Investigative Ophthalmology & Visual Science

PurposeElevated intraocular pressure (IOP) due to increased outflow resistance through the trabecular meshwork (TM) is a major risk factor for primary open-angle glaucoma. Outflow through the TM is segmental, consisting of high flow (HF) and low flow (LF) regions. Here, we investigate how ocular hypertension impacts segmental outflow using a dexamethasone (DEX) mouse model and compare TM stiffness between HF and LF regions. MethodsNanoparticles containing DEX or vehicle were injected twice weekly in 2-4-month-old C57BL/6J mice (n=14), and IOP was measured weekly. At week 4, mouse eyes were perfused in vivo with fluorescent nanospheres to assess flow patterns and the circumferential percentage of high, intermediate, and low flow regions in each eye. Sagittal sections were collected from HF and LF regions, and atomic force microscopy (AFM) was used to measure tissue stiffness. Immunofluorescent labeling was used to compare fibronectin and -SMA protein levels. ResultsDEX treatment significantly elevated IOP by an average of 33.3% and altered tracer distribution but not the percentage of HF and LF regions around the circumference. No significant differences in TM stiffness were detected between DEX-treated and control mice, or between HF and LF regions. Increased fibronectin in LF regions of DEX-treated eyes suggested subtle TM structural changes that were not detected by AFM. ConclusionsDexamethasone alters segmental flow distribution and may impact cell contractility rather than ECM stiffness to cause IOP elevation in young mice. These findings better characterize the nature of segmental outflow and TM mechanics in this model of steroid-induced glaucoma.

PurposeWe aimed to assess optic nerve head (ONH) deformations following acute intraocular pressure (IOP) elevations and horizontal eye movements (adduction and abduction) in control eyes, highly myopic (HM) eyes, HM eyes with glaucoma (HMG), and eyes with pathologic myopia alone (PM) or PM with staphyloma (PM+S). MethodsWe studied 282 eyes, comprising of 99 controls, 51 HM, 35 HMG, 21 PM and 75 PM+S eyes. For each eye, we imaged the ONH using spectral-domain optical coherence tomography (OCT) under the following conditions: (1) primary gaze, (2) 20{degrees} adduction, (3) 20{degrees} abduction and (4) primary gaze with acute IOP elevation (to ~35 mmHg) achieved through ophthalmodynamometry. For each OCT volume, we automatically segmented the ONH tissues using deep learning. We performed digital volume correlation (DVC) analysis to compute IOP- and gaze-induced ONH displacements and effective strains (i.e. local deformations). All biomechanical quantities were compared across groups. ResultsUnder IOP elevation, we found that HM eyes exhibited significantly lower strains (3.9 {+/-} 2.4 %) than PM eyes (6.9 {+/-} 5.0%, p < 0.001), HMG eyes (4.7 {+/-} 1.8%, p = 0.04) and PM+S eyes (7.0 {+/-} 5.2%, p < 0.001). Under adduction, we found that HM eyes exhibited significantly lower strains (4.8% {+/-} 2.7%) than PM+S eyes (6.0 {+/-} 3.1%, p = 0.02). We also found significant associations between axial length (or refractive error) and strains - eyes with higher axial length and greater myopia were associated with higher strains. IOP-induced strains were also positively correlated with adduction-induced strains. ConclusionWe found that HMG eyes experienced significantly higher strains under IOP elevations as compared to HM eyes. Additionally, PM+S eyes experienced highest ONH strains as compared to other groups under all biomechanical loads. Our preliminary findings suggest the possibility of using a simple biomechanical test to tease out the susceptibility of HM eyes to further develop glaucoma and/or staphyloma.

Glaucoma is the leading cause of irreversible blindness with trabecular meshwork (TM) dysfunction resulting in elevated intraocular pressure and retinal ganglion cell (RGC) damage leading to vision loss. In this study, we discovered that secretome, derived from human TM stem cells, via minimal invasive periocular injection, can reduce intraocular pressure, restore TM homeostasis, protect RGC, and restore RGC function in both steroid-induced and genetic myocilin mutant mouse models of glaucoma. The secretome upregulated the COX2-PGE2 axis via mitochondrial TMEM177 and led to activation of endogenous stem cells and TM regeneration. Inhibition of COX2 abolished the protective effect of secretome on TM cells. Secretome treatment also enhanced RGC survival and function. Proteomic analysis revealed that the secretome is enriched with proteins involved in extracellular matrix modulation leading to the remodeling of TM to restore homeostasis. This study highlights the feasibility of stem cell-free therapy for glaucoma with minimal invasive administration and the involvement of multiple novel pathways for a cumulative regenerative effect on the TM to protect RGC. Brief summaryThis study describes a cell-free treatment using stem cell secretome in two animal models of glaucoma and explores the potential mechanisms

Piezo channels are associated with neuropathology in diseases like traumatic brain injury and glaucoma, but pathways linking tissue stretch to aberrant neural signaling remain unclear. The present study demonstrates that Piezo1 activation increases action potential frequency in response to light and the spontaneous dark signal from mouse retinal explants. Piezo1 stimulation was sufficient to increase cytoplasmic Ca2+ in soma and neurites, while stretch increased spiking activity in current clamp recordings from of isolated retinal ganglion cells (RGCs). Axon-marker beta-tubulin III colocalized with both Piezo1 and Piezo2 protein in the mouse optic nerve head, while RGC nuclear marker BRN3A colocalized with Piezo channels in the soma. Piezo1 was also present on GFAP-positive regions in the optic nerve head and colocalized with glutamine synthetase in the nerve fiber layer, suggesting expression in optic nerve head astrocytes and Muller glia end feet, respectively. Human RGCs from induced pluripotent stem cells also expressed Piezo1 and Piezo2 in soma and axons, while staining patterns in rats resembled those in mice. mRNA message for Piezo1 was greatest in the RPE/choroid tissue, while Piezo2 levels were highest in the optic nerve, with both channels also expressed in the retina. Increased expression of Piezo1 and Piezo2 occurred both 1 and 10 days after a single stretch in vivo; this increase suggests a potential role in rising sensitivity to repeated nerve stretch. In summary, Piezo1 and Piezo2 were detected in the soma and axons of RGCs, and stimulation affected the light-dependent output of RGCs. The rise in RGCs excitability induced by Piezo stimulation may have parallels to the early disease progression in models of glaucoma and other retinal degenerations. HighlightsO_LIActivation of Piezo1 excites retinal ganglion cells, paralleling the early neurodegenerative progression in glaucoma mouse models and retinal degeneration. C_LIO_LIPiezo1 and Piezo2 were expressed in axons and soma of retinal ganglion cells in mice, rats, and human iPSC-RGCs. C_LIO_LIFunctional assays confirmed Piezo1 in soma and neurites of neurons. C_LIO_LISustained elevation of Piezo1 and Piezo2 occurred after a single transient stretch may enhance damage from repeated traumatic nerve injury. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/599602v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@7bb1a0org.highwire.dtl.DTLVardef@cab0dcorg.highwire.dtl.DTLVardef@121690eorg.highwire.dtl.DTLVardef@7848fa_HPS_FORMAT_FIGEXP M_FIG Graphical abstract Piezo1 and Piezo2 channels in retinal ganglion cells and the impact of Piezo1 stimulation on light-dependent neural activity. Puttipong Sripinun, Lily P. See, Sergei Nikonov, Venkata Ramana Murthy Chavali, Vrathasha Vrathasha, Jie He, Joan M. OBrien, Jingsheng Xia, Wennan Lu, Claire H. Mitchell*. Activation of Piezo channels through mechanical or pharmacological stimulation leads to an influx of Ca2+ and other cations into RGCs, depolarizing the membrane and increasing the action potential frequency to modulate the visual signal. Created with Biorender.com C_FIG

Glaucoma is an optic neuropathy often referred to as "the silent thief of sight", due to its late diagnosis, which is generally made when degeneration of the optic nerve and retinal ganglion cells is already well under way. It is thus of utmost importance to have a better understanding of the disease, and to investigate more deeply the early causes of glaucoma. The transcriptional coactivator YAP recently emerged as an important regulator of eye homeostasis and is drawing attention in the glaucoma research field. Here we show that Yap conditional knockout mice (Yap cKO), in which the deletion of Yap is induced in both Muller glia (i.e. the only retinal YAP-expressing cells) and the non-pigmented epithelial cells of the ciliary body, exhibit breakdown of the aqueous-blood barrier accompanied by progressive collapse of the ciliary body as we observed in human uveitic patients. In addition, aged Yap cKO mice harbor glaucoma features, including alteration of glutamate recycling, deregulation of key homeostatic Muller-derived proteins, retinal vascular defects, optic nerve degeneration, and retinal ganglion cell death. Together, our findings reveal the essential role of YAP in preserving the ciliary body and the retinal ganglion cells, thereby preventing the onset of glaucoma features.

PurposeTo evaluate changes in monkey optic nerve head (ONH) morphology under acutely controlled intraocular pressure (IOP) and intracranial pressure (ICP). MethodsSeven ONHs from six monkeys were imaged via optical coherence tomography while IOP and ICP were maintained at one of 16 conditions. These conditions were defined by 4 levels for each pressure: low, baseline, high and very high. Images were processed to determine scleral canal area, aspect ratio, and planarity and anterior lamina cribrosa (ALC) shape index and curvature. Linear mixed effect models were utilized to investigate the effects of IOP, ICP and their interactions on ONH morphological features. The IOP-ICP interaction model was compared with one based on translaminar pressure difference (TLPD). ResultsWe observed complex, eye-specific, non-linear patterns of ONH morphological changes with changes in IOP and ICP. For all ONH morphological features, linear mixed effects models demonstrated significant interactions between IOP and ICP that were unaccounted for by TLPD. Interactions indicate that the effects of IOP and ICP depend on the other pressure. The IOP-ICP interaction model was a higher quality predictor of ONH features than a TLPD model. ConclusionsIn vivo modulation of IOP and ICP causes nonlinear and non-monotonic changes in monkey ONH morphology that depend on both pressures and is not accounted for by a simplistic TLPD. These results support and extend prior findings. Translational Relevance: A better understanding of ICPs influence on the effects of IOP can help inform the highly variable presentations of glaucoma and effective treatment strategies.

Myopia, projected to affect half of the global population by 2050, is a growing healthcare concern. Chronotype, an output of the human biological clock, and sleep parameters have been associated with several diseases, including myopia. We explored the connection between refractive errors and sleep and circadian rhythm parameters by employing a sample of 71,016 adults who completed the Munich Chronotype Questionnaire in the Estonian Biobank. After accounting for possible confounders, such as age, sex, education level, and duration of daylight exposure, we observed that individuals with late chronotype, characterised by a delayed sleep-wake pattern on free days, had higher odds for myopia. In contrast, early chronotype was associated with hyperopia. Furthermore, increased social jet lag and reduced sleep duration were associated with both myopia and hyperopia. These results emphasise the complex interplay between circadian rhythms and sleep in refractive development, with potential implications for public health and clinical practice.

Non-exudative age-related macular degeneration (AMD) involves retinal pigment epithelium (RPE) dysfunction and has been linked to altered intraocular immunity. Our investigation focuses on immune cell subsets and inflammation-associated factors in the eyes with early and intermediate AMD. We observed elevated levels of activated natural killer (NK) cells and interferon-{gamma}, concurrent with reduced myeloid-derived suppressor cells (MDSCs) and adenosine in AMD eyes. Aqueous humor from AMD patients had diminished ability to dampen NK cell activation, an effect rescued by adenosine supplementation. The Cryba1 cKO mouse model recapitulated these immune alterations, and single-cell RNA-sequencing identified NK cell-related genes and NK cell-RPE interactions. Co-culture of activated NK cells with RPE cells induced barrier dysfunction and Gasdermin-E driven pyroptosis providing a functional link relevant to AMD. These findings suggest a double-hit model where elevated immune activation and loss of immune dampening mechanisms drive AMD progression. Resetting the intraocular immune balance may be a promising therapeutic strategy for managing early and intermediate AMD. O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/634301v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1516bfaorg.highwire.dtl.DTLVardef@87f505org.highwire.dtl.DTLVardef@1e725caorg.highwire.dtl.DTLVardef@622717_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Illustra tion of pro posed mechanism underlying NK cell-RPE interaction in early AMD pathogenesis. Dysregulated NK cell communicate s with stressed RPE in early AMD immunopathology. Aberrant AMD aqueous humor and retina shows increased NK cells and NK effector molecules like IFN{gamma} with reduced MDSC and adenosine in human subjects, don or eye and animal model. Activated NK cells interaction with RPE causes dysfunction and pyroptotic cell death via Gasdermin-E pathway in AMD.. Created with BioRender.com C_FIG

Purpose: To assess optic nerve head (ONH) deformations and strains during adduction, abduction, and intraocular pressure (IOP) elevation in subjects with high-tension glaucoma (HTG) and normal-tension glaucoma (NTG). Design: Clinic-based cross-sectional study. Participants: 114 HTG subjects and 114 NTG subjects. Methods. We recruited 228 subjects (114 subjects with HTG [pre-treatment IOP > 21mmHg] and 114 with NTG [pre-treatment IOP < 21mmHg]). For each subject, we imaged the ONH using spectral-domain optical coherence tomography (OCT) under the following conditions: (1) primary gaze, (2) 20 degree adduction, (3) 20 degree abduction, and (4) primary gaze with acute IOP elevation (to approximately 33 mmHg) achieved through ophthalmodynamometry. For each OCT volume, we automatically segmented the prelaminar tissue (PLT), the choroid, the sclera and the lamina cribrosa (LC) using a deep learning algorithm. We also digitally aligned the OCT volumes obtained from (2)-(4) to the primary gaze volume (1) before performing digital volume correlation (DVC) analysis to quantify IOP- and gaze-induced ONH tissues three-dimensional displacements and effective strain (a local measure of tissue deformation) for all scenarios. Main Outcome Measures: Three-dimensional ONH displacements and strains. Results: Across all subjects, adduction generated high effective strain (4.2 {+/-} 1.4%) in the ONH tissues with no significant difference (p>0.05) with those induced by IOP elevation (4.5 {+/-} 1.5%); while abduction generated significantly lower (p = 0.014) effective strain (3.8 {+/-} 1.1%). Interestingly, the LC of HTG subjects exhibited significantly higher effective strain than those of NTG subjects under IOP elevation (HTG:4.6 {+/-} 1.7% vs NTG:4.1 {+/-} 1.5%, p = 0.047). Conversely, the LC tissue of NTG subjects exhibited significantly higher effective strain than those of HTG subjects under adduction (NTG: 4.9 {+/-} 1.9% vs HTG: 4.0 {+/-} 1.4%, p = 0.041). Conclusion: We found that adduction produced comparable strains and displacements as IOP elevation. We also found that NTG subjects experienced higher strains due to adduction than HTG subjects, while HTG subjects experienced higher strain due to IOP elevation than NTG subjects - and that these differences were most pronounced in the LC tissue.

The cornea is the clear window that lets light into the eye. It is composed of five layers: epithelium, Bowmans layer, stroma, Descemets membrane and endothelium. The maintenance of its structure and transparency are determined by the functions of the different cell types populating each layer. Attempts to regenerate corneal tissue and understand disease conditions requires knowledge of how cell profiles vary across this heterogeneous tissue. We performed a single cell transcriptomic profiling of 19,472 cells isolated from eight healthy donor corneas. Our analysis delineates the heterogeneity of the corneal layers by identifying cell populations and revealing cell states that contribute in preserving corneal homeostasis. We identified that the expression of CAV1, CXCL14, HOMER3 and CPVL were exclusive to the corneal epithelial limbal stem cell niche, CKS2, STMN1 and UBE2C were exclusively expressed in highly proliferative transit amplifying cells, and NNMT was exclusively expressed by stromal keratocytes. Overall, this research provides a basis to improve current primary cell expansion protocols, for future profiling of corneal disease states, to help guide pluripotent stem cells into different corneal lineages, and to understand how engineered substrates affect corneal cells to improve regenerative therapies.

PurposeFuchs Endothelial Corneal Dystrophy (FECD), a degenerative corneal disorder, is marked by the thickening of Descemets membrane and a progressive loss of corneal endothelial cells, ultimately leading to vision loss. A feature associated with the disease is the reduced stiffness of Descemets membrane. However, the effects of this change in Descemets membrane, on corneal endothelial cell health are not well understood. To explore this, we used in vitro, in vivo, and ex vivo studies to investigate how changes in substrate stiffness and the signaling pathways associated with these changes influence corneal endothelial functions. MethodsFor in-vitro studies, we cultured bovine corneal endothelial cells for 96 hours on stiff (32 kPa) and soft (8 kPa) substrate CytoSoft plates. By using Jess immunoassay and traditional western blotting, we evaluated changes in integrin signaling components, endothelial-to-mesenchymal transition, apoptosis, autophagy, and ubiquitin-proteasome pathway markers. Mitochondrial health and mitochondrial superoxide levels were assessed using commercial kits. We assessed the protein levels of the above-mentioned markers in the Col8a2Q455K/Q455K FECD mouse model. To evaluate whether FAK signaling contributes to the FECD pathogenesis, we injected the Col8a2Q455K/Q455K mice with an FAK inhibitor and assessed the corneal phenotypes. ResultsWe observed increased levels of phosphorylated FAK, integrins 4 and 5 in bovine corneal endothelial cells cultured on soft substrate. We also found upregulated endothelial-to-mesenchymal transition (EndMT) markers, mitochondrial dysfunction, and apoptosis in cells grown on soft substrate. In the Col8a2Q455K/Q455K mouse model of FECD, there was increased pFAK Y397 levels coincident with the onset of phenotypes. Intraperitoneal injections of a pFAK inhibitor improved antioxidant protein expression and decreased EndMT; however, it did not improve FECD-associated disease progression. ConclusionIn this study, we explored how changes in the physical characteristics of the Descemets membrane impact corneal endothelial cell health. While we discovered activation of Focal adhesion kinase as a result of stiffness changes, its inhibition alone was insufficient to improve cell health in an FECD mouse model.

PURPOSETo evaluate the duration-dependent and synergetic impact of high-intensity light (HL) and optical refocus (RF) on lens-induced myopia (LIM) development in chickens. METHODSMyopia was induced in one eye in chicks (10 groups, n=126) from day 1 post- hatching (D1) until D8 using -10D lenses. Fellow eyes remained uncovered as controls. Nine groups were exposed daily to continuous 2 hours (h), 4h, or 6h of either HL (15,000 lux); RF (removal of -10D lens); or both (HL+RF). One group served as the LIM group without any interventions. Ocular axial length (AL), refractive error, and choroidal thickness were measured on D1, D4, and D8. Outcome measures are expressed as inter-ocular difference (IOD= experimental - control eye) {+/-}SEM. RESULTSBy D8, LIM increased AL (0.36{+/-}0.04 mm), myopic refraction (-9.02{+/-}0.37D), and choroidal thinning (-90.27{+/-}16.44 {micro}m) in the LIM group (all, P<0.001). Compared to the LIM group, exposure to 2h, 4h, or 6h of HL, RF, or HL+RF reduced myopic refraction in a duration-dependent manner, with RF being more effective than HL (P<0.05). Only 6h of HL+RF (not 2h or 4h) prevented LIM and was more effective than RF (P=0.004) or HL (P<0.001) in reducing myopic refraction, and more effective than HL (P<0.001) in reducing axial elongation. CONCLUSIONDaily exposure to 2h, 4h, or 6h of HL, RF, or HL+RF reduced lens-induced myopic refraction in a duration-dependent manner in chickens. Only 6h of HL+RF completely stopped LIM development. The synergetic effect of HL and RF is dependent on the duration of the interventions.

Conjunctival in vitro models present a valuable system to investigate conjunctival tissue homeostasis and pathologies. Combinations of collagen and fibroblasts as a stroma equivalent and the supplementation with serum have been reported to promote the differentiation of epithelial cells. However, how the individual factors affect differentiation of ocular surface cells is insufficiently understood. In this study, we analyzed the effect of serum concentration, a collagen matrix, and fibroblasts on conjunctival differentiation in a 3D in vitro model. For this purpose, we developed a computational analysis pipeline for the quantification of optical coherence tomography (OCT) data sets, allowing a time resolved, non-invasive assessment of conjunctiva epithelium differentiation, including goblet cell density. High-resolution dynamic full-field OCT (D-FFOCT) was employed to verify the identity of goblet cells. Conjunctival markers were further analyzed via histology, real-time quantitative PCR, and ELISA to confirm the data of the OCT analysis pipeline. We found that serum is required to induce epithelial differentiation while higher concentrations of 5 - 10% impaired epithelial development. The culture on a collagen matrix increased conjunctival markers upon stimulation with serum, while the co-culture with fibroblasts increased epithelial stratification. Increased serum concentration resulted in the increased occurrence of goblet cells of up to 20 cells/mm{superscript 2}. Altogether, the complementary analyses confirmed the quantified OCT data. Summarized, we identified the combination of serum (3%), collagen, and fibroblasts as a condition resulting in the highest physiological resemblance. Altogether, our study emphasizes the need for fine-tuning of culture conditions for 3D in vitro models. SIGNIFICANCE STATEMENTPhysiologically relevant in vitro conjunctiva models are essential for studying ocular surface homeostasis and disease. Our study refines 3D conjunctival culture conditions to more closely resemble native tissue by systematically identifying serum concentration, collagen scaffolds and fibroblasts as key drivers of epithelial differentiation. By applying non-invasive optical coherence tomography analysis, we enable longitudinal assessment of tissue maturation, addressing the need for non-destructive, repeatable tissue analysis. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=164 HEIGHT=200 SRC="FIGDIR/small/698626v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@13efafaorg.highwire.dtl.DTLVardef@1cf4529org.highwire.dtl.DTLVardef@c5c63forg.highwire.dtl.DTLVardef@5a85c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Primary congenital glaucoma (PCG) is a severe, early-onset eye disease most often caused by abnormal development of the aqueous humor outflow pathway (AHOP). Located circumferentially at the anterior chamber angle where the iris meets the cornea, the AHOP comprises the trabecular meshwork (TM) and Schlemms canal (SC), which together regulate intraocular fluid drainage. Malformation of this pathway leads to elevated intraocular pressure, painful ocular enlargement, and retinal damage that can result in blindness. While single-gene mutations account for approximately 25% of PCG cases across diverse populations, most molecular causes remain unknown and are likely due to rare or complex genetic factors. Progress in identifying these mechanisms has been limited by a lack of detailed gene expression data during AHOP development. To address this gap, we generated the first single-cell RNA sequencing dataset from developing AHOP tissue, using rat eyes at three key stages of TM and SC formation. This high-resolution dataset contains the transcriptomic profiles of 29,626 genes across 86,653 cells clustered into 13 general cell types, which included over 10,000 cells related to TM/SC subtypes. Analysis of 44 genes previously linked to Mendelian childhood glaucoma showed that 36 (82%) were expressed in these TM/SC-related populations, validating the datasets relevance. Notably, this study identified 395 genes selectively upregulated in developing TM/SC subtypes, revealing numerous candidates potentially involved in the formation and function of TM/SC structures. This resource will support the discovery of rare Mendelian disease genes and inform the development of polygenic risk scores for complex genetics underlying early-onset forms of glaucoma.

PurposeRetinal neurodegeneration is difficult to monitor due to insensitive disease endpoints. Mitochondrial dysfunction and oxidative stress are promising early biomarkers of retinal ganglion cell (RGC) degeneration. This study investigates dynamics of flavoprotein fluorescence (FPF), a non-invasive mitochondrial oxidative stress measure, and sensitivity to early neurodegeneration and neuroprotection in vitro and in vivo. MethodsFPF activity in response to neurodegeneration and neuroprotection were characterized in vitro in wild-type (WT) and SARM1 knockout (SARMKO) human embryonic stem cell-derived RGCs with and without Vacor treatment over 24 hours and confirmed with mitochondrial reactive oxygen species (ROS) measures. Further FPF evaluation was explored in vivo using the optic nerve crush (ONC) model in WT and SARMKO mice to compare early RGC stress detection within rodent retinas. ResultsIn vitro FPF intensities in WT RGCs increased within 8 hours of degeneration induction, preceding significant mitochondrial ROS production. Neuroprotective SARMKO RGCs maintained comparable FPF and ROS levels following insult. In vivo FPF changes were not observed in WT and SARMKO mice over 4 days following ONC, while only early retinal thickening was observed from OCT. Early FPF and OCT changes were not reflective of late RGC survival observed from ex vivo RGC soma and axon counts. ConclusionsThese findings highlight differences in FPF sensitivity to mitochondrial stress between simplified in vitro systems and complex in vivo rodent retinas. This study demonstrates the potential of FPF as an early neurodegeneration and neuroprotection endpoint in vitro while identifying limitations and areas of development for its translatability to preclinical in vivo assessment.

Postnatal eye growth is critical for healthy vision and yet, how this process is regulated at the cellular level is still unclear. The choroid is thought to play a crucial role in relaying signals from the retina to the sclera to modulate eye growth, but which cells are targeted and how they interact is not known. Using primary cultures of pediatric and adult human choroid and sclera stromal fibroblasts, we investigated the effect of choroid-conditioned medium (CCM) on scleral fibroblasts contractile activity. We show that CCM activates pediatric scleral fibroblast contraction, with age and antero-posterior location differences. Upon exposure to dopamine, a known negative regulator of eye growth, pediatric - but not adult - choroid cells lose their ability to stimulate scleral fibroblasts. Using RNA-Sequencing, we show that dopamine stimulates pathways linked to ribosomal activation, translation and exosome release exclusively in pediatric choroid cells. Removing exosomes from the CCM rescued the ability of dopamine-treated choroid cells to stimulate scleral fibroblasts. We further identify retinoic acid as the active exosome-associated compound preventing scleral fibroblast activation. Mechanistically, we show that CCM stimulates actomyosin-mediated protrusive activity in scleral fibroblasts. This is prevented when scleral cells are exposed to dopamine-CCM, and fully rescued upon exosome removal or ALDH1 inhibition. We thus propose that pediatric choroid stromal cells specifically relay dopamine-mediated retinal signals to the sclera during post-natal eye growth, operating through changes in secretome - including the production of exosome-associated retinoic acid - rather than gene expression changes. SIGNIFICANCEEye size is critical to optimal vision but the exact cellular and molecular mechanisms regulating it are still unknown. Failure to properly regulate postnatal eye growth leads to elongated eyes and myopia. Myopia is expected to affect half of the world population by 2050, with many at risk of blinding complications. Eye growth and homeostasis are supported by the sclera and the choroid, but how the two tissues interact to regulate eye growth is unknown. We use here human cells to demonstrate direct, age-specific, interactions between the two cell types and provide a rationale for the known negative effect of dopamine on eye growth, with significant implications for future studies as well as for the understanding and treatment of myopia.

In response to central nervous system (CNS) injury, tissue resident immune cells such as microglia and circulating systemic neutrophils are often first responders. The degree to which these cells interact in response to CNS damage is poorly understood, and even less so, in the neural retina which poses a challenge for high resolution imaging in vivo. In this study, we deploy fluorescence adaptive optics scanning light ophthalmoscopy (AOSLO) to study microglia and neutrophils in mice. We simultaneously track immune cell dynamics using label-free phase-contrast AOSLO at micron-level resolution. Retinal lesions were induced with 488 nm light focused onto photoreceptor (PR) outer segments. These lesions focally ablated PRs, with minimal collateral damage to cells above and below the plane of focus. We used in vivo (AOSLO, SLO and OCT) imaging to reveal the natural history of the microglial and neutrophil response from minutes-to-months after injury. While microglia showed dynamic and progressive immune response with cells migrating into the injury locus within 1-day after injury, neutrophils were not recruited despite close proximity to vessels carrying neutrophils only microns away. Post-mortem confocal microscopy confirmed in vivo findings. This work illustrates that microglial activation does not recruit neutrophils in response to acute, focal loss of PRs, a condition encountered in many retinal diseases.

The epithelial layer of the cornea is a critical physical and ocular immune barrier for maintaining tissue integrity, homeostasis, and transparency for proper vision. Corneal injury can trigger inflammation, impair wound healing and compromise immune privilege and avascularity, leading to vision loss. Moreover, injury to the cornea can disrupt the engine of epithelial repair and restoration, the limbal stem cell (LSC) niche. Here we used a corneal suture model to induce epithelial damage, sustained inflammation and neovascularization, to examine the impact on LSCs. Using single-cell transcriptomics, we analyzed corneal cell state changes and additionally evaluated the potential of duloxetine, an FDA-approved medicine, to promote wound healing and corneal homeostasis. Single-cell RNA-seq analysis revealed loss of homeostatic limbal stem cells, basal and differentiated epithelial cells and an increase in distinct limbal-like, conjunctival, inflammatory, and vascular cell states, suggesting a coordinated wound healing response in different tissue layers. Importantly, duloxetine treatment promoted epithelial homeostasis, enhanced stem cell-like and stromal repair processes, and suppressed immune and vascular responses. Examination of corneal cell perturbation and transformations at the single-cell level thorough marker profile annotations can improve the understanding of LSC plasticity and function while yielding potential biomarkers of corneal repair processes.

Glaucoma is a blinding disease where the retinal ganglion cells and their axons degenerate. Degradation of axonal microtubules is thought to play a critical role in the pathogenesis, but the mechanism is unknown. Here we investigate whether microtubule disruption in glaucoma can be alleviated by metabolic rescue. The morphology and integrity of microtubules of the retinal nerve fibers were evaluated by second-harmonic generation microscopy in a mouse model of glaucoma, DBA/2, which received a dietary supplement of nicotinamide to reduce metabolic stress. It was compared with control DBA/2, which did not receive nicotinamide, and non-glaucomatous DBA/2-Gpnmb+. We found that morphology but not microtubules are significantly protected by nicotinamide. Furthermore, from co-registered images of second-harmonic generation and immunofluorescence, it was determined that microtubule deficit was not due to a shortage of tubulins. Microtubule deficit colocalized with the sectors in which the retinal ganglion cells were disconnected from the brain, indicating that microtubule disruption is associated with axonal transport deficit in glaucoma. Together, our data suggests significant role axonal microtubules play in glaucomatous degeneration, offering a new opportunity for neuroprotection.

PurposeMacrophage migration inhibitory factor (MIF) is a pleiotropic cytokine implicated in many inflammatory and fibrotic diseases; however, its role in primary open-angle glaucoma (POAG) and trabecular meshwork (TM) dysfunction remains unknown. In this study, we investigated whether MIF-CD74 signaling regulates TM pathobiology through modulation of the transcription factor, Blimp-1, and downstream cytoskeletal reorganization and extracellular matrix (ECM) remodeling. MethodPrimary human TM cells (HTMC) were exposed to glaucomatous stressors, including TGF-{beta}2, rMIF, or a pro-inflammatory milieu. Expression of MIF, its receptor CD74, and Blimp-1 was measured by qPCR and immunoblotting. ECM proteins and phosphorylated myosin-light chain (pMLC) were evaluated by immunofluorescence staining. In vivo, MIF-CD74 and Blimp-1 expression were examined in the TM/anterior segment (AS) tissue of Tg.CreMYOCY437H and lentiviral (LV)-TGF-{beta}2-induced ocular hypertension (OHT) mouse models. Functional involvement of MIF signaling in TM pathobiology was examined using the irreversible MIF inhibitor 4-IPP and the immunomodulatory metabolites agmatine and thiamine. ResultsGlaucomatous stressors significantly upregulated MIF and CD74 expression with concomitant suppression of Blimp-1 in HTMC. Similarly, TM/AS tissue from both OHT models (Tg.CreMYOCY437H and LV-TGF-{beta}2) demonstrated increased MIF-CD74 expression accompanied by reduced Blimp-1 levels. Activation of MIF-CD74 signaling triggered pro-inflammatory and cell death pathways and promoted ECM remodeling, characterized by increased fibrotic protein expression and enhanced RhoA/ROCK-mediated MLC phosphorylation, indicating modulation of TM contractility. Pharmacological inhibition of MIF attenuated inflammatory signaling, reduced ECM deposition and cytoskeletal remodeling, and suppressed RhoA/ROCK/MLC activation, restoring a protective TM phenotype. ConclusionOur findings identify MIF-CD74 signaling as a previously unrecognized regulator of TM dysfunction in POAG. MIF-mediated suppression of Blimp-1 mechanistically links inflammatory signaling to cytoskeletal contractility and fibrotic ECM remodeling, key determinants of aqueous humor outflow resistance. Targeting the MIF-CD74/Blimp-1 axis may represent a novel therapeutic strategy to restore TM homeostasis and reduce intraocular pressure in glaucoma.