Name: a middle cerebral artery occlusion technique for inducing post-stroke depression in rats
Uploaded: 2019-05-22T13:00:00
Description: Watch this Scientific Journal Video about A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats at JoVE.com

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 wate...

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 mastic...

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.

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			<td height="21" style="height:21px;width:216px;">Black lusterless perspex box</td>
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			<td>(120 cm &#215; 60 cm &#215; 60 cm), divided into a 25% central zone and the surrounding border zone</td>
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			<td>150 ml bottles filled with 100 ml of water and 100 ml 1%(w/v) sucrose solution</td>
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			<td height="21" style="height:21px;width:216px;">Electric Shock</td>
			<td>Heat System Ultasonic Inc.</td>
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			<td height="21" style="height:21px;width:216px;">Imaging System</td>
			<td style="width:71px;">Kodak</td>
			<td style="width:119px;">-</td>
			<td>For imaging and quantification</td>
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			<td height="21" style="height:21px;width:216px;">Monofilament</td>
			<td style="width:71px;">-</td>
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			<td>-</td>
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			<td height="21" style="height:21px;width:216px;">Paper towels</td>
			<td style="width:71px;">Pharmacy</td>
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			<td>Dry towels used for keeping rats dry after immersing them in water</td>
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			<td height="21" style="height:21px;width:216px;">Pexiglass cylinder</td>
			<td style="width:71px;">-</td>
			<td style="width:119px;">-</td>
			<td>a 100 cm tall and 40 cm in diameter cylinder used for carrying out the forced swim test</td>
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			<td height="21" style="height:21px;width:216px;">Purina Chow</td>
			<td style="width:71px;">Purina</td>
			<td align="right" style="width:119px;">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>
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			<td height="42" style="height:42px;width:216px;">Rat Cages</td>
			<td style="width:71px;">Techniplast</td>
			<td style="width:119px;">2000P</td>
			<td>Conventional housing for rodents. Was used for housing rats throughout the experiment</td>
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			<td height="21" style="height:21px;width:216px;">Scanner&#160;</td>
			<td>Canon CanoScan</td>
			<td style="width:119px;">4200F</td>
			<td>-</td>
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			<td height="63" style="height:63px;width:216px;">Video Camera</td>
			<td style="width:71px;">ETHO-VISION (Noldus)</td>
			<td style="width:119px;">-</td>
			<td>Digital video camera for high definition recording of rat behavior under open field test</td>
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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 obtaine...

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 ...

Our protocol proposes a new medial cerebral artery occlusion technique in rats to help study the mechanism of post-stroke depression development and to find a more efficient therapy. The main advantage of our technique is its ability to reduce the variability in weight changes, brain edema, and infarct volume, and to minimize procedure-related deaths. After confirming a lack of response to tail pinch in a male 300 to 350-gram Sprague Dawley rat, place the animal on a 37 degrees Celsius heating plate and use a rectal probe for monitoring the core body temperature.Apply ointment to the animal's eyes and shave the hair from the neck. Then, disinfect the exposed skin with 70 percent alcohol chlorhexidine, and cover the rat with the sterile surgical drape before performing the occlusion according to standard techniques. To obtain the neurological severity score, place an occluded rat onto a flat surface and allow the animal to move about freely.After one minute, gently pull the rat backward by the tail and grade the animal's gait response. To measure the infarct volume, scan the prepared stained brain slices at a 1, 600 by 1, 600 dots per inch resolution and crop and standardize the scale for all of the images. Then, load the standardized images into an appropriate image analysis software program and use a tracing tool to measure the area of marked pallor in six consecutive two-millimeter slices to determine the infarct size as a percentage of the whole brain slice.30 to 33 days after the surgery, transfer the animals to a closed, quiet, light-controlled room in individual cages, and place a bottle containing 100 milliliters of 1%sucrose solution into each cage for 24 hours. The next day, remove the bottles as well as the food and drinking water for 12 hours. At the end of the deprivation period, place one bottle containing 100 milliliters of 1%sucrose solution and one bottle containing 100 milliliters of tap water into each cage for four hours.Then, record the volume of the sucrose solution and water consumed to allow calculation of the affinity to sucrose preference for each animal. For Porsolt's forced-swim test, 30 to 33 days after the surgery, first place each rat in a vertical plexiglass cylinder containing 80 centimeters of 25 degrees Celsius water for 15 minutes. At the end of the habituation period, allow the rat to dry for 15 minutes in the heated enclosure at 32 degrees Celsius before returning the animal to its home cage.The next day, return the rat to the cylinder for a final five minutes, recording the five-minute test to allow calculation of the total duration of immobility during that period. Histological analysis reveals a statistically significant infarct volume as a percentage of the total brain volume post-MCAO when compared to animals in the sham control group. A statistically-significant brain edema is also observed in the experimental animals compared to the sham control group.MCAO animals demonstrate lower neurological performances compared to sham-operated animals at 50 minutes, 24 hours, and seven days post-surgery. MCAO rats also consume a significantly lower amount of sucrose and exhibit a longer immobility during Porsalt's forced-swim tests compared to the sham-operated animals. This is an important model for behavioral stroke studies and could serve as a tool for assessing future therapies or providing critical preclinical data on the efficacy of therapeutic substances.

Research

JoVE Journal

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