Name: Innovative Techniques and Future Directions in Stroke Research (Video) | JoVE
Uploaded: 2023-05-05T14:50:00
Description: Watch this Scientific Journal Video about Innovative Techniques and Future Directions in Stroke Research at JoVE.com

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.

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

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

Innovative Techniques and Future Directions in Stroke Research (Video) | JoVE

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. ...