Abstract
Purpose :
Ibudilast, an allosteric macrophage migration inhibitory factor (MIF) and phosphodiesterase inhibitor, is in clinical trials in the US for evaluation as a neuroprotective agent in neurological diseases. Noxious insult to the retina triggers macrophage/microglia (MØ/µglia) activation that manifests as an amoeboid morphology and migration to the site of injury, contributing to disease pathogenesis. We evaluated the effects of Ibudilast on MØ/µglia morphology and distribution in the retina following N-methyl-D-aspartate (NMDA) damage, which simulates glutamate excitotoxicity involved in retinal ischemic disease.
Methods :
Under an IACUC-approved protocol, white leghorn chicks were treated with intravitreal injection of NMDA (500nmol/20ul) ± Ibudilast (1mg/ml). Chicks were sacrificed at one-day post-injection (D1, n=6) or nine days post-injection (D9, n=8). MØ/µglia were evaluated using CD45 immunofluorescence. D1 confocal images were analyzed using ImageJ to calculate average mean intensity and area above a threshold. D9 confocal images were analyzed using NIS-Elements software. To investigate differences in MØ/µglia distribution, the retina was divided into three layers: Photoreceptor + Outer Nuclear Layer (PR-ONL), Outer Plexiform + Inner Nuclear Layer (OPL-INL), and Inner Plexiform + Ganglion Cell + Nerve Fiber Layer (IPL-GCL-NFL). Areas, Form factor 1 (FF1), aspect ratio (AR) morphology measures, and cell counts were identified by thresholding the CD45 signal. Statistics were performed in JMP with Wilcoxon Rank Sums.
Results :
In the D1 group, MØ/µglia were solely located in the inner plexiform layer (IPL) 24h after damage in both treated and control groups. There were no significant differences in CD45 intensity (p=0.82) or area above a threshold (p=0.45). In the D9 group, the number of CD45 positive cells in the PR-ONL layer decreased with Ibudilast treatment (16.69 ± 12.00 vs. 9.31 ± 4.87, p=0.029). All other measurements in all three layers were not found to exhibit significant differences.
Conclusions :
The decrease in MØ/µglia in the photoreceptor layer may indicate that Ibudilast treatment inhibits MIF’s ability to bind to CCR4, thereby reducing MØ/µglia migration to the outer retina. Further studies involving additional markers are required to fully elucidate the effect of Ibudilast treatment on MØ/µglia dynamics and activation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.