Name: oxygen-glucose deprivation and reoxygenation as an in vitro ischemia-reperfusion injury model for studying blood-brain barrier dysfunction
Uploaded: 2015-05-07T15:00:00
Description: Watch this Scientific Journal Video about Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction at JoVE.com

We acknowledge Scott and White Hospital Research Grants Program for financial support and Texas A&#38;M Health Science Center College of Medicine Integrated Imaging Laboratory for the use of the confocal laser microscope. We acknowledge Mr. Glen Cryer for help with manuscript editing.

1. Seeding of Endothelial Cells
<ol>
	<li>Obtain primary cultures of RBMEC&#8217;s from adult Sprague Dawley rats (or obtain them commercially).</li>
	<li>Cultivate RBMECs in 100 cm fibronectin (50 &#181;g/ml) coated petri dishes using the rat brain endothelial cell growth medium. Change the medium every two days, until confluency is reached.</li>
	<li>On reaching 80-90% confluency, gently wash the cells in 5 ml&#160;phosphate buffered saline (PBS) by swirling. The cells are then detached by exposing them to 1 ml&#160;...

<ol>
	<li>Eltzschig, H. K., Eckle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia+and+reperfusion--from+mechanism+to+translation.">Ischemia and reperfusion--from mechanism to translation.</a> Nat Med. 17 (11), 1391-1401 (2011).</li><li>Kalogeris, T., Baines, C. P., Krenz, M., Korthuis, 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=Cell+biology+of+ischemia/reperfusion+injury.">Cell biology of ischemia/reperfusion injury.</a> Int Rev Cell Mol Biol. 298, 229-317 (2012).</li><li>Yang, X., 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=Lycium+barbarum+polysaccharides+reduce+intestinal+ischemia/reperfusion+injuries+in+rats.">Lycium barbarum polysaccharides reduce intestinal ischemia/reperfusion injuries in rats.</a> Chem Biol Interact. 204 (3), 166-172 (2013).</li><li>Kaur, C., Ling, E. 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+in+hypoxic-ischemic+conditions.">Blood brain barrier in hypoxic-ischemic conditions.</a> Curr Neurovasc Res. 5 (1), 71-81 (2008).</li><li>Khatri, R., McKinney, A. M., Swenson, B., Janardhan, V. <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,+reperfusion+injury,+and+hemorrhagic+transformation+in+acute+ischemic+stroke.">Blood-brain barrier, reperfusion injury, and hemorrhagic transformation in acute ischemic stroke.</a> Neurology. 79 (13), S52-S57 (2012).</li><li>Hossmann, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+for+the+investigation+of+brain+ischemia.">Experimental models for the investigation of brain ischemia.</a> Cardiovasc Res. 39, 106-120 (1998).</li><li>Ogawa, S., Gerlach, H., Esposito, C., Pasagian-Macaulay, A., Brett, J., Stern, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+modulates+the+barrier+and+coagulant+function+of+cultured+bovineendothelium.+Increased+monolayer+permeability+and+induction+of+procoagulant+properties.">Hypoxia modulates the barrier and coagulant function of cultured bovineendothelium. Increased monolayer permeability and induction of procoagulant properties.</a> J Clin Invest. 85 (4), 1090-108 (1990).</li><li>Zulueta, J. J., Sawhney, R., Yu, F. S., Cote, C. C., Hassoun, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+generation+of+reactive+oxygen+species+in+endothelial+cellsexposed+to+anoxia-reoxygenation.">Intracellular generation of reactive oxygen species in endothelial cellsexposed to anoxia-reoxygenation.</a> Am J Physiol. 272 (5 Pt 1), L897-L902 (1997).</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>Abramov, A. Y., Scorziello, A., Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+distinct+mechanisms+generate+oxygen+free+radicals+in+neurons+and+contribute+to+cell+death+during+anoxia+and+reoxygenation.">Three distinct mechanisms generate oxygen free radicals in neurons and contribute to cell death during anoxia and reoxygenation.</a> J Neurosci. 27 (5), 1129-1138 (2007).</li><li>Gundimeda, U., 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=Green+tea+polyphenols+precondition+against+cell+death+induced+by+oxygen-glucose+deprivation+via+stimulation+of+laminin+receptor,+generation+of+reactive+oxygen+species,+and+activation+of+protein+kinase+Cepsilon.">Green tea polyphenols precondition against cell death induced by oxygen-glucose deprivation via stimulation of laminin receptor, generation of reactive oxygen species, and activation of protein kinase Cepsilon.</a> J Biol Chem. 287 (41), 34694-34708 (2012).</li><li>Mehta, S. L., Manhas, N., Raghubir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+targets+in+cerebral+ischemia+for+developing+novel+therapeutics.">Molecular targets in cerebral ischemia for developing novel therapeutics.</a> Brain Res Rev. 54 (1), 34-66 (2007).</li><li>Alluri, 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=Reactive+Oxygen+Species-Caspase-3+Relationship+in+Mediating+Blood-Brain+Barrier+Endothelial+Cell+Hyperpermeability+Following+Oxygen-Glucose+Deprivation+and+Reoxygenation.">Reactive Oxygen Species-Caspase-3 Relationship in Mediating Blood-Brain Barrier Endothelial Cell Hyperpermeability Following Oxygen-Glucose Deprivation and Reoxygenation.</a> Microcirculation. 21 (2), 187-195 (1111).</li><li>Sun, H., Breslin, J. W., Zhu, J., Yuan, S. Y., Wu, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+and+ROCK+signaling+in+VEGF-induced+microvascular+endothelial+hyperpermeability.">Rho and ROCK signaling in VEGF-induced microvascular endothelial hyperpermeability.</a> Microcirculation. 13 (3), 237-247 (2006).</li><li>Doggett, T. M., Breslin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+actin+cytoskeleton+in+live+endothelial+cells+expressing+GFP+actin.">Study of the actin cytoskeleton in live endothelial cells expressing GFP actin.</a> J Vis Exp. (57), (2011).</li></ol>

OGD-R as an in vitro model for ischemia-reperfusion injury has been well established for studying neurons10,11. There are also studies showing the effect of OGD on brain endothelial cells and alterations in permeability and TJ integrity9. However, our study shows the effect of OGD as well as reoxygenation, which is a closer representation of ischemic reperfusion injury in in vivo conditions that occur following ischemic stroke.
Hypoxic-ischemic condition...

Ischemia-Reperfusion (IR) injury is found to be the frequent cause of various debilitating complications and deaths associated with stroke, myocardial infarction, trauma, peripheral vascular disease and traumatic brain injury1,2. IR injury in cerebral vessels is an early secondary injury leading to inflammation and edema3. One of the serious complications that occurs as a result of oxidative and metabolic stress following inflammation is loss of homeostatic balance leading to free radical formation, alterations in the blood-brain barrier (BBB) tight junctions (TJs) and microvascular permeability4,5.
Currently, in vivo models used to study the effects of IR injury on the BBB include middle cerebral artery occlusion (MCAO), microembolism, and transgenic or knockout animals. However, each has its drawbacks and limitations as discussed by Hossmann6. MCAO model is used to study the effects of redox stress, changes in junctional communications of the BBB and the interactions between brain and immune cells. However, they present various technical challenges such as the need for precise microsurgical procedures and the difficulties therein. Microembolism instantaneously breaks down the BBB while use of transgenic or knockout animals to study cerebral ischemia may have challenges like gene-dependent molecular influences on&#160;infarct formation, changes in vascular anatomy and varying body weights6. Hence, in vitro models of ischemia have found increasing interest in recent times mainly due to their applicability in performing mechanistic studies for drugs. However, the results of in vitro studies may not fully represent an in vivo study and must be interpreted with caution6.
Counteractive effect of low oxygen concentrations on endothelial cell monolayers and microvascular permeability have been studied by Ogawa7. Rat brain microvascular endothelial cells (RBMECs) were used to develop the in vitro BBB. The oxygen-glucose deprivation and reoxygenation (OGD-R) technique presented in this protocol has been adapted from studies by Zulueta et al and Zhu et al8,9. We exposed brain endothelial cells to OGD-R by placing them in a hypoxia/anoxia chamber containing 0% O2, 5% CO2 and 95% N2. Cells were later assessed for alterations in TJ integrity and stress fiber formation using immunofluorescence localization and rhodamine phalloidin labeling respectively. Immunofluorescence staining for zonula occludens-1 (ZO-1) is performed to determine TJ integrity, as ZO-1 is an important scaffolding membrane bound TJ protein. Rhodamine Phalloidin labeling determines the filamentous actin (f-actin) in the cell cytoskeleton and is a clear indication of actin stress fiber formation in endothelial cells.
The goal of this method is to provide insight into developing OGD-R as an in vitro IR model for studying BBB endothelial cell TJ integrity and f-actin stress fiber formation. The results will provide information on the fate of TJ protein, ZO-1 and stress fiber formation following OGD-R. Understanding these relationships will provide an opportunity to determine the underlying molecular mechanisms that are triggered following OGD-R and develop potential therapeutic strategies to enhance the BBB disruption following OGD-R treatment.

<table border="0" cellpadding="0" cellspacing="0" width="917">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Name of Material/ Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:129px;">Catalog Number</td>
			<td style="width:251px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Proox model 110</td>
			<td style="width:167px;">Biospherix</td>
			<td style="width:129px;">Model 110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">DMEM, no glucose</td>
			<td style="width:167px;">Gibco, Life technologies</td>
			<td style="width:129px;">11966-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Rhodamine Phalloidin</td>
			<td style="width:167px;">Life technologies</td>
			<td style="width:129px;">R415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">ZO-1 Rabbit Polyclonal Antibody</td>
			<td style="width:167px;">Life technologies</td>
			<td align="right" style="width:129px;">617300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc Lab Tek II-CC 8&#160;well sterile, glass&#160;slides&#160;</td>
			<td style="width:167px;">Thermo scientific</td>
			<td align="right" style="width:129px;">177402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC-tagged anti-rabbit secondary antibody&#160;</td>
			<td style="width:167px;">Santa cruz</td>
			<td style="width:129px;">sc-2090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">DPBS 1X</td>
			<td style="width:167px;">Thermo scientific</td>
			<td style="width:129px;">SH 30028.03</td>
			<td>Any other PBS available can be used</td>
		</tr>
	</tbody>
</table>

oxygen-glucose deprivation and reoxygenation as an in vitro ischemia-reperfusion injury model for studying blood-brain barrier dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Cells cultured on fibronectin precoated Nunc II chamber slides were subjected to OGD-R by placing in a Biospherix ProOx model 110 chamber. After subjecting cells to OGD-R, they were processed&#160;for ZO-1 junctional staining using immunofluorescence technique as shown in Figure 2 and cytoskeletal assembly indicating F-actin stress fiber formation using rhodamine phalloidin stain label as shown in Figure 3. Control cells that were not subjected to OGD-R showed continual junctional integr...

Watch this Scientific Journal Video about Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction at JoVE.com

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

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