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Aniridia is a panocular condition characterized by impaired eye development and vision, which is mainly due to the haploinsufficiency of the paired-box-6 (PAX6) gene. Like what is seen in aniridia patients, Pax6-deficient mice Pax6(Sey-Neu/+) exhibit a varied degree of ocular damage and impaired vision. Our previous studies showed that theses phenotypes were partially rescued by PD0325901, a mitogen-activated protein kinase kinase (MEK or MAP2K) inhibitor. In this study, we assessed the long-term efficacy of PD0325901 treatment in retinal health and visual behavior. At about one year after the postnatal treatment with PD0325901, Pax6(Sey-Neu/+) mice showed robust improvements in retina size and visual acuity, and the elevated intraocular pressure (IOP) was also alleviated, compared to age-matched mice treated with vehicles only. Moreover, the Pax6(Sey-Neu/+) eyes showed disorganized retinal ganglion cell (RGC) axon bundles and retinal layers, which we termed as hotspots. We found that the PD treatment reduced the number and size of hotspots in the Pax6(Sey-Neu/+) retinas. Taken together, our results suggest that PD0325901 may serve as an efficacious intervention in protecting retina and visual function in aniridia-afflicted subjects.

Retinal ganglion cells (RGCs) exhibit compartmentalized organization, receiving synaptic inputs through their dendrites and transmitting visual information from the retina to the brain through the optic nerve. Little is known about the structure of RGC axon bundles extending from individual RGC somas to the optic nerve head (ONH) and how they respond to disease insults. We recently introduced visible-light optical coherence tomography fibergraphy (vis-OCTF), a technique for directly visualizing and analyzing mouse RGC axon bundlesin vivo. In this study, we validated vis-OCTF's ability to quantify RGC axon bundles with an increased number of RGCs using mice deficient in BCL2-associated X protein (BAX−/−). Next, we performed optic nerve crush (ONC) injury on wild-type (WT) mice and showed that the changes in RGC axon bundle width and thickness were location-dependent. Our work demonstrates the potential of vis-OCTF to longitudinally quantify and track RGC damage at single axon bundle level in optic neuropathies.SIGNIFICANCE STATEMENTNearly all clinical and preclinical studies measure the retinal nerve fiber (RNFL) thickness as the sole indicator of retinal ganglion cell (RGC) damage without investigating RGC axon bundles directly. We demonstrated visible-light optical coherence tomography fibergraphy (vis-OCTF) to directly quantify global and regional RGC axon bundle organizationsin vivoas a new biomarker for RGC health. We validatedin vivovis-OCTF measures using both confocal microscopy of the immunostained flat-mounted retina and numerical simulations. Vis-OCTF for monitoring RGC axon bundle organization has the potential to bring new insight into RGC damage in optic neuropathies.

Purpose To develop a practical technique for visualizing and quantifying retinal ganglion cell (RGC) axon bundles in vivo. Methods We applied visible-light optical coherence tomography (vis-OCT) to image the RGC axon bundles, referred to as vis-OCT fibergraphy, of healthy wild-type C57BL/6 mice. After vis-OCT imaging, retinas were flat-mounted, immunostained with anti-beta-III tubulin (Tuj1) antibody for RGC axons, and imaged with confocal microscopy. We quantitatively compared the RGC axon bundle networks imaged by in vivo vis-OCT and ex vivo confocal microscopy using semi-log Sholl analysis. Results Side-by-side comparison of ex vivo confocal microscopy and in vivo vis-OCT confirmed that vis-OCT fibergraphy captures true RGC axon bundle networks. The semi-log Sholl regression coefficients extracted from vis-OCT fibergrams (3.7 ± 0.8 mm-1) and confocal microscopy (3.6 ± 0.3 mm-1) images also showed good agreement with each other (n = 6). Conclusions We demonstrated the feasibility of using vis-OCT fibergraphy to visualize RGC axon bundles. Further applying Sholl analysis has the potential to identify biomarkers for non-invasively assessing RGC health. Translational relevance Our novel technique for visualizing and quantifying RGC axon bundles in vivo provides a potential measurement tool for diagnosing and tracking the progression of optic neuropathies.

C5a is a crucial terminal effector of the C cascade, mostly involved in pain and neuroinflammatory conditions. DF3016A is a novel potent and selective C5a receptor (C5aR) inhibitor that crosses the blood-brain barrier (BBB) and may have pharmacological properties. We have previously demonstrated a protective effect of DF3016A on injured primary cortical neurons by oxygen-glucose deprivation-reoxygenation (OGD/R) model to mimic the neuroinflammatory process. Here, we investigated the molecular pathway and factors involved in the neuroprotection previously reported. Our findings show that DF3016A protects against the neuroinflammatory insult by activating brain-derived neurotrophic factor (BDNF) transcription pathway, which involves methyl CpG-binding protein 2 (MeCP2) and microRNA-132 (miR-132) regulatory factors, both required in nociceptive signaling and neuroinflammation. Further in vivo investigations will confirm the functionality of the DF3016A molecule as a therapeutic resource in neuroinflammation and pain injuries.