This work was funded by the National Institute of Health, the National Heart Lung and Blood Institute (NIH and NHLBI; HL125572-01A1) and the LSUHSC-S start up fund to F.N.E. Gavins.

This protocol and the experiments reported in the video were approved by the LSUHSC-S Institutional Animal Care and Use Committee and are in compliance with the guidelines of NIH.
NOTE: Male C57BL/6 mice weighing 25 - 29 g were used in this study. The mice were maintained on a standard chow pellet diet with free access to water, under a 12 hr light/dark cycle in individually ventilated cages. The procedure will be performed under sterile conditions using sterile techniques (e.g., sterile gloves, sterile instruments).
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<ol>
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N., Perretti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+annexin+1+counter-regulatory+circuit+affords+protection+in+the+mouse+brain+microcirculation.">Activation of the annexin 1 counter-regulatory circuit affords protection in the mouse brain microcirculation.</a> FASEB J. 21 (8), 1751-1758 (2007).</li><li>Chen, J., 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=Atorvastain+induction+of+VEGF+and+BDNF+promotes+brain+plasticity+after+stroke+in+mice.">Atorvastain induction of VEGF and BDNF promotes brain plasticity after stroke in mice.</a> J Cereb Blood Flow Metab. 25 (2), 281-290 (2005).</li><li>Li, Y., 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=Intrastriatal+transplantation+of+bone+marrow+nonhematopoietic+cells+improves+functional+recovery+after+stroke+in+adult+mice.">Intrastriatal transplantation of bone marrow nonhematopoietic cells improves functional recovery after stroke in adult mice.</a> J Cereb Blood Flow Metab. 20 (9), 1311-1319 (2000).</li><li>Liesz, 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=The+spectrum+of+systemic+immune+alterations+after+murine+focal+ischemia;+the+immunodepression+versus+immunomodulation.">The spectrum of systemic immune alterations after murine focal ischemia; the immunodepression versus immunomodulation.</a> Stroke. 40 (8), 2849-2858 (2009).</li><li>Beckmann, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+magnetic+resonance+angiography+non-invasively+reveals+mouse+strain+differences+in+the+cerebrovascular+anatomy+in+vivo.">High resolution magnetic resonance angiography non-invasively reveals mouse strain differences in the cerebrovascular anatomy in vivo.</a> Magn Reson Med. 44 (2), 252-258 (2000).</li><li>Barone, F. C., Knudsen, D. J., Nelson, A. H., Feuerstein, G. Z., Willette, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+strain+differences+in+susceptibility+to+cerebral+ischemia+are+related+to+cerebral+vascular+anatomy.">Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy.</a> J Cereb Blood Flow Metab. 13 (4), 683-692 (1993).</li><li>Burk, J., Burggraf, D., Vosko, M., Dichgans, M., Hamann, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+of+cerebral+microvasculature+after+moderate+hypothermia+following+experimental+focal+cerebral+ischemia+in+mice.">Protection of cerebral microvasculature after moderate hypothermia following experimental focal cerebral ischemia in mice.</a> Brain Res. (1226), 248-255 (2008).</li><li>Noor, R., Wang, C. X., Shuaib, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+hyperthemia+on+infarct+volume+in+focal+embolic+model+of+cerebral+ischemia+in+rats.">Effects of hyperthemia on infarct volume in focal embolic model of cerebral ischemia in rats.</a> Neurosci Lett. 349 (2), 130-132 (2003).</li><li>Shin, H. K., 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=Mild+induced+hypertension+improves+blood+flow+and+oxygen+metabolism+in+transient+focal+cerebral+ischemia.">Mild induced hypertension improves blood flow and oxygen metabolism in transient focal cerebral ischemia.</a> Stroke. 39 (5), 1548-1555 (2008).</li><li>Richter, S. H., Garner, J. P., W&#252;rbel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+standardization:+cure+or+cause+of+poor+reproducibility+in+animal+experiments?.">Environmental standardization: cure or cause of poor reproducibility in animal experiments?.</a> Nat Methods. 6 (4), 257-261 (2009).</li><li>Holloway, P. 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=Both+MC1+and+MC3+receptors+provide+protection+from+cerebral+ischemia-reperfusion-induced+neutrophil+recruitment.">Both MC1 and MC3 receptors provide protection from cerebral ischemia-reperfusion-induced neutrophil recruitment.</a> Arterioscler Thromb Vasc Biol. 35, (2015).</li><li>Vandeputte, 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=Characterization+of+the+inflammatory+response+in+a+photothrombotic+stroke+model+by+MRI:+implications+for+stem+cell+transplantation.">Characterization of the inflammatory response in a photothrombotic stroke model by MRI: implications for stem cell transplantation.</a> Mol Imaging Biol. 13 (4), 663-671 (2010).</li><li>Iwae, Y., 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=Glial+cell-mediated+deterioration+and+repair+of+the+nervous+system+after+traumatic+brain+injury+in+a+rat+model+as+assessed+by+positron+emission+tomography.">Glial cell-mediated deterioration and repair of the nervous system after traumatic brain injury in a rat model as assessed by positron emission tomography.</a> J Neurotrauma. 27 (8), 1463-1475 (2010).</li><li>Morris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+of+a+water-maze+procedure+for+studying+spatial+learning+in+the+rat.">Developments of a water-maze procedure for studying spatial learning in the rat.</a> J Neurosci Methods. 11 (1), 47-60 (1984).</li><li>Mouzon, B., 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=Repetitive+mild+traumatic+brain+injury+in+a+mouse+model+produces+learning+and+memory+deficits+accompanied+by+histological+changes.">Repetitive mild traumatic brain injury in a mouse model produces learning and memory deficits accompanied by histological changes.</a> J Neurotrauma. 29 (18), 2761-2773 (2012).</li><li>Fleming, 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=Early+and+progressive+sensorimotor+anomalies+in+mice+overexpressing+wild-type+human+&#945;-synuclein.">Early and progressive sensorimotor anomalies in mice overexpressing wild-type human &#945;-synuclein.</a> J Neurosci. 24 (42), 9434-9440 (2004).</li><li>Sedelis, M., Schwarting, R. 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The authors have nothing to disclose.

Since its conception 20 years ago, the MCAo model for human stroke involving insertion of a filament has been used in a huge number of studies. This is mainly due to the fact that it mimics what happens clinically in the most common form of stroke (i.e., ischemic stroke). The striatum is more sensitive to ischemia than the cerebral cortex, and as such, the length of ischemic time will translate into whether both the striatum and the dorsolateral cortex will be affected, or just the striatum. Both infarct and rep...

Stroke is the fifth leading cause of death worldwide, with one person dying from the disease every 4 minutes. Over 800,000 Americans suffer a stroke every year, which is not only devastating for the patient, but also for their families. Stroke is the main cause of adult disability and the annual expenditure is estimated to be in the order of $ 36.5 billion1 despite very few treatment options being available.
Tissue plasminogen activator (tPA) is the only Food and Drug Administration (FDA) licensed drug for ischemic stroke. However, it is only effective if administered to patients within 3-6 hours from the onset of the stroke, and in these cases it benefits only 4% of patients2. Therefore, it is imperative that reproducible, clinically relevant animal models of stroke are used to aid in the development of potential therapeutic strategies and treatments for this disease. It is important to note that in vitro models, whilst useful in modeling certain aspects of cerebral dysfunction, are not capable of recapitulating the complex physiological interactions that occur in brain and periphery following a stroke. Consequently, in vivo models are essential.
The most common type of stroke is ischemic in origin, accounting for 87% of total strokes. Other strokes are intracerebral hemorrhage (9%) and subarachnoid hemorrhage (4%), and are caused most often by an emboli to the middle cerebral artery (MCA). This is attributable to the prominent curve at the root of MCA, which causes laminar blood flow entering the brain to become disrupted. The MCA arises from the internal carotid artery (ICA) and routes along the lateral sulcus, where it branches and projects to the basal ganglia and the lateral surfaces of the frontal, parietal and temporal lobes, including the primary motor and sensory cortex. The Circle of Willis is created by posterior cerebral arteries being connected to the cerebral arteries and the posterior communicating arteries.
The intraluminal filament or suture model of MCAo is one of the most widely used in stroke research. However, there are a couple of different variations to this model, and these are based on whether the microfilament is inserted into the external carotid artery (ECA, termed the Longa method)3, or whether it is inserted into the ICA (termed the Koizumi method)4. In Koizumi&#39;s method, the common carotid artery (CCA) on the side of the surgery must be permanently tied if the filament is removed to prevent bleeding from the incision in the CCA, while in Longa&#39;s method it is the ECA that must be permanently tied5. Here the Longa method will be used as we feel this is a far superior and a more clinically relevant surgical model of ischemic stroke. Furthermore, the use of a silicon-tipped monofilament, especially with the Longa method, produces very reproducible MCAo as opposed to the flame-blunted monofilaments, which often produce incomplete occlusion and/or subarachnoid hemorrhage6.
The intraluminal filament method can be used as a model of permanent or transient occlusion4,6. To perform the transient model, the filament is removed after a period of ischemia (e.g., 30 min, 60 min, or 2 hr), and reperfusion is allowed to happen. This model, to some extent, simulates the restoration of blood flow after spontaneous or therapeutic intervention (e.g., tPA administration) to lyse a thromboembolic clot in humans. For the permanent model, the filament is simply left in place for a period of time (e.g., 24 hr), so no reperfusion occurs. Another advantage of the intraluminal filament method is the fact that a craniotomy does not need to be performed, allowing the skull to be left intact and avoiding any changes in intracranial pressure and temperature.
In this video we demonstrate how to perform the Longa intraluminal filament method to induce MCAo and reperfusion. We also show how to perform the 18-point neurological score and determine the infarct volume using 2,3,5-triphenyltetrazalium chloride (TTC) staining.

<table border="0" cellpadding="0" cellspacing="0" width="1521">
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		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Male C57BL/6 mice</td>
			<td style="width:291px;">Jackson Laboratory, Bar Harbor, ME</td>
			<td style="width:397px;">#000664</td>
			<td style="width:397px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Ketamine Hydrochloride</td>
			<td style="width:291px;">Morris &#38; Dickson, Shreveport, LA</td>
			<td>67457-108-10</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Xylazine</td>
			<td style="width:291px;">Akorn, Inc, Lake Forest, IL</td>
			<td>NADA# 139-236</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">DC temperature control system</td>
			<td style="width:291px;">FHC, Bowdoin, ME</td>
			<td>40-90-8D</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Mini rectal thermistor probe</td>
			<td style="width:291px;">FHC, Bowdoin, ME</td>
			<td>40-80-5D-02</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Heating pad</td>
			<td style="width:291px;">FHC, Bowdoin, ME</td>
			<td>40-90-2-06</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Clippers</td>
			<td style="width:291px;">Amazon, Bellevue, WA</td>
			<td>#64800</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">70% ethanol</td>
			<td style="width:291px;">Worldwide Medical Products, Bristol, PA</td>
			<td>#51011023</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Dissecting microscope</td>
			<td style="width:291px;">Olympus, Center Valley, PA</td>
			<td>SZ40</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Iris scissors (straight)</td>
			<td style="width:291px;">Fine Science Tools, Foster City, CA</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Dumont forceps (45&#176; bent tip)</td>
			<td style="width:291px;">Fine Science Tools, Foster City, CA</td>
			<td>11297-00</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Micro vessel clip</td>
			<td style="width:291px;">Fine Science Tools, Foster City, CA</td>
			<td>18055-05</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Micro dissecting spring scissors (straight)</td>
			<td style="width:291px;">Fine Science Tools, Foster City, CA</td>
			<td>14088-10</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Retractors (blunt)</td>
			<td style="width:291px;">Fine Science Tools, Foster City, CA</td>
			<td>18200-11 (Helen used 17022-13)</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Cotton tipped applicators</td>
			<td style="width:291px;">Fisher Scientific, Waltham, MA</td>
			<td>23-400-100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Gauze sponges</td>
			<td style="width:291px;">Covidien, Mansfield, MA</td>
			<td>#9023</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">6-0 silk braided surgical suture</td>
			<td style="width:291px;">Roboz, Gaithersburg, MD</td>
			<td>SUT-1073-11</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">0.9% sodium chloride</td>
			<td style="width:291px;">Morris &#38; Dickson, Lake Forest, IL</td>
			<td>0409-4888-20</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">6-0 medium MCAO suture (silicon rubber coated monofilament)</td>
			<td style="width:291px;">Doccol Corporation, Sharon, MA</td>
			<td>6023PKRe</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Sofsilk 6-0 silicone coated braided silk</td>
			<td style="width:291px;">Covidien, Mansfield, MA</td>
			<td>SUT-14-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Carprofen</td>
			<td style="width:291px;">Pfizer, New York, NY</td>
			<td>NADA# 141-199</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Puralube</td>
			<td style="width:291px;">Dechra, Norwich, UK</td>
			<td>NDC 17033-211-38</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Physitemp temperature controller</td>
			<td style="width:291px;">Harvard Apparatus, Holliston, MA</td>
			<td>TCAT-2AC</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Heat lamp</td>
			<td style="width:291px;">Harvard Apparatus, Holliston, MA</td>
			<td>HL-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Laser doppler probe</td>
			<td style="width:291px;">AD Instruments, Colorado Springs, CO</td>
			<td>MSP100XP</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">24-well plates</td>
			<td style="width:291px;">Fisher Scientific, Waltham, MA</td>
			<td>#353226</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Phosphate buffered saline (PBS)</td>
			<td style="width:291px;">Life Technologies, Carlsbad, CA</td>
			<td>20012-050</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Single edge razor blades</td>
			<td style="width:291px;">Fisher Scientific, Waltham, MA</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">2,3,5-triphenyltetrazalium chloride (TTC)</td>
			<td style="width:291px;">Sigma Aldrich, St. Louis, MO</td>
			<td>T8877-50G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Mouse brain matrix slicer</td>
			<td style="width:291px;">Braintree Scientific, Braintree, MA</td>
			<td>BS-A 5000C</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Water bath</td>
			<td style="width:291px;">VWR, Radnor, PA</td>
			<td>#182</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">10% formalin</td>
			<td style="width:291px;">Sigma Aldrich, St. Louis, MO</td>
			<td>HT501128-4L</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Image J analysis software</td>
			<td style="width:291px;">NIH, Bethesda, MD</td>
			<td>free download</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Retractor</td>
			<td style="width:291px;">Medical Device Purchase, Newcastle, CA</td>
			<td>MP-740</td>
			<td></td>
		</tr>
	</tbody>
</table>

surgical approach for middle cerebral artery occlusion and reperfusion induced stroke in mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

Mice underwent 30-min MCAo-induced brain ischemia (Figure 1) followed by a period of reperfusion (24 hr and 1 wk are presented here, but the length of reperfusion can be varied). The mortality during MCAo was minimal (approximately 2%). Post ischemia, the mortality rate (within the first 24 hr) was around 26%.
Laser Doppler flowmetry was used to confirm blood flow perfusion in the MCA territory before and after ...

Watch this Scientific Journal Video about Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice at JoVE.com

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

The overall goal of the Longa method is to reproduce ischemia at the root of the middle cerebral artery, or MCA, since ischemic strokes in humans most commonly occur due to the occlusion of the MCA. This method can help us answer key questions in the stroke field such as how to find a suitable therapeutic target for the treatment of this disease. The main advantage of the Longa method as opposed to alternative techniques is the survival rate.There's also an increase in leukocyte-endothelial cell interactions and infarct volume with this technique. Demonstrating this procedure will be Shantel Vital, the manager for my laboratory. After inducing anesthesia in a male C57 black six mouse according to the text protocol, monitor the depth of anesthesia by a foot pinch and apply a sterile ocular ointment to prevent dryness.Then, shave the animal's neck. Place the mouse in a supine position on a temperature-regulated mat and insert a rectal probe to monitor and maintain a body temperature of 36.5 plus or minus 0.1 degrees Celsius. Then, use 70%ethyl alcohol to disinfect the skin.Next using Iris straight scissors, make a midline incision and retract the soft tissues to expose the vessels. Using Dumont forceps, dissect the common carotid artery, or CCA, and the external carotid artery, or ECA, from the surrounding tissue without damaging the vagus nerve. With 7-0 silk, make a temporary suture by tying a loose knot around the CCA.Then, make a permanent suture around the ECA and the smaller vessels extending from it by tightly ligating the vessels. Now, make a suture around the ECA proximal to the CCA bifurcation. Then, place a microvessel clip around the internal carotid artery, or ICA, and the pterygopalatine artery, or PPA.Using microdissecting spring scissors, make a small incision in the ECA as close to the permanent suture as possible and insert a 180-micrometer silicone-tipped monofilament. With the filament inserted, tighten the temporary suture around the ECA and remove the microvessel clip. Use the microdissecting spring scissors to cut the ECA between the permanent distal suture and the entry point of the filament.Guide the filament through the ICA until resistance is felt in the MCA. Refer to additional details in the text protocol. After a 30-minute occlusion period, remove the filament by using Dumont forceps to gently pull it back and secure the suture around the open end of the ECA.Remove the temporary suture around the CCA by carefully loosening the ligature to allow blood flow to resume through the CCA. Remove the retractors, and then use a continuous surgical suture to close the incision. Inject the mouse with one milliliter of saline subcutaneously for volume replenishment and with the analgesic carprofen for relief of pain and discomfort from the surgical procedure.Observe the mouse throughout the recovery from the anesthesia in a dedicated cage heated to 30 degrees Celsius and place mashed chow in a Petri dish on the floor of the cage to encourage eating. Using an 18-point scoring system, neurologically evaluate mice after the appropriate reperfusion period assessing the general, motor, sensory and proprioception scores. A higher neurological score corresponds to decreased neurological function.To measure bran infarct volume, after inducing anesthesia and monitoring the depth according to the text protocol, place the mouse in a supine position and use Iris straight scissors to cut the skin from the abdomen to the neck before cutting through the peritoneum. Using Dumont forceps, lift up the sternum and cut the ribs. Then, use two pairs of hemostats to open the left and right sides of the chest exposing the heart.Insert a 26-gauge needle attached to a five-millimeter syringe into the left ventricle and cut the right atrium. Then with room temperature normal saline, or PBS, perfuse the heart until the fluid becomes clear. With the Iris straight scissors and forceps, remove the animal's head and carefully remove the brain from the skull.Then, use a razor blade to cut off the olfactory bulb and the cerebellum so that the brain will fit into the matrix. After chilling the matrix, place the brain inside and set it on ice. Then with a razor blade, make a first cut in the brain two millimeters from the top and leave the blade in place.With a second razor blade, make another two-millimeter cut behind the first. Then, remove the first razor blade with the tissue attached. Repeat this process until all the tissue has been sliced.Place each segment in the order in which they were cut into one well of a 24-well plate containing 2%two, three, five-triphenyltetrazolium chloride, or TTC, and place the plate in a shallow water bath at 37 degrees Celsius for 20 minutes. After incubating for 10 minutes, turn over the slices. Then, place a small amount of 10%formalin into the wells of a new 24-well plate and transfer the segments into the wells of formalin in the order in which they were cut.After scanning the segments into the computer, use ImageJ analysis software to analyze the infarct size as a percentage of the whole brain slice by outlining the infarcted area to generate an area measurement. Next, measure the entire contralateral hemisphere to determine the area. Finally, divide the infarct area by the area of the contralateral hemisphere and multiply it by 100 to determine the infarct volume.As shown here, laser doppler flowmetry was used to confirm blood flow perfusion in the MCA territory before and after MCA reperfusion. When the temporary CCA tie is released after 30 minutes of ischemia for filament removal and reperfusion to occur, there is a surge in perfusion which reached just under 100%of baseline perfusion five minutes into reperfusion. Listed in this table are the neurological scores pertaining to the assessment of general, sensory, motor and proprioceptive deficits.This graph illustrates that the score for 27 mice at 24 hours was 12.56 plus or minus 0.7 and remained high one week later at 11.50 plus or minus 1.5. A higher neurological score represents decreased neurological function. Infarct volumes showed similar patterns to the neurological score at 24 hours.As presented here, 24 hours after MCAo, mice had large infarct volumes which were heightened one week post stroke. Once mastered, this technique can be performed within 15 minutes if it's done properly. While attempting this procedure, it's important to maintain the temperature at 36.5 degrees Celsius.Following this procedure, other methods can also be performed such as cecal ligation and puncture. This enables us to answer additional questions such as the effects of sepsis before and after stroke. After watching this video, you should have a good understanding of how to induce ischemia by inserting a monofilament into the middle cerebral artery and removing it to allow for reperfusion.Don't forget that working with instrumentation such as Iris scissors and forceps can be dangerous and precautions should always be taken.

surgical-approach-for-middle-cerebral-artery-occlusion-reperfusion

Research

JoVE Journal

Medicine

21.1K Views.  Louisiana State University. The overall goal of the Longa method is to reproduce ischemia at the root of the middle cerebral artery, or MCA, since ischemic strokes in humans most commonly occur due to the occlusion of the MCA. This method can help us answer key questions in the stroke field such as how to find a suitable therapeutic target for the treatment of this disease. The main advantage of the Longa method as opposed to alternative techniques is the survival rate.There's also an increase in leukocyte-endoth...