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>
	<li>Jin, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protecting+against+cerebrovascular+injury:+contributions+of+12/15-lipoxygenase+to+edema+formation+after+transient+focal+ischemia.">Protecting against cerebrovascular injury: contributions of 12/15-lipoxygenase to edema formation after transient focal ischemia.</a> Stroke. 39 (9), 2538-2543 (2008).</li><li>Lo, E. H., Dalkara, T., Moskowitz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms,+challenges+and+opportunities+in+stroke.">Mechanisms, challenges and opportunities in stroke.</a> Nat Rev Neurosci. 4 (5), 399-415 (2003).</li><li>Tam, S. J., Watts, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connecting+vascular+and+nervous+system+development:+angiogenesis+and+the+blood-brain+barrier.">Connecting vascular and nervous system development: angiogenesis and the blood-brain barrier.</a> Annu Rev Neurosci. 33, 379-408 (2010).</li><li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+use+of+H102+peptide-loaded+dual-functional+nanoparticles+in+the+treatment+of+Alzheimer's+disease.">The potential use of H102 peptide-loaded dual-functional nanoparticles in the treatment of Alzheimer's disease.</a> J Control Release. , (2014).</li><li>Obermeier, B., Daneman, R., Ransohoff, R. 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A., Pourseif, M. M., Omidi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+transport+machineries+and+targeted+therapy+of+brain+diseases.">Blood-brain barrier transport machineries and targeted therapy of brain diseases.</a> Bioimpacts. 6 (4), 225-248 (2016).</li><li>Kaya, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnesium+sulfate+attenuates+increased+blood-brain+barrier+permeability+during+insulin-induced+hypoglycemia+in+rats.">Magnesium sulfate attenuates increased blood-brain barrier permeability during insulin-induced hypoglycemia in rats.</a> Can J Physiol Pharmacol. 79 (9), 793-798 (2001).</li><li>Pasban, E., Panahpour, H., Vahdati, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+oxygen+therapy+does+not+protect+the+brain+from+vasogenic+edema+following+acute+ischemic+stroke+in+adult+male+rats.">Early oxygen therapy does not protect the brain from vasogenic edema following acute ischemic stroke in adult male rats.</a> Sci Rep. 7 (1), 3221 (2017).</li><li>Haghnejad Azar, A., Oryan, S., Bohlooli, S., Panahpour, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-Tocopherol+Reduces+Brain+Edema+and+Protects+Blood-Brain+Barrier+Integrity+following+Focal+Cerebral+Ischemia+in+Rats.">Alpha-Tocopherol Reduces Brain Edema and Protects Blood-Brain Barrier Integrity following Focal Cerebral Ischemia in Rats.</a> Med Princ Pract. 26 (1), 17-22 (2017).</li><li>Belayev, L., Busto, R., Zhao, W., Ginsberg, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+blood-brain+barrier+permeability+following+middle+cerebral+artery+occlusion+in+rats.">Quantitative evaluation of blood-brain barrier permeability following middle cerebral artery occlusion in rats.</a> Brain Res. 739 (1-2), 88-96 (1996).</li><li>Bodsch, W., Hossmann, K. 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(48), (2011).</li><li>Hungerhuber, E., Zausinger, S., Westermaier, T., Plesnila, N., Schmid-Elsaesser, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+bilateral+laser+Doppler+fluxmetry+and+electrophysiological+recording+during+middle+cerebral+artery+occlusion+in+rats.">Simultaneous bilateral laser Doppler fluxmetry and electrophysiological recording during middle cerebral artery occlusion in rats.</a> J Neurosci Methods. 154 (1-2), 109-115 (2006).</li><li>Panahpour, H., Nouri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-Ischemic+Treatment+with+candesartan+protect+from+cerebral+ischemic/reperfusioninjury+in+normotensive+rats.">Post-Ischemic Treatment with candesartan protect from cerebral ischemic/reperfusioninjury in normotensive rats.</a> Int J Pharm Pharm Sci. 4 (4), 286-289 (2012).</li><li>Panahpour, H., Dehghani, G. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+tight+junction+permeability+and+ischemic+stroke.">Blood-brain barrier tight junction permeability and ischemic stroke.</a> Neurobiol Dis. 32 (2), 200-219 (2008).</li><li>Zhu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baicalin+reduces+the+permeability+of+the+blood-brain+barrier+during+hypoxia+in+vitro+by+increasing+the+expression+of+tight+junction+proteins+in+brain+microvascular+endothelial+cells.">Baicalin reduces the permeability of the blood-brain barrier during hypoxia in vitro by increasing the expression of tight junction proteins in brain microvascular endothelial cells.</a> J Ethnopharmacol. 141 (2), 714-720 (2012).</li><li>Kucuk, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+losartan+on+the+blood-brain+barrier+permeability+in+long-term+nitric+oxide+blockade-induced+hypertensive+rats.">Effects of losartan on the blood-brain barrier permeability in long-term nitric oxide blockade-induced hypertensive rats.</a> Life Sci. 71 (8), 937-946 (2002).</li><li>Uyama, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+vascular+permeability+in+the+gerbil+brain+after+transient+ischemia+using+Evans+blue+fluorescence.">Quantitative evaluation of vascular permeability in the gerbil brain after transient ischemia using Evans blue fluorescence.</a> J Cereb Blood Flow Metab. 8 (2), 282-284 (1988).</li><li>Kleinig, T. 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The authors have nothing to disclose.

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.

<table border="0" cellpadding="0" cellspacing="0" width="829">
	<tbody>
		<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>
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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...

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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 ...

The overall goal of this procedure is to present a highly reproducible method of an in vivo assessment of the blood-brain barrier disruption in a react model of ischemic stroke. This is accomplished by induction of ischemic stroke using intraluminal filament for middle cerebral artery occlusion in the left side of the brain. The next step of the procedure is cannulation of the jugular vein external branch and injection of Evans Blue dye at the end of the middle cerebral artery occlusion and beginning of the reperfusion period.Twenty-four hours later under deep anesthesia, the animal's circulation system is washed out from Evans Blue and the brain is removed. Then two cerebral hemispheres are separated, homogenized, and centrifuged, and ultimately supernatants of tissues is used to measure Evans Blue concentration with a spectrophotometer. Anesthesia and flowmetry.Induce anesthesia using 4%isoflurane and maintain it with isoflurane 1-1.5%in a mixture of 70%nitrous oxide and 13%oxygen for the duration of the surgery. Place anesthetized animal in the prone posture on feedback control heating blanket and maintain the body temperature at normal physiological range by means of a rectal probe connected to this heating unit. Shave surgery region in the left side of the skull and apply Betadine solution using a gauze pad to disinfect the skin.Replace with a sterile pad containing 70%ethanol and repeat both steps with three cycles. Make a one centimeter long skin incision on the skull extending from the lateral canthus of the left eye to the base of the left ear and dissect left temporalis muscle to expose the skull. Then cut a small hole at five millimeter lateral and one millimeter posterior to the bregma to ease placement of the tip of the Doppler flowmeter pencil probe.Turn the rat from prone to supine position and then put the laser pencil probe into the previously drilled skull hole to monitor the regional cerebral blood flow. Record each animal's baseline regional cerebral blood flow and consider it as 100%Induction of the focal cerebral ischemia. Shave the neck region and disinfect the skin with Betadine solution and 70%ethanol.Inject 0.2 milliliter of 0.5%bupivacaine subcutaneously in the surgery site for analgesia. Make a two centimeter long surgical incision in the ventral surface of the neck to access the left common carotid artery. Isolate the common carotid artery from the neighboring fascia and the vagus nerve to access bifurcation of the external carotid artery and the internal carotid artery.Ligate permanently either the left common carotid artery or the external carotid artery employing a 5-0 sac suture and dissect internal carotid artery to the level of the pterygopalatine artery. Loosely place a 5-0 sac suture around common carotid artery and then temporarily clamp internal carotid artery with a vascular micro clip. Make a small incision on the common carotid artery prior to previously placed loose tie and then insert a 4-0 silicone-coated nylon suture into the luminal space of internal carotid artery and tighten the suture around the common carotid artery to secure the nylon suture and prevent blood leaking.Remove the vascular micro clip from the internal carotid artery. Then advance silicone-coated intraluminal filament until observing a marked decline in the regional cerebral blood flow that indicates occlusion of the middle cerebral artery origin. Notably, medial cerebral artery occlusion is started where a decrease in the regional cerebral blood flow of more than 80%is detected.In this model, we temporarily block the pterygopalatine artery using a micro clamp to guide the filament correctly into the atrial path. Note, this step is very important for the success of the medial cerebral artery occlusion. Jugular vein cannulation and Evans Blue injection.Make a one centimeter longitudinal incision in the neck to the left side of the midline and then bluntly dissect away superficial fascia to access the external branch of the left jugular vein. Then permanently ligate the cornual end of the jugular vein with 5-0 sac suture. Loosely place two ties around the vein and then temporarily clamp the cardiac end of the jugular vein with vascular micro clamp.Make a small incision on the jugular vein with two sutures and then insert heparinized serum-filled catheter into the luminal space of the vein and advance it approximately 10 millimeters. Afterward, tighten the ligatures around the vein to secure the catheter and prevent bleeding. Inject small amount of serum to avoid collapsing of the vein.At the end of ischemic period, that is 90 minutes, start reperfusion by withdrawing the intraluminal suture. At the beginning of reperfusion period, slowly inject Evans Blue dye one milliliter per kilogram of 2%solution in saline over a five-minute period. Subsequently, wash the cannula by injection of 0.5 milliliter normal saline and withdraw the injection cannula from the vein.Note the color change of the tissue after Evans Blue injection. Finally, suture the neck and head incisions and place the animal in a recovery cage. Assessment of the blood-brain barrier permeability.After 24 hours of reperfusion, deeply anesthetize animal with sodium thiopental. Then open thoracic cavity by making a small hole beneath the sternum. Make a small opening in the right atrium and inject 250 milliliters pre-warmed saline through the left ventricle for 15 minutes to wash Evans Blue away from the circulation until the normal saline which is left out from the atrium becomes colorless.Immediately remove the brain from the skull and place in the brain matrix. Dissect olfactory bulb and cerebellum and then using a clean razor blade, separate the right hemisphere of the brain from the left along the midline. Weigh each hemisphere and homogenize them separately in 2.5 milliliter phosphate buffered saline and then mix it with 2.5 milliliters of 16%trichloroacetic acid for two minutes using a vortex machine.Centrifuge brain samples for 30 minutes and allow them to cool down in the fridge for 10 minutes. Use the supernatants to estimate the Evans Blue absorbance by a spectrophotometer at 610 nanometers. Representative results.In sham-operated animals, Evans Blue concentration of tissues prepared for both hemispheres of the brain are very small and there is no significant difference in comparison with each other. Induction of ischemia over 90 minutes followed by 24 reperfusion caused a significant increase in Evans Blue levels in the lesioned hemisphere of the brain in ischemic rats. Collectively, these findings indicate that under normal conditions, Evans Blue cannot readily cross the blood-brain barrier into cerebral parenchyma and cerebral ischemic insults induced the extravasation of Evans Blue through an in-house permeability of blood-brain barrier.The photograph shows the brain in the sham-operated and ischemic animals. Note, the intensity of Evans Blue extravasation in the brain tissue that is blue color arises from extent of the blood-brain barrier disruption in the lesioned hemisphere.Conclusion. In vivo assessment of the blood-brain barrier allows researcher to study possible pathophysiological mechanisms of ischemia-induced vasogenic brain edema and to find new therapeutic interventions.This technique is simple and reliable. A researcher needs to take into account some technical points to further enhance the performance of the technique and ensure upon its accuracy. Constant recording of the regional cerebral blood flow with laser Doppler flowmeter and using of silicone-coated filament can increase the MCAO success rate and reduce the mortality rate.dosage and time point of injection are two essential parameters for obtaining reliable results due to the dynamic nature of blood-brain barrier following ischemic insults.

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11.2K Views.  Ardabil University of Medical Sciences. The overall goal of this procedure is to present a highly reproducible method of an in vivo assessment of the blood-brain barrier disruption in a react model of ischemic stroke. This is accomplished by induction of ischemic stroke using intraluminal filament for middle cerebral artery occlusion in the left side of the brain. The next step of the procedure is cannulation of the jugular vein external branch and injection of Evans Blue dye at the end of the middle cerebral artery occlusion and b...