María Josefa González Riquelme¹

(1) Universidad de Murcia, 30565, España (Región de Murcia)

Microglia cells respond to injury and infection, and act in the first line to defense tissue and promote the destruction of invading pathogens. To this end, M1 microglial cells release pro-inflammatory factors and various neurotoxic mediators that if cronified, may cause neuronal death., Inflammation is resolved by M2 microglial cells, with an antiinflammatory phenotype that leads to wound healing and brings back tissue homeostasis. The main purpose of this work is to analyze the temporal course of microglial activation and M2 appearance in the mouse retina after optic nerve crush (ONC), a well characterized model of neuronal degeneration. In this model, retinal ganglion cell (RGC) death occurs in two phases, a quick one where RGC loss is first significant at day 3 and proceeds lineally until day 9. During this quick phase 50% of RGC have died at day 5, and 85% at day 9. Thereafter the second phase of RGC death commences, and they die slowly but continuously and by 45 days post lesion, only 2% of them survive.

ONC was performed in the left optic nerve of adult female C57/BL6 mice. Both retinas of each animal were dissected as flat mounts at 3, 5, 9 and 45 days after the axotomy (n= 4-6 retinas/time point). As control, intact retinas were analyzed in parallel. Flat mounts were subjected to double Immunodetection of Iba1 (microglial cells) and CD2006+ (M2 state), photographed and the total number of M2 cells quantified and plotted to assess their topographical distribution. The average density of Iba1 + microglia is also calculated, quantifying them in 12 areas of the retina.

In the injured retinas, there is a significant increase of CD206+cells at 5 and 9 days post-lesion. At 45 days, however, their number has decreased below the number found intact retinas. With respect to the microglia Iba1+ we observed that indeed, there is a significant increase at 5, 9 and 45 days compared to control animals. In the contralateral uninjured retinas, the number of M2 cells decreases significantly compared to intact ones at all time points of analyses. We observe that in the contralateral uninjured retinas, there is a decrease in the density of Iba 1+ at 5 days compared to intact animals. In intact and contralateral retinas M2 cells are few, and they are found scattered across the retina. After injury, they increase around the optic nerve disk, and the retinal periphery.

In the injured retina, optic nerve crush induces an increase in total microglia and M2 microglia at 5 and 9 days, reflecting the course of RGC death. While the M2 microglia decreases at 45 days, the number of microglia Iba1+ remains higher than in intact retinas at all time points. In the contralateral retinas there is a transient decrease of microglia Iba 1+ at 5 days and a decrease over the time of M2 microglia.