Name: an in vivo assessment of blood-brain barrier disruption in a rat model of ischemic stroke
Uploaded: 2018-03-11T14:00:00
Description: Watch this Scientific Journal Video about An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke at JoVE.com

The authors are thankful to the Vice Chancellor for Research of the Ardabil University of Medical Sciences (Ardabil, Iran) for the financial support (grant No: 9607).

All procedures were performed in accordance with the guidelines of Ardabil University of Medical Sciences Research Council for conducting animal studies (Ethical ID number: IR.ARUMS.REC.1394.08). In this visualized study, we used adult male Sprague-Dawley rats (300-350g) obtained from Pasture Institute (Tehran, Iran).
1. Anesthesia and Flowmetry
<ol>
	<li>Induce anesthesia using 4% isoflurane and maintain it with isoflurane (1-1.5%) in a mixture of nitrous oxide (70% v/v) and oxygen (30% v/v...

<ol>
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Thus far, various methods such as autoradiography and detection of the radioactive tracers24,25, immunofluorescence microscopy26,27, and EB extravasation technique20,23 have been used to evaluate the blood-brain barrier damage. EB dye is strongly able to bind to the serum albumin and is used as a tracer for detecting vascular leakage and quantifyi...

Vasogenic brain edema due to blood-brain barrier (BBB) disruption remains an important complication of the ischemic stroke and a major determinant of the survival rate in the stroke patients1,2. The blood-brain barrier (BBB), which is formed by brain capillary endothelial cells (BCECs) and composed of distinct neurovascular components (e.g., tight junctions among BCECs, pericytes, astroglial, and neuronal cells3), provides a specialized and dynamic interface between the central nervous system (CNS) and peripheral blood-circulation4,5. Insults such as ischemia-reperfusion injuries could disrupt the functional integrity of the BBB and lead to subsequent penetration of circulating leukocytes into the brain parenchyma that ultimately trigger cerebral inflammation and primary brain injuries6,7. Animal models are needed for the exact detection of the dysfunction of BBB following occurrence of a stroke. Such models are of great importance for studying underlying pathophysiological mechanisms and introducing new neuroprotective strategies. In vitro cell culture-based models of the BBB have been highly developed and used for molecular study of the BBB physiopathology8,9,10. Nevertheless, in vivo animal models, which produce ischemic damage of the BBB similar to human clinical conditions, are also very worthwhile in this regard. Quantitative detection of the extravasation of Evans blue (EB) is a well-accepted and sensitive technique that has been used for assessment of the BBB integrity and function in neurodegenerative diseases, including ischemic stroke11,12,13,14. This method is cost-effective, feasible, reproducible, and completely applicable in any experimental laboratory. Its implementation does not require advanced equipment, such as radioactive tracers15 or magnetic resonance imaging (MRI)16, that are prerequisites for other methods. In this article, we comprehensively demonstrate basic technical processes of BBB assessment using EB extravasation in rat models of ischemic stroke.

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		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Isoflurane</td>
			<td style="width:203px;">Piramal</td>
			<td style="width:225px;">AWN 34041100</td>
			<td style="width:193px;">20 - 25 &#176;C</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">2,3,5-Triphenyltetrazolium chloride (TTC)</td>
			<td>Molekula</td>
			<td>31216368</td>
			<td>4 years</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Sprague&#8211;Dawley rats&#160;</td>
			<td>Pasture Institute (Tehran, Iran)</td>
			<td></td>
			<td>300-350g</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Evans Blue&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>314-13-6</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Trichloroacetic acid&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>76-03-9</td>
			<td>2 years</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Bupivacaine HCl (0.5%)</td>
			<td>Delpharm Tours</td>
			<td></td>
			<td>below&#160; 25 &#176;C</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Bupernorphine</td>
			<td>Exir (Iran)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Sodium Carbonate</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>497-19-8</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Sodium chloride&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Di- Sodium hydrogen phosphate</td>
			<td>EMD Millipore&#160;</td>
			<td>231-448-7</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Potassium chloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>&#160;7447-40-7</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Ethanol&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">silicone(Xantopren)</td>
			<td>Heraeus</td>
			<td>EN ISO 4823</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Activator universal plus</td>
			<td>Heraeus</td>
			<td>66037445</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Micro-Dissecting forceps</td>
			<td>Stoelting</td>
			<td>52100-41</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Spring Scisors</td>
			<td>Stoelting</td>
			<td>52130-00</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Operating&#160; Scissors</td>
			<td>Roboz</td>
			<td>52140-70</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Brain matrix&#160;</td>
			<td>Stoelting</td>
			<td>51390</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Anesthesia Machine for Small Animals |</td>
			<td>&#160;Kent Scientific</td>
			<td>SS-01</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Power Lab system</td>
			<td>AD Instruments</td>
			<td>ML880</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Laser Doppler flowmeter</td>
			<td>AD Instruments</td>
			<td>ML191</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Heating feed back system</td>
			<td>Harvard Appratus</td>
			<td>72-7560</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Vascular micro clamp</td>
			<td>FineScience Tools</td>
			<td>18055-03</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Silk 5-0 suture thread</td>
			<td>Ethicon</td>
			<td>682G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Ethilon 4-0 suture thread&#160;</td>
			<td>Ethicon</td>
			<td>EH6740G</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vivo assessment of blood-brain barrier disruption in a rat model of ischemic stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

There was no significant difference in EB levels in the right hemisphere versus the left hemisphere of the sham-operated rats (1.06 &#177; 0.1 &#181;g/g and 1.1 &#177; 0.09 &#181;g/g, respectively). As shown in Figures 2A-2B, induction of transient ischemia (90 min ischemia/ 24 h reperfusion) caused a significant difference in EB levels (10.41 &#177; 0.84 &#181;g/g, p &#60;0.001) in the left hemisphere of ischemic rats, as compared to the respective hemisphere in...

Watch this Scientific Journal Video about An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke at JoVE.com

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

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Neuroscience

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