Name: surgical approach for middle cerebral artery occlusion and reperfusion induced stroke in mice
Uploaded: 2016-10-20T13:00:00
Description: Watch this Scientific Journal Video about Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice at JoVE.com

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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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. 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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.

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			<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>
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		<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>
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			<td height="22" style="height:22px;width:436px;">Clippers</td>
			<td style="width:291px;">Amazon, Bellevue, WA</td>
			<td>#64800</td>
			<td></td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<td height="22" style="height:22px;width:436px;">Gauze sponges</td>
			<td style="width:291px;">Covidien, Mansfield, MA</td>
			<td>#9023</td>
			<td></td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...