The primate retinal model established in this study we offering the investigation of the comprehensive mechanism and the therapeutic development of cGMP-PKG dependent retinal deterioration. This individual nonhuman primate model reserves retinal tissue confirmation and intercellular interactions which enables better simulation of in vivo conditions. It also reduces the animal use and it shortens the experimental duration.
Providing a control experimental approach for investigating retinal development or degeneration. Begin the preparation of the retinal explants by washing the eyeballs isolated from wild type macaques with 10 milliliters of 5%povidone iodine for 30 seconds. To avoid bacterial contamination, dip the eyeballs into a 5%penicillin streptomycin PBS solution for one minute before incubation in 10 milliliters of R16 medium for five minutes.
Then immerse the eyeballs in 10 milliliters of protein Ace-K solution preheated at 37 degrees Celsius and incubated for two minutes. Perform next incubation in 10 milliliters of one to one R16 plus fetal bovine serum solution for five minutes. Give a final wash in 10 milliliters of fresh R16.
Once done, separate the sclera, cornea, iris, lens and vitreous body. Then cut the retina from four sides with tweezers and scissors. Next, use a four milliliter tree fine blade to cut the retinal explant at a 1.5 millimeter from the nasal side, four millimeter from the temporal side and a two millimeter from the superior and inferior sides of the optic nerve head.
Then using a six millimeter tree fine blade cut the central side of the retinal explant containing the optic disc and macula. Using a wide pipet, pull the retina explant containing the retina and choroid and place it in the middle of the insert with the photoreceptor side up. Fully submerge the retina in one milliliter of the complete medium on the plate below the membrane.
Culture the retinal explants and place them in a 37 degree Celsius incubator with humidified 5%carbon dioxide. Give the appropriate drug concentration treatment to explants for four days. Use at least three explants per treatment condition and use the explants without the drug as a positive control.
For fixing and cryo-sectioning, treat the retinal explants with one milliliter of paraform aldehyde for 45 minutes. After a brief rinsing with one milliliter of PBS serially incubate the explants in 10%sucrose for 10 minutes, 20%sucrose for 20 minutes, and 30%sucrose for 30 minutes. Cut the retinal explants uniformly with four quadrants in the periphery and six millimeters at the center.
Then transfer the retinal explants into a tin box of about 1.5 by 1.5 by 1.5 centimeters with an optimal cutting temperature compound embedding medium covering the explant. Immediately, freeze the tissue sections in liquid nitrogen and place them in a 20 degrees refrigerator for storage. Next, trim the agar into a small block containing the tissue sample.
Section the blocks into 10 micrometer blocks and place the sections on adhesion microscope slides using a soft brush. Then dry the sections for 45 minutes at 40 degrees Celsius and store them at minus 80 degrees Celsius until use. For cyclic guanosine monophosphate or cGMP staining, draw a hydrophobic ring around the dried retinal sections with a liquid blocker pen and cover the samples.
Immerse the slides in 200 microliters of 0.3%PBS and Triton-X100 or PBST for 10 minutes followed by a one hour treatment in 200 microliters of blocking solution at room temperature. Next, incubate the sample slide overnight with the 200 microliters of primary antibody prepared in the blocking solution at four degrees Celsius and rinse it thrice with PBS for 10 minutes. Incubate the slide in 200 microliters of secondary antibody for one hour at room temperature.
After three washes of PBS for 10 minutes cover the samples with an antifade mounting medium containing DAPI and store at four degrees Celsius for at least 30 minutes before imaging. Dry the slide at room temperature for 15 minutes and draw a water repellent barrier ring around the retinal tissue specimen with a liquid blocker pen. After incubation with 40 milliliters of PBS for 15 minutes treat the sections with 42 milliliters of 0.05 molar tris-buffer or TBS at 37 degrees Celsius.
Aspirate the TBS, incubate the samples with six microliters of proteinase K for five minutes and wash slides thrice with PBS for five minutes each. Expose the slides to 40 milliliters of ethanol acetic acid solution in the chaplin. Cover it with a transparent sheet and incubate at minus 20 degrees Celsius for five minutes.
Wash the slides thrice with TBS for five minutes each time. Expose the slides to 200 to 300 microliters of blocking solution or 1%BSA and incubate them in a humidity chamber for one hour. At approximately 130 microliters of TMR red solution per slide and after one hour, give two washes of 200 microliters of PBS for five minutes each.
Place cover slides containing one to two drops of antifade mounting medium with DAPI over the samples and incubate the slides at four degrees Celsius for at least 30 minutes before imaging. After sequential staining with tunnel and cGMP proceed for light and fluorescence microscopy by adjusting the camera parameters. Capture the images of four fields from each section using the software with a digital camera at 20x magnification.
Obtain representative pictures from the central areas of the retina using DAPI, EGFP and TMR with z-stack scanning and an interval of one micrometer and optional at 1.251 micrometers. In the retinal explants treated with different concentrations of the PDE6 inhibitors Zaprinast the number of tunnel and cGMP positive cells were strongly increased in the outer nuclear layer as compared with the control group. The high proportion of tunnel positive cells suggested that the increase in cGMP activity may enhance the pre nest induced retinal cell degeneration and cGMP accumulation plays a role in photoreceptor degeneration.
Explant preparation must be carried out as soon as possible. Op animal scatter fives and any accumulation because it improves cell survival and enhance the status of fiber of bulk stack series. To a greatest extent visible the vitreous should be cleaned with steroids.
Avoid contacting the retinal nerve fiber layers when transferring retinal explants to prevent mechanical cell injury. Please take note, to issue a smooth transfer of retinal explants to culture inserts the pet can be pre-soaked in something to keep it moist before transferring retinal tissues. Additionally, the entire process must be carried out in the steroid atmosphere to avoid contamination during in the process.
