We thank Professor Olena Severynovska of the Department of Physiology, Faculty of Biology, Ecology, and Medicine, Oles Honchar Dnipro University, Dnipro, Ukraine for her support and helpful contributions to our discussions. The data obtained are part of R.K.&#8217;s PhD thesis.

The Animal Care Committee of the Ben-Gurion University of the Negev, Israel approved all treatment and testing procedures used in this protocol.
1. Preparation of Rats for the Experimental Procedure
NOTE: Select adult male Sprague-Dawley rats weighing 300-350 g.
<ol>
	<li>House the rats, four per cage, in a vivarium at 22 &#176;C and 40% humidity, with a reversed 12 h light/dark cycle (lights off at 8:00 a.m.) and unlimited access to food and water.</li>
	<li>Randomly assign the rats to two groups, namely an MCAO group (N = 24) and a Sham group (N = 19).</li>
</ol>
2. Preparation of Rats for Surgery
<ol>
	<li>To prepare the rats for the modified MCAO procedure, anesthetize each rat for 30 min with a mixture of isoflurane (4% for induction, 2% for surgery, and 1.3% for maintenance) in 24% oxygen (2 L/min) in an induction chamber and allow it to breathe spontaneously29,30. 
	NOTE:&#160;Stimulate pedal withdrawal reflex by pinching the skin between the toes and/or toe pads using&#160;blunt forceps. Consider the depth of anesthesia adequate when the reflex disappears.</li>
	<li>Maintain the core body temperature at 37 &#176;C with a heating plate.</li>
	<li>Measure the body temperature of all rats through a probe placed in the rat&#8217;s rectum before beginning the surgical procedure.</li>
	<li>Keep the body temperature constant (37 &#176;C) for all rats to minimize any hypothermic effect on the neurological outcome and any neurological injury.</li>
	<li>Apply artificial tears ointment to both of each animal&#8217;s eyes while they lie on the prep table.</li>
	<li>Shave the neck of each rat and disinfect the skin with 70% alcohol chlorhexidine (70% alcohol and 0.5% chlorhexidine gluconate). Repeat the disinfection step two more times.&#160;</li>
	<li>Cover the rats with sterilized surgical drapes.</li>
</ol>
3. Surgery (the MCAO Technique)
NOTE: Perform surgery as described by Boyko et al.28 and use the instruments provided by McGarry et al.29 and Ulu&#231; et al.30.
<ol>
	<li>Perform a ventral midline incision and dissect the superficial fascia.</li>
	<li>Carefully perform a sharp and blunt dissection within the three triangle-shaped muscle (the sternohyoid, the digastric, and the sternomastoid muscle) to identify the carotid arteries (the ECA, the ICA, and the common carotid artery [CCA]).</li>
	<li>Carefully dissect and expose the right CCA and the ICA.</li>
	<li>Separate the right CCA and ICA from the vagus nerve. 
	NOTE: Upon identifying the carotid arteries as stipulated in step 3.2, the ICA would be recognized as one-half of the CCA branches alongside the ECA.</li>
	<li>Insert a catheter (heat-blunted or silicone-coated 4-0 nylon) monofilament directly through the ICA, ~18.5-19 mm from the bifurcation point of the right CCA into the circle of Willis until reaching a mild resistance, to occlude MCA. 
	NOTE: The heat-blunted filament and the silicone-coated 4-0 nylon play the same role and are the more preferred monofilaments in recent times, given that they provide better occlusion than the plain nylon thread28,30.</li>
	<li>Tie a 4-0 silk suture around the ICA just above the right CCA (pterygopalatine artery) bifurcation to block the ICA proximal to the filament insertion point permanently and distal to it temporarily.</li>
	<li>Tie the ICA around the intraluminal thread to fasten the silk suture to prevent bleeding.</li>
	<li>Subject the sham-operated rats to the same surgical procedure as the MCAO rats but insert a nylon thread instead28. 
	NOTE: The nylon thread, like the silicon-coated nylon, occludes the ICA, but it is not as effective as the latter.</li>
	<li>Close the wound on the rat&#8217;s neck after occluding the MCA, turn off the anesthesia, and place the rat in an incubator under observation until it wakes up. 
	NOTE: The need for the proximal ligation is to occlude the ICA, and that of the additional distal ligation is to reduce the bleeding around the filament and secure it in place. Also, the rat should wake up a few minutes after its wound has been closed.</li>
</ol>
4. Post-surgical Recovery
<ol>
	<li>Administer 5 mL of 0.9% saline solution to each rat intraperitoneally, immediately after surgery, to prevent dehydration.</li>
	<li>Administer analgesia unconditionally on the first day post-operation; use meloxicam 1 mg/kg SQ q24 h.&#160;Give diluted dipyrone (0.5 g dissolved in 400 mL of drinking water) to rats showing symptoms of pain during the first 3 days.</li>
	<li>Sacrifice any rats with seizures (seizures are caused by the increased intracranial pressure from cerebral edema or cerebral hemorrhage28).</li>
</ol>
5. Neurological Severity Score31
NOTE: This procedure is performed by two observers who do not take part in the surgical proceedings; they test the neurological deficits and grade motor deficits on a cumulative score of 0-432. The evaluation of this score may be performed at different time intervals; in this investigation, it was performed 50 min, 24 h, 7, 15, and 30 days post-surgery. Find below the steps for evaluating the NSS. Although not a necessity in this situation, this score is required to ascertain stroke in rodents in order to administer treatment.
<ol>
	<li>Place the rat on a ceramic floor and allow it to move about freely for 1 min.</li>
	<li>Gently pull the rat backward by the tail and grade as follows.
	<ol>
		<li>Grade the rat with score 0 for no neurological deficit.</li>
		<li>Grade the rat with score 1 for forelimb flexion.</li>
		<li>Grade the rat with score 2 for contralateral weak forelimb grip.</li>
		<li>Grade the rat with score 3 for circling to the paretic side when pulled by the tail.</li>
		<li>Grade the rat with score 4 for spontaneous circling32. 
		NOTE: If more than one of the reactions are observed, preference is given to the action with a higher score.</li>
	</ol>
	</li>
</ol>
6. Determination of the Infarct Volume (Hhistologic Examination)
<ol>
	<li>Measurement of the infarct volume 
	NOTE: Perform this procedure as described previously33,34. Measure brain infarct volume using 2,3,5-triphenyltetrazolium chloride (TTC) staining 24 h after reperfusion.
	<ol>
		<li>Euthanize five rats from each group, 24 h after the last NSS, by exposing them to an isoflurane overdose in an induction chamber.</li>
		<li>Decapitate the rats and quickly isolate their brains using small scissors and forceps.</li>
		<li>Wash the isolated brains in 0.9% saline.</li>
		<li>Examine bleeding points on the brains to exclude the mice that underwent subarachnoid hemorrhage at the circle of Willis.</li>
		<li>Place each brain on a clean glass slide on a -20 &#176;C ice pack and then place them in a -20 &#176;C fridge for 5 min to make the brain easier to slice.</li>
		<li>Take the glass slide with the brain on it out of the -20 &#176;C fridge, put it back on the -20 &#176;C ice pack, and dissect the frontal pole and the cerebellum with blade and forceps.</li>
		<li>Slice the brain sections horizontally into 2 mm thickness with a blade to produce six slices.</li>
		<li>Prepare a 0.05% TTC solution by adding 1.25 g of TTC powder to 500 mL of normal saline solution prior to sacrifice, transfer the solution to a 24-well plate (1 mL per well) covered in foil, and store it at 4 &#176;C. 
		NOTE: TTC and tissue stained with TTC are light sensitive.</li>
		<li>Using forceps, transfer the brain slices to the 24-well plate containing the TTC solution (one slice per well) and stretch out the slices in the solution.</li>
		<li>Incubate the plate content at 37 &#176;C in a shallow water bath for 30 min.</li>
		<li>Aspirate the TTC solution from the plate with a pipette, wash them with a brainwashing fluid, and incubate at room temperature for 30 min.</li>
		<li>Place the slices in the order in which they were cut on a laboratory glass and examine the segments with a scanner.</li>
		<li>Analyze the infarct size as a percentage of the whole brain slice, using ImageJ analysis software, based on visual identification.</li>
	</ol>
	</li>
	<li>Analysis of the infarct volume35
	<ol>
		<li>Quantify the infarct volume by using a standard image analysis software (ImageJ) and analyze the infarct brain volume as a percentage of the whole brain size33.</li>
		<li>Place the brain slices on glass microscope slides and scan them with an optical scanner at a high resolution (1,600 x 1,600 dpi) for an adequate analysis.</li>
		<li>Crop the images and standardize the scale for all images, using the metric ruler included in the scanned image.</li>
		<li>Measure the area of marked pallor in six consecutive 2 mm coronal sections using a Wand (tracing) tool and freehand selection on the ImageJ 1.37v software36.</li>
		<li>Calculate the indirect infarct volume using the following formula37.</li>
	</ol>
	</li>
</ol>
7. Measurement of Brain Edema38
NOTE: Brain edema was measured 24 h after the last MCAO.&#160;&#160;Euthanize animals with severe neurological deficit interfering with eating and/or drinking for at least three days, more than 20% weight loss, hemiplegia or seizures.
<ol>
	<li>Assess the magnitude of the edema of the right hemisphere by using the summation of coronally sliced areas to calculate the volumes of the right and left hemispheres in arbitrary units (pixels). 
	NOTE: Use the ImageJ 1.37v software for this calculation, after optical scanning (resolution: 1,600 x 1,600 dpi). Select the area of interest and use the Measure function from the Analysis menu. Macros were used in our investigation.</li>
	<li>Express the brain edema area as a percentage of the standard areas in the unaffected contralateral hemisphere.</li>
	<li>Calculate the degree of swelling using the equation developed previously39.</li>
</ol>
8. Behavioral Paradigms
<ol>
	<li>Perform behavioral tests between days 30 and 33 after surgery in a closed, quiet, and light-controlled room.</li>
	<li>Assign investigators blinded to all experimental procedures to videotape all behavioral tests with a commercially available program.</li>
	<li>Sucrose preference test40,41
	<ol>
		<li>Place the rats in individual cages in the same room as where they are housed during the dark cycle.</li>
		<li>For the next 24 h, place one bottle of 100 mL of 1% (w/v) sucrose solution in each cage with the rat to be tested and allow for the adaptation of the rat.</li>
		<li>After 24 h, deprive the rats of food and water for 12 h by removing the bottles.</li>
		<li>Then after 12 h, place two bottles in each cage for 4 h, one containing 100 mL of tap water and another containing 100 mL of sucrose solution (1% [w/v]).</li>
		<li>Record the amount of sucrose solution and water consumed by rats in milliliters. Calculate the affinity to sucrose preference as follows.</li>
	</ol>
	</li>
	<li>Porsolt forced swim test42 
	NOTE: The Porsolt forced swim test was carried out as described in a previous protocol published by Zeldetz et al.40 and Boyko et al.42. The principle of this test states that, when rats are forced to swim in a restricted area from whence, they cannot escape, they eventually become immobile, ceasing any attempts to escape the water43,44. This test was carried out in a different room during the dark cycle.
	<ol>
		<li>Place each rat in a vertical plexiglass cylinder (height: 100 cm; diameter: 40 cm) containing 80 cm of water at 25 &#176;C for 15 min for habituation.</li>
		<li>Take the rat out and allow it to dry for 15 min in a heated enclosure (32 &#176;C).</li>
		<li>Return the rat to its home (original) cage.</li>
		<li>Repeat step 8.4.1 24 h later, this time for the investigation, for 5 min.</li>
		<li>Videotape the 5 min test and calculate the total duration of immobility during that period.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

One of the ways in which the MCAO technique presented here could be deemed safer than the original MCAO model is illustrated by the fact that the ECA and its branches, including the occipital artery, the terminal lingual, and the maxillary artery, are not compromised when occluding the MCA via the ICA. The original MCAO model&#39;s offset of the ECA (and its branches), by distally dissecting and coagulating them46, causes impaired mastication, owing to a compromise to the vascular supply to masticating muscles47. The destruction of the muscle could eventually cause the release of additional computational fluid dynamics. Contrary to the original MCAO technique in which access to the MCA occurs through the ECA, the technique described here is modified for stroke, with no occurrence of blood flow interruption in the ECA and its tributaries.
The most important arguments for preferring the novel MCAO model to the original MCAO rest in its ability to reduce variability in brain edema, infarct volume, and weight changes, significantly, as well as decrease MCAO-related mortality. Mortality in MCAO procedures is an important factor48; a 20% mortality rate is deemed reasonable49,50. The mortality rates (20% for original MCAO, 12.5% for novel MCAO, and 0% for control) in the current investigation were all within range of the acceptable rate, but the novel MCAO technique fared better than the original MCAO.
Rats undergoing the novel MCAO procedure lost less weight right after surgery and gained more weight by the end of the investigation than rats subjected to the original MCAO technique28. More weight loss and less weight gain in rats subjected to the original MCAO could result from ligating the ECA during surgery, which causes distal hypoperfusion in the facial, lingual, and maxillary arteries, as well as ischemia-related damages to the muscles that support mastication. If mastication is impaired, oral intake lessens, which, when coupled with catabolism, could account for weight loss, and in the long run, for a poor neurological outcome, morbidity, and death43. Access to the MCA via the ICA does not require interfering with the ECA and its branches. Thus, no impairments are triggered during the novel MCAO procedure, and no weight, morbidity, or mortality problems are experienced by rats undergoing the procedure.
The tests used to assess underlying depressive factors, such as depressive behavior, anhedonia, immobility, and learning and memory impairment, are standard procedures applied in animal models of depression51,52. A behavioral test, like the Porsolt forced swim test, is commonly affected by unusual motor abilities following MCAO. Here, this test was applied from days 30 to 33 after the surgical procedure to ascertain that the induction of PSD in the modified MCAO rats did not overly affect their motor abilities. According to the results, rats from the experimental group showed a comparable total escape behavior to that of sham-control rats. MCAO rats had significantly more escape failures, a significantly raised duration of immobility, and a reduced preference for sucrose when compared with sham-operated animals. This would suggest that this MCAO technique is a capable alternative to the original MCAO method.
The difference between inclusion and exclusion of rats in the MCAO procedure for a broader experimental evaluation depends very much on the outcome of the operation. With the MCAO-related PSD-inducing model, some unwanted side-effects of MCAO could be curtailed, leaving room for the inclusion of smaller, and possibly fragile rats in the MCAO procedure. The animal model of MCAO presented here provides a scenario for lessening unintended outcomes after MCAO-induced PSD because it has the potential to reduce variability in weight changes, brain edema, and infarct volume, as well as MCAO-related deaths. This technique could potentially serve as a tool for assessing future PSD therapies and provide preclinical data on the efficacy of therapeutic substances, as well as monitor other factors modifying modalities on the clinical outcome of stroke and PSD.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/58875">A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats</a>. The Authors section was updated.
One of the author names was updated from:
Dmitri Frank
to
Dmitry Frank

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/58875">A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats</a>. The Authors section was updated.

Erratum: A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

Stroke is fourth on the list of death-perpetrating diseases in the United States1,2,3, while it causes the majority of disabilities in adults in developed countries4; this makes stroke a leading contender among the world&#39;s most significant health issues. Normalcy in stroke-surviving patients is rare, with about 15%-40% of survival victims suffering permanent disability, 20% requiring institutional care 3 months after stroke onset5, and about a third of 6-month survivals needing others to help them live through each day6. Stroke reportedly also accounts for the rising national health expenditures7. Estimates from the American Heart Association has stroke-related costs in the United States at over $50 billion in 20108.
Not only does stroke cause individuals&#39; long-term damages, but some survivors tend to suffer emotional and behavioral disorders, such as dementia, fatigue, anxiety, depression, delirium, and aggression9,10,11,12,13,14. The most recurrent psychological sequel after a stroke is post-stroke depression (PSD), diagnosed in about 40%-50% of survivals15,16,17. Stroke-induced depression results in increased morbidity and mortality18,19,20,21,22. The pathophysiology of PSD is not known completely, but it is apparently caused by multiple factors and is linked to disability, cognitive impairment, and lesion site23.
The rat model of focal brain ischemia, created by MCAO, is the most widespread animal model of stroke24,25,26,27. In demonstrating the induction of PSD in rats by occluding the MCA via the ICA, techniques that minimize mortality and variability in the MCAO model are employed28.
The primary objective of this protocol is to outline the steps for inducing PSD in rats by occluding the MCA via the ICA, a modified model of MCAO, which reduces mortality and the outcome of variability28. Specific aims include performing neurological and histological examinations (determining the neurological severity score [NSS], the volume of the infarction zone, and brain edema) to verify the efficacy of MCAO and using behavioral tests to examine the influence of this MCAO procedure on the development of emotional disorders, mainly PSD.

<table><tbody><tr><td>Absorbent pad</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Black lusterless perspex box</td><td>-</td><td>-</td><td>(120 cm &amp;times; 60 cm &amp;times; 60 cm), divided into a 25% central zone and the surrounding border zone</td></tr><tr><td>Bottles</td><td>Techniplast</td><td>ACBT0262SU</td><td>150 mL bottles filled with 100 mL of water and 100 mL 1%(w/v) sucrose solution</td></tr><tr><td>Electric Shock</td><td>Heat System Ultasonic Inc.</td><td>-</td><td>-</td></tr><tr><td>Horizon-XL</td><td>Mennen Medical Ltd</td><td></td><td></td></tr><tr><td>Imaging System</td><td>Kodak</td><td>-</td><td>For imaging and quantification</td></tr><tr><td>Monofilament</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Paper towels</td><td>Pharmacy</td><td>-</td><td>Dry towels used for keeping rats dry after immersing them in water</td></tr><tr><td>Pexiglass cylinder</td><td>-</td><td>-</td><td>a 100 cm tall and 40 cm in diameter cylinder used for carrying out the forced swim test</td></tr><tr><td>Purina Chow</td><td>Purina</td><td>5001</td><td>Rodent laboratory chow given to rats, mice and hamster is a life-cycle nutrition that has been used in biomedical researc for over 5 decades. Provided to rats ad libitum in this experiment</td></tr><tr><td>Rat Cages</td><td>Techniplast</td><td>2000P</td><td>Conventional housing for rodents. Was used for housing rats throughout the experiment</td></tr><tr><td>Scanner&amp;nbsp;</td><td>Canon CanoScan</td><td>4200F</td><td>-</td></tr><tr><td>Video Camera</td><td>ETHO-VISION (Noldus)</td><td>-</td><td>Digital video camera for high definition recording of rat behavior under open field test</td></tr></tbody></table>

a middle cerebral artery occlusion technique for inducing post-stroke depression in rats

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of all ischemic stroke patients suffer from PSD, a disorder considered to be an ischemic stroke-related precursor for increased death and degradation in health. The pathophysiology of PSD is still obscure. To study the mechanism of development and occurrence of PSD further, and to find out a therapy, we attempted to develop a new protocol that requires occluding the middle cerebral artery (MCA) via the internal carotid artery (ICA) in rats. This protocol describes a model of PSD induced in rats through the middle cerebral artery occlusion (MCAO). Also used in the experiment are the Porsolt forced swim test and the sucrose preference test to confirm and evaluate the depressive mood of the rats under investigation. Rather than inserting the catheter through the external carotid artery (ECA), as stipulated for the original procedure, this MCAO technique has the monofilament passing directly through the ICA. This MCAO technique was developed a few years ago and leads to a reduction in mortality and variability. It is generally accepted that the criteria used are preferred in the selection of biological models. The data obtained with this protocol show that this model of MCAO could be a way of inducing PSD in rats and could potentially lead to the understanding of the pathophysiology and the future development of new drugs and other neuroprotective agents.

Histological findings (Table 1) revealed a statistically significant infarct volume as a percentage of total brain (p &#60; 0.0001) post-MCAO when compared to animals in the sham-control group. Also reported was a statistically significant brain edema when the evaluation from the experimental group (p &#60; 0.0003) was put side by side with that of the sham-control group.
The NSS scores obtained, as represented in Table 2, show lower neurological performances in the experimental group (MCAO) compared to higher figures for the sham-control group following Mann-Whitney tests: p &#60; 0.001 after 50 min, p &#60; 0.05 after 24 h, and p &#60; 0.05 after 7 days.
Findings from the sucrose preference evaluations revealed that MCAO rats also consumed a significantly less amount of sucrose (p &#60; 0.0001, Figure 2A) and had a longer immobility duration (p &#60; 0.0001, Figure 2B) compared to the sham-operated rats.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58875/58875fig1.jpg" /> 
Figure 1: Graphic demonstration of the protocol timeline. The various tests run on rats at different times are shown on the scheme: MCAO = middle cerebral artery occlusion at the beginning of the experiment; NSS = neurological severity score, 50 min, 24 h, and 7 and 30 days post-MCAO; and behavioral tests (sucrose preference and Porsolt forced swim tests) from days 30 to 33 post-MCAO. This figure has been modified from Ifergane et al.45. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58875/58875fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58875/58875fig2.jpg" /> 
Figure 2: Sucrose preference test performed from days 30 to 33 with post-MCAO (n = 16) and sham-control rats (n = 14). Percentage (%) of sucrose preference. MCAO rats consumed less sucrose (p &#60; 0.0001) than the sham-control rats, with a significant difference in sucrose consumption between the two groups shown in the figure. MCAO = middle cerebral artery occlusion. All data represent the group mean &#177; SEM. This figure has been modified from Ifergane et al.45. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58875/58875fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58875/58875fig3.jpg" /> 
Figure 3: Porsolt forced swim test performed from days 30 to 33 post-MCAO (n = 16) and post-sham procedure (n = 14). Immobility duration (in seconds). The immobility time in the forced swim test was significantly longer in the MCAO group than in the sham group (p &#60; 0.0002). MCAO = middle cerebral artery occlusion. All data represent the group mean &#177; SEM. This figure has been modified from Ifergane et al.45. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58875/58875fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Histological Findings</td>
			<td>Middle cerebral artery occlusion group </td>
			<td>Sham-operated group</td>
		</tr>
		<tr>
			<td>Total brain (Infarct volume)</td>
			<td>8.8% &#177; 6.5</td>
			<td>0.3% &#177; 0.1</td>
		</tr>
		<tr>
			<td>Brain Edema</td>
			<td>10.2% &#177; 4.6</td>
			<td>2.6% &#177; 1.2</td>
		</tr>
		<tr>
			<td colspan="2">All data represent group mean &#177; S.E.M</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Histological findings for infarct volume and brain edema. MCAO = middle cerebral artery occlusion (n = 5); sham (n = 5).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Neurological Severity Score</td>
			<td>Middle cerebral artery occlusion group</td>
			<td>Sham-operated group</td>
		</tr>
		<tr>
			<td>50 min post-surgery</td>
			<td>2.75 &#177; 0.14</td>
			<td>0.0 &#177; 0.0</td>
		</tr>
		<tr>
			<td>24 h post-surgery</td>
			<td>3.2 &#177; 0.15</td>
			<td>0.0 &#177; 0.0</td>
		</tr>
		<tr>
			<td>7 days post-surgery</td>
			<td>0.91 &#177; 0.2</td>
			<td>0.0 &#177; 0.0</td>
		</tr>
		<tr>
			<td colspan="2">All data represent group mean &#177; S.E.M</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Neurological severity score (NSS) for MCAO and sham-operated rats. MCAO = middle cerebral artery occlusion (n = 16); sham (n = 14). This table is taken from Ifergane et al.45.

Watch this Scientific Journal Video about A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats at JoVE.com

A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

Here we present a protocol to induce post-stroke depression in rats by occluding the middle cerebral artery via the internal carotid artery. We use the Porsolt forced swim test and the sucrose preference test to confirm and evaluate induced depressive moods.

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of ...

a-middle-cerebral-artery-occlusion-technique-for-inducing-post-stroke

Nanyang Technological University

Research

JoVE Journal

Behavior

10.2K Views.  Ben-Gurion University of the Negev. Ons protocol stelt een nieuwe mediale cerebrale slagader occlusie techniek bij ratten om te helpen bij het bestuderen van het mechanisme van post-stroke depressie ontwikkeling en het vinden van een meer efficiënte therapie. Het belangrijkste voordeel van onze techniek is de mogelijkheid om de variabiliteit in gewichtsveranderingen, hersenoedeem en infarctvolume te verminderen en om proceduregerelateerde sterfgevallen te minimaliseren. Na bevestiging van een gebrek aan reactie op staart snuif...

Video: A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

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

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

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

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

Neuroscience

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 ...

Medicine

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

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.

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 ...

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 ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...

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. ...

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

Transient Middle Cerebral Artery Occlusion Model of Stroke

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

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

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

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

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 ...

Single-Step Surgery for Inducing Cerebral Ischemic Coma in Rats

Cerebral Ischemic Coma Model Induced by Modified Four-Vessel Occlusion

Coma caused by cerebral ischemia is the most serious complication of cerebral ischemia. Four-vessel occlusion can establish a cerebral ischemic coma model for disease research and drug development. However, the commonly used four-vessel occlusion method mainly involves inserting an electrocoagulation pen into the bilateral pterygoid foramen of the first cervical vertebra behind the neck to electrocoagulate the vertebral arteries. This process carries the risk of incomplete electrocoagulation, bleeding, and damage to the brainstem and spinal cord. Twenty-four hours after surgery, re-anesthetized rats undergo carotid artery ligation in front of the neck. Two surgeries expose the rats to a higher risk of infection and increase the experimental period. In this study, during a single surgical procedure, an anterior cervical incision was used to locate the key site where the vertebral artery penetrates the first cervical vertebra. The bilateral vertebral arteries were electrocauterized under visual conditions, while the bilateral common carotid arteries were separated to place loose knots. When the limbs of the rats began twitching, the bilateral common carotid arteries were quickly ligated to induce ischemic coma. This method can avoid the risk of infection caused by two surgical operations and is easy to perform with a high success rate, providing a useful reference for relevant practitioners.

Author Spotlight: Enhancing Cerebral Ischemia Research with a Simplified Rat Model

This protocol describes the process for inducing a cerebral ischemic coma model using a modified four-vessel occlusion method.

Coma caused by cerebral ischemia is the most serious complication of cerebral ischemia. Four-vessel occlusion can establish a cerebral ischemic coma model ...

A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

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