Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

The overall goal of this procedure is to assess the phagocytic function of retinal microglia in vivo. This method can help answer key questions in the ophthalmology field, such as whether certain compounds alter microglia phagocytic function in a physiological setting. The main advantage of this technique is that it uses flow cytometry, allowing a fast and precise quantitative analysis of microglial phagocytosis.

Helping me to demonstrate the procedure will be Susumu Sakimoto, a postdoc from my laboratory. Begin by loading a 33-gauge needle and syringe with 0.5 microliters of fluorescently labeled particle solution. Then place an anesthetized mouse sideways on soft material under a surgical microscope and confirm the appropriate level of sedation by a lack of response to toe pinch.

Next, use forceps to carefully apply pressure around one eyelid, so that the eye ball pops slightly out of the socket. Then grasp the head with two fingers just above the ear and by the animal’s jaw, and gently stretch the skin parallel to the eyelids to keep the eye slightly bulged out of the socket. Intravitreal injections are challenging, and if not performed correctly will lead to biased and variable results.

To reduce trauma to the eye, hold the animal in a stable position with minimal head movement. To puncture the eyeball, gently grasp the mouse with one hand. Then locate the corneal limbus where the cornea and sclera connect, which is visible as a gray circle in pigmented mice.

Holding the syringe in the other hand, insert the needle into the limbus. Then retract the needle slightly to expel a small volume of vitreous fluid, and slowly depress the plunger to inject the particles. When all of the beads have been delivered, slowly withdraw the syringe to avoid reflux of the injected material and apply moisturizing drops to keep the eye hydrated once the eye has retracted back into place.

Then place the animal on a heating pad in its own cage with monitoring until it is fully recovered. Three hours after intravitreal injection, use 45 degree angle forceps to gently press against the eyelid to proptose the eyeball. Position the forceps behind the eyeball and pull.

Then transfer the eyeball to the dry area of a petri dish, containing a small volume of PBS with calcium and magnesium under a dissecting microscope. And use one tip of a superfine forceps to perforate the eye in the corneal limbus. Next, holding the eyeball with fine 45-degree angled forceps, use spring scissors to cut around the corneal limbus until roughly half of the circumference is cut.

Transfer the eyeball into PBS and use a second pair of fine 45-degree angled forceps to tear the cornea and sclera apart. The lens and retina will come out intact. Ensure the lens and retina are separated and transfer the retina to a 5.4 milliliter polystyrene test tube, containing two milliliters of PBS with calcium and magnesium.

To obtain a single cell suspension of the retinal cells, use a neuronal tissue dissociation kit according to the manufacturer’s instructions and resuspend the cells in 200 microliters of staining buffer. Then transfer the sample to one well of a 96-well U-bottom plate. After centrifugation, invert the plate to discard the supernatant and block the FC receptors with 25 microliters of stain buffer containing CD16, CD32 antibody per well for five minutes at room temperature.

Next label the cells with the antibodies of interest for 15 minutes at room temperature in the dark. Then pellet the cells and follow this by a wash in 200 microliters of fresh staining buffer per well. Now resuspend the pellets in 200 microliters of stain buffer and viability dye and transfer the samples to 1.2 milliliter microtiter tubes.

Then wash the wells with an additional 100 microliters of staining buffer and viability dye and pool the washes with the corresponding samples for analysis by flow cytometry. This method can be used in 10 to 20-day old postnatal or adult mice and can be adapted to test the effect of compounds and/or the genetic manipulation of microglial phagocytosis. For example, in this experiment after intraperitoneal challenge with varying doses of LPS, a 1.42 milligram per kilogram dose of LPS was determined to induce a statistically significant increase in the percentage of phagocytic microglia, compared to vehicle challenged controls.

Once mastered, this technique can be completed in six hours if it is performed properly. Following this procedure, other methods like cell sorting followed by qPCR or proteomic analysis, can be used to answer further questions about the differences between phagocytic and non phagocytic microglia in the retina. In developing this technique, it’s our expectation that we can now make it possible for vision and neuro scientists to further explore microglial phagocytic function in situ and in a physiologically relevant context.