This protocol can help researchers easily separate the retinas of mice and observe the dynamic changes in the retinal vasculature of the OIR mouse model. The main advantages of this method are preservation of the integrity of the separated retinas and allowing each mouse to serve as its own control, which makes the results more comparable. Researchers trying this method for the first time may struggle while taking the five orientation imagings because it is a bit difficult to place pups in the correct position.
To collect the tissue for retinal whole mounting, use curved scissors to disconnect the eyeballs from the orbital tissue of a euthanized C57-Black/6 pup. Then, insert curved forceps into the posterior part of the eyeball, clamping the optic nerve and quickly lifting the eye out from the orbit. Place the eyeballs in pre-cooled PBS to remove any hair and blood from the surface.
When all of the eyeballs have been collected, transfer the cleaned eyeballs to a two-milliliter microcentrifuge tube containing 4%paraformaldehyde, and incubate for 15 minutes at room temperature and 12 to 15 revolutions per minute. At the end of the incubation, add a drop of PBS to the center of a culture dish under a dissecting microscope, and place one eyeball into the drop. Holding the eyeball with a pair of forceps, use a one-milliliter syringe needle to carefully puncture the cornea at the corneal limbus.
Insert the tip of a pair of scissors into the puncture, and cut the cornea along the corneal limbus, being careful not to cut the retina. Use forceps to remove the iris and lens, and place the remaining eyecup in 4%paraformaldehyde for a 45-minute incubation at room temperature on the shaker. At the end of the incubation, place the fixed eyecup in a fresh drop of PBS at the center of a new culture dish.
Holding the eyecup with one pair of forceps, use a second pair of forceps to gently separate the retina and sclera layers. Place the tip of the scissors between the retina and sclera layers, and cut the sclera toward the optic nerve. Peel the sclera off of the retina to obtain the retinal cup.
Using forceps, release the connection between the radial hyaloid vessels and peripheral retina. Use a two-milliliter pipette with the tip cut off to transfer the retinal cup into one well of a 48-well plate for three five-minute washes in room temperature PBS on the shaker. After the last wash, incubate the cup in 1%Triton X-100 and 5%normal donkey serum in PBS overnight at four degrees Celsius.
To label the retinal vasculature with isolectin B4, incubate the retina in one well of a 48-well plate in 400 microliters 0.1%normal donkey serum supplemented with isolectin B4-594 overnight at 4 degrees Celsius on a shaker. If necessary, another primary antibody can be added at the same time. On the following day, wash the retina with three 20-minute washes in 0.1%PBST on the shaker.
After the last wash, incubate the retina with an appropriate high-affinity secondary antibody for one hour at room temperature before labeling the sample with DAPI for 20 to 25 minutes. At the end of the incubation, wash the retina with three 30-minute washes in 0.1%PBST at room temperature with shaking. After the last wash, transfer the retinal cup to a clean slide with the opening facing up, and use the microscope to cut the retina radially at the three, six, nine, and 12 o'clock positions from the periphery toward the center, up to one to 1.5 millimeters from the optic nerve head.
Rinse the retina three times with a few drops of PBS per wash before using air-laid paper to dry and flatten the retina. Add a drop of mounting medium to the center of the coverslip until the diameter of the droplet covers half of the coverslip. Then quickly place the coverslip mounting medium side down onto the outspread retina.
Once all of the retinal flat mounts have been prepared, the samples can be imaged by confocal fluorescence microscopy. For in vivo imaging, add 20 microliters of mydriatic eye drops to achieve long-lasting pupil dilation. After five minutes, place the pups in a stable position on a small heating pad in front of the imaging device.
To maintain moisture in the corneas, regularly add artificial tears throughout the imaging procedure. Next, click Infrared Fundus Imaging on the device to adjust the optic nerve head to the center of the screen. Intraperitoneally inject 150 microliters of 0.5%fluorescein sodium salt solution, and immediately click the FA and Injection buttons on the touch panel of the imaging device to start the timer.
After three minutes, when the blood circulation of the retina has entered the venous phase, acquire the first image of the central retina. Move the lens of the imaging device horizontally to the nasal side of the eye until the optic nerve head is located at the midpoint of one side of the image acquisition area, and take the second image. Continue to move the lens and acquire images of the temporal, superior, and inferior retina in the same manner.
To process the retinal images in an appropriate imaging processing program, open the File menu and click New to create a new canvas with a black background. Open the image of the central retina. Then click File to add a second image.
Adjust the opacity of the second image to 60%and move and resize the image until the two images overlap. Click Switch Between Free Transform and Warp Modes, and make subtle adjustments to the vessels if necessary. Then set the opacity of the second image back to 100%Select both images, and click Auto-Blend Layers.
Check Panorama as the blend method, and also check the boxes for Seamless Tones and Colors and Content Aware Fill Transparent Areas. Click OK to complete the stitching of the first two images. Next, add the third image, and stitch this image to the first two images as demonstrated until all five of the images from a single retina have been stitched.
When all of the images from the analysis have been processed, use the Crop Tool to crop the FFA images to a uniform size to facilitate observation of the changes in the retinal vasculature dynamics at different developmental time points. After five days of exposure to hyperoxia, the central retinas of postnatal day 12 pups demonstrate a large non-perfusion area. Upon transfer to five days of hypoxic conditions, the retinas gradually neovascularize, exhibiting an increase in fluorescence signal stronger than that observed in the surrounding normal vessels.
After postnatal day 17, the pathological neovascularization regresses rapidly and the retina demonstrates a completely normal appearance by postnatal day 25. A good repeatability and stability of oxygen-induced retinopathy model can be achieved by controlling the litter size and postnatal weight gain of the pups. As observed, the vaso-obliteration typically peaks at postnatal day 12 and disappears at postnatal day 25 while the neovascularization peaks at postnatal day 17 and regresses by postnatal day 25.
In postnatal day 15 to 25 oxygen-induced retinopathy mice, the retinal blood vessel diameter increases and becomes highly torturous compared to that observed for the retinas of normal mice. The forceps in the left hand mainly play in a supporting role. Take care not to squeeze the eyeball, which can deform or rupture it.
