Name: an acute retinal model for evaluating blood retinal barrier breach and potential drugs for treatment
Uploaded: 2016-09-13T15:00:00
Description: Watch this Scientific Journal Video about An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment at JoVE.com

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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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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		<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 ...

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