Optic Nerve

BackgroundThe resident astrocyte-retinal ganglion cell (RGC) lipoxin circuit is impaired during retinal stress, which includes ocular hypertension-induced neuropathy. Lipoxin B4 produced by homeostatic astrocytes directly acts on RGCs to increase survival and function in ocular hypertension-induced neuropathy. RGC death in the retina and axonal degeneration in the optic nerve are driven by the complex interactions between microglia and macroglia. Whether LXB4 neuroprotective actions include regulation of other cell types in the retina and/or optic nerve is an important knowledge gap.MethodsCellular targets and signaling of LXB4 in the retina were defined by single-cell RNA sequencing. Retinal neurodegeneration was induced by injecting silicone oil into the anterior chamber of mouse eyes, which induced sustained and stable ocular hypertension. Morphological characterization of microglia populations in the retina and optic nerve was established by MorphOMICs and pseudotime trajectory analyses. The pathways and mechanisms of action of LXB4 in the optic nerve were investigated using bulk RNA sequencing. Transcriptomics data was validated by qPCR and immunohistochemistry. Differences between experimental groups were assessed by Student's t-test and one-way ANOVA.ResultsSingle-cell transcriptomics identified microglia as a primary target for LXB4 in the healthy retina. LXB4 downregulated genes that drive microglia environmental sensing and reactivity responses. Analysis of microglial function revealed that ocular hypertension induced distinct, temporally defined, and dynamic phenotypes in the retina and, unexpectedly, in the distal myelinated optic nerve. Microglial expression of CD74, a marker of disease-associated microglia in the brain, was only induced in a unique population of optic nerve microglia, but not in the retina. Genetic deletion of lipoxin formation correlated with the presence of a CD74 optic nerve microglia population in normotensive eyes, while LXB4 treatment during ocular hypertension shifted optic nerve microglia toward a homeostatic morphology and non-reactive state and downregulated the expression of CD74. Furthermore, we identified a correlation between CD74 and phospho-phosphoinositide 3-kinases (p-PI3K) expression levels in the optic nerve, which was reduced by LXB4 treatment.ConclusionWe identified early and dynamic changes in the microglia functional phenotype, reactivity, and induction of a unique CD74 microglia population in the distal optic nerve as key features of ocular hypertension-induced neurodegeneration. Our findings establish microglia regulation as a novel LXB4 target in the retina and optic nerve. LXB4 maintenance of a homeostatic optic nerve microglia phenotype and inhibition of a disease-associated phenotype are potential neuroprotective mechanisms for the resident LXB4 pathway.

Glaucoma is characterized by progressive retinal ganglion cell (RGC) loss and optic nerve head (ONH) changes, but the roles of glial activation and immune responses remain unclear. This study examines gliosis, microglial diversity, and inflammation in postmortem retinal tissues. Postmortem retinal and ONH samples (total n = 50) from patients with open-angle glaucoma (G, n = 18) were compared with those from age-matched controls (n = 32), including healthy individuals (Ctrl) and disease controls (patients with early age-related macular degeneration [AMD] and diabetes mellitus [DM]). Immunostaining was performed to assess glial activation, blood–retinal barrier (BRB) integrity, and immune infiltration, which were quantified via ImageJ and Zen lite. Generalized estimating equations (GEEs) with Bonferroni correction accounted for intrapatient variability. G retinae presented significant RGC loss accompanied by widespread gliosis, with activation of microglia (Iba1), astrocytes (GFAP), and Müller cells (Vimentin). This gliotic response differed across conditions, with astrocyte activation being more prominent in DM and microglial activation predominating in AMD. In glaucoma, gliosis is evident even in early-stage disease, regardless of the severity of retinal ganglion cell (RGC) loss or structural changes in the ONH. Furthermore, microglia showed a marked shift in morphological diversity, transitioning to hyperramified, bushy, and amoeboid forms, along with an increased distribution of activation markers such as CD45, CD11b, and CD163. Additionally, biochemical evidence of alterations to the BRB integrity, characterized by reduced tight junction protein expression, facilitates immune cell infiltration, as indicated by the minimal and inconsistent presence of CD3/CD4+ T cells. Gliosis persisted regardless of RGC loss severity, suggesting that gliosis progresses independently of neuronal degeneration. Unlike AMD and DM, where specific glial subtypes dominate, glaucoma exhibits widespread gliosis. Microglial heterogeneity indicates the existence of a continuum of functional states. Furthermore, dysregulation of the BRB, inconsistent immune infiltration, and multimodal microglial activation indicate that the inflammatory response in glaucoma patients is driven primarily by resident microglia, with limited interactions with infiltrating immune cells. These findings highlight the need for further research into glial modulation as a potential therapeutic strategy.

BackgroundAxon regeneration within the mammalian central nervous system is extremely limited. In optic neuropathy conditions like glaucoma, the inability of retinal ganglion cell (RGC) axons to regenerate is a major impediment to functional recovery. In contrast, adult teleost fish such as zebrafish can fully regenerate RGC axons enabling visual recovery from optic nerve (ON) injury making it an ideal model to probe the mechanisms of successful axon regeneration.MethodsLaser Capture Microdissection followed by RNA-sequencing (LCM-seq) was used to identify genes and pathways differentially expressed in RGCs during ON regeneration. We validate these findings by in situ hybridization and qRT-PCR. Using loss- and gain-of-function experiments we demonstrate the necessity of srebf2 for efficient ON regeneration and recovery of visual function. Finally, we use LCM-seq coupled with experimental manipulations to identify downstream srebf2 target genes and test the role of hmgcra/b and mevalonate in this process. Statistical analysis was performed using Student's t-test, two-way ANOVA, or repeated measures with appropriate post-hoc tests where applicable.ResultsLCM-seq comparison of uninjured versus 3-day post ON injury RGCs identified significant upregulation of the cholesterol synthesis pathway during axon regeneration. The master regulator of this pathway, the transcription factor srebf2, is upregulated throughout the regeneration phase. Chemical inhibition or morpholino-based gene knockdown of srebf2 decreased axon regeneration into the ON and optic tectum and delayed recovery of visual behavior over the course of normal optic nerve regeneration without causing a significant loss of RGCs. Constitutively active srebf2 can fully rescue axon regeneration and visual behavior losses caused by inhibition of endogenous srebf2 but does not accelerate regeneration compared to the control group. LCM-seq confirms the expected regulation of predicted srebf2 target genes after loss- or gain-of-function in vivo. Downstream of srebf2, hmgcra/b knockdown or simvastatin treatment delayed axon regeneration and this effect was rescued by supplemental mevalonate. Mevalonate treatment alone was sufficient to accelerate ON regeneration.ConclusionsThese results demonstrate that srebf2 and the downstream mevalonate synthesis pathway plays an important role in regulating efficient axon regeneration in the zebrafish visual system. Involvement of this pathway should be closely examined in failed mammalian ON regeneration.

PurposeTo investigate the ability of flavoprotein fluorescence (FPF) imaging to quantify disease burden in optic disc drusen (ODD).DesignCross-sectional study.ParticipantsOne hundred and fifty-seven ODD eyes (94 participants, ages 7-89 years) and 69 control eyes (53 participants, ages 10-78 years).MethodsComprehensive examination, visual function testing, and multimodal ophthalmic imaging. Statistical analysis was performed using parametric and nonparametric tests, ANOVA, and Spearman correlation.Main outcome measuresLogMAR, static perimetry mean deviation, optic disc and macular FPF, enhanced-depth imaging optical coherence tomography (EDI-OCT), OCT peripapillary retinal nerve fiber layer (pRNFL), and macular ganglion cell complex (mGCC) thickness.ResultsOptic disc FPF signal corresponded with superficial and buried drusen visualized on 97 EDI-OCT B-scans. Compared with controls, ODD eyes have significantly elevated disc FPF (P ConclusionsDisc FPF is an objective imaging technique to quantify disease burden in ODD, reflecting a combination of drusen autofluorescence signal and metabolic stress from axonopathy. Disc FPF is correlated with structural and functional changes and has high predictive power of visual field loss in ODD, supporting its use as an outcome measure in prospective natural history and treatment studies in ODD.