An Assay to Detect Protection of the Retinal Vasculature from Diabetes-Related Death in Mice

Our goal is to comprehend the reason behind the gradual onset of diabetic retinopathy, which occurs decades after the onset of diabetes. We're investigating the pathogenesis of diabetic retinopathy's two phases, loss of protection and damage accumulation. Our goal is to investigating the protection of retinal vessels against diabetes induced insults through a protection assay.

Our protection assay unveils the dual phases of protection loss and accumulation of damage related to diabetic retinopathy. And so this innovative approach provide a valuable tool for understanding diabetic retinopathy pathogenesis, and the potential application could be extended beyond DR.It could apply to a broader physiological and pathological context. While existing literature predominantly focuses on the pathological alterations in diabetic retinopathy, there remains a notable gap in our knowledge concerning protective factors.

This protection assay plays a crucial role in bridging the lack and helps us understand the role of protective factors involved in the retinal vasculature's resilience to damage induced by diabetes. This novel protection assay enhances our understanding of diabetic retinopathy progression, providing a targeted tool to investigate the delay between diabetes onset and DR manifestation in mice. It advances our research by dissecting the interplay between protection and damage, offering potential for targeted therapeutics and broader applications.

Our future research will concentrate on delineating the specific cell types, endothelial cells, parasites, and neuronal cells in the mouse retina, and the molecular mechanisms underlying this protection. Additionally, we aim to identify the potential genes behind this protective mechanism. To begin, take isolated retinas in a 24 well dish filled with double distilled water.

Then replace the double distilled water with 800 microliters of YL trypsin solution and incubate at 37 degrees Celsius with gentle to no shaking for four hours and 15 minutes. Using the glass transfer pipette dipped in YL trypsin solution, transfer the retina to a 35 millimeter Petri dish containing lint-free double distilled water. To remove the outer nuclear layer, flip the retina semi sphere downward and use a straight single hair brush to gently press it to the bottom of the dish.

Employ a loop brush to gently brush away the photoreceptors of the retina. Next to remove the vitreous, flip the retina semi sphere upward. Use curved forceps to grasp the vitreous under a dissecting microscope.

Using another curved forceps, grasp the end of the vitreous where it connects the optic nerve. Pull the two forceps away from each other to remove and discard the vitreous. Then flip the retina downward again and use the straight brush to gently press it to the bottom of the dish.

Afterward, use the loop brush to brush over the vasculature from the optic nerve head towards the periphery to remove remaining neural tissue. Spin the retina slowly with the straight brush and use the loop brush to remove all small chunks of neural tissue until the vascular network is clean. To mount the retinal vasculature, use the loop brush to flip the retina semi sphere facing upward and gently push the vasculature down onto the glass slide.

Then use the hair to tack down the opened vasculature onto the center of the slide. Brush the bull shaped retinal vessel from the optic nerve to the periphery to flat mount the vasculature. Repeat brushing in all directions until the vasculature sticks to the slide completely.