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/pubmed?term=((Ischemia+and+reperfusion--from+mechanism+to+translation%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">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/pubmed?term=((Cell+biology+of+ischemia%2Freperfusion+injury%5BTitle%5D)+AND+%22Int+Rev+Cell+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((Lycium+barbarum+polysaccharides+reduce+intestinal+ischemia%2Freperfusion+injuries+in+rats%5BTitle%5D)+AND+%22Chem+Biol+Interact%22%5BJournal%5D)">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/pubmed?term=((Blood+brain+barrier+in+hypoxic-ischemic+conditions%5BTitle%5D)+AND+%22Curr+Neurovasc+Res%22%5BJournal%5D)">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/pubmed?term=((Blood-brain+barrier%2C+reperfusion+injury%2C+and+hemorrhagic+transformation+in+acute+ischemic+stroke%5BTitle%5D)+AND+%22Neurology%22%5BJournal%5D)">Blood-brain barrier, reperfusion injury, and hemorrhagic transformation in acute ischemic stroke.</a> Neurology. 79 Suppl 1. (13), S52-S57 (2012).</li>
<li>Hossmann, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+models+for+the+investigation+of+brain+ischemia%5BTitle%5D)+AND+%22Cardiovasc+Res%22%5BJournal%5D)">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/pubmed?term=((Hypoxia+modulates+the+barrier+and+coagulant+function+of+cultured+bovineendothelium.+Increased+monolayer+permeability+and+induction+of+procoagulant+properties%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">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/pubmed?term=((Intracellular+generation+of+reactive+oxygen+species+in+endothelial+cellsexposed+to+anoxia-reoxygenation%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22J+Ethnopharmacol%22%5BJournal%5D)">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/pubmed?term=((Three+distinct+mechanisms+generate+oxygen+free+radicals+in+neurons+and+contribute+to+cell+death+during+anoxia+and+reoxygenation%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Green+tea+polyphenols+precondition+against+cell+death+induced+by+oxygen-glucose+deprivation+via+stimulation+of+laminin+receptor%2C+generation+of+reactive+oxygen+species%2C+and+activation+of+protein+kinase+Cepsilon%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">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/pubmed?term=((Molecular+targets+in+cerebral+ischemia+for+developing+novel+therapeutics%5BTitle%5D)+AND+%22Brain+Res+Rev%22%5BJournal%5D)">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/pubmed?term=((Reactive+Oxygen+Species-Caspase-3+Relationship+in+Mediating+Blood-Brain+Barrier+Endothelial+Cell+Hyperpermeability+Following+Oxygen-Glucose+Deprivation+and+Reoxygenation%5BTitle%5D)+AND+%22Microcirculation%22%5BJournal%5D)">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/pubmed?term=((Rho+and+ROCK+signaling+in+VEGF-induced+microvascular+endothelial+hyperpermeability%5BTitle%5D)+AND+%22Microcirculation%22%5BJournal%5D)">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/pubmed?term=((Study+of+the+actin+cytoskeleton+in+live+endothelial+cells+expressing+GFP+actin%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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><tbody><tr><td>Proox model 110</td><td>Biospherix</td><td>Model 110</td><td></td></tr><tr><td>DMEM, no glucose</td><td>Gibco, Life technologies</td><td>11966-025</td><td></td></tr><tr><td>Rhodamine Phalloidin</td><td>Life technologies</td><td>R415</td><td></td></tr><tr><td>ZO-1 Rabbit Polyclonal Antibody</td><td>Life technologies</td><td>617300</td><td></td></tr><tr><td>Nunc Lab Tek II-CC 8&amp;nbsp;well sterile, glass&amp;nbsp;slides&amp;nbsp;</td><td>Thermo scientific</td><td>177402</td><td></td></tr><tr><td>FITC-tagged anti-rabbit secondary antibody&amp;nbsp;</td><td>Santa cruz</td><td>sc-2090</td><td></td></tr><tr><td>DPBS 1X</td><td>Thermo scientific</td><td>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

17.9K 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

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

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 ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Neuroscience

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is characterized by an interruption in blood supply to the brain leading to cell death and cognitive dysfunction. During and after ischemic stroke, blood-brain barrier (BBB) dysfunction facilitates injury progression and contributes to poor patient recovery. Current BBB models primarily include endothelial monocultures and double co-cultures with either astrocytes or pericytes.
Such models lack the ability to fully imitate a dynamic brain microenvironment, which is essential for cell-to-cell communication. Additionally, commonly used BBB models often contain immortalized human endothelial cells or animal-derived (rodent, porcine, or bovine) cell cultures that pose translational limitations. This paper describes a novel well-insert-based BBB model containing only primary human cells (brain microvascular endothelial cells, astrocytes, and brain vascular pericytes) enabling the investigation of ischemic brain injury in vitro. 
The effects of oxygen-glucose deprivation (OGD) on barrier integrity were assessed by passive permeability, transendothelial electrical resistance (TEER) measurements,and direct visualization of hypoxic cells. The presented protocol offers a distinct advantage inmimicking the intercellular environment of the BBB in vivo, serving as a more realistic in vitro BBB model for developing new therapeutic strategies in the setting of ischemic brain injury.

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Here, we describe the method for establishing a triple cell culture model of the blood-brain barrier based on primary human brain microvascular endothelial cells, astrocytes, and pericytes. This multicellular model is suitable for studies of neurovascular unit dysfunction during ischemic stroke in vitro or for the screening of drug candidates.

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is ...

Biochemistry

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

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.

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.

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

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) ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

Fluorescent Dye-Based Visualization of Blood-Brain Barrier Breakdown in Mice

Evaluation of Blood-Brain Barrier Breakdown in a Mouse Model of Mild Traumatic Brain Injury

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is a complex process influenced by several factors, such as the concentration of dyes in the blood, permeability of brain vessels, duration of dye extravasation, and reduction in dye concentration in the tissue due to degradation and diffusion. In a mild traumatic brain injury model, exposure to blast-induced shock waves (BSWs) triggers BBB breakdown within a limited time window. To determine the precise sequence of BBB breakdown, Evans blue, and fluorescein isothiocyanate-dextran were injected intravascularly and intracardially into mice at various time points relative to BSW exposure. The distribution of dye fluorescence in brain slices was then recorded. Differences in the distribution and intensity between the two dyes revealed the spatiotemporal sequence of BBB breakdown. Immunostaining of the brain slices showed that astrocytic and microglial responses correlated with the sites of BBB breakdown. This protocol has broad potential for application in studies involving different BBB breakdown models.

A method was developed to visualize dye extravasation due to blood-brain barrier (BBB) breakdown by administering two fluorescent dyes to mice at different time points. The use of glycerol as a cryoprotectant facilitated immunohistochemistry on the same sample.

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is ...

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

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