Quantification of Diabetes-induced Adherent Leukocytes in Retinal Vasculature

The focus of research in our laboratory is to learn what causes retinopathy in diabetes and how it can be prevented. Several laboratories have shown the leukostasis, the overaccumulation of white blood cells, in the small vessels of the eye is increasing diabetes. And this might contribute to the occlusion and degeneration of the vessels in the retina in diabetes. This technique allows the visualization and quantification of the severity of the leukostasis in the retinal vasculature. And it can be used in the studies that want to evaluate the efficacy of different therapies, including drugs.

[Instructor] To set up the pressure infuser, connect in series the 0.9% saline bag. the intravenous catheter set, a four-way stopcock valve, and the gavage needle. Open the perfusion system and flush the lines and ports with saline for two minutes at a flow rate of 18 to 20 milliliters per minute to remove any air bubbles. Place the anesthetized mouse supine on the perfusion stage. Identify the xiphoid process visually. With the hemostat in the dominant hand, secure and lock the skin. Then transfer the hemostat to the nondominant hand and lift the skin. Next, using scissors, cut a patch of skin at a 90-degree angle to the spine to expose the outer abdominal wall. With the xiphoid process and rib cage visible, dissect bilaterally through the abdominal wall, carefully avoiding any organs or major vessels. Now, visualize the heart ventricles and lungs through the diaphragm. Using the scissor tip, make an incision in the diaphragm on one side near the spine. Expose the heart in the thoracic cavity after bisecting the sternum. With forceps in the non-dominant hand, grasp the heart near its apex. Using the dominant hand, hold the gavage needle attached to the intravenous catheter and puncture the apex of the heart. Ensure the ball tip of the gavage needle is slightly protruding from the puncture site to avoid fully perforating the left ventricle or entering the pulmonary vasculature. Next, open the stopcock to allow the flow of 0.9% saline. Simultaneously, use scissors to cut open the right atrium. Continue perfusion for two to three minutes, moving the needle gently from side to side and up and down to prevent vasculature kinking and increase blood drainage from the heart. After perfusion, turn the stopcock handle to shut off the saline flow and allow flow from the syringe to the gavage needle. Then manually perfuse 10 milliliters of concanavalin A solution within 30 to 35 seconds. After perfusing the heart of an anesthetized mouse with concanavalin A solution, turn the mouse on its side. Place the index finger of the nondominant hand on the superior eyelid and the thumb on the inferior eyelid. Gently retract the eyelids and surrounding skin to proptose the eye, allowing it to partially bulge out of the socket. Next, using the curved scissors in the dominant hand, scoop under the eye at a 45-degree angle. Cut the muscular attachments and the optic nerve. Using the scissors as a spatula, transfer the eye to a small container. Place the eye onto dental wax to prepare for globe opening. Under the dissecting microscope, using micro forceps, hold the scleral fold or muscle remnants attached to the posterior of the eye and orient the eye so that the cornea faces to one side. Make an incision behind and parallel to the limbus. Use micro scissors to separate the anterior segment of the eye, including the lens around the perimeter of the eye cup, ensuring the retina is fully detached from the sclera. Scoop the retina out from the sclera with the micro spatula. If the retina is still attached by the optic nerve, insert micro scissors between the retina and sclera to sever the optic nerve. Place the unfixed retina on a slide with a drop of PBS. Make four to five radial cuts into the retina, creating a clover leaf pattern so that it lies flat. Observe the flat mounted retina under the microscope at 100x magnification using a 10x objective. Fluorescent microscopy of the retinal vasculature showed that diabetic retinas displayed mild leukostasis with typically 3 to 12 leukocytes per retina compared to 1 to 3 in nondiabetic controls. Significant leukocyte clusters were observed in lipopolysaccharides-challenged inflammatory models in contrast to the mild clustering in diabetic samples.