Name: surgical approach for middle cerebral artery occlusion and reperfusion induced stroke in mice
Uploaded: 2016-10-20T13:00:00.000+00:00
Duration: 10 min 30 s
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).
1. Pre-surgical Preparations
<ol>
	<li>Induce anesthesia using a combination of ketamine (150 mg/kg) and xylazine (10 mg/kg) injected intraperitoneally (i.p.). Monitor depth of anesthesia by foot pinch initially every 3 - 5 min and every 10 min once anesthesia is achieved. While the animal is under anesthesia, administer a sterile ocular ointment to prevent dryness.&#160;The initial dose of ketamine/xylazine usually lasts about 30-40 minutes.&#160; An additional dose can be administered if needed, which is verified by the animal&#8217;s response to a foot pinch.</li>
	<li>Place mice in a supine position on a temperature regulated heat mat and maintain body temperature at 36.5 &#177; 0.1 &#176;C, which is verified using a rectal probe.</li>
	<li>Shave the neck and disinfect the skin with 70% ethyl alcohol.&#160;</li>
</ol>
2. Occlusion of the MCA (Figure 1)
<ol>
	<li>Make a midline neck incision using Iris straight scissors and retract (using retractors) the soft tissues to expose the vessels.</li>
	<li>Dissect the CCA and the ECA from surrounding tissue using Dumont forceps without damaging the vagus nerve.</li>
	<li>Make a temporary suture by tying a loose knot around the CCA using 6-0 silk.</li>
	<li>Make a permanent suture around the ECA and the smaller vessels extending from it by tightly ligating the vessels (distal to the bifurcation of the CCA).</li>
	<li>Make a suture around the ECA proximal to the CCA bifurcation.</li>
	<li>Place a microvessel clip around the internal carotid artery (ICA) and the pterygopalatine artery (PPA).</li>
	<li>Make a small incision in the ECA using micro dissecting spring scissors and insert a 180 &#956;m silicon-tipped monofilament. Make the cut as close to the permanent suture as possible for easier manipulation of the filament.</li>
	<li>Tighten the temporary suture around the ECA with the filament inserted and remove the microvessel clip.</li>
	<li>Cut the ECA between the permanent, distal suture and the entry point of the filament using micro dissecting spring scissors.</li>
	<li>Guide the filament through the ICA until resistance is felt (approx. 9 - 10 &#956;m beyond the bifurcation of the CCA) in the MCA. 
	NOTE: In the event too much resistance is felt while the majority of the filament is still visible, the filament may have entered the PPA. If this happens, pull the filament back to the bifurcation, gently push forward into the ICA using Dumont forceps, and advance the filament until it can be visualized in the ICA.</li>
</ol>
3. Reperfusion
<ol>
	<li>After a 30-min occlusion period, remove the filament by gently pulling it back using Dumont forceps and secure the suture around the open end of the ECA.</li>
	<li>Remove the temporary suture around the CCA by carefully loosening the ligature using Dumont forceps and blood flow is resumed through the CCA.</li>
	<li>Close the incision with a continuous surgical suture.&#160;Closure of the skin can be accomplished by either continuous or interrupted sutures.&#160; Skin staples are also an acceptable method.&#160;</li>
	<li>Inject mice with 1 ml of saline subcutaneously as volume replenishment and with the analgesic carprofen (5 mg/Kg, s.c.) for relief of pain and discomfort from the surgical procedure, Signs that indicate additional pain relief is needed include hunched back, ungroomed coat, decreased activity, abnormal posturing, and decreased appetite.</li>
	<li>Observe mice throughout the recovery from the anesthesia in a heated 30 &#176;C cage using a heat lamp regulated using a temperature controller and place mashed chow in a petri dish on the floor of the cage to encourage eating. The mice will be housed one per cage during the reperfusion period.</li>
</ol>
4. Sham Surgery
<ol>
	<li>Subject mice to the same procedure without the monofilament insertion.</li>
</ol>
5. Post-operative Neurological Scores (Table 1; Figure 2)
<ol>
	<li>Neurologically evaluate mice after the appropriate reperfusion period using an 18-point scoring system assessing the General; Motor; Sensory; Proprioception. A higher neurological score corresponds to decreased neurological function. 
	NOTE: Mice that are judged unresponsive and unable to walk will be euthanized. Other criteria for humane euthanasia will include a weight loss of greater than 20%, respiratory distress, and infection around the surgical area. A CO2 chamber will be used for euthanasia and the physical method to confirm death will be cervical dislocation or thoracotomy.</li>
</ol>
6. Measurement of Brain Infarct Volume (Figure 3)
<ol>
	<li>Induce anesthesia using a combination of ketamine (150 mg/kg) and xylazine (10 mg/kg) injected i.p. Monitor depth of anesthesia by foot pinch initially every 3 - 5 min and every 10 min once anesthesia is achieved.</li>
	<li>Place mice in a supine position and cut the skin from the abdomen to the neck followed by the peritoneum using iris straight scissors.</li>
	<li>Lift up the sternum using Dumont forceps and cut the ribs to expose the heart.</li>
	<li>Open the left and the right side of the chest using two pairs of hemostats, exposing the heart.</li>
	<li>Insert a 26 G needle attached to a 5 ml syringe into the left ventricle and cut the right atrium. Perfuse with room temperature normal saline or phosphate buffered saline (PBS) until the fluid becomes clear (usually 3 - 5 min).</li>
	<li>Remove the skull carefully away from the brain using iris straight scissors and Dumont forceps.</li>
	<li>Cut off the olfactory bulbs and the cerebellum using a razor blade so that the brain will fit in the matrix. Use this matrix to slice the brain into even segments. Chill the matrix before use to keep the tissue cool. Place the brain into the matrix and set it on ice.</li>
	<li>Slice the brain into 2 mm coronal segments using two razor blades. Make the first cut using a razor blade beginning 2 mm from the top and leave this razor blade in place. Make another 2 mm cut behind the first. Remove the first razor blade with the tissue attached. Repeat this process until all tissue has been sliced. 
	NOTE: There should be 4 - 5 segments when finished.</li>
	<li>Place the segments into 24-well plate containing 2% 2,3,5-triphenyltetrazalium chloride (TTC), which is then placed in a shallow water bath at 37 &#176;C for 20 min. Placing each segment into an individual well helps to keep them in the order in which they were cut. Ensure that the TTC solution completely covers the tissue segments. After 10 min turn all the slices over.</li>
	<li>Place a small amount of 10% formalin into the wells of a new 24-well plate and transfer the segments to this plate in the order in which they were cut.</li>
	<li>Scan the segments into the computer and analyze the infarct size as a percentage of the whole brain slice6 using ImageJ analysis software (NIH 1.57 Image Software)6.
	<ol>
		<li>Outline the infarcted area to generate an area measurement. Next measure the entire contralateral hemisphere to determine the area. Divide the infarct area by the area of the contralateral hemisphere and multiply by 100 to determine the infarct volume.</li>
	</ol>
	</li>
</ol>

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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 reperfusion times can be varied accordingly, and this offers the researcher the ability to be able to study pathological effects associated with transient ischemic attacks (TIAs) to much larger infarcts.
Over the years modifications and troubleshooting of the filament method of MCAo has shown the importance of the procedure being performed in sterile conditions to avoid the risk of infection9. Results may vary between rodent species, strain (anatomical variations have been shown)10,11, weight, and anesthetics used, but the trends in the results presented here are likely to be reflected in other laboratories where MCAo is performed. Other critical steps to take into account are body temperature, blood pressure and blood gases. It is well established that body temperature affects neurological damage, with smaller lesions associated with hypothermia12 and more severe deficits being presented in hyperthermia13. As such, temperature should be measured and controlled, for example, via the use of animal temperature controllers with a heat pad, so as to maintain body temperature at 36.5 &#176;C. Volume replenishment and the encouragement of eating by placing soften chow at the bottom of the cage are also additional factors to observe. Blood pressure and blood gases6,14 can both easily be measured and monitored using readily available equipment from commercial suppliers.
Limitations of the MCAo technique include properly dissecting the vagus nerve from the arteries without damage and avoiding advancing the filament into the PPA, which can affect not only the ability to induce stroke effectively, but also the survival rate of the mouse. The most important significance with respect to alternative in vivo MCAo methods is the survival rate when using the Longa method described here. We have also found that this corresponds to increased leukocyte-endothelial cell interactions and increased infarct volumes, which make this model, once mastered, an excellent tool for studying potential therapeutic targets for the treatment of stroke.
Although we have not reported any here, a number of behavioral tests can also be performed such as Morris water maze, Rotarod, pole test, gait analysis, sticky label test, corner test, cylinder test, staircase test, and ladder test (see Table 2 for details and references). Sham animals have no problems with completing these behavioral tests; however, stroke animals perform these tests much less successfully. Following on from this, one area of debate is whether environmental enrichment affects not only the reproducibility of the MCAo model, but also whether it will affect the outcome of behavioral tests15. This is unclear, but it is worth standardizing this within the laboratory, and throughout the study.
There are a plethora of different tests available for measuring stroke outcome. We have presented the employment of laser doppler for blood flow measurements, an in-depth 18-point neurological assessment score, and also infarct volume measurements. However, additional options are also useful, such as imaging. We routinely employ the use of intravital fluorescence microscopy to study cellular interactions (characteristic of an inflammatory response)6 within the cerebral microcirculation in real-time, in anesthetized animals5,6,16. MCAo and reperfusion produces a heightened cerebral inflammatory response versus sham animals5,6,16. Other imaging modalities such as magnetic resonance imaging17 and positron emission tomography18 can also be used, as they provide the opportunity for longitudinal studies that are clinically relevant.
Histological analysis of brain tissue, plasma and serum samples should be routinely performed as they allow further characterization of the pathophysiological responses to stroke and the mechanisms involved, but also, and perhaps more importantly, they allow researchers to study the effects of compounds on the outcome of stroke, thus providing important data for potential therapeutic targets for this debilitating and devastating disease. Finally, we believe it is highly advisable for researchers to consider not only the most appropriate stroke model based on the requirements for their study, but also that GLP (&#34;Good Laboratory practice&#34;) is made compulsory. See Table 3 for GLP suggestions.

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><tbody><tr><td>Male C57BL/6 mice</td><td>Jackson Laboratory, Bar Harbor, ME</td><td>#000664</td><td></td></tr><tr><td>Ketamine Hydrochloride</td><td>Morris &amp;amp; Dickson, Shreveport, LA</td><td>67457-108-10</td><td></td></tr><tr><td>Xylazine</td><td>Akorn, Inc, Lake Forest, IL</td><td>NADA# 139-236</td><td></td></tr><tr><td>DC temperature control system</td><td>FHC, Bowdoin, ME</td><td>40-90-8D</td><td></td></tr><tr><td>Mini rectal thermistor probe</td><td>FHC, Bowdoin, ME</td><td>40-80-5D-02</td><td></td></tr><tr><td>Heating pad</td><td>FHC, Bowdoin, ME</td><td>40-90-2-06</td><td></td></tr><tr><td>Clippers</td><td>Amazon, Bellevue, WA</td><td>#64800</td><td></td></tr><tr><td>70% ethanol</td><td>Worldwide Medical Products, Bristol, PA</td><td>#51011023</td><td></td></tr><tr><td>Dissecting microscope</td><td>Olympus, Center Valley, PA</td><td>SZ40</td><td></td></tr><tr><td>Iris scissors (straight)</td><td>Fine Science Tools, Foster City, CA</td><td>11251-20</td><td></td></tr><tr><td>Dumont forceps (45&amp;deg; bent tip)</td><td>Fine Science Tools, Foster City, CA</td><td>11297-00</td><td></td></tr><tr><td>Micro vessel clip</td><td>Fine Science Tools, Foster City, CA</td><td>18055-05</td><td></td></tr><tr><td>Micro dissecting spring scissors (straight)</td><td>Fine Science Tools, Foster City, CA</td><td>14088-10</td><td></td></tr><tr><td>Retractors (blunt)</td><td>Fine Science Tools, Foster City, CA</td><td>18200-11 (Helen used 17022-13)</td><td></td></tr><tr><td>Cotton tipped applicators</td><td>Fisher Scientific, Waltham, MA</td><td>23-400-100</td><td></td></tr><tr><td>Gauze sponges</td><td>Covidien, Mansfield, MA</td><td>#9023</td><td></td></tr><tr><td>7-0 silk braided surgical suture</td><td>Braintree Scientific, Braintree, MA</td><td>SUT-S103</td><td></td></tr><tr><td>0.9% sodium chloride</td><td>Morris &amp;amp; Dickson, Lake Forest, IL</td><td>0409-4888-20</td><td></td></tr><tr><td>6-0 medium MCAO suture (silicon rubber coated monofilament)</td><td>Doccol Corporation, Sharon, MA</td><td>6023PKRe</td><td></td></tr><tr><td>Sofsilk 6-0 silicone coated braided silk</td><td>Covidien, Mansfield, MA</td><td>SUT-14-1</td><td></td></tr><tr><td>Carprofen</td><td>Pfizer, New York, NY</td><td>NADA# 141-199</td><td></td></tr><tr><td>Puralube</td><td>Dechra, Norwich, UK</td><td>NDC 17033-211-38</td><td></td></tr><tr><td>Physitemp temperature controller</td><td>Harvard Apparatus, Holliston, MA</td><td>TCAT-2AC</td><td></td></tr><tr><td>Heat lamp</td><td>Harvard Apparatus, Holliston, MA</td><td>HL-1</td><td></td></tr><tr><td>Laser doppler probe</td><td>AD Instruments, Colorado Springs, CO</td><td>MSP100XP</td><td></td></tr><tr><td>24-well plates</td><td>Fisher Scientific, Waltham, MA</td><td>#353226</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Life Technologies, Carlsbad, CA</td><td>20012-050</td><td></td></tr><tr><td>Single edge razor blades</td><td>Fisher Scientific, Waltham, MA</td><td>12-640</td><td></td></tr><tr><td>2,3,5-triphenyltetrazalium chloride (TTC)</td><td>Sigma Aldrich, St. Louis, MO</td><td>T8877-50G</td><td></td></tr><tr><td>Mouse brain matrix slicer</td><td>Braintree Scientific, Braintree, MA</td><td>BS-A 5000C</td><td></td></tr><tr><td>Water bath</td><td>VWR, Radnor, PA</td><td>#182</td><td></td></tr><tr><td>10% formalin</td><td>Sigma Aldrich, St. Louis, MO</td><td>HT501128-4L</td><td></td></tr><tr><td>ImageJ analysis software</td><td>NIH, Bethesda, MD</td><td>free download</td><td></td></tr><tr><td>Retractor</td><td>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 MCAo/reperfusion. Figure 4 demonstrates clearly that when the temporary CCA tie is released after 30 min of ischemia for filament removal and reperfusion to occur, there is a surge in perfusion, which reached &#60; 100% of baseline perfusion (i.e., prior to surgery) 5 min into reperfusion.
24 hr after stroke onset (prior to measurement of infarct volume) neurological scores pertaining to general, sensory, motor and proprioceptive deficits were assessed (Table 1). This scoring system is intended to provide objective (&#39;yes or no&#39;) criteria for assessment. Figure 2 shows that the score for mice (n= 27) at 24 hr was 12.56 &#177; 0.7, and remained high 1 week later (11.50 &#177; 1.5). A higher neurological score corresponds to decreased neurological function.
Infarct volumes mirrored similar patterns to the neurological score at 24 hr, i.e., greater than sham animals. 24 hr after MCAo, mice had large infarct volumes (Figure 3: 15.9 &#177; 2.6%), which were heightened 1 week post stroke (28.5 &#177; 1.9%).
<img alt="Figure 1" src="/files/ftp_upload/54302/54302fig1.jpg" /> 
Figure 1: Schematic Showing the Location of Middle Cerebral Artery Occlusion (MCAo) Surgery and Large-vessel Vasculature of the Cerebral Circulation. A. Surgical MCAo is achieved via insertion of a filament into the external carotid artery and then into the proximal middle carotid artery. B-G. Steps for inducing MCAo. Anterior communication artery (ACA); middle cerebral artery (MCA); posterior communication artery (PcomA); posterior cerebral artery (PCA); basilar artery (BA); internal carotid artery (ICA); external carotid artery (ECA); pterygopalatine artery (PPA); internal carotid artery (ICA). (Modified with permission from Smith et al., reference #5.) <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54302/54302fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54302/54302fig2.jpg" /> 
Figure 2: The 18-point Stroke Score. This comprehensive stroke score assesses functional improvements in general, and sensory, motor and proprioceptive aspects of mouse behavior following a stroke. Data are mean&#160;&#177;&#160;SEM. *P &#60; 0.05. n = 3 mice/group. The data was analyzed using ANOVA plus a Bonferroni post hoc test.&#160;(Modified with permission from Smith et al., reference #5.) <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54302/54302fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54302/54302fig3.jpg" /> 
Figure 3: Infarct Volumes of C57BL/6 mice 24 hr after MCAo. Mice were subjected to 30-min MCAo and 24 hr or 1 wk of reperfusion. A) Brains were removed, sectioned and stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC). In living tissue, dehydrogenases enzymatically reduce TTC to 1,3,5-triphenylformazaon (TPF), which is a red color, but in ischemic tissue the enzyme is not functional, so the tissue remains white. B) The graph shows an increase in infarct volume in mice having had a stroke. Data are mean&#160;&#177;&#160;SEM. **P&#160;&#60;&#160;0.002, ****P&#160;&#60;&#160;0.0001 versus sham; n = 4 mice/group. The data was analyzed using ANOVA plus a Bonferroni post hoc test.&#160;Scale bar = 1 cm (Modified with permission from Smith et al., reference #5.) <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54302/54302fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54302/54302fig4.jpg" /> 
Figure 4: Perfusion of MCA Territory Throughout MCAo and Reperfusion. Mice were subjected to 30-min MCAo, and &#60; 5 min reperfusion. Baselines were normalized to 100%. As expected, perfusion 5 min into the reperfusion period was higher than MCAo, i.e., when no perfusion of the tissue occurred. Data are mean&#160;&#177;&#160;SEM. *P &#60; 0.05. n = 3 mice/group. The data was analyzed using ANOVA plus a Bonferroni post hoc test.&#160;(Modified with permission from Smith et al., reference #5.) <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54302/54302fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/54302/54302table1.jpg" /> 
Table 1: Neurological Scoring System. One point given for a yes answer to each of the tests. Marks out of 18 7,8.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Test</td>
			<td>Brief Description</td>
			<td>What does it test for?</td>
			<td>References</td>
		</tr>
		<tr>
			<td>Morris water maze</td>
			<td>An open-field water-maze procedure where rodents learn to escape from water onto a hidden platform</td>
			<td>Spatial memory, movement control, and cognitive mapping</td>
			<td>19</td>
		</tr>
		<tr>
			<td>Rotarod</td>
			<td>A horizontal rotating rod where rodents must walk forward so as not to fall off</td>
			<td>Motor coordination, balance, and grip strength</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Pole</td>
			<td>Rodents placed on a pole and observed for sliding or falling as they make their way down into a cage</td>
			<td>Movement disorders caused by cortical damage</td>
			<td>21, 22</td>
		</tr>
		<tr>
			<td>Gait analysis</td>
			<td>As rodents cross a glass plate their paw prints are captured to examine their movements</td>
			<td>Walking patterns (paw pressure, stride length, width and frequency, toe spread, gait angle, and body rotation) and motor coordination</td>
			<td>23, 24</td>
		</tr>
		<tr>
			<td>Sticky label test</td>
			<td>Strips of tape applied to the hairless part of forepaw of the rodents to record time to contact each paw, order of contact, time of removal, and order of removal</td>
			<td>Forepaw sensitivity and sensorimotor deficits</td>
			<td>25, 26</td>
		</tr>
		<tr>
			<td>Corner</td>
			<td>Rodents placed between two boards forming a 30&#176; angle; when entering deep into the corner both sides of the vibrissae are stimulated and the rodent rears and turns back to face the open end&#160;</td>
			<td>Neurodevelopmental disorders and repetitive behaviors (monitoring turns in one direction versus the opposite direction)</td>
			<td>27, 28</td>
		</tr>
		<tr>
			<td>Cylinder</td>
			<td>Rodents are placed in a glass cylinder with the forelimb activity while rearing against of the wall is recorded</td>
			<td>Locomotor asymmetry and evaluate poststroke limb use</td>
			<td>28</td>
		</tr>
		<tr>
			<td>Staircase</td>
			<td>Rodents placed on a platform in a box with a baited double staircase; rodents are food restricted to promote movement up the stairs to collect the bait&#160;</td>
			<td>Reaching abilities for each forelimb independently requiring sensory capacities, dexterity, and motor coordination</td>
			<td>25, 29</td>
		</tr>
		<tr>
			<td>Ladder</td>
			<td>Rodents walk across a horizontal ladder to reach their cage; foot slips while walking across the ladder are recorded</td>
			<td>Foot faults during locomotion; forelimb and hindlimb function and coordination</td>
			<td>30</td>
		</tr>
	</tbody>
</table>
Table 2: Examples of Murine Behavioral Tests.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Suggestion for GLP is as follows:&#160;</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; Specific details of which model of stroke has been chosen (e.g., Longa vs. Koizumi), and the strain, age, sex, weight reported clearly.</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; Studies performed in a double-blinded fashion.</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; Power analysis reported.</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; Criteria for inclusion and exclusion in the study decided prior to the study, and reported.</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; Animals monitored daily (basic appearance, body weight and behavior) for any signs of distress, pain or sickness.</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; Reporting of animals excluded from the study.</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; Randomization of animals into groups.</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; Reporting of negative and positive results.</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; Reporting of length of surgery, anesthetic, body temperature and blood gases.</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; Reporting of environmental enrichment used, if any.</td>
		</tr>
	</tbody>
</table>
Table 3: Suggestions for Good Laboratory practice.

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Medicine

21.5K Views.  Louisiana State University. 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.

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

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 ...

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

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 ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

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 ...

Neuroscience

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 ...

Establishing a Transcranial Middle Cerebral Artery Occlusion Mice Model

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

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

Author Spotlight: Innovative Techniques and Future Directions in Stroke Research 

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

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

Occlusion of Distal Middle Artery to Develop an Ischemic Stroke Mouse Model

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic stroke patients are eligible to receive intravascular thrombolysis or mechanical thrombectomy therapy within the optimal time window. Among those stroke survivors, around two-thirds suffer neurological dysfunctions over an extended period. Establishing a stable and repeatable experimental ischemic stroke model is extremely significant for further investigating the pathophysiological mechanisms and developing effective therapeutic strategies for ischemic stroke. The middle cerebral artery (MCA) represents the predominant location of ischemic stroke in humans, with the MCA occlusion serving as the frequently employed model of focal cerebral ischemia. In this protocol, we describe the methodology of establishing the distal MCA occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6 mice. Since the occlusion site is located at the cortical branch of MCA, this model generates a moderate infarcted lesion restricted to the cortex. Neurological behavioral and histopathological characterization have demonstrated visible motor dysfunction, neuron degeneration, and pronounced activation of microglia and astrocytes in this model. Thus, this dMCAO mouse model provides a valuable tool for investigating the ischemiastroke and worth of popularization.

Author Spotlight: Establishing a Reliable Distal MCA Occlusion Model in Mice for Stroke Research

Here, we present a protocol to establish a distal middle cerebral artery occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6J mice and evaluate the subsequent neurological behavior and histopathological features.

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic ...