Bringhurst Meats (Berlin, NJ) is acknowledged for their genuine help in providing the swine eyeballs.

All protocols were carried out in compliance with the policies of the Institutional Animal Care and Use Committee where applicable.
1. Preparation
<ol>
	<li>Prepare the stabilization media with 75% Dulbecco&#39;s Modified Eagle Medium (DMEM), 25% Hanks&#39; Balanced Salt Solution (HBSS). Mix well, aliquot and store at -20 &#176;C until use.</li>
	<li>Prepare the Phosphate Buffered Saline (PBS) and sterilize by autoclaving. Store the solution at room temperature. Warm the PBS to 37 &#176;C before the experiment (5 ml per well).</li>
	<li>Prepare 10%, 20% and 30% sucrose in PBS. Filter all the solutions individually over separate 0.22 &#956;m filters for sterilization. Save the solutions at 4 &#176;C.</li>
	<li>Prepare the histamine stock solution in PBS to a concentration of 90 mM. Sterilize the solution by filtering over a 0.22 &#181;M filter. Aliquot and save the solution at -80 &#176;C. Avoid multiple freeze-and-thaw cycles.</li>
	<li>Make fresh 4% paraformaldehyde (PFA) in PBS before the experiment. Place it on ice. 
	Caution: Keep PFA in a fume hood and wear appropriate protective equipment.</li>
	<li>Thaw the stabilization medium (20 ml per retina). Add Penicillin-Streptomycin to a final concentration of 100 U/ml. Warm part of the medium (15 ml per retina) to 37 &#176;C in incubator before the experiment. Leave the rest of the medium on ice.</li>
	<li>Place the HBSS (10 ml per retina) on ice.</li>
</ol>
2. Retinal Organotypic Culture
<ol>
	<li>Transfer the samples to a tissue culture hood. Open the eyecup by cutting around the lens. Using a brush, gently remove the vitreous humor without pulling on the retina. Gently detach the retina from the cut edge with a brush to expose the optic disc. Severe the optic nerve with a razor and release the retina.</li>
	<li>Transfer the retina into a Petri dish and carefully rinse the retina once using cold HBSS. Place the sample in HBSS on ice. Dissect as many retinas out as needed by repeating Step 2.1 and this step.</li>
	<li>Cut the retina in half symmetrically with a razor. Gently transfer samples to a 6-well plate with 3 ml of stabilization media per well. Allow equilibration for 30 min at 37 &#176;C in an incubator with the atmosphere containing 5% CO2.</li>
	<li>Prepare the following reagents during the incubation at Step 2.3: Dilute the histamine stock solution to the desired concentration (450 &#956;M) in warm medium. Dissolve LXA4 (stored under Argon) into the medium to a final concentration of 250 ng/ml. 
	NOTE: When measuring the effect of LXA4, one half from a single retina is used for histamine alone, while the other half is used for histamine with LXA4.</li>
	<li>Aspirate the stabilization medium carefully and add the prepared medium to each well (3 ml per well). Incubate the samples at 37 &#176;C for 1 hr in an incubator with the atmosphere containing 5% CO2.</li>
	<li>Rinse the samples once in warm, sterile PBS.</li>
</ol>
3. Prepare the Samples for Immunostaining
<ol>
	<li>Aspirate PBS from the plates. Add 4% PFA (3 ml per well) and incubate for 15 min at room temperature.</li>
	<li>Quickly aspirate the PFA. Rinse the samples once using PBS. Transfer the samples to 10% sucrose (5 ml per well) and incubate for 2 to 4 hr at 4 &#176;C.</li>
	<li>Transfer the samples to a 30% sucrose (5 ml per well) and incubate overnight at 4 &#176;C. Mix one part of the commercial freezing medium with two parts of 20% sucrose to make the working freezing medium. Leave it at 4 &#176;C overnight or longer until the air bubbles disappear.</li>
	<li>Transfer the samples to the working freezing medium and allow equilibration for 5 min at room temperature. Trim each retina into rectangles (roughly 3 mm by 5 mm).</li>
	<li>Carefully transfer the samples into the working freezing media contained in a cylindrical container (about 1 cm radius x 2 cm height) devised from aluminum foil. Ensure that the retinal slices are vertical (oriented to provide a cross-section of the retina upon sectioning) and freeze them in liquid nitrogen. Save the blocks at -80 &#176;C until use.</li>
	<li>Section each block on a cryostat into 14 &#956;m thick slices and mount the sections on glass slides. Store the slides at -80 &#176;C until use.</li>
</ol>
4. Immunostaining
<ol>
	<li>Wash the sections with 5% sucrose phosphate buffer (SPB, 5% sucrose in PBS, pH 7.4) once. Block the non-specific binding sites by incubating the sections in blocking buffer (5% SPB with 10% normal goat serum and 0.5% Triton X-100) for 1 hr at room temperature.</li>
	<li>Incubate the sections (on slides) with the appropriate primary antibody (GFAP or MAP2, diluted at 1:500 in blocking buffer) for 1 hr. Aspirate the solution. Wash three times in 5% SPB. For staining the endogenous IgG, skip this step.</li>
	<li>Incubate the sections with the corresponding secondary antibodies (Cyanine3/FITC conjugated donkey anti-mouse/anti-rabbit IgG, diluted at 1:800 in the blocking buffer) for 1 hr.</li>
	<li>Wash the sections thoroughly with 5% SPB three times. Prepare the samples for microscopy by mounting them in mounting medium containing DAPI and covering with coverslips.</li>
	<li>Using a laser confocal microscope, capture images through a 20X lens under identical parameters such as laser intensity, gain value, dwell time, etc. across groups. Set the excitation/emission wavelengths for the blue channel (to detect DAPI) as 408/447 nm, for the red channel (to detect Cyanine3) as 561/785 nm and for the green channel (to detect FITC) as 488/525 nm.</li>
</ol>
5. Image Analysis
<ol>
	<li>Quantify percentage of leaky blood vessels according to the following criteria12: Include only blood vessels with endothelial cell nuclei within the plane of section in the count; consider a blood vessel leaky if it shows a gradient of immunostaining surrounding the vessel.</li>
	<li>To determine the expression level, select regions of interest and measure the mean grey value using the image analysis software.</li>
	<li>To measure the width of the process, choose an area of the section where the processes are distinct (the outer nuclear layer, ONL, for example). Then choose an optical field where such processes are visible and continuous. Set the scale bar according to the microscopic setting, draw a line across the process and perform the measurement.</li>
	<li>To analyze the continuity of the process, select regions of interest, apply the filter called variance which enhances the edges in the image by replacing each pixel with its neighborhood variance, automatically adjust the contrast and brightness, count the lines and normalize the count by the area.</li>
	<li>Calculate statistical significances using a two-tailed Student&#39;s t-test. *, P &#60; 0.05; **, P &#60; 0.01; ***, P &#60; 0.001. Plot the result as Mean &#177; SEM.</li>
</ol>

<ol>
	<li>Cunha-Vaz, J., Bernardes, R., Lobo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-retinal+barrier.">Blood-retinal barrier.</a> J Ophthalmol. 21, 3 (2011).</li><li>Kim, J. 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=Blood-neural+barrier:+intercellular+communication+at+glio-vascular+interface.">Blood-neural barrier: intercellular communication at glio-vascular interface.</a> J Biochem Molec Biol. 39, 339-345 (2006).</li><li>Kumagai, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+transport+in+brain+and+retina:+implications+in+the+management+and+complications+of+diabetes.">Glucose transport in brain and retina: implications in the management and complications of diabetes.</a> Diabetes Metab Res Rev. 15, 261-273 (1999).</li><li>Runkle, E. A., Antonetti, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-retinal+barrier:+structure+and+functional+significance.">The blood-retinal barrier: structure and functional significance.</a> Methods Molec Biol. 686, 133-148 (2011).</li><li>Takata, K., Hirano, H., Kasahara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+of+glucose+across+the+blood-tissue+barriers.">Transport of glucose across the blood-tissue barriers.</a> Int Rev Cytol. 172, 1-53 (1997).</li><li>Goncalves, A., Ambrosio, A. F., Fernandes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+claudins+in+blood-tissue+barriers+under+physiological+and+pathological+states.">Regulation of claudins in blood-tissue barriers under physiological and pathological states.</a> Tissue Barriers. 1, 24782 (2013).</li><li>Patton, N., 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=Retinal+image+analysis:+concepts,+applications+and+potential.">Retinal image analysis: concepts, applications and potential.</a> Prog Ret Eye Res. 25, 99-127 (2006).</li><li>Di Marco, L. Y., 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=Vascular+dysfunction+in+the+pathogenesis+of+Alzheimer's+disease--A+review+of+endothelium-mediated+mechanisms+and+ensuing+vicious+circles.">Vascular dysfunction in the pathogenesis of Alzheimer's disease--A review of endothelium-mediated mechanisms and ensuing vicious circles.</a> Neurobiol Dis. 82, 593-606 (2015).</li><li>Nagele, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-reactive+autoantibodies+prevalent+in+human+sera+increase+intraneuronal+amyloid-beta(1-42)+deposition.">Brain-reactive autoantibodies prevalent in human sera increase intraneuronal amyloid-beta(1-42) deposition.</a> J Alzheimer's Dis: JAD. 25, 605-622 (2011).</li><li>Goldwaser, E., Acharya, N., Nagele, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrovascular+and+blood-brain+barrier+compromise:+A+mechanistic+link+between+vascular+disease+and+Alzheimer's+disease+subtypes+of+neurocognitive+disorders.">Cerebrovascular and blood-brain barrier compromise: A mechanistic link between vascular disease and Alzheimer's disease subtypes of neurocognitive disorders.</a> J Parkinsons Dis Alzheimer's Dis. 2, 10 (2015).</li><li>Horton, N. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+three-photon+microscopy+of+subcortical+structures+within+an+intact+mouse+brain.">In vivo three-photon microscopy of subcortical structures within an intact mouse brain.</a> Nat Photonics. 7, (2013).</li><li>Sedeyn, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histamine+Induces+Alzheimer's+Disease-Like+Blood+Brain+Barrier+Breach+and+Local+Cellular+Responses+in+Mouse+Brain+Organotypic+Cultures.">Histamine Induces Alzheimer's Disease-Like Blood Brain Barrier Breach and Local Cellular Responses in Mouse Brain Organotypic Cultures.</a> Biomed Res Int. 2015, 937148 (2015).</li><li>Avdeef, A., Deli, M. A., Neuhaus, W., Di, L., Kerns, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Blood-Brain Barrier in Drug Discovery: Optimizing Brain Exposure of CNS Drugs and Minimizing Brain Side Effects for Peripheral Drugs. , 188 (2014).</li><li>Eigenmann, D. E., 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=Comparative+study+of+four+immortalized+human+brain+capillary+endothelial+cell+lines,+hCMEC/D3,+hBMEC,+TY10,+and+BB19,+and+optimization+of+culture+conditions,+for+an+in+vitro+blood-brain+barrier+model+for+drug+permeability+studies.">Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies.</a> Fluids Barriers CNS. 10, 33 (2013).</li><li>Takeshita, Y., 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=An+in+vitro+blood-brain+barrier+model+combining+shear+stress+and+endothelial+cell/astrocyte+co-culture.">An in vitro blood-brain barrier model combining shear stress and endothelial cell/astrocyte co-culture.</a> J Neurosci Methods. 232, 165-172 (2014).</li><li>Alberghina, M., Lupo, G., Anfuso, C. D., Moro, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitate+transport+through+the+blood-retina+and+blood-brain+barrier+of+rat+visual+system+during+aging.">Palmitate transport through the blood-retina and blood-brain barrier of rat visual system during aging.</a> Neurosci Lett. 150, 17-20 (1993).</li><li>Hosoya, K., Yamamoto, A., Akanuma, S., Tachikawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipophilicity+and+transporter+influence+on+blood-retinal+barrier+permeability:+a+comparison+with+blood-brain+barrier+permeability.">Lipophilicity and transporter influence on blood-retinal barrier permeability: a comparison with blood-brain barrier permeability.</a> Pharm Res. 27, 2715-2724 (2010).</li><li>Minamizono, A., Tomi, M., Hosoya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+dehydroascorbic+acid+transport+across+the+rat+blood-retinal+and+-brain+barriers+in+experimental+diabetes.">Inhibition of dehydroascorbic acid transport across the rat blood-retinal and -brain barriers in experimental diabetes.</a> Biol Pharm Bull. 29, 2148-2150 (2006).</li><li>Serlin, Y., Levy, J., Shalev, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+pathology+and+blood-brain+barrier+disruption+in+cognitive+and+psychiatric+complications+of+type+2+diabetes+mellitus.">Vascular pathology and blood-brain barrier disruption in cognitive and psychiatric complications of type 2 diabetes mellitus.</a> Cardiovasc Psychiatry Neurol. 2011, 609202 (2011).</li><li>Kolb, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+retina+works+-+Much+of+the+construction+of+an+image+takes+place+in+the+retina+itself+through+the+use+of+specialized+neural+circuits.">How the retina works - Much of the construction of an image takes place in the retina itself through the use of specialized neural circuits.</a> Am Sci. 91, 28-35 (2003).</li><li>Ikram, M. K., Cheung, C. Y., Wong, T. Y., Chen, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+pathology+as+biomarker+for+cognitive+impairment+and+Alzheimer's+disease.">Retinal pathology as biomarker for cognitive impairment and Alzheimer's disease.</a> J Neurol Neurosurgery Psychiatry. 83, 917-922 (2012).</li><li>Tan, Z., Ge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid-beta,+the+retina,+and+mouse+models+of+Alzheimer+disease.">Amyloid-beta, the retina, and mouse models of Alzheimer disease.</a> Am J Pathol. 176, 2055 (2010).</li><li>Serhan, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-resolving+lipid+mediators+are+leads+for+resolution+physiology.">Pro-resolving lipid mediators are leads for resolution physiology.</a> Nature. 510, 92-101 (2014).</li><li>Bucolo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+topical+indomethacin,+bromfenac+and+nepafenac+on+lipopolysaccharide-induced+ocular+inflammation.">Effects of topical indomethacin, bromfenac and nepafenac on lipopolysaccharide-induced ocular inflammation.</a> J Pharm Pharmacol. 66, 954-960 (2014).</li><li>Edelman, J. L., Lutz, D., Castro, 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=Corticosteroids+inhibit+VEGF-induced+vascular+leakage+in+a+rabbit+model+of+blood-retinal+and+blood-aqueous+barrier+breakdown.">Corticosteroids inhibit VEGF-induced vascular leakage in a rabbit model of blood-retinal and blood-aqueous barrier breakdown.</a> Exp Eye Res. 80, 249-258 (2005).</li></ol>

The authors have nothing to disclose.

In this report, we present a powerful ex vivo acute retinal model of BRB dysfunction using the swine retina. This model system does not require special instruments and can be easily adapted under most laboratory settings. However, to obtain a successful result, several steps require close attention. After obtaining the eyeballs from the source, they must be kept at 4 &#176;C or on ice and processed as soon as possible. When the effect of a treatment is being analyzed, two halves of the same retina must be used -...

A growing body of evidence supports the existence of blood retinal barrier (BRB)1-5 and its similarity to the blood brain barrier (BBB)6,7. Compromise of the BBB has been tightly linked causally or as a diagnostic marker to chronic neurodegenerative diseases such as Alzheimer&#39;s disease (AD)8,9 and acute conditions such as delirium10. Mechanistic insights into these pathologies and discoveries for potential drug targets are generally hampered by the limited accessibility and network intricacy of the brain. Alternatives such as in vivo imaging11, brain organotypic culture12, primary cell cultures13,14 and co-culture systems15 have been generated. However, most of these models require special instruments, long experimental periods or multiple markers to identify cells. Functional and structural similarities between BBB and BRB as well as a correlation between the dysfunctions of the two have been argued16-19. In addition, easier access, well-defined cell types and a layered structure have allowed the well-characterized retina as a window to the brain. The structural and functional identities of the BBB and BRB remain to be compared in detail. However, retinal pathologies, especially the BRB breach, have also been tightly associated with the progression of various diseases, including diabetes18-19 and AD21,22. Thus, it is of interest to establish a BRB dysfunction system not only to delineate the mechanism but also to screen potential drugs. In this report, a protocol enabling BRB dysfunction using a simple acute retinal culture is developed and presented.
Increased BBB permeability and AD-like pathological changes have been established in a brain organotypic culture incubated with histamine, a pro-inflammatory mediator12. Therefore, in the presented system, histamine was applied to the ex vivo retinal culture to induce BRB dysfunction. Retinas from several species, such as Mus musculus and Bos Taurus, have been tested. Due to their commercial availability and resemblance to human tissue, fresh swine eyeballs were utilized to provide the data reported here. After incubating with histamine and/or other drugs, the retinas were processed for evaluation by immunostaining for several proteins12, such as Immunoglobulin G (IgG), one of the major components of the blood; glial fibrillary acidic protein (GFAP), a well-known marker for glial activation; and microtubule-associated protein 2 (MAP2), a neuron-specific cytoskeletal protein essential for microtubule assembly. Furthermore, the layered structure of the retina allows a detailed analysis of the processes of M&#252;ller cells and ganglion cells, such as changes in their width and continuity. Thus several additional parameters are available to assess the consequences of the BRB breach at an early stage and to evaluate the reversal effects of potential treatments as well.
In this protocol, potential reversal effects of screened drugs are evaluated from three perspectives: the leakage of blood vessels (BVs), the activation of glial cells and the damage-response of neuronal cells. Several quantification methods are utilized, for instance, the expression level shown by intensity of the immunostaining, width measurement of a process and continuity of neuronal processes shown by an enhancement filter. To better illustrate the method and to help interpret results, lipoxin A4 (LXA4), a compound endogenously synthesized in response to inflammatory injury and attenuating endothelial dysfunction23, has been chosen for demonstration purposes.

<table border="0" cellpadding="0" cellspacing="0" width="984">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">DMEM</td>
			<td style="width:269px;">Life Technologies&#160;</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:305px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">HBSS</td>
			<td style="width:269px;">Life Technologies&#160;</td>
			<td style="width:119px;">14170-112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Sucrose</td>
			<td style="width:269px;">J.T.Baker</td>
			<td style="width:119px;">4072-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Histamine&#160;</td>
			<td style="width:269px;">Sigma</td>
			<td style="width:119px;">H7125-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:269px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">PFA</td>
			<td style="width:269px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Freezing Media&#160;</td>
			<td style="width:269px;">Triangle Biomedical Sciences</td>
			<td style="width:119px;">TFM-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Normal Goat Serum&#160;</td>
			<td style="width:269px;">Rockland</td>
			<td style="width:119px;">D104-00-0050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Triton X-100</td>
			<td style="width:269px;">Sigma</td>
			<td style="width:119px;">T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">GFAP Antibody</td>
			<td style="width:269px;">Millipore</td>
			<td style="width:119px;">AB5804</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">MAP2 Antibody</td>
			<td style="width:269px;">EMD Millipore</td>
			<td style="width:119px;">MAB3418</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">FITC conjugated Donkey anti-rabbit IgG</td>
			<td style="width:269px;">Jackson ImmunoResearch Laboratories, Inc.</td>
			<td style="width:119px;">711-095-152</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Cy3 conjugated Donkey anti-mouse IgG</td>
			<td style="width:269px;">Jackson ImmunoResearch Laboratories, Inc.</td>
			<td style="width:119px;">715-165-150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">mounting medium containing DAPI</td>
			<td style="width:269px;">Vector Laboratories, Inc.</td>
			<td style="width:119px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Laser Confocal Microscope</td>
			<td style="width:269px;">Nikon</td>
			<td style="width:119px;">Eclipse Ti microscope</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">ImageJ</td>
			<td style="width:269px;">National Institutes of Health</td>
			<td style="width:119px;">1.45s</td>
			<td></td>
		</tr>
	</tbody>
</table>

an acute retinal model for evaluating blood retinal barrier breach and potential drugs for treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

We present a low-cost, time-efficient and easy-to-use system to evaluate potential treatments that could protect against the BRB breach induced by histamine. IgG is constrained within the vessels in control retina (Figure 1A), but leaks out of the blood vessels upon histamine exposure (Figure 1B), confirming that the model is successfully established.
LXA4 was chosen to demonstrate the screening...

Watch this Scientific Journal Video about An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment at JoVE.com

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

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7.1K Views.  Rowan University. A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, Müller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

Video: An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

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.

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.

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

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.

The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy.

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

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) ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...