Isolation of Mouse Retinal Capillaries and Subendothelial Matrix for Stiffness Measurement Using Atomic Force Microscopy

Vascular inflammation is known to cause degeneration of retinal capillaries in early diabetic retinopathy, which is a major vision threatening microvascular complication of diabetes. Our lab is investigating whether and how mechanical cues in the form of vascular stiffness can contribute to these abnormal retinal vascular changes in early diabetes. There's a growing interest in developing ways to prevent diabetic retinopathy at the early stage, with a particular focus on inhibiting retinal inflammation that occurs early on in diabetes and contributes to retinal neurovascular dysfunction.

Our recent work indicates that in addition to genetic and biochemical factors, mechanical cues such as vascular stiffness can promote retinal inflammation in early diabetes. Our protocol will allow researchers to isolate intact retinal vessels and subendothelial matrix for subsequent stiffness measurements using atomic force microscope. These measurements will help determine the role of vascular stiffness and endothelial mechanobiology in the development of vascular defects associated with diabetic retinopathy and macular degeneration.

By applying minute nano two picton level iteration forces, atomic force microscopy offers a sensitive, accurate, and reliable technique to directly measure the stiffness of soft vial samples such as blood vessels, cells, and accessible metrics. To our knowledge, such gentle measurements are uniquely possible using an atomic force microscope. Our recent work has shown that retinal capillaries become stiffer in diabetes, which leads to retinal vascular inflammation and degeneration.

A deeper understanding of the mechanical regulation of the diabetic retinopathy, has a potential to identify microbiology based anti-inflammatory targets for more effective therapies in the future. To begin, place a formal and fixed mouse eye on a piece of wax paper under a dissecting microscope. With a pair of micro forceps, hold the remnants of the muscle and optic nerve.

Then orient the eye so that the cornea faces one side. Now use a surgical blade to make a one to two millimeter long incision behind and parallel to the limbus. Apply a downward force with the blade while holding the eye in place until the anterior segment separates from the posterior end.

Then discard the anterior segment and lens. Transfer the posterior segment into a six centimeter dish filled with PBS. Use a micro spatula to scoop out the retina, while simultaneously holding the optic nerve with micro forceps.

To rinse the retina, transfer it into a well of a 12 well plate that has six wells filled with double distilled water. Use a P 1000 pipette to carefully pipette water up and down, adjacent to the retina, while blowing the water to cause gentle agitation. Next, use an inverted glass pipette to transfer the retina to the second well.

To digest the retina, transfer the rinsed retina into Trypsin solution in a 12 well plate with an inverted glass pipette. Center in a Trypsin soaked P 200 pipette tip on the optic nerve. Then gently pipette the entire vascular network up and down to dissociate the nonvascular tissue.

Now use an inverted glass pipette pre-rinsed in Trypsin to transfer the retinal vasculature into a well containing double distilled water, in a six well plate. Swirl the plate, then pipette the vasculature up and down with an inverted Trypsin rinsed glass pipette to remove any residual nonvascular tissue. Carefully transfer the retinal vasculature to the center of the marked region on a charged surface microscopy slide.

Then use a P 200 pipette to carefully remove the excess water from one edge. Place the slide in a bio safety cabinet close to the front side, to let the residual water evaporate. When the vasculature is almost dry, transfer it under a phase contrast microscope.

Use a P 200 pipette to slowly add double distilled water on one side of the marked region to carefully rehydrate the vasculature. Fixation of the isolated retinal vasculature resulted in structurally robust and sufficiently durable tissue suitable for AFM measurements. In contrast, the retinal vessels isolated from unfixed or briefly fixed eyes became fragmented and collapsed.

To begin, rinse glutaraldehyde cross-linked gelatin coated cover slips in PBS containing calcium magnesium, about five times on an orbital shaker. During the first three rinses, lift the cover slips using a sterile tweezer to allow thorough rinsing of the glutaraldehyde. Next, replace the culture medium of mouse RECs with fresh medium supplemented with sterile filtered ascorbic acid.

After 15 days of treatment, rinse the cells with calcium magnesium free PBS. Incubate the cells in 250 microliters of warm decellularization buffer for two to three minutes. Confirm the decellularization of the cells under a phase contrast microscope.

Gently pipette out the buffer without disturbing the REC secreted subendothelial matrix. Then add 0.5 milliliters of PBS into each well. After removing the PBS, add 200 microliters of our N-ace free DN-ace 1.

Incubate the mixture at 37 degrees Celsius for 30 minutes to remove all cellular debris. Next, view the macroscale fibrous subendothelial matrix under a phase contrast microscope. Then use a confocal microscope to view the finer nano scale matrix fibers at 100 x magnification.

Subendothelial matrix aggregates were observed on the glass cover slips following decellularization of 10 day or 15 day cultures of human or mouse RECs. Confocal microscopy of the decellularized matrices showed a dense nano fibrillar collagen four and fibronectin matrix. To begin, fix the cantilever holder of an AFM onto the cantilever changing stand.

Use watchmaker forceps to mount the selected cantilever on the holder. Tighten the screw on the holder to secure the cantilever. Place and lock the cantilever holder on the AFM head that is resting on the stand.

Use the step motor function in the AFM software to withdraw the AFM head to the highest point, and set that position as point zero. Carefully lift the AFM head from its stand, then mounted on the sample stage by placing the legs in their respective slots. To calibrate the cantilever, use the step motor function to bring it close to the sample surface.

Then click on approach in the contact mode for spectroscopy window. Bring down the cantilever in small increments of 15 micrometers. Press acquire to capture a force curve.

Then select the force curve. Open it in the calibration manager window and select contact based mode in the method section. Now use the select fit range function and select the cantilever retraction curve for a linear curve fit.

Next, check the sensitivity checkbox to convert the force unit from volt to Newton. Lift the cantilever by 100 to 200 micrometers in the liquid and select the thermal noise function. Again, click on select fit range and fit the thermal noise bell curve with a Lawrence curve.

After curve fitting, choose the spring constant K box. Confirm that the spring constant is close to the manufacturer's value and note it down for future reference. Next place the slide mounted sample of rodent eye retinal vessels or subendothelial matrix on the AFM stage and visualize the retinal vessels.

Select contact mode for spectroscopy in the software's experiment section. Then set the set point force to 0.5 nano newtons and click approach. Use the stage screws on the AFM stage to carefully position the cantilever probe at a desired location on the sample.

Click approach to position the cantilever closer to the sample, and then click acquire to capture the force curves. Finally, save all force curves for analysis. The approach and retraction curves obtained from a retinal capillary isolated from a diabetic mouse were steeper than those obtained from a non-diabetic mouse.