Abstract
Purpose :
Our preliminary data show that the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is upregulated in a murine N-methyl-D-aspartate (NMDA) excitotoxic model. We evaluated the effect of MIF genetic depletion on retinal ganglion cell (RGC) death and retinal thickness in a murine NMDA model, which simulates glutamate excitotoxicity involved in retinal ischemic disease.
Methods :
Under an IACUC approved protocol, MIF-knockout (MIFKO) mice and C57BL/6 background controls (WT) were treated with intravitreal injection of NMDA (0.1M/2ul) in the left eye and vehicle in the right. Eyes were enucleated and fixed 24h after treatment (n=6 WT, 11 MIFKO) for analysis of dying cells with TUNEL or at day 9 for ganglion cell layer (GCL) thickness measurements (n=7 WT, 6 MIFKO). GCL thickness measurements were obtained using fluorescent microscopy of DAPI stained sections. Analysis was performed with NIS Elements software using the MCT threshold method for cell death and RGC density calculations. Statistics were performed in JMP with Wilcoxon Signed Rank and Wilcoxon Rank Sum tests. Averages ± standard error of the mean were calculated.
Results :
Within each WT and MIFKO group at 24h post damage, the proportion of dying cells in the GCL was significantly higher with NMDA treatment compared to vehicle (WT: 0.35 ± 0.05 vs 0.01 ± 0.01, p=0.0313; MIFKO: 0.15 ± 0.03 vs 0.001 ± 0.001, p=0.0010). The proportion of dying cells in the GCL was significantly reduced 58.6% in damaged MIFKO compared to damaged WT retina (p=0.0077). At day 9 post damage, MIFKO eyes did not lose RGC density compared to undamaged controls (1138.01 ± 235.69 vs 1102.74 ± 126.10, p=1.00) while the WT mice had significantly lower RGC density after NMDA treatment (737.62 ± 120.72 vs 1253.56 ± 143.98, p=0.0313). The ratio of damaged to undamaged RGC densities at D9 showed preservation of RGCs in the MIFKO compared to WT mice (1.13 ± 0.27 vs 0.60 ± 0.13, p=0.05). There was no difference in TUNEL and GCL thickness between vehicle-treated MIFKO and WT groups.
Conclusions :
Our results indicate that MIF has a significant impact on cell survival in excitotoxic damage as genetic depletion reduced cell death and preserved the retinal layer thickness. Future studies should further evaluate MIF inhibition as a potential therapeutic for excitotoxic retinal damage and preservation of retinal neurons.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.