The overall goal of this procedure is to isolate the retinal vascular network. This is accomplished by first performing a series of water washes on the isolated retina. In the second step, the retina is digested in trypsin at 37 degrees Celsius for about one to one and a half hours, depending on the size of the retina.
Next, the vasculature is isolated via additional water washes and dissection under a microscope. Ultimately, pathologic vascular changes such as micro aneurysms, capillary degeneration, and abnormal endothelial cell to parasite ratio can be visualized. So the main advantage of try and digest over existing methods like preparing retinal flat mounts is that the entire vascular network can be highlighted in great clarity.
Generally, individuals new to this method will struggle because it is technically challenging and difficult to perform consistently. Visual demonstration of this method is critical as the vascular isolation steps are difficult to learn by words alone. This is a very valuable tool in any laboratory that's performing retinal vascular biology studies such as some diseases such as diabetic retinopathy, demonstrating this technique will be Jonathan Chow, a talented medical student from my laboratory.
After fixing the enucleated mouse eye and 10%neutral buffered formin for 24 hours, place the eye in a Petri dish and cover it with a small amount of water. Begin the dissection by using a pair of dissection scissors to carefully make an initial cut in the cornea. Then, while holding the cut area with straight forceps, use a pair of scissors to cut along the border of the cornea and the sclera removing the cornea.
Next, use fine, curved, and straight forceps. To slowly and carefully peel the sclera and choroid away from the retina towards the optic nerve. Then remove the lens and any excess iris and debris from the retina to help separate the neural retinal layers from the blood vessels.
At approximately 500 microliters of 45 micrometer filtered water to a 24 well plate and place the retina in a well then shake gently for four to five hours at room temperature, changing the water every 30 to 60 minutes. Continue to wash the retina in water overnight with gentle shaking At room temperature the following day, remove the water and at 3%trypsin, check under a microscope to ensure that no debris is found in the well. To avoid having the vasculature adhere to it, then incubate the tissue at 37 degrees Celsius with gentle or no shaking for one to one and a half hours.
When the tissue begins to show signs of disintegration, place the plate on the microscope stage and then avoiding the retina. Carefully remove the trypsin to separate the vasculature. First, add filtered water to the retina using forceps.
Peel off the internal limiting membrane, which appears as a thin, transparent layer. Shake the plate with mild agitation. After five minutes, place the plate on the microscope stage again and carefully replace the water, avoiding damage to the retina to help free the network of vessels from the adherent retinal tissue.
Wash the retina as just demonstrated until there is little to no debris remaining in the water after the last wash. So typically removing all the nonvascular tissue is the trickiest part of the procedure. The following techniques can either be used in sequence or individually To facilitate isolation of the vascular network, one important tip is to dip all tools and trypsin before manipulating the retina.
This can help avoid a tissue from adhering to the equipment. Coat a 200 microliter micro pipette tip with reserved trypsin, and then carefully pipette water up and down, adjacent to and blowing towards the retinal vessels, causing gentle agitation. If the nonvascular tissue is still resistant center, a trypsin coated micro pipette tip at the optic nerve and carefully pipette the entire vessel network up and down to help break down the nonvascular tissue.
Finally, fine. Forceps can be used to gently lift the vasculature out of the water to dislodge any adherence nonvascular components. The end result should be an intact debris free retinal vasculature network.
To visualize the vasculature, first place a drop of water onto a clean slide. Then transfer the digested vessels into the water under a scope check. If the VAs flap mount is folded or unfolded, folded vessels lead to poor visibility.
When stained, if the tissue is folded, add additional water and gently shake the slide to facilitate unfolding. Then use a micro pipette to slowly remove any excess water. The final product of a successful procedure is a flat mount of the entire network of the mouse retinal vasculature with the architecture maintained and stained with hemat, toin, and eoin.
As shown here, as seen in this image, a clear differentiation between the endothelial cells and the pericytes can be observed in the retina. The nuclei of the endothelial cells as indicated by the white arrow, are oval or elongated and lie entirely within the vessel wall. The parasite nuclei is indicated by the black arrow are small, spherical stain densely, and generally have a protuberant position along the capillary wall.
Trypsin and digest is a common procedure used to analyze vascular pathology in diabetic animal models. This image shows an example of capillary degeneration as indicated by the white arrow as observed in a DB DB mouse, a model for type two diabetes. Although this procedure has been optimized for mouse retinas, similar results can also be obtained in rat retinas.
It is important to note that rat retinas as shown in these images, are larger than mouse retinas and require 12 rather than 24 while plates. The vasculature network of the rat retina is also less fragile than its mouse counterpart. It is extremely crucial to remove as much of the nonvascular tissue as possible.
Any remnants will lead to non-specific staining and lead to poor visualization of the vasculature. In this image, for example, an unsuccessful removal of the entire nonvascular tissue can be observed. Likewise, it is also important to have the retina be unfolded when mounting so as to improve the visualization of the vasculature.
This image shows an unsuccessful unfolding of the vascular flat mount. After mastering this technically difficult procedure. Trips and digests can be an extremely valuable method that allows a detailed visualization of the entire vascular network.
By improving this technique in the mouse eye, researchers can take advantage of genetically manipulated mice to study various pathways that can take control of the retinal vasculature and allow us to understand the pathophysiology of various retinal diseases. As a result of this, we can improve our understanding of disease pathologies such as diabetic retinopathy. By studying mice models like the DVDB mouse, this can ultimately allow the opportunity to find new potential therapeutic interventions targeting the retinal vasculature.
