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 be...

<ol>
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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.

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	<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>
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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 ...

The overall goal of this ex vivo retinal model system is to screen drugs, that ameliorate the blood neural barrier breach. This method can answer several key questions in the field of neural degenerative diseases, like what are the early events, and how can we screen for drugs that can help treat those patients. The main advantage of this technique is that it is low-cost, easy to use, fast and adaptable.Generally individuals new to this method will struggle because obtaining reliable and reproducible data depends on the technical skills to generate good samples. First swine retinas, obtained from a commercial source, are used here. After procurement from the source, the eyeballs must be kept at four degree, or on ice, and processed as soon as possible.Begin the dissection in a tissue culture hood by cutting around the lens to open the eye cap. Then using a brush, gently remove the vitreous humor, without pulling on the retina. Use a brush to gently detach the retina from the cut edge, to expose the optic disc.Sever the optic nerve with a razor and release the retina. Transfer the retina into a petri dish and carefully rinse the retina once using cold HBSS. Place the sample in HBSS on ice.Next, use a razor to cut the retina in half symmetrically. When treatments are required, one half of the retina is treated, while the other half of the same retina must be processed to set the baseline and to correct for the animal variations. Gently transfer the samples to a six-well plate containing three milliliters of stabilization medium per well.Equilibrate the retinas for 30 minutes at 37 degrees Celsius in an incubator with the atmosphere containing 5%carbon dioxide. After the incubation, carefully aspirate the stabilization medium, and add three milliliters of medium containing histamine and LXA4 to each well. Incubate the samples for another hour.After rinsing with warm sterile PBS, add three milliliters of 4%PFA to each well and incubate for 15 minutes at room temperature. Once the incubation time has elapsed, quickly aspirate the PFA and rinse the samples once with PBS. Add 10%sucrose to the wells and incubate for two to four hours at four degrees Celsius.Next, replace the 10%sucrose with 30%sucrose and incubate overnight at four degrees Celsius. Then mix one part commercial freezing medium with two parts 20%sucrose, to make working freezing medium. Leave at four degrees Celsius overnight or longer until the air bubbles disappear.The next day, replace the 30%sucrose with working freezing medium, and allow it to equilibrate for five minutes at room temperature. After equilibration, trim each retina into three millimeter by five millimeter rectangles, then freeze the retina slices by placing them into freezing medium, contained in a cylinder of aluminium foil. Ensure that the retinal slices are vertical to provide a cross-section of the retina upon sectioning, and freeze them in liquid nitrogen.Section each block on a cryostat into 14 micron slices, and mount the sections on glass slides. Store the slides at negative 80 degree Celsius until use. Begin immunostaining by washing the sections with 5%sucrose phosphate buffer, or SPB.Then block the non-specific binding sites, by incubating the sections and blocking buffer for one hour at room temperature. Next incubate the sections with the appropriate primary antibody for one hour. Once the hour has elapsed, aspirate the solution and wash the sections three times in 5%SPB.Then incubate the sections with the corresponding secondary antibodies for one hour, while protected from light. After washing the sections three times with 5%SPB, prepare the samples for microscopy by mounting them in medium containing DAPI, and covering with cover slips. Finally, capture images using a confocal microscope.To measure the width of the process, use the magnifying glass tool to choose an area of the section where the processes are gesting, such as the outer nuclear layer. Then choose an optical field where such processes are visible and continuous. Click the analyze tool in the main menu and then click set scale on the submenu, to set the scale bar according to the microscopic setting.Select the line function in the main menu and draw a line across the process. Click analyze in the main menu, and then click measure in the submenu, to perform the measurement. To analyze the continuity of the process, return to the main menu and use the polygon function to select the inner plexiform layer as the region of interest.Measure the area by clicking analyze and then measure. Click process, filter and then variants, to enhance the edges in the image by replacing each pixel with its neighborhood variant. Then automatically adjust the contrast and brightness by clicking image, adjust, brightness and contrast, and then auto in the submenu.Then count the lines. In these images, IGG immunoreactivity is seen in red. In control groups, IGG is restricted within the blood vessels.In the histamine group, IGG is detected out of the blood vessels, where it forms leakage clouds indicated by dotted circles. In the LXA4 treated groups, IGG is again restricted within the blood vessels. Further addition of LXA4 rescues the vessels'function induced by histamine.This histogram shows the percentage of leaky blood vessels across the tested groups. In these images immunostaining for GFAP, which stains Muller cells, is presented. Positive staining is obtained in the processes of Muller cells, across the retina, and around the blood vessels.The width of the Muller cell processes, from all groups are presented. Treatment with histamine, or LXA4 alone, reduced process width. But when both reagents were applied together, process width was rescued.In these images, immunostaining for MAP2 is presented. Positive staining from the processes and cell bodies of ganglion cells is observed. The density of continuous MAP2 positive ganglion cell processes from all groups are presented.Treatment with histamine decreases the continuity of dendritic processes, while LXA4 has no significant effects. Once mastered, this experiment can be done in three to five days, if performed properly, with five minutes per retina for dissection, six minutes for treatment, followed by sectioning, immunostaining, and analysis. While attempting this procedure, it is important to use fresh tissue and proper controls.Following this procedure, other methods such as live imaging, can be added, to track real-time changes in levels of calcium and mitochondrial properties. After watching this video, you should have a good understanding of how to use this ex vivo acute retinal culture model, to create a BRB dysfunction, to assess the damage, and to screen for potential drugs. Thank you very much for watching and all the best with your experiments.

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JoVE Journal

Medicine

7.1K Görüntüleme.  Rowan University. The overall goal of this ex vivo retinal model system is to screen drugs, that ameliorate the blood neural barrier breach. This method can answer several key questions in the field of neural degenerative diseases, like what are the early events, and how can we screen for drugs that can help treat those patients. The main advantage of this technique is that it is low-cost, easy to use, fast and adaptable.Generally individuals new to this method will struggle because obtaining reliable a...