Enrichment of Bruch's Membrane from Human Donor Eyes

The overall goal of this tissue dissection technique is to enrich Brooks membrane from a postmortem human eye and provide scientists with the specific material necessary for analyzing its protein and carbohydrate content. This method can help us understand mechanisms such as the Immunoregulation on Brook's membrane that underlie a range of ocular diseases, including the most prevalent form of blindness in the Western world, age-related macular degeneration. The main advantage of this technique is it allows the analysis of just the acellular Brooks membrane without the contamination of blood components or the retinal pigment, epithelial cells that could otherwise lead to apparent results.

The visual demonstration of this method is critical because the handling of human tissue is difficult and the isolation of a rich brooks membrane is difficult to mentally envisage from a written methodology alone. Demonstrating the procedure will be Dr.Selena Mcca, a postdoc from our laboratory To begin clean the workspace around and underneath the microscope with disinfectant, and then wipe the area down with 70%ethanol. Next, gather together the tools needed for the procedure when ready, place the eye globe into a disposable Petri dish, retaining the lid for later use.

Use a new disposable scalpel to remove any residual optic nerve which may interfere with creating a flat mount of the tissue. Next, make four incisions equally spaced apart to create four quadrants. Avoid cutting through the macular region only when enriching the macular brooks membrane while penetrating through the entire depth of the eye.

Continue the incision all the way around from the margin of the eye cup to the optic nerve. This will allow the eye cup to be flattened. Next, hold one quadrant of the flattened eye cup with one tweezer while using a larger set to grip the vitreous and underlying tissue.

Pull this tissue away from the retinal pigment, epithelium or RPE starting at the periphery and moving towards the center to the opposing side. If the vitreous and neurosensory retina do not detach as one, use a scalpel to excise any residual tissue anchored at the optic disc while holding one quadrant of tissue in place. Use a sterile cell scraper to gently dislodge the RPE monolayer from the entire inside surface of the eye cup.

These cells readily detach and can be seen as dark brown pigmented clumps. Rinse with PBS to gently wash away dislodged RPE cells. Next, gently peel off the overlying dark red tissue from all four eye quadrants.

Place the excised tissue into a clean dish. Add a few drops of PBS, flatten the tissue using cell and forceps if required. Using the flattened edge of one cell scraper to hold the excised tissue in place, use the second scraper to gently scrape the tissue, turn the tissue over and repeat, removing as much excess material as possible.

Place the tissue into a clean Petri dish. Once the material is removed, the translucent membrane that remains is the crudely enriched Brooks membrane. Work under a dissecting microscope to remove the choroid and further enrich the membrane.

Briefly rinse the brooks membrane with ultrapure water to remove residual choroid and blood before placing the membrane into a micro centrifuge tube. After suspending the membrane in approximately one milliliter of ultrapure water vortex briefly to help lice any contaminating cellular matter. Centrifuge the sample to palate and aspirate off the supernatant.

The enriched brooks membrane that remains is now ready for solubilization or can be kept in storage at minus 80 degrees Celsius. For later use to extract protein from the enriched brooks membrane. Immerse the tissue in 500 microliters of eight molar urea for six hours.

At room temperature, centrifuge the solubilized samples to pellet any remaining extracellular matter. Remove the snat and transfer it to a dialysis membrane. Place the sample in one liter of PBS to dialyze for 16 hours at four degrees Celsius.

For each sample, add 40 microliters of protein isolation beads and rotate the mixture at room temperature for 30 minutes. Pellet the beads by centrifugation and remove the supernatant. Solubilize the protein bound to the beads in 30 microliters of two x sample loading buffer.

After a 10 minute incubation, pellet the beads and collect the supernatant. Load the released proteins onto a precast SDS page. Gradient gel for protein separation.

Follow standard procedures to visualize the separated protein bands if desired. Perform a western blot following standard laboratory procedures To enrich the macular Brooks membrane. Follow the same procedures as demonstrated earlier, except that while the neurosensory retina is still in place, locate the yellow foveal coloration that represents the central macular region.

Place a sterile six millimeter biopsy punch above the foveal region and firmly cut into the macula, keeping the biopsy punch in place. Make a separate incision with a scalpel from the edge of the eye cup to the edge of the biopsy punch blade. Peel away the surrounding eye material to achieve a clean biopsy.

Remove the six millimeter disc of tissue from the punch and place it on the clean Petri dish lid reserved earlier. Place the Petri dish lid and macular punch under the dissecting microscope. Check for yellow foveal staining to infer the accuracy of the biopsy.

Next, use a sterile cell scraper to gently remove the RPE cells. Secure the macular disc via the sclera using tweezer tips and slowly peel off the choroid and brooks membrane complex. If required, use a scalpel blade to separate brooks membrane from the sclera.

Again, use the cell scraper to remove unwanted choroid and blood matter from Brooks membrane. Place the macular Brooks membrane into a tube and suspend it in one milliliter of ultrapure water Vortex briefly to help lice any contaminating cellular matter. Centrifuge the sample to pellet the membrane and aspirate off the snat.

The enriched brooks membrane isolated from the macula can now be used for proteomic analysis using the desired digestion protocols. These h and d stained tissue sections of human macular tissue before and after scraping away the outer choroidal structures demonstrate the enrichment of brooks membrane and acellular chorio capis from the choroidal stroma. This SDS page gel demonstrates a lack of albumin contamination from blood in both the neat and diluted brooks membrane samples.

This representative western blot was probed to detect the immune regulator factor H like protein, one from three individual donors. After watching this video, you should have a good understanding of how to enrich Brooks membrane from a human eye in such a way as to provide a valuable resource for the subsequent biochemical and biophysical analysis of this unique acellular structure. Don't forget that working with human tissue and sharp implements can be extremely hazardous and precautions such as the use of full personal protective equipment and hepatitis B vaccinations.

Before handling the human tissue must always be taken.