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

Summary

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.

Abstract

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 Müller 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 Müller 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.

Introduction

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'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 cult....

Protocol

All protocols were carried out in compliance with the policies of the Institutional Animal Care and Use Committee where applicable.

1. Preparation

1. Prepare the stabilization media with 75% Dulbecco's Modified Eagle Medium (DMEM), 25% Hanks' Balanced Salt Solution (HBSS). Mix well, aliquot and store at -20 °C until use.
2. Prepare the Phosphate Buffered Saline (PBS) and sterilize by autoclaving. Store the solution at room temperature. Warm the PBS to 37 °C before the experiment (5 ml per well).
3. Prepare 10%, 20% and 30% sucrose in PBS. Filter all the solutions individually over separate 0.22 μ....

Results

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.......

Discussion

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 °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 -.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Life Technologies	11965-092
HBSS	Life Technologies	14170-112
Sucrose	J.T.Baker	4072-05
Histamine	Sigma	H7125-1G
Penicillin-Streptomycin	Invitrogen
PFA	Electron Microscopy Sciences	15710
Freezing Media	Triangle Biomedical Sciences	TFM-5
Normal Goat Serum	Rockland	D104-00-0050
Triton X-100	Sigma	T8787
GFAP Antibody	Millipore	AB5804
MAP2 Antibody	EMD Millipore	MAB3418
FITC conjugated Donkey anti-rabbit IgG	Jackson ImmunoResearch Laboratories, Inc.	711-095-152
Cy3 conjugated Donkey anti-mouse IgG	Jackson ImmunoResearch Laboratories, Inc.	715-165-150
mounting medium containing DAPI	Vector Laboratories, Inc.	H-1200
Laser Confocal Microscope	Nikon	Eclipse Ti microscope
ImageJ	National Institutes of Health	1.45s