Abstract
Purpose :
Retinal ganglion cell (RGC) death presents as a hallmark feature of various ocular diseases, thus understanding avenues of RGC neuroprotection is imperative. Broadly known to play an immunomodulatory and neurotrophic role, locally synthesized calcitriol may also provide insight into attenuating RGC degeneration. Employing a transgenic knock out mouse model for CYP27B1, the calcitriol synthesizing enzyme, we hypothesize that calcitriol deficiency will result in greater loss of RGCs upon mechanical crush injury to the optic nerve.
Methods :
Using a unilateral optic nerve crush (ONC) model, differences in progression of injury at 3, 7, 14-day timepoints post crush was investigated in 8 – 12-week-old CYP27B1+/+ (WT) and CYP27B1-/- (KO) mice. Structural analysis of optic nerves and retinas was conducted using immunofluorescent staining. Onset of RGC death and survival with progression of injury was determined by staining retinal wholemounts with antibodies against Brn3a and cleaved caspase 3 (N = 4-6/ group). Injury mediated immune responses were also characterized within the optic nerve and the retina (N= 4-6/ group) using flow cytometry. Parenchymal and non-parenchymal macrophage populations were targeted using CD45, CD11b, CD11c, F4/80 and Ly6G antibodies. Immunofluorescence staining analysis was performed using ImageJ. A 2-way ANOVA and unpaired t-tests were used to determine statistical significance.
Results :
No statistically significant differences were observed in RGC and immune populations at baseline between CYP27B1 WT and KO animals. However, upon injury, a significantly greater loss of RGCs in the KO animals was observed at 3-day (p<0.01), 7-day (p<0.05) and 14-day (p<0.01) timepoints. Caspase 3 stained apoptosis revealed a similar pattern (p<0.001). When comparing microglial and macrophage localization within the optic nerve, KO animals demonstrated peak localization at an earlier timepoint (3-day) compared to their WT counterparts (7-day). Lastly, both microglial and macrophage localization within the retina was greater in the calcitriol deficient KO animals when compared to WT animals.
Conclusions :
These results suggest that the locally produced calcitriol plays an instrumental role in aiding RGC survival during acute injury. We aim to further investigate its role by conducting gene expression experiments that target the molecular mechanism through which neuroprotection is exerted.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.