This method helps to understand phenotypic differences of RP cells across the entire human retina. The dissection technique that Davide Ortolan has developed is highly reproducible in generating a detachment between RP and the retina. And the REShAPE software is really appreciable in analyzing large images of RP flat mounts.
The method that Davide has developed can be used to study regional differences in the RP phenotype from patients with different types of retinal degenerative diseases. To begin, cut the 50 milliliter conical tube slightly below the 5 milliliter mark. Using hot glue, attach the tube tip to the bottom of the weighing boat.
Then mix the two components of the silicone elastomer kit at a 10-to-1 ratio avoiding entrapping air. Pour the mix into the weighing boat containing this spherical piece of the round bottom tube. Cure the silicone at room temperature overnight.
Remove the weighing boat and round bottom tube from the cured silicone mold. First, fill a 1 milliliter syringe with 1700 millimoles of D-Mannitol solution and attach it to a 21-gauge needle. Insert the needle through the pars plana to avoid puncturing the anterior chamber of the eye and inject 400 microliters of the solution into the vitreous.
Leave the eye at room temperature for 45 minutes. Using a pair of fine scissors and forceps, cut open the anterior chamber at the level of the pars plana. Fill the posterior eye chamber with DPBS containing calcium and magnesium.
Under the stereo microscope, localize the macula visualized as a yellow spot on the retina. Cut the eye into quadrants, namely nasal, temporal, superior, and inferior, while ensuring to preserve the macular region. Remove the needles if they're in the way.
Transfer the butterfly to posterior chamber of the eye into a 100-millimeter Petri dish containing DPBS with calcium and magnesium. Before removing the retina, mark the petal that contains the macula by making a V-shaped cut in the ciliary margin. Lift and cut all the vitreous that lays on the retina.
Cut the retina from the ciliary margins in all the petals ensuring not to scratch the RPE. Place the tissue in 4%PFA and incubate for one hour. Wash three times with DPBS containing calcium and magnesium.
Transfer the tissue into a container filled with the same buffer and store it at 4 degrees Celsius. Next, transfer the sample to a 100-millimeter Petri dish containing DPBS with calcium and magnesium. Punch out the optic nerve head with a 1.5-millimeter biopsy punch.
Collect the retina and store it in the same buffer at 4 degrees Celsius. To remove the sclera from the RPE choroid, gently lift the RPE choroid layer from the periphery. Then with a pair of Vannas Spring Scissors, cut the choroidal vessels and connective tissue that are between the sclera and the RPE.
After complete separation from the sclera, collect the RPE choroid layer. Transfer the tissue into a container filled with DPBS containing calcium and magnesium, and store it at 4 degrees Celsius. Transfer the RPE choroid to one well of a six-well plate.
Block and permeabilize the sample for one hour at room temperature. Incubate the sample for one hour at room temperature with phalloidin conjugated with 647 fluorophore at a 1-to-250 dilution in the permeabilization buffer. Wash three times in DPBS containing calcium and magnesium.
Transfer the RPE choroid sample to a 50 x 75 millimeter glass slide and flatten it. Cut each petal into two to make the sample flatter. Draw a contour of the flat mount with a hydrophobic pen.
To quench the lipofuscin autofluorescence, add 500 microliters of the autofluorescence quencher solution, and incubate at room temperature for two minutes. Wash thoroughly in DPBS containing calcium and magnesium. Remove the DPBS and add the mounting medium.
Place a cover glass on the flat mount and seal with nail polish. Open the software. In the directories tab, select the input and output folders.
In the output directory, let the software automatically change the path to the input directory or process. Alternatively, change the output directory manually. To generate heat maps, select all from the dropdown menu in create color images tab.
Check the box no in the use manual limits feature to let the software use the minimum and maximum values detected in each image. Check the box yes to manually adjust the range of values for each shape metric heat map. Then click on the set limits button and insert values in the text boxes to choose ranges for the individual parameters.
After changing the values of interest, click on save. Click on low defaults to reset all the limits. To select a cell size threshold in lower cell size, insert the size of the smallest cell to be included in the analysis.
In upper cell size, insert the size of the largest cell to be included. In convert pixels to real units, check no to run the analysis in pixel units, check yes to run the analysis in micrometers. In length of scale bar pixels, enter the pixel value in the text box.
In length of scale bar microns, enter the corresponding distance in micrometers. To start the analysis, press go for it. This protocol results in a single-plane image of a flat mount where the cell location is identified by REShAPE-generated segmentation of the RPE cell borders.
Also, 30 shape metrics, including the cell area of the individual RPE cells, are measured for every correctly identified RPE cell. Black tiles resulting from residual pieces of retina or other bright objects can be removed by choosing one of the filtering options available in the RT filter dropdown menu. Using RBG images for the reshape analysis will produce entirely black binary images.
If this occurs, converting the RGB images to gray scale will produce correctly-segmented binary images. If the staining is not optimal or if the sample is damaged by a scratch, then large clumps of cells may be identified as a single very large cell. In this case, large objects can be excluded from the analysis by changing the cell size threshold.
To obtain a flat tissue, the RP and choroid need to be separated from the sclera even if these steps can take a long time. Following the generation of the RP flat mount, it is possible to stain for additional RPE markers so that you can study different regions of the RP monolayer. Using this method, we have discovered five different RP populations that are differentially sensitive to different retinal degenerative diseases.
