The overall goal of these techniques is to evaluate the retinal pathophysiology in streptozotocin-induced diabetic retinopathy rat model. The diabetic retinopathy is one of the leading cause of blindness. The continuous search is going on to discover pharmacological agents with better efficacy and longer action to treat diabetic retinopathy.
The process of discovering pharmacological agents in pre-clinical animal models plays a vital role. These animal models helps to understand the structural and functional alterations of the posterior segment tissues of the eye. Therefore, we'll be demonstrating three different techniques to study these alterations in the posterior segment issues.
We are demonstrating histology to study morphological changes of retinal cells in layers, BRB breakdown assay to study compromised BRB by quantifying leaked protein in the vitreous from plasma, and also fluoresce angiography to visualize retinal vasculature using FITC-dextran dye. Euthanize rat using high dose of pentobarbital and confirm its death with no detectable heartbeat. Enucleate the eye by making incisions using a scalpel blade on the nasal and temporal regions of the eye.
Then cut along its edges to remove the eye from the orbital socket. Remove the excess fat surrounding the eye and place it in 5 mL of fixative solution. Incubate the eye in fixative solution for 24 to 48 hours.
After fixation, remove the eye from the fixative solution and transfer to Petri dish filled with 10 mL PBS. Using micro forceps, hold the eye with optic nerve and make a nick at pars plana with the help of micro scissors. Cut through the entire margin of the cornea to separate the anterior cup and posterior cup of eye.
Remove the lens and cut the optic nerve. Cut the posterior cup into two halves, passing through the optic nerve. Transfer each half into the separate cassettes.
Dehydrate the tissue by gradually increasing the concentration of ethanol from 50%to 100%ethanol. And finally, clear the ethanol with xylene. Impregnate the tissue with pre-heated paraffin at 60 degrees Celsius to replace xylene in the tissue.
Prepare the paraffin blocks using a steel mold to hold the tissue and cassette as its base. While preparing the block, the optic disc portion of the eye should face the bottom of the steel mold. Fill the cassette with paraffin and transfer it to a cool surface.
Mount the blade and cassette onto the respective holders on microtome. Using a microtome, trim the excess paraffin wax and cut a ribbon of five to six sections with a thickness of 5 micrometer. Transfer the ribbon onto the surface of pre-warmed water bath to unfold the ribbon.
Collect the sections on a glass slide coated with albumin. Allow the slides to dry overnight at 37 degrees Celsius. To perform Hematoxylin and Eosin staining, preheat the slides at 60 degrees Celsius for 1 hour to melt the paraffin.
Follow the staining procedure as given in manuscript. Begin with the rehydration of tissue, and then stain with Hematoxylin followed by Eosin. Now, dehydrate the tissue and add mounting medium on the sections.
Place the cover slip on sections and visualize under light microscope. To perform blood-retinal barrier breakdown assay, anesthetize the rat using ketamine and xylazine and confirm the anesthetic state by toe pinch. Locate the heart and insert 1 mL syringe with 24-gauge needle to draw the blood by cardiac puncture.
Once the sufficient blood is collected, transfer it to EDTA-coated tube. Mix it gently to prevent hemolysis. Enucleate the eye and immediately transfer it to dry ice.
Under frozen conditions, begin the dissection of eye by removal of cornea and lens. Pull the vitreous from posterior segment of eye using forceps without any contamination from retina. Transfer it to homogenizing tube with three to four beads.
Homogenize the samples using bead homogenizer at medium speed for 10 seconds. Transfer blood and vitreous humor samples into microcentrifuge tubes to process further. Centrifuge both the samples at 5, 200 g for 10 minutes at 4 degrees Celsius.
Using a multichannel pipette, add 250 microliters of Bradford reagent into wells. Dilute vitreous samples 10 times and plasma samples 20 times using PBS. Add 5 microliters of diluted samples to Bradford reagent and mix well.
Incubate the samples for 20 to 30 minutes, and then measure the absorbance at 590 nanometer using a 96-well plate reader. Intensity of sample is directly proportional to the concentration of protein. To perform fluorescence angiography, anesthetize the rat using 3%isoflurane and confirm the anesthetic state by toe punch.
Dip the tail in warm water before injecting the dye. Inject 1 mL of 50 mg per mL FITC-dextran solution into lateral tail vein. Allow the dye to circulate for 5 to 10 minutes.
Euthanize the rat using pentobarbital and confirm its death by no detectable heartbeat. Enucleate the eye and proceed for flat mount preparation. To prepare retinal flat mount, place the eye on a fiber-free Kimwipe and cut the anterior portion of eye.
Slowly pull the lens, along with the vitreous humor, from the posterior segment of eye. Transfer the posterior segment into Petri dish filled with PBS and cut the optic nerve. Now, place the tip of the pointed forcep between sclera and retina.
Gently move it all along the rim of the cup to make sure that retina is not attached to sclera at any point. Cut near the optic disc, such that retina completely detaches from the sclera. Once it is confirmed that retina is not attached to sclera on any side, push it slowly into the PBS solution.
With the help of a forcep, transfer retina onto a clean glass slide without causing any damage to it. Using micro scissor or scalpel blade, make four cuts on the retina, as shown, to divide it into four quadrants. Remove excess PBS from retinal surroundings and add anti-fading mounting medium on the retinal flat mount.
Cover it with a coverslip and visualize the flat mount under confocal microscope. It is important to note here that the entire process of fluorescence angiography is performed under minimum light to avoid the quenching of fluorescence from FITC-dextran dye. In diabetic rat, the retinal cells undergo degeneration, which leads to further complications.
These structural alterations can be visualized by histology technique to determine the cell number and thickness of retina. In case of diabetic rats, the thickness of retinal layers increases due to edema. Hence, this technique helps to screen various potential pharmacological agents to treat diabetic retinopathy.
Metabolic changes in retina due to diabetes causes loss of pericytes and breakdown of blood-retinal barrier. As there is leakage of protein and other biomolecules into retina and vitreous, the levels of total protein increase in the vitreous of diabetic rats. Preventing the breakdown of blood-retinal barrier could significantly hamper the progress of diabetic retinopathy.
In diabetic retinopathy, expression of vascular endothelial growth factor increases that lead to formation of new vessels. These vessels disturb the normal retinal structure, and therefore its innervation is of primary concern to treat diabetic retinopathy. Fluorescence angiography technique is utilized to visualize new blood vessels and also leakage of blood vessels, as highlighted here.
However, angiogenesis is prominent only after six to 12 months of diabetes induction. This technique helps us to quantify the effect of drug treatment on innervation of angiogenesis and vascular leakage. The future treatment of diabetic retinopathy needs more efficient pharmacological agents, but the future treatments can only be achieved through better understanding of the disease pathogenesis.
Mastering these techniques could help researchers to have a better understanding about pathogenesis of diabetic retinopathy, which could lead to a discovery of better pharmacological interventions.
