Study supported by grant PID2020-113202RB-I00 funded by Ministerio de Ciencia e Innovaci&#243;n (MCIN)/Agencia Estatal de Investigaci&#243;n (AEI), Gobierno de Espa&#241;a/10.13039/501100011033 and &#34;European Regional Development Fund (ERDF). A way of making Europe&#34;. NCC and MAR had predoctoral fellowships (PRE2021-099481 and PRE2018-085737, respectively) funded by MCIN/AEI/ 10.13039/501100011033 and by &#34;European Social Fund (ESF) Investing in your future&#34;.&#160;We thank Francisca Ruiz-Ja&#233;n and Leonardo M&#225;rquez-Kisinousky for their technical support. We acknowledge the support of the MRI imaging facility of Institut d&#39;Investigacions Biom&#232;diques August Pi i Sunyer (IDIBAPS). The Centres de Recerca de Catalunya (CERCA) Program of Generalitat de Catalunya supports IDIBAPS.

Animal work was conducted following the Catalan and Spanish laws (Real Decreto 53/2013) and the European Directives, with approval of the ethical committee (Comit&#233; &#200;tic d&#39;Experimentaci&#243; Animal, CEEA) of the University of Barcelona, and the local regulatory bodies of the Generalitat de Catalunya. Studies are reported in compliance with the ARRIVE guidelines. This procedure is designed to be performed in adult mice, starting at 8 weeks of age, with no age limit. Examples of the surgical procedure developed in C57BL/6 mice of 10-12 weeks of age are provided here. Anatomical differences depending on mouse strain should be considered.
1. Animal preparation
<ol>
	<li>Before starting the surgical procedure, gather and sterilize all the required materials and tools. Set up the operating table with all the necessary surgical materials (listed in the Table of Materials).</li>
	<li>Anesthetize the animal using isoflurane inhalation in a mixture of oxygen and nitrous oxide (30%/70%).</li>
	<li>Administer buprenorphine (see Table of Materials) subcutaneously at a dosage of 0.05 mg/kg BW to provide analgesia and alleviate any pain and discomfort. 
	NOTE: Analgesia is mandatory, but different protocols are accepted. Pain and discomfort signs must also be controlled during the first days after MCAo (see step 4). Apply corrective solutions when necessary.</li>
	<li>Place the animal in an anesthesia induction box (see Table of Materials) with 5% isoflurane until it reaches a state of deep anesthesia (loss of reflex in paw puncture and ocular reflex).</li>
	<li>Position the mouse on the operating table and decrease the level of isoflurane to 1.5%, delivered by face mask. Apply vet ointment to avoid eye dryness during the procedure.</li>
	<li>Maintain body temperature at 37 &#177; 0.5 &#176;C controlled by a rectal probe connected to a heating pad (see Table of Materials).</li>
	<li>Shave the ventral part of the neck and the head (calvaria) with an electric razor. Carefully remove fur debris and disinfect the skin areas three times in circular movements with iodine-based disinfectant and 70% alcohol. 
	.</li>
</ol>
2. Cerebral blood flow (CBF) assessment with laser Doppler flowmetry (LDF)
<ol>
	<li>With scissors, make an incision on the skin of the head, in the direction of the sagittal suture, from the ears to the area between the eyes.</li>
	<li>Retract the skin and remove the periosteum on the right side of the skull.</li>
	<li>Find the coordinates (2.5 mm lateral from Bregma) and attach the Doppler holder (see Table of Materials) using cyanoacrylate. After the glue has dried, connect the Doppler probe and check for the correct signal readout.</li>
</ol>
3. Transient middle cerebral artery occlusion (tMCAo)
<ol>
	<li>Turn the mouse over to the supine position, and fix it to the surgical table with medical tape.</li>
	<li>Make a midline incision on the neck. Laterally pull back the skin and salivary glands using retractors (see Table of Materials) to expose the carotid territory.</li>
	<li>Identify the vascular anatomy of the common carotid artery (CCA), the ICA, and the ECA, as well as the different arteries derived from them (maxillary and lingual, superior thyroid, occipital, and pterygopalatine) (Figure 1A).</li>
	<li>Detach the main arteries from the adjacent connective tissue so that they can be handled. 
	NOTE: Take special care not to damage the nerves, especially the vagus nerve, which runs parallel to the CCA.</li>
	<li>Wrap a 6-0 silk suture (see Table of Materials) around the ECA at the maxillary/lingual bifurcation. Tightly secure a knot to permanently interrupt the circulation.</li>
	<li>Pass a second suture around the same artery, between the first knot and the CCA bifurcation, and keep this knot loose.</li>
	<li>Place a third thread around the CCA and tie a slip knot that can be easily untied. 
	NOTE: This can also be carried out with a vascular clip, but the thread allows more movement and flexibility. At this stage it is possible to observe a first decrease in CBF in the LDF signal.</li>
	<li>Place a vascular clip (see Table of Materials) interrupting blood circulation from the ICA.</li>
	<li>Make a small incision in the ECA, close to the area where the tight knot is located.</li>
	<li>Insert the monofilament until the thick coating has completely entered the arterial lumen.</li>
	<li>Tighten the second knot to hold the monofilament inside the artery and prevent the pressure exerted by the blood from pushing it out (Figure 1B).</li>
	<li>Remove the vascular clip from the ICA.</li>
	<li>Cut the ECA below the first knot and rotate the stump to orient it in the direction of the ICA (Figure 1C).</li>
	<li>Advance the monofilament via the ICA until the point where the MCA branches out. 
	NOTE: The occlusion is reflected in an abrupt blood flow drop in the LDF readout. We consider a successful occlusion when the drop in CBF is greater than 70% from the basal value. If CBF measurement systems are not available, the point of occlusion can be noted by the resistance to advance, which in adult mice is usually about 11 mm from the bifurcation of the CCA.
	<ol>
		<li>If anesthesia is continued during the occlusion period, monitor the mouse and keep it under constant observation for 45 min.</li>
		<li>In case the mouse is awakened during the occlusion period, suture the skin of the neck with several stitches. Without disconnecting the LDF probe, place the mouse in the temperature-controlled box, allowing recovery from anesthesia. 
		NOTE: It is common for the mouse to exhibit spontaneous circling behavior during this period, indicative of successful occlusion.</li>
		<li>After 40 min, anesthetize the mouse again following the same anesthesia and disinfection procedures as indicated in points 1.4, 1.5 and 1.7.&#160;Place it back on the surgical table, and remove the stitches from the neck.</li>
	</ol>
	</li>
	<li>After 45 min of occlusion, loosen the knot holding the monofilament in place. Pull slowly and gently on the filament and check that tissue recanalization occurs.</li>
	<li>Pull out the filament and tighten the knot to prevent blood loss.</li>
	<li>Untie the CCA knot. Ensure that there is no arterial wall damage.</li>
	<li>Remove the retractors and re-position the muscles, glands, and skin. Suture the skin (6-0) and apply disinfectant.</li>
	<li>Disconnect the Doppler probe, and detach the holder. Suture and disinfect the skin of the head.</li>
	<li>During the recovery period from anesthesia, leave the mouse in a cage provided with a heater to maintain the temperature. Keep it under constant observation until it is fully recovered from anesthesia. After recovery, the mouse can be returned to its cage. 
	&#8203;NOTE: Housing with social enrichment is highly recommended. However, never mix operated mice with non-operated mice in the same cage without any physical separation in order to prevent aggression.</li>
</ol>
4. Post-operative care
<ol>
	<li>Periodically supervise the animals following the procedures and regulations established according to the local regulations. Provide analgesic treatment on the appropriate schedule to minimize pain after the surgery. 
	NOTE: In the present study, the same analgesic was applied as at the beginning of the intervention (Buprenorphine 0.05mg/kg BW) at 6 h and 24 h after surgery.</li>
	<li>Perform euthanasia when the supervision parameters indicate so, following the institutionally approved protocols.</li>
	<li>Daily monitor the weight of the animals. Provide soft food to the animals during the first few days after surgery. In addition, hydrate them by subcutaneous injection of saline (200 &#181;L) immediately after surgery and periodically thereafter if it is observed that the mouse does not hydrate on its own. Arrange food and water in a way that is easily accessible to the animal.</li>
	<li>Once the in vivo study is completed, anesthetize the mice, euthanize them, and remove the brain tissue for further histopathological analysis (if needed).</li>
</ol>

A Model of Transient Cerebral Ischemia by Occlusion of Middle Cerebral Artery

<ol>
	<li>Siddiqi, A. Z., Wadhwa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+acute+stroke:+current+practices+and+future+horizons.">Treatment of acute stroke: current practices and future horizons.</a> Cardiovascular Revascularization Medicine. 49, 56-65 (2023).</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+ischemia+in+the+rat:+1.+Description+of+technique+and+early+neuropathological+consequences+following+middle+cerebral+artery+occlusion.">Focal cerebral ischemia 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, 53-60 (1981).</li><li>Koizumi, J., Nakazawa, T., Ooneda, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+studies+of+ischemic+brain+edema.+A+new+experimental+model+of+cerebral+embolism+in+rats+in+which+recirculation+can+be+introduced+in+the+ischemic+area.">Experimental studies of ischemic brain edema. A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area.</a> Japanese Journal of Stroke. 8, 1-8 (1986).</li><li>Longa, E. Z., Weinstein, P. R., Carlson, R., 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>Hossmann, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+ischemia:+Models,+methods,+and+outcomes.">Cerebral ischemia: Models, methods, and outcomes.</a> Neuropharmacology. 55, 257-270 (2008).</li><li>Seto, A., 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=Induction+of+ischemic+stroke+in+awake+freely+moving+mice+reveals+that+isoflurane+anesthesia+can+mask+the+benefits+of+a+neuroprotection+therapy.">Induction of ischemic stroke in awake freely moving mice reveals that isoflurane anesthesia can mask the benefits of a neuroprotection therapy.</a> Frontiers in Neuroenergetics. 6, 1 (2014).</li><li>D&#237;az-Marugan, L., 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=Poststroke+lung+infection+by+opportunistic+commensal+bacteria+is+not+mediated+by+their+expansion+in+the+gut+microbiota.">Poststroke lung infection by opportunistic commensal bacteria is not mediated by their expansion in the gut microbiota.</a> Stroke. 54 (7), 1875-1887 (2023).</li><li>Xie, L., Kang, H., Nedergaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+model+of+transient+occlusion+of+the+middle+cerebral+artery+in+awake+mice.">A novel model of transient occlusion of the middle cerebral artery in awake mice.</a> Journal of Natural Sciences. 2 (2), e176 (2016).</li><li>Arbaizar-Rovirosa, 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=Aged+lipid-laden+microglia+display+impaired+responses+to+stroke.">Aged lipid-laden microglia display impaired responses to stroke.</a> EMBO Molecular Medicine. 15 (2), e17175 (2023).</li><li>Orsini, F., 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=Targeting+mannose-binding+lectin+confers+long-lasting+protection+with+a+surprisingly+wide+therapeutic+window+in+cerebral+ischemia.">Targeting mannose-binding lectin confers long-lasting protection with a surprisingly wide therapeutic window in cerebral ischemia.</a> Circulation. 126 (12), 1484-1494 (2012).</li><li>Majid, A., 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=Differences+in+vulnerability+to+permanent+focal+cerebral+ischemia+among+3+common+mouse+strains.">Differences in vulnerability to permanent focal cerebral ischemia among 3 common mouse strains.</a> Stroke. 31, 2707-2714 (2000).</li><li>Rogers, D. C., Campbell, C. A., Stretton, J. L., Mackay, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+motor+impairment+and+infarct+volume+after+permanent+and+transient+middle+cerebral+artery+occlusion+in+the+rat.">Correlation between motor impairment and infarct volume after permanent and transient middle cerebral artery occlusion in the rat.</a> Stroke. 28, 2060-2065 (1997).</li><li>Hedna, V. 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=Validity+of+Laser+Doppler+flowmetry+in+predicting+outcome+in+murine+intraluminal+middle+cerebral+artery+occlusion+stroke.">Validity of Laser Doppler flowmetry in predicting outcome in murine intraluminal middle cerebral artery occlusion stroke.</a> Journal of Vascular and Interventional Neurology. 8 (3), 74-82 (2015).</li><li>Yin, L., 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=Laser+speckle+contrast+imaging+for+blood+flow+monitoring+in+predicting+outcomes+after+cerebral+ischemia-reperfusion+injury+in+mice.">Laser speckle contrast imaging for blood flow monitoring in predicting outcomes after cerebral ischemia-reperfusion injury in mice.</a> BMC Neuroscience. 23, 80 (2022).</li><li>Thakkar, P. C., 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=Therapeutic+relevance+of+elevated+blood+pressure+after+ischemic+stroke+in+the+hypertensive+rats.">Therapeutic relevance of elevated blood pressure after ischemic stroke in the hypertensive rats.</a> Hypertension. 75 (3), 740-747 (2020).</li><li>Yu, X., Feng, Y., Liu, R., Chen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothermia+protects+mice+against+ischemic+stroke+by+modulating+macrophage+polarization+through+upregulation+of+interferon+regulatory+factor-4.">Hypothermia protects mice against ischemic stroke by modulating macrophage polarization through upregulation of interferon regulatory factor-4.</a> Journal of Inflammation Research. 14, 1271-1281 (2021).</li><li>Denorme, F., Portier, I., Kosaka, Y., Campbell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperglycemia+exacerbates+ischemic+stroke+outcome+independent+of+platelet+glucose+uptake.">Hyperglycemia exacerbates ischemic stroke outcome independent of platelet glucose uptake.</a> Journal of Thrombosis and Haemostasis. 19, 536-546 (2021).</li></ol>

The authors declare no conflicts of interest.

The intraluminal tMCAo procedure is the most commonly used model of focal brain ischemia with reperfusion in basic research. Currently, mice are the preferred animal model due to the availability of genetically modified strains. However, it&#39;s essential to acknowledge that genetically modified mice and their genetic backgrounds can impact brain vascularization. The presence of collateral circulation and anastomoses between different arterial territories can significantly influence the outcomes of experimental procedur...

Stroke is a devastating disease that affects approximately 15 million people worldwide annually, according to the WHO. Around one-third of patients succumb to the condition, while another third experience permanent disability. Stroke is a complex pathology involving various cell types, such as neural and peripheral immune cells, vasculature, and systemic responses1. The intricate network of reactions triggered by stroke at the systems level cannot be currently replicated using in vitro models. Thus, experimental animal models are essential to delve into the disease&#39;s mechanisms and to develop and test new therapies. Currently, early tissue reperfusion is the only approved intervention, either through thrombolysis with tissue-type plasminogen activator (tPA) or endovascular thrombectomy1.
Occlusions of the middle cerebral artery (MCA) are frequent in stroke patients. Consequently, rodent models of transient MCA occlusion (tMCAo) were initially developed in rats2,3,4. Nowadays, genetically modified mice are the most commonly used animals in experimental stroke models. In this study, we describe a minimally invasive model of intraluminal tMCAo in mice. The approach is performed via the carotid artery at the neck level, without craniectomy.
The duration of the occlusion period is a critical factor that determines the extent of the ischemic lesion. Even short occlusions of 10 min can cause selective neuronal death without an apparent infarction, while longer occlusions, typically lasting 30 to 60 min, result in some degree of cerebral infarction. Unlike the proximal and distal branches of the MCA that supply the cortex and have collaterals, the lenticulo-striatal arteries providing blood to the striatum lack collaterals5. As a consequence, there is a greater reduction of blood flow in the striatum than in the cortex after tMCAo. Thus, occlusions of 30 min or less generally affect the striatum but not the cortex, whereas longer occlusions, from 45 min onwards, often generate an ischemic lesion in the entire MCA territory, including the striatum and dorsolateral cortex.
To ensure the well-being of the mice, we administer analgesics prior to the procedure and use anesthesia during surgery. Nevertheless, anesthesia can potentially introduce artificial alterations in the physiology of the mouse and affect some outcome measures6. The surgical intervention, when performed by experienced personnel, usually lasts about 15 min for inducing MCAo. Subsequently, the total time under anesthesia depends on the occlusion period. For experiments where minimizing anesthesia is crucial, an alternative step in the procedure involves discontinuing anesthesia during the occlusion period and limiting it only to the surgical steps for inserting and withdrawing the filament occluding the MCA. This approach reduces the duration of anesthesia and minimizes its potential artifactual effects on the experimental model7,8. Therefore, the method of inducing transient focal ischemia is presented by intraluminal occlusion of the MCA with two variants: with the mouse anesthetized during the entire occlusion period or with the mouse awake during this period. In either case, a sham surgery should be performed in parallel with the intervention carried out on the ischemic mice. Additionally, data on outcome assessment is provided as measured by behavioral tests and MRI at various time points after reperfusion. Finally, the main factors to consider when implementing the experimental procedure are discussed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6/0 suture&#160;</td>
			<td>Arago</td>
			<td></td>
			<td>Vascular ligatures</td>
		</tr>
		<tr>
			<td>6/0 suture with curved needle</td>
			<td>Arago</td>
			<td></td>
			<td>Skin sutures</td>
		</tr>
		<tr>
			<td>9 mg/mL Saline</td>
			<td>Fresenius Kabi</td>
			<td>CN616003 EC</td>
			<td>For hydration</td>
		</tr>
		<tr>
			<td>Anaesthesia system</td>
			<td>SurgiVet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt retractors, 1 mm wide</td>
			<td>Fine Science Tools</td>
			<td>18200-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorfine</td>
			<td>Buprex</td>
			<td></td>
			<td>For pain relief</td>
		</tr>
		<tr>
			<td>Clamp applying forceps</td>
			<td>Fine Science Tools</td>
			<td>S&#38;T CAF4</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont mini forceps</td>
			<td>Fine Science Tools</td>
			<td>M3S 11200-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>91106-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Glue</td>
			<td>Loctite</td>
			<td></td>
			<td>To stick LDF probe to the skull</td>
		</tr>
		<tr>
			<td>Grip Strength Meter</td>
			<td>IITC Life Science Inc.</td>
			<td>#2200</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>B-Braun</td>
			<td>CN571105.8</td>
			<td></td>
		</tr>
		<tr>
			<td>LDF Perimed</td>
			<td>Perimed</td>
			<td>Periflux System 5000</td>
			<td></td>
		</tr>
		<tr>
			<td>LDF Probe Holders</td>
			<td>Perimed</td>
			<td>PH 07-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MRI magnet</td>
			<td>Bruker BioSpin, Ettlingen, Germany</td>
			<td></td>
			<td>BioSpec 70/30 horizontal animal scanner&#160;</td>
		</tr>
		<tr>
			<td>Needle Holder with Suture Cutter</td>
			<td>Fine Science Tools</td>
			<td>12002-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon filament</td>
			<td>Doccol</td>
			<td>701912PK5Re</td>
			<td></td>
		</tr>
		<tr>
			<td>Recovery cage with heating pad</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sirgical scissors</td>
			<td>Fine Science Tools</td>
			<td>91401-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Small vessel cauterizer kit</td>
			<td>Fine Science Tools</td>
			<td>18000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope and cold light</td>
			<td>Leica</td>
			<td>M60</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture tying forceps</td>
			<td>Fine Science Tools</td>
			<td>18025-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostat, rectal probe and mouse pad</td>
			<td>Letica Science Instruments</td>
			<td>LE 13206</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas spring scissors (4mm cutting edge)</td>
			<td>Fine Science Tools</td>
			<td>15019-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Vascular clamps</td>
			<td>Fine Science Tools</td>
			<td>00396-01</td>
			<td></td>
		</tr>
	</tbody>
</table>

transient middle cerebral artery occlusion model of stroke

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models.
In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

There are different approaches to evaluate the outcome of the tMCAo procedure. In vivo neuroimaging methods (MRI) and behavioral testing are utilized here.
Mice develop ischemic lesions in the brain, mainly affecting the territory supplied by the MCA ipsilateral to the occlusion, such as the striatum and dorsolateral cortex. Several methods exist to determine the extent of the lesion, including 2,3,5-triphenyltetrazolium chloride (TTC) tissue staining, histological staining (hematoxyl...

Watch this Scientific Journal Video about Assessing Ischemic Stroke Damage Through Middle Cerebral Artery Occlusion Model at JoVE.com

Author Spotlight: Assessing Ischemic Stroke Damage Through Middle Cerebral Artery Occlusion Model 

This protocol describes the model of transient focal cerebral ischemia in mice through intraluminal occlusion of the middle cerebral artery. Additionally, examples of outcome assessment are shown using magnetic resonance imaging and behavioral tests.

Transient Middle Cerebral Artery Occlusion Model of Stroke

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies ...

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1.6K Views.  Spanish National Research Council (CSIC). This protocol describes the model of transient focal cerebral ischemia in mice through intraluminal occlusion of the middle cerebral artery. Additionally, examples of outcome assessment are shown using magnetic resonance imaging and behavioral tests.

Video: Author Spotlight: Assessing Ischemic Stroke Damage Through Middle Cerebral Artery Occlusion Model 

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume three-dimensionally. While MRI is an extremely sensitive tool for detecting tissue abnormalities, association of signal changes with an underlying pathological process is usually not straightforward. In the central nervous system, for example, inflammation, demyelination, axonal damage, gliosis, and neuronal death may all induce similar findings on MRI. As such, interpretation of MRI scans depends on the context, and radiological-histopathological correlation is therefore of the utmost importance. Unfortunately, traditional pathological sectioning of brain tissue is often imprecise and inconsistent, thus complicating the comparison between histology sections and MRI. This article presents novel methodology for accurately sectioning primate brain tissues and thus allowing precise matching between histology and MRI. The detailed protocol described in this article will assist investigators in applying this method, which relies on the creation of 3D printed brain slicers. Slightly modified, it can be easily implemented for brains of other species, including humans.

The overall goal of this protocol is to accurately align magnetic resonance imaging (MRI) image volumes with histology sections via the creation of customized 3D-printed brain holders and slicer boxes.

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

A Magnetic Resonance Imaging Protocol for Stroke Onset Time Estimation in Permanent Cerebral Ischemia

MRI provides a sensitive and specific imaging tool to detect acute ischemic stroke by means of a reduced diffusion coefficient of brain water. In a rat model of ischemic stroke, differences in quantitative T1 and T2 MRI relaxation times (qT1 and qT2) between the ischemic lesion (delineated by low diffusion) and the contralateral non-ischemic hemisphere increase with time from stroke onset. The time dependency of MRI relaxation time differences is heuristically described by a linear function and thus provides a simple estimate of stroke onset time. Additionally, the volumes of abnormal qT1 and qT2 within the ischemic lesion increase linearly with time providing a complementary method for stroke timing. A (semi)automated computer routine based on the quantified diffusion coefficient is presented to delineate acute ischemic stroke tissue in rat ischemia. This routine also determines hemispheric differences in qT1 and qT2 relaxation times and the location and volume of abnormal qT1 and qT2 voxels within the lesion. Uncertainties associated with onset time estimates of qT1 and qT2 MRI data vary from &plusmn; 25 min to &plusmn; 47 min for the first 5 hours of stroke. The most accurate onset time estimates can be obtained by quantifying the volume of overlapping abnormal qT1 and qT2 lesion volumes, termed 'Voverlap' (&plusmn; 25 min) or by quantifying hemispheric differences in qT2 relaxation times only (&plusmn; 28 min). Overall, qT2 derived parameters outperform those from qT1. The current MRI protocol is tested in the hyperacute phase of a permanent focal ischemia model, which may not be applicable to transient focal brain ischemia.

A protocol for stroke onset time estimation in a rat model of stroke exploiting quantitative magnetic resonance imaging (qMRI) parameters is described. The procedure exploits diffusion MRI for delineation of the acute stroke lesion and quantitative T1 and T2 (qT1 and qT2) relaxation times for timing of stroke.

MRI provides a sensitive and specific imaging tool to detect acute ischemic stroke by means of a reduced diffusion coefficient of brain water. In a rat ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent ...

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The brain's ability to self-heal after ischemic stroke is limited by inadequate blood supply in the impacted area. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis in patients with moyamoya disease. It involves craniotomy with placement of a vascular temporalis muscle graft on the ischemic brain surface. EMS has never been studied in the setting of acute ischemic stroke in mice. The hypothesis driving this study is that EMS enhances cerebral angiogenesis at the cortical surface surrounding the muscle graft. The protocol shown here describes the procedure and provides initial data supporting the feasibility and efficacy of the EMS approach. In this protocol, after 60 min of transient middle cerebral artery occlusion (MCAo), mice were randomized to either MCAo or MCAo + EMS treatment. The EMS was performed 3-4 h after occlusion. The mice were sacrificed 7 or 21 days after MCAo or MCAo + EMS treatment. Temporalis graft viability was measured using nicotinamide adenine dinucleotide reduced-tetrazolium reductase assay. A mouse angiogenesis array quantified angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize graft bonding with brain cortex and change in vessel density. The preliminary data here suggest that grafted muscle remained viable 21 days after EMS. Immunostaining showed successful graft implantation and increase in vessel density near the muscle graft, indicating increased angiogenesis. Data show that EMS increases fibroblast growth factor (FGF) and decreases osteopontin levels after stroke. Additionally, EMS after stroke did not increase mortality suggesting that protocol is safe and reliable. This novel procedure is effective and well-tolerated and has the potential to provide information of novel interventions for enhanced angiogenesis after acute ischemic stroke.

The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The ...