治療のために血液網膜関門の違反と潜在的な薬を評価するための急性網膜モデル

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.