The authors thank Kathy Gage for her editorial support. Scheme figures were created with BioRender.com. This study was supported by funds from the Department of Anesthesiology (Duke University Medical Center) and NIH grants (NS099590, HL157354, NS117973, and NS127163).

All procedures described in this work are conducted in accordance with the NIH guidelines for the care and use of animals in research, and the protocol was approved by the Duke Institute Animal Care and Use Committee (IACUC). Young (8-10 weeks old) and aged (22 months old) male C57Bl/6 mice were used for the present study. An overview of this protocol is illustrated in Figure 1.
1. Surgical preparation
<ol>
	<li>Examine the mouse for gross abnor...

Establishing a Transcranial Middle Cerebral Artery Occlusion Mice Model

<ol>
	<li>Tsao, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+disease+and+stroke+statistics-2022+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics-2022 update: a report from the American Heart Association.</a> Circulation. 145 (8), e153 (2022).</li><li>Nogueira, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombectomy+6+to+24+hours+after+stroke+with+a+mismatch+between+deficit+and+infarct.">Thrombectomy 6 to 24 hours after stroke with a mismatch between deficit and infarct.</a> The New England Journal of Medicine. 378 (1), 11-21 (2018).</li><li>Fisher, M., Savitz, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+brain+cytoprotection+in+acute+ischaemic+stroke-renewed+hope+in+the+reperfusion+era.">Pharmacological brain cytoprotection in acute ischaemic stroke-renewed hope in the reperfusion era.</a> Nature Reviews Neurology. 18 (4), 193-202 (2022).</li><li>Longa, E. Z., Weinstein, P. R., Carlson, S., Cummins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats.">Reversible middle cerebral artery occlusion without craniectomy in rats.</a> Stroke. 20 (1), 84-91 (1989).</li><li>Knauss, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+semiquantitative+non-invasive+measurement+of+PcomA+patency+in+C57BL/6+mice+explains+variance+in+ischemic+brain+damage+in+filament+MCAo.">A semiquantitative non-invasive measurement of PcomA patency in C57BL/6 mice explains variance in ischemic brain damage in filament MCAo.</a> Frontiers in Neuroscience. 14, 576741 (2020).</li><li>Yang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-ischemia+common+carotid+artery+occlusion+worsens+memory+loss,+but+not+sensorimotor+deficits,+in+long-term+survived+stroke+mice.">Post-ischemia common carotid artery occlusion worsens memory loss, but not sensorimotor deficits, in long-term survived stroke mice.</a> Brain Research Bulletin. 183, 153-161 (2022).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+O-GlcNAcylation+is+neuroprotective+in+young+and+aged+brains+after+ischemic+stroke.">Increasing O-GlcNAcylation is neuroprotective in young and aged brains after ischemic stroke.</a> Experimental Neurology. 339, 113646 (2021).</li><li>Jiang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XBP1+(X-box-binding+protein-1)-dependent+O-GlcNAcylation+Is+neuroprotective+in+ischemic+stroke+in+young+mice+and+its+impairment+in+aged+mice+is+rescued+by+thiamet-G.">XBP1 (X-box-binding protein-1)-dependent O-GlcNAcylation Is neuroprotective in ischemic stroke in young mice and its impairment in aged mice is rescued by thiamet-G.</a> Stroke. 48 (6), 1646-1654 (2017).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptomic+analysis+of+the+immune+cell+landscape+in+the+aged+mouse+brain+after+ischemic+stroke.">Single-cell transcriptomic analysis of the immune cell landscape in the aged mouse brain after ischemic stroke.</a> Journal of Neuroinflammation. 19 (1), 83 (2022).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+effects+of+neuronal+ATF6+activation+in+permanent+ischemic+stroke.">Beneficial effects of neuronal ATF6 activation in permanent ischemic stroke.</a> Frontiers in Cellular Neuroscience. 16, 1016391 (2022).</li><li>Tamura, A., Graham, D. I., McCulloch, J., Teasdale, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+cerebral+ischaemia+in+the+rat:+2.+Regional+cerebral+blood+flow+determined+by+[14C]iodoantipyrine+autoradiography+following+middle+cerebral+artery+occlusion.">Focal cerebral ischaemia in the rat: 2. Regional cerebral blood flow determined by [14C]iodoantipyrine autoradiography following middle cerebral artery occlusion.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 1 (1), 61-69 (1981).</li><li>Tamura, A., Graham, D. I., McCulloch, J., Teasdale, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+cerebral+ischaemia+in+the+rat:+1.+Description+of+technique+and+early+neuropathological+consequences+following+middle+cerebral+artery+occlusion.">Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 1 (1), 53-60 (1981).</li></ol>

The first transcranial MCA occlusion model was established in rats in 198111,12, and replaced by the no-craniectomy MCAO model in 19894. The initial transcranial MCA occlusion had a wide surgical field, such that the entire zygomatic arch was removed and the muscles pulled laterally. Local tissues were swollen after surgery, causing stress and decreased food intake for the animals. In our modified transcranial MCAO model, the incision is l...

Nearly 800,000 people suffer a stroke in the US every year, and most of these strokes are ischemic in nature1. Timely restoration of the cerebral blood flow with tissue plasminogen activator (tPA) and/or thrombectomy is currently the most effective treatment for stroke patients; however, the full recovery of neurologic functions in the long term is rare2,3. Thus, searching for novel stroke therapy that targets functional improvement is an intense area of research that requires clinically relevant animal models of stroke. 
The most common ischemic stroke model in rodents uses intraluminal middle cerebral artery occlusion (MCAO) to induce stroke. In this model, initially developed by Zea Longa in 1989, a nylon filament is introduced into the internal carotid artery (ICA) to block the blood flow to the middle cerebral artery (MCA)4. However, this model has limitations. First, when the filament is inserted into the ICA, the blood flow to the posterior cerebral artery (PCA) could be partially blocked as well, especially in mice. Critically, the posterior communicating artery (PcomA), a small artery that connects anterior and posterior cerebral circulation, is frequently underdeveloped in some mouse strains, such as C57Bl/6, the strain predominantly used in experimental stroke research. This patency of the PcomA is believed to contribute to the variability in lesion size in mice after stroke5. Indeed, when blood flow to the PCA drops precipitously during MCAO, and the PcomA is unable to provide sufficient collateral blood flow, the stroke infarct can expand into the territory of the PCA. Moreover, in this model, a long duration of ischemia leads to a higher chance of mortality in mice. Consequently, a short MCAO duration of 30-60 min is typically used in mice. However, most stroke patients experience a few hours of ischemia before reperfusion treatment. Thus, a mouse stroke model with an extended duration of ischemia is of high clinical relevance. 
The overall goal of this procedure is to model ischemic stroke in mice that have a medium-sized infarct and an excellent survival rate. This transcranial MCAO model addresses critical attributes of clinical stroke, as prolonged ischemia can be performed, and aged mice tolerate this model well, allowing for the long-term assessment of functional recovery.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% bupivacaine</td>
			<td>Hospira</td>
			<td>NDC 0409-1159-18</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% sodium chloride</td>
			<td>ICU Medical</td>
			<td>NDC 0990-7983-03</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3,5-Triphenyltetrazolium Chloride (TTC)&#160;</td>
			<td>Sigma or any available vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20 G IV catheter</td>
			<td>BD</td>
			<td>381534</td>
			<td>20 GA 1.6 IN</td>
		</tr>
		<tr>
			<td>30 G needle</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 silk suture</td>
			<td>Look</td>
			<td>SP116</td>
			<td>Black braided silk</td>
		</tr>
		<tr>
			<td>8-0 suture with needle&#160;</td>
			<td>Ethilon</td>
			<td>2822G</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol swabs</td>
			<td>BD</td>
			<td>326895</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia induction box</td>
			<td>Any suitable vendor</td>
			<td></td>
			<td>Pexiglass make&#160;</td>
		</tr>
		<tr>
			<td>Electrical grinder</td>
			<td>JSDA</td>
			<td>JD 700</td>
			<td></td>
		</tr>
		<tr>
			<td>High temperature cautery loop tip</td>
			<td>Bovie</td>
			<td>AA03</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Covetrus</td>
			<td>NDC 11695-6777-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser doppler perfusion monitor</td>
			<td>Moor Instruments</td>
			<td>moorVMS-LDF1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lubricant eye ointment</td>
			<td>Bausch + Lomb</td>
			<td>339081</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse rectal probe</td>
			<td>Physitemp</td>
			<td>RET-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrous Oxide</td>
			<td>Airgas</td>
			<td>UN1070</td>
			<td></td>
		</tr>
		<tr>
			<td>Otoscope</td>
			<td>Welchallyn</td>
			<td>728</td>
			<td>2.5 mm Speculum</td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>Airgas</td>
			<td>UN1072</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td>CVS</td>
			<td>955338</td>
			<td></td>
		</tr>
		<tr>
			<td>Recovery box</td>
			<td>Brinsea&#160;</td>
			<td>TLC eco</td>
			<td></td>
		</tr>
		<tr>
			<td>Rimadyl (carprofen)</td>
			<td>Zoetis</td>
			<td>6100701</td>
			<td>Injectable 50 mg/mL</td>
		</tr>
		<tr>
			<td>Rodent ventilator</td>
			<td>Harvard</td>
			<td>Model 683</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Physitemp</td>
			<td>TCAT-2DF&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Triple antibioric &#38; pain relief</td>
			<td>CVS</td>
			<td>NDC 59770-823-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporizer</td>
			<td>RWD</td>
			<td>R583S</td>
			<td></td>
		</tr>
	</tbody>
</table>

a modified transcranial middle cerebral artery occlusion model to study stroke outcomes in aged mice

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

Author Spotlight: Innovative Techniques and Future Directions in Stroke Research 

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice

Stroke affects nearly 800, 000 people annually in the US.Around 87%of this, ischemic, presenting as a severe medical condition. However, there are limited treatment options available especially during the chronic stroke phase. Our research aims to develop a recovery enhancing therapy to reduce disability and improve the quality of life for stroke patients.They widely use the filament, the middle cerebral artery occlusion model, as a drawback where the inserted filament can block a blood flow to the posterior cerebral artery. The C57BL/6 in mice often have an undisturbed posterior communicating artery leading to a high mortality in the later chronic stroke recovery phase. Our modified transcranial middle cerebral artery occlusion model partially across the MCA at the trunk generating brain damage in the cortex and the striatum via a small cranial window.This model achieves a high long-term survival rate, detectable neurological deficits, and works well even in aged mice. Our transcranial MCAO model can incorporate the critical attributes of clinical stroke, such as delayed reperfusion, neurological deficits, and aging factor. This model can be used to evaluate the therapeutical potential of new stroke treatments on long-term function recovery.

With a direct view under a surgical microscope, it can be visually confirmed that MCA blood flow is blocked during ischemia. Our previous study showed a &#62;80% blood flow reduction in the ischemic area using a laser Doppler monitor6. In order to determine post-MCAO blood flow changes, LSCI can be used to further confirm the ischemic insult and reperfusion (Figure 1). Indeed, in Figure 3A, it is observed that the blood supply was reduced...

Watch this Scientific Journal Video about Innovative Techniques and Future Directions in Stroke Research at JoVE.com

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional outcomes.

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. ...

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Neuroscience

1.7K ビュー.  Duke University Medical Center. 脳卒中は、米国で年間約800, 000人が罹患しています。このうち約87%が虚血性で、重篤な病状を呈する。しかし、特に慢性脳卒中期には、治療の選択肢が限られています。私たちの研究は、脳卒中患者の障害を軽減し、生活の質を向上させるための回復促進療法を開発することを目的としています。彼らは、挿入されたフィラメントが後大脳動脈への血流を妨げる可?...

ビデオ: 著者スポットライト:脳卒中研究における革新的な技術と将来の方向性 

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional...

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a rat dry eye model with lacrimal gland dysfunction induced by scopolamine

Author Spotlight: Challenges in Developing Dry Eye Animal Models and Future Research Directions

Here, we establish a rat model of lacrimal gland dysfunction to provide a basis for the study of aqueous-deficient dry...

Scopolamine-Induced Lacrimal Gland Dysfunction in Rats

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measurement of healthy and injured triceps surae morphology

Author Spotlight: Exploring the Complexities of Achilles Tendon Injuries &amp;#8212; Research and Future Directions

Ultrasound imaging is becoming more accessible in clinical and research settings, and a consistent protocol will be beneficial for comparison between studies and for clinical interpretations. This protocol for ultrasound evaluation is a valid and reliable method to evaluate Achilles tendon morphology in healthy, tendinopathic, and ruptured tendons.

Ultrasound Evaluation of Achilles Tendon Morphology

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an experimental approach to investigating effects of artificial light at night on free-ranging animals: implementation, results, and directions for future research

An Experimental Approach to Investigating Effects of Artificial Light at Night on Free-Ranging Animals: Implementation, Results, and Directions for Future Research

Artificial light at night (ALAN) has wide-reaching biological effects. This article describes a system for manipulating ALAN inside nest boxes while monitoring behavior, consisting of LED lights coupled to a battery, timer, and audio-capable infrared video camera.  Researchers could employ this system to explore many outstanding questions regarding the effects of ALAN on...

alternative methods for the detection of superoxide anion generation in platelets 

Author Spotlight: Innovative Techniques for ROS Detection and Implications for Platelet Research

The generation of superoxide anion is essential for the stimulation of platelets and, if dysregulated, critical for thrombotic diseases. Here, we present three protocols for the selective detection of superoxide anions and the study of redox-dependent platelet...

Detection of Superoxide Anion in Platelets Using Fluorescence Imaging and EPR

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generating human pancreatic tissue slices to study endocrine and exocrine pancreas physiology

Author Spotlight: Advancing Type 1 Diabetes Research Using Innovative Pancreatic Slice Platforms

This protocol describes how to generate human pancreas slices from deceased organ donors to study cell function under near-physiological conditions. This innovative approach enables the investigation of normal and structurally damaged islets and the intricate interplay between endocrine and exocrine compartments.

Slicing Human Pancreas for Islet Function Research

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operation procedure and precautions of thread-embedding acupuncture therapy in alzheimer

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Improving Cognitive Functions in Rats via Thread-Embedding Acupuncture Therapy

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social threat-safety test uncovers psychosocial stress-related phenotypes

Author Spotlight: Unveiling Mechanisms of Stress Resilience - Significant Findings, Advancements, and Future Research

The social threat-safety test allows a simultaneous assessment of social avoidance development as a measurement of aversive conditioned learning and social threat-safety discrimination ability, both utilized to identify stress-susceptible and stress-resilient individuals within a single group of chronically socially defeated male...

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a rat model of middle cerebral artery occlusion/reperfusion without damaging the anatomical structure of cerebral vessels

Author Spotlight: Development of a Rat Model for Ischemic Stroke with Preserved Cerebral Vascular Integrity Using Microsurgical Techniques

The current protocol describes a rat model of middle cerebral artery occlusion/reperfusion that preserves the anatomical structure of cerebral vessels without causing...

Middle Cerebral Artery Occlusion in Rats with Preserved Vascular Anatomy

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blood sampling and hormone measurement for determining the stage in the ovarian cycle in marmosets

Author Spotlight: Advancing Primatology Through Germ Cell Research and Genetic Modification Techniques

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Assessing Ovarian Cycle in Marmosets by Blood Collection and Hormone Analysis

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green synthesis of quinoline-based ionic liquid

Author Spotlight: Advancing Antimicrobial Resistance Research with Innovative Approaches and Synthetic Compounds

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periorbital placement of a laser doppler probe for cerebral blood flow monitoring prior to middle cerebral artery occlusion in rodent models

Author Spotlight: Advancing Stroke Research with Periorbital Doppler Monitoring of Regional Cerebral Blood Flow in Rodent Models

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live calcium imaging of virus-infected human intestinal organoid monolayers using genetically encoded calcium indicators

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

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free-hand intracerebroventricular injections in mice

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a rat model of pouchitis following proctocolectomy and ileal pouch-anal anastomosis using dextran sulfate sodium

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Experimental Rat Model of Pouchitis for Studying Ulcerative Colitis Pathogenesis and Treatment

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developing a clinically relevant hemorrhagic shock model in rats

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Vascular Cannulation with Hemorrhagic Shock Induction in Rats

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targeted knockdown of genes in the choroid plexus

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methods for tattooing xenopus laevis with a rotary tattoo machine

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Middle Cerebral Artery Occlusion (MCAO) to Induce Stroke in a Murine Model

To begin, lay an eight to 10 weeks anesthetized C57 black six male mouse on the surgical bench. Using blunt forceps, pull out the tongue and hold it with fingers. Insert a laryngoscope into the mouth and visualize the vocal cord.Stabilize the mouse chin on the laryngoscope and hold the 20 gauge intravenous, or IV catheter with a guide wire inserted in it. Then slightly insert the guide wire into the vocal cord and slowly push the 20 gauge IV catheter into the trachea until its wing part is even with the nose tip. Once the mouse is connected to the ventilator, keep it in a lateral position with the right temporal area facing up.Maintain the rectal temperature at 37 degrees Celsius using a heating pad and a heat lamp. After locating the zygomatic arch and incising the temporal muscle, use two forceps to dissect the underlying zygomatic arch and expose the joint between the maxilla and zygomatic bones. Then using scissors, cut a three millimeter portion of the zygomatic arch and remove it.Finally, separate the masseter muscle from the skull base. Position four small retractors in different directions and expose the cranial skull base with one retractor pulling the trigeminal nerve branches laterally. Apply a saline drop to the skull above the middle cerebral artery, or MCA trunk and proximal to the rhinal cortex branch.Use an electrical grinder to thin the skull until a small fracture is visible. And with the tip of the forceps, remove the thin skull. Place a single-strand loop of black braided silk on top of the MCA.Then insert an 8-O microsurgical needle to lift the MCA trunk and tie the suture under the needle, leaving the needle's two ends on top of the silk thread loop knot. For transient middle cerebral artery occlusion, or MCAO, slightly tighten the silk thread knot under the needle to block arterial blood flow, representing MCAO onset. Use the forceps to hold the suture and slowly remove the needle at the end of the ischemia.The laser speckle contrast imaging confirmed the reduced blood supply in the right MCA. For transient MCAO after suture removal, the cerebral blood flow reperfusion was evident, and further improved after 24 hours. TTC staining of the brain after 24 hours of stroke demonstrated the generation of infarcted tissue in both the cortical and lateral striatum areas in young and aged mice.The infarct size was moderate compared to filament MCAO.