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;of warm 0.25% trypsin- ethylenediaminetetraacetic acid (EDTA) solution, equilibrated to 37 &#176;C.</li>
	<li>Incubate the cells at 37 &#176;C for 2-5 min&#160;until the cells are detached and dispersed. 
	NOTE: Tap the culture dish to detach the cells. View cells under the microscope to confirm complete detachment of cells from the surface of the dish.</li>
	<li>Add 5 ml&#160;complete media to the petri dish in order to neutralize trypsin. Pipette out the medium containing detached cells and collect it into a 15 ml&#160;centrifuge tube. .</li>
	<li>Centrifuge the media containing endothelial cells at 220 x g for 5 min.</li>
	<li>Aspirate the supernatant and preserve the pellet containing cells.Suspend the pellet into 3-5 ml&#160;fresh rat brain endothelial cell growth medium by gently mixing it up and down with pipette Cells will be then be counted using automated cell counter or a hemocytometer.</li>
	<li>Transfer the cell suspension into fibronectin (50 &#956;g/ml) precoated 8&#160;well sterile chamber slide system, 0.7 cm2/well with a seeding density ranging between 10,000-15,000 cells per well. Grow the cells at 37 &#176;C until confluence is achieved 
	NOTE: For performing western blots or other experiments,cells can be grown in 10 cm cell culture dishes or special dishes as required by the experiment.</li>
</ol>
2. Oxygen and Glucose Deprivation-Reoxygenation In Vitro Model
NOTE: The following protocol has been adapted from Zulueta et al. 1997; Zhu H et al. 20128,9.
<ol>
	<li>Use the hypoxia cell culture system to study the effects of of OGD-R on RBMECs in (see Figure 1). Setup and calibrate the hypoxia cell culture system before beginning the experiment, according to manufacturer&#8217;s instructions.</li>
	<li>Remove the confluent chamber slides (step 1.8) from the 37&#160;&#176;C incubator. Replace the complete medium in the chamber slide with deoxygenated, no glucose, Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) and placed in hypoxia chamber with 95%, N2 and 5% CO2 for 2 h&#160;at 37 &#176;C, to represent OGD condition.</li>
	<li>Move the cells back to the incubator with 95% O2, 5% CO2 at 37 &#176;C&#160;and provided with fresh rat brain endothelial cell complete medium and incubate for another 1 hr at 37 &#176;C. 
	NOTE: This step represents a reoxygenation situation.</li>
	<li>Use the chamber slides for immunofluorescence localization and rhodamine phalloidin labeling (see section 3).</li>
</ol>
3. Immunofluorescence Localization of ZO-1 and f-actin Labeling Using Rhodamine Phalloidin
<ol>
	<li>Expose the chamber slides containing RBMEC monolayers to 100 &#956;l of opti-MEM/reduced serum media/well for 1 hr. Wash the chamber slides 3 times in 100 &#956;l of phosphate-buffered saline (PBS, pH 7.0-7.2).</li>
	<li>Fix the cells using 100 &#956;l of 4% paraformaldehyde in PBS (pH 7.0-7.2) for 15 min and wash the chamber slides for 3 more times in PBS (pH 7.0-7.2).</li>
	<li>Permeabilize the cells using 100 &#956;l of 0.5% Triton X-100 in PBS, (pH 7.0-7.2) for another 15 min. Block with 100 &#956;l of 2% bovine serum albumin (BSA) in PBS for an hour. After this step either stain/label cells either for ZO-1 or f-actin.
	<ol>
		<li>For immunofluorescence staining incubate the cells with an anti-rabbit primary antibody against ZO-1 in 1:150, prepared in 2% BSA-PBS for O/N at 4 &#176;C. Wash the cells 3 times in PBS,(pH 7.0-7.2). Incubate with 100 &#956;l of Fluorescein isothiocyanate (FITC)-tagged anti-rabbit secondary antibody for 1 hr at RT.</li>
		<li>For Rhodamine Phalloidin labeling, following blocking, expose the cells to 100 &#956;l of rhodamine phalloidin in 1:50 dilution, prepared in 2% BSA-PBS, for 20 min.</li>
		<li>Wash the cells from immunofluorescence staining and rhodamine phalloidin labeling in PBS, (pH 7.0-7.2). Mount the chambers using mounting media containing anti-fade reagent with DAPI.</li>
	</ol>
	</li>
	<li>Visualize the cells using a 60 X water immersion lens and cells are scanned in a single optical plane under a confocal microscope.</li>
</ol>

<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>

The authors have nothing to disclose.

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 ...

oxygen-glucose-deprivation-reoxygenation-as-an-vitro-ischemia

Research

JoVE Journal

Medicine

17.8K Views.  Texas A&M University Health Science Center College of Medicine. 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. 

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

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...