hubei Province (2022CFC057).

The experimental protocol was approved by the Institutional Animal Care and Use Committee of Jianghan University and was conducted in accordance with Experimental Animals Ethical Guidelines issued by the Center for Disease Control of China. Adult male C57BL/6J mice, 10 weeks old, weighted 24-26 g, were used in this protocol. All mice were housed under a 12-h light/dark cycle controlled environment with food and water ad libitum.
1. Preoperative preparation
<p class=...

Occlusion of Distal Middle Artery to Develop an Ischemic Stroke Mouse Model

<ol>
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The authors have no conflicting interests to disclose.

In the present protocol of the craniotomy electrocoagulation dMCAO model, the surgical procedures are conducted with minimal invasiveness, wherein only a portion of the temporalis muscle is separated to mitigate the adverse effects on masticatory function. The mice all recovered well after the procedure, with no observed instances of feeding difficulties. The MCA can be easily discerned in the temporal bone of the mouse, thereby facilitating precise identification of suitable craniotomy locations. This dMCAO model-induce...

Stroke is a common acute cerebrovascular disease characterized by high incidences of disability and fatality1. Of all stroke cases, nearly 80% belong to ischemic stroke2. Up to now, intravenous thrombolysis remains one of a limited number of productive approaches for the treatment of acute ischemic stroke. However, the effectiveness of thrombolytic treatment is restricted by the narrow effective time window and the occurrence of hemorrhagic transformation3. In the long-term rehabilitation phase following an ischemic stroke, a considerable number of patients are likely to experience durable neurological dysfunctions4. Further investigation is urgently required to unravel the underlying pathophysiological mechanisms of ischemic stroke, as well as to facilitate the development of novel therapeutic strategies targeting ischemic stroke. The establishment of a dependable and replicable model of ischemic stroke is crucial for basic research as well as subsequent translational research in the field of ischemic stroke.
In 1981, Tamura et al. developed a focal cerebral ischemia model by employing transcranial electrocoagulation at the proximal site of the middle cerebral artery (MCA)5. Since then, numerous researchers have utilized various methodologies such as ligation, compression, or clipping to induce distal MCA occlusion (dMCAO) for establishing transient or permanent ischemic stroke models6,7,8. Compared to the filament model, the dMCAO model exhibits notable advantages such as smaller infarct size and higher survival rate, rendering it more suitable for investigating long-term functional recovery subsequent to ischemic stroke9. In addition, the dMCAO model demonstrates a higher survival rate in aged rodents compared to the filament model, making it an advantageous tool for investigating ischemic stroke in elderly and comorbid animal models10. The photothrombotic (PT) stroke model has been demonstrated to possess the characteristics of less surgical invasiveness and a significantly low mortality rate. However, the PT model exhibits a greater degree of cellular necrosis and tissue edema compared to the dMCAO model, leading to the absence of collateral circulation11. Furthermore, it is noteworthy that the ischemic lesions observed in the PT model predominantly arise from microvascular occlusion, which differs substantially from the cerebral ischemia induced by large vessel embolism in the dMCAO model12.
In this paper, we present the methodology for inducing the murine dMCAO model by coagulating the distal MCA via small bone window craniotomy. Additionally, we conducted histological examinations and behavioral evaluations to comprehensively characterize the ischemic insults and stroke outcomes in this experimental model. We aim to acquaint researchers with this model and facilitate further investigations into the pathologic mechanisms of ischemic stroke.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2,3,5-Triphenyltetrazolium 
			Chloride (TTC)</td>
			<td>Sigma-Aldrich</td>
			<td>108380</td>
			<td>Dye for TTC staining</td>
		</tr>
		<tr>
			<td>24-well culture plate</td>
			<td>Corning (USA)</td>
			<td>CLS3527</td>
			<td>Vessel for TTC staining</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Wuhan Servicebio Technology 
			Co., Ltd.</td>
			<td>G1101</td>
			<td>Tissue fixation</td>
		</tr>
		<tr>
			<td>5% bovine serum albumin</td>
			<td>Wuhan BOSTER Bio Co., Ltd.</td>
			<td>AR004</td>
			<td>Non-specific antigen blocking</td>
		</tr>
		<tr>
			<td>5-0 Polyglycolic acid suture</td>
			<td>Jinhuan Medical Co., Ltd</td>
			<td>KCR531</td>
			<td>Material for surgery</td>
		</tr>
		<tr>
			<td>Anesthesia machine</td>
			<td>Midmark Corporation</td>
			<td>VMR</td>
			<td>Anesthetized animal</td>
		</tr>
		<tr>
			<td>Antifade mounting medium</td>
			<td>Beyotime Biotech</td>
			<td>P0131</td>
			<td>Seal for IF staining</td>
		</tr>
		<tr>
			<td>Automation-tissue-dehydrating&#160; 
			machine</td>
			<td>Leica Biosystems (Germany)</td>
			<td>TP1020</td>
			<td>Dehydrate tissue</td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet (France)</td>
			<td>20220328</td>
			<td>Material for surgery</td>
		</tr>
		<tr>
			<td>Diclofenac&#160;sodium&#160;gel</td>
			<td>Wuhan Ma Yinglong Pharmaceutical 
			&#160;Co., Ltd.</td>
			<td>H10950214</td>
			<td>Analgesia for animal</td>
		</tr>
		<tr>
			<td>Drill tip (0.8 mm)</td>
			<td>Rwd Life Science Co., Ltd.</td>
			<td></td>
			<td>Equipment for surgery</td>
		</tr>
		<tr>
			<td>Eosin staining solution</td>
			<td>Wuhan Servicebio Technology 
			Co., Ltd.</td>
			<td>G1001</td>
			<td>Dye for H&#38;E staining</td>
		</tr>
		<tr>
			<td>Eye ointment</td>
			<td>Guangzhou Pharmaceutical Co., Ltd</td>
			<td>H44023098</td>
			<td>Material for surgery</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Olympus (Japan)</td>
			<td>BX51</td>
			<td>Image acquisition</td>
		</tr>
		<tr>
			<td>GFAP Mouse monoclonal antibody</td>
			<td>Cell Signaling Technology Inc. 
			(Danvers, MA, USA)</td>
			<td>3670</td>
			<td>Primary antibody for IF staining</td>
		</tr>
		<tr>
			<td>Goat anti-mouse Alexa 
			488-conjugated IgG</td>
			<td>Cell Signaling Technology Inc. 
			(Danvers, MA, USA)</td>
			<td>4408</td>
			<td>Second antibody for IF staining</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa 
			594-conjugated IgG</td>
			<td>Cell Signaling Technology Inc. 
			(Danvers, MA, USA)</td>
			<td>8889</td>
			<td>Second antibody for IF staining</td>
		</tr>
		<tr>
			<td>Grip strength meter</td>
			<td>Shanghai Xinruan Information Technology Co., Ltd.</td>
			<td>XR501</td>
			<td>Equipment for behavioral test</td>
		</tr>
		<tr>
			<td>Hematoxylin staining solution</td>
			<td>Wuhan Servicebio Technology 
			Co., Ltd.</td>
			<td>G1004</td>
			<td>Dye for H&#38;E staining</td>
		</tr>
		<tr>
			<td>Iba1 Rabbit monoclonal antibody</td>
			<td>Abcam</td>
			<td>ab178846</td>
			<td>Primary antibody for IF staining</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Rwd Life Science Co., Ltd.</td>
			<td>R510-22-10</td>
			<td>Anesthetized animal</td>
		</tr>
		<tr>
			<td>Laser doppler blood flow meter</td>
			<td>Moor Instruments (UK)</td>
			<td>moorVMS</td>
			<td>Blood flow monitoring</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Boehringer-Ingelheim</td>
			<td>J20160020</td>
			<td>Analgesia for animal</td>
		</tr>
		<tr>
			<td>Microdrill</td>
			<td>Rwd Life Science Co., Ltd.</td>
			<td>78001</td>
			<td>Equipment for surgery</td>
		</tr>
		<tr>
			<td>Microsurgical instruments set</td>
			<td>Rwd Life Science Co., Ltd.</td>
			<td>SP0009-R</td>
			<td>Equipment for surgery</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Thermo Fisher Scientific (USA)</td>
			<td>HM325</td>
			<td>Tissue section production</td>
		</tr>
		<tr>
			<td>Microtome blade</td>
			<td>Leica Biosystems (Germany)</td>
			<td>819</td>
			<td>Tissue section production</td>
		</tr>
		<tr>
			<td>Monopolar electrocoagulation generator</td>
			<td>Spring Scenery Medical Instrument 
			Co., Ltd.</td>
			<td>CZ0001</td>
			<td>Equipment for surgery</td>
		</tr>
		<tr>
			<td>Mupirocin ointment</td>
			<td>Tianjin Smith Kline &#38; French 
			Laboratories Ltd.</td>
			<td>H10930064</td>
			<td>Anti-infection for animal</td>
		</tr>
		<tr>
			<td>NeuN Rabbit monoclonal antibody</td>
			<td>Cell Signaling Technology Inc. 
			(Danvers, MA, USA)</td>
			<td>24307</td>
			<td>Primary antibody for IF staining</td>
		</tr>
		<tr>
			<td>Neutral balsam</td>
			<td>Absin Bioscience</td>
			<td>abs9177</td>
			<td>Seal for H&#38;E staining</td>
		</tr>
		<tr>
			<td>Paraffin embedding center</td>
			<td>Thermo Fisher Scientific (USA)</td>
			<td>EC 350</td>
			<td>Produce paraffin blocks</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Sigma-Aldrich</td>
			<td>P3761</td>
			<td>Euthanized animal</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Shanghai Beyotime Biotech Co., Ltd</td>
			<td>C0221A</td>
			<td>Rinsing for tissue section</td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Shenzhen Codos Electrical Appliances 
			Co.,Ltd.</td>
			<td>CP-9200</td>
			<td>Equipment for surgery</td>
		</tr>
		<tr>
			<td>Sodium citrate solution</td>
			<td>Shanghai Beyotime Biotech Co., Ltd.</td>
			<td>P0083</td>
			<td>Antigen retrieval for IF staining</td>
		</tr>
	</tbody>
</table>

induction of acute ischemic stroke in mice using the distal middle artery occlusion technique

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic stroke patients are eligible to receive intravascular thrombolysis or mechanical thrombectomy therapy within the optimal time window. Among those stroke survivors, around two-thirds suffer neurological dysfunctions over an extended period. Establishing a stable and repeatable experimental ischemic stroke model is extremely significant for further investigating the pathophysiological mechanisms and developing effective therapeutic strategies for ischemic stroke. The middle cerebral artery (MCA) represents the predominant location of ischemic stroke in humans, with the MCA occlusion serving as the frequently employed model of focal cerebral ischemia. In this protocol, we describe the methodology of establishing the distal MCA occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6 mice. Since the occlusion site is located at the cortical branch of MCA, this model generates a moderate infarcted lesion restricted to the cortex. Neurological behavioral and histopathological characterization have demonstrated visible motor dysfunction, neuron degeneration, and pronounced activation of microglia and astrocytes in this model. Thus, this dMCAO mouse model provides a valuable tool for investigating the ischemiastroke and worth of popularization.

Author Spotlight: Establishing a Reliable Distal MCA Occlusion Model in Mice for Stroke Research

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique

In the present research, we aim to provide a comprehensive and detailed protocol for establish middle cerebral artery occlusion model in C57BL/6J mice using transcranial electrocoagulation and we evaluated the subsequent neurological behavior and histopathological features. The middle cerebral artery or MCA is recognized as the primary site of ischemic stroke in humans. The MCA occlusion methods, including technique, photochemical introduction, and occlusion have been extensively employed to induce focal cerebral ischemia in rodents.Compared to other ischemia stroke models, this model has the advantage of less surgical invasiveness, smaller infarct size, and higher survival rate, rendering it more suitable for investigation the long-term functional recovery after ischemic stroke. Overall, the current approach effectively generates a reliable experimental ischemic stroke model that exhibits high survivability and excellent repeatability. Consequently, this methodology serves as an invaluable tool for both foundation and translational research in the field of stroke.In the future, we will employ behavior paradigms such as novel object recognition and water maze to investigate the dynamic alterations in long-term learning and memory capabilities among distal MCA-occluded mice. This will enable us to ascertain the viability of utilizing them as animal model for cognition-impairment studies post-stroke.

The key instruments used to perform the dMCAO are the microsurgical instruments set, the isoflurane vaporizer, and the monopolar microsurgical electrocoagulation generator shown in Figure 1. The experimental procedure of this study is illustrated in Figure 2.&#160;In brief, a small bone window craniotomy was employed to expose the distal MCA, which was subsequently coagulated to induce permanent focal cerebral ischemia in C57BL/6 mice. Furthermore, the ischemic ...

Watch this Scientific Journal Video about Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique at JoVE.com

Here, we present a protocol to establish a distal middle cerebral artery occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6J mice and evaluate the subsequent neurological behavior and histopathological features.

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic ...

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Research

JoVE Journal

Neuroscience

Distal Middle Artery Occlusion Procedure to Induce Ischemic Stroke in Mice

To begin, wipe the heating surgery board with 75%ethanol and set the temperature to 37 degrees Celsius. Place the anesthetized mouse in a right lateral position on the board with the head positioned away. To protect the cornea from drying, apply eye ointment.After shaving the fur from the left orbit to the left ear canal, use a depilatory cream to remove the remaining fur. Disinfect the surgical area three times with povidone iodine and 75%ethanol. Perform a toe-pinch assessment.After making a vertical incision between the left orbit and the left ear canal, retract the soft skin tissue to expose the temporal muscle. Using straight micro forceps, separate the apical and dorsal segments of the temporal muscle from the skull. Identify the Y-shaped bifurcation of the middle cerebral artery, or MCA, beneath the temporal bone.Then using an electric cranial drill, thin out the skull to make a bone window until the dura mater becomes visible. Remove the dura mater above the MCA with curved micro forceps. Using electric coagulation forceps, coagulate the artery at the site's proximal and distal to the bifurcation.Monitor the blood flow of the left MCA cortical branch in a laser speckle blood-flow meter. Then suture the muscle and the skin separately with polyglycolic acid sutures, and apply diclofenac sodium gel and mupirocin ointment to the skin incision. Place the mouse in a recovery chamber.

Neurological Behavioral Tests in Distal Middle Artery Occluded Mice to Study Ischemic Stroke Outcomes

To perform the grip strength test in the previously developed distal MCAO model, grasp the posterior section of a mouse tail and gradually lower the mouse until it holds the horizontal bar with both forepaws. Keeping the body horizontal, pull the mouse backward at a constant speed of two centimeters per second. When the mouse releases its forepaws from the bar, record the peak force in grams.For the pole test, place the mouse vertically on the top of a wooden pole. Record the time taken by the mouse to turn around and the total time to climb down the pole with a 60-second cutoff time. To conduct the adhesive test, attach a patch of adhesive tape to the right forepaw of the mouse.Put the mouse back into the rearing cage and record the time taken by the mouse to remove the adhesive with a 60-second cutoff time. To conduct a cylinder test, wipe an open top clear glass cylinder with 75%ethanol, and place the mouse in it. Using a camera, record its spontaneous standing exploratory behavior for three minutes.The distal MCAO mice showed a significant reduction in grip strength and contralateral forepaw usage rate as compared to the sham operated group. Further, the occluded mice showed a significant increase in the descent time in the pole test, and in the time taken to remove the adhesive.

Mouse Brain Histology to Study the Pathological Mechanisms of Ischemic Stroke Induced by Distal Middle Artery Occlusion

To begin, obtain the dissected brain of the euthanized distal MCAO mouse model after behavioral testing, and place it in a minus 20 degrees Celsius freezer for 20 minutes. Then position the brain into a one-millimeter brain matrix. And using microtone blades, slice the brain into two-millimeter-thick coronary sections.Transfer the brain sections to a 24-well culture plate and add 2%TTC to cover the brain tissue. Incubate the plate at 37 degrees Celsius for 30 minutes. Then carefully fix the brain section in 4%Paraldehyde solution for 15 minutes.Using a scanister, perform optical scanning of the brain sections. The histological analysis of the brain revealed the disordered arrangement of neuron cells and a significant reduction in the density of NeuN positive cells in the peri-infarct area of the distal MCA occluded mice.

1.0K Views.  Jianghan University.  Here, we present a protocol to establish a distal middle cerebral artery occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6J mice and evaluate the subsequent neurological behavior and histopathological features.

Occlusion of Distal Middle Artery to Develop an Ischemic Stroke Mouse Model

Summary