Early inner plexiform layer thinning and retinal nerve fiber layer thickening in excitotoxic retinal injury using deep learning-assisted optical coherence tomography.

Excitotoxicity from the impairment of glutamate uptake constitutes an important mechanism in neurodegenerative diseases such as Alzheimer's, multiple sclerosis, and Parkinson's disease. Within the eye, excitotoxicity is thought to play a critical role in retinal ganglion cell death in glaucoma, diabetic retinopathy, retinal ischemia, and optic nerve injury, yet how excitotoxic injury impacts different retinal layers is not well understood. Here, we investigated the longitudinal effects of N-methyl-D-aspartate (NMDA)-induced excitotoxic retinal injury in a rat model using deep learning-assisted retinal layer thickness estimation. Before and after unilateral intravitreal NMDA injection in nine adult Long Evans rats, spectral-domain optical coherence tomography (OCT) was used to acquire volumetric retinal images in both eyes over 4 weeks. Ten retinal layers were automatically segmented from the OCT data using our deep learning-based algorithm. Retinal degeneration was evaluated using layer-specific retinal thickness changes at each time point (before, and at 3, 7, and 28 days after NMDA injection). Within the inner retina, our OCT results showed that retinal thinning occurred first in the inner plexiform layer at 3 days after NMDA injection, followed by the inner nuclear layer at 7 days post-injury. In contrast, the retinal nerve fiber layer exhibited an initial thickening 3 days after NMDA injection, followed by normalization and thinning up to 4 weeks post-injury. Our results demonstrated the pathological cascades of NMDA-induced neurotoxicity across different layers of the retina. The early inner plexiform layer thinning suggests early dendritic shrinkage, whereas the initial retinal nerve fiber layer thickening before subsequent normalization and thinning indicates early inflammation before axonal loss and cell death. These findings implicate the inner plexiform layer as an early imaging biomarker of excitotoxic retinal degeneration, whereas caution is warranted when interpreting the ganglion cell complex combining retinal nerve fiber layer, ganglion cell layer, and inner plexiform layer thicknesses in conventional OCT measures. Deep learning-assisted retinal layer segmentation and longitudinal OCT monitoring can help evaluate the different phases of retinal layer damage upon excitotoxicity.

Lysyl oxidase like-1 deficiency in optic nerve head astrocytes elicits reactive astrocytosis and alters functional effects of astrocyte derived exosomes.

Glaucoma is a multifactorial progressive ocular pathology that manifests clinically with damage to the optic nerve (ON) and the retina, ultimately leading to blindness. The optic nerve head (ONH) shows the earliest signs of glaucoma pathology, and therefore, is an attractive target for drug discovery. The goal of this study was to elucidate the effects of reactive astrocytosis on the elastin metabolism pathway in primary rat optic nerve head astrocytes (ONHA), the primary glial cell type in the unmyelinated ONH. Following exposure to static equibiaxial mechanical strain, we observed prototypic molecular and biochemical signatures of reactive astrocytosis that were associated with a decrease in lysyl oxidase like 1 (Loxl1) expression and a concomitant decrease in elastin (Eln) gene expression. We subsequently investigated the role of Loxl1 in reactive astrocytosis by generating primary rat ONHA cultures with ∼50% decreased Loxl1 expression. Our results suggest that reduced Loxl1 expression is sufficient to elicit molecular signatures of elastinopathy in ONHA. Astrocyte derived exosomes (ADE) significantly increased the length of primary neurites of primary neurons in vitro. In contrast, ADE from Loxl1-deficient ONHA were deficient of trophic effects on neurite outgrowth in vitro, positing that Loxl1 dysfunction and the ensuing impaired elastin synthesis during reactive astrocytosis in the ONH may contribute to impaired neuron-glia signaling in glaucoma. Our data support a role of dysregulated Loxl1 function in eliciting reactive astrocytosis in glaucoma subtypes associated with increased IOP, even in the absence of genetic polymorphisms in LOXL1 typically associated with exfoliation glaucoma. This suggests the need for a paradigm shift toward considering lysyl oxidase activity and elastin metabolism and signaling as contributors to an altered secretome of the ONH that may lead to the progression of glaucomatous changes. Future research is needed to investigate cargo of exosomes in the context of reactive astrocytosis and identify the pathways leading to the observed transcriptome changes during reactive astrocytosis.

Association of fractal dimension and other retinal vascular network parameters with cognitive performance and neuroimaging biomarkers: The Multi-Ethnic Study of Atherosclerosis (MESA).

INTRODUCTION: Retinal vascular network changes may reflect the integrity of the cerebral microcirculation, and may be associated with cognitive impairment. METHODS: Associations of retinal vascular measures with cognitive function and MRI biomarkers were examined amongst Multi-Ethnic Study of Atherosclerosis (MESA) participants in North Carolina who had gradable retinal photographs at Exams 2 (2002 to 2004, n = 313) and 5 (2010 to 2012, n = 306), and detailed cognitive testing and MRI at Exam 6 (2016 to 2018). RESULTS: After adjustment for covariates and multiple comparisons, greater arteriolar fractal dimension (FD) at Exam 2 was associated with less isotropic free water of gray matter regions (ß = -0.0005, SE = 0.0024, p = 0.01) at Exam 6, while greater arteriolar FD at Exam 5 was associated with greater gray matter cortical volume (in mm3 , ß = 5458, SE = 20.17, p = 0.04) at Exam 6. CONCLUSION: Greater arteriolar FD, reflecting greater complexity of the branching pattern of the retinal arteries, is associated with MRI biomarkers indicative of less neuroinflammation and neurodegeneration.

Reverse translation of artificial intelligence in glaucoma: Connecting basic science with clinical applications.

Artificial intelligence (AI) has been approved for biomedical research in diverse areas from bedside clinical studies to benchtop basic scientific research. For ophthalmic research, in particular glaucoma, AI applications are rapidly growing for potential clinical translation given the vast data available and the introduction of federated learning. Conversely, AI for basic science remains limited despite its useful power in providing mechanistic insight. In this perspective, we discuss recent progress, opportunities, and challenges in the application of AI in glaucoma for scientific discoveries. Specifically, we focus on the research paradigm of reverse translation, in which clinical data are first used for patient-centered hypothesis generation followed by transitioning into basic science studies for hypothesis validation. We elaborate on several distinctive areas of research opportunities for reverse translation of AI in glaucoma including disease risk and progression prediction, pathology characterization, and sub-phenotype identification. We conclude with current challenges and future opportunities for AI research in basic science for glaucoma such as inter-species diversity, AI model generalizability and explainability, as well as AI applications using advanced ocular imaging and genomic data.

Internalization of Angiotensin-(1-12) in Adult Retinal Pigment Epithelial-19 Cells.

Purpose: Angiotensin-(1-12) [Ang-(1-12)] serves as a primary substrate to generate angiotensin II (Ang II) by angiotensin-converting enzyme and/or chymase suggests it may be an unrecognized source of Ang II-mediated microvascular complication in hypertension-mediated retinopathy. We investigated Ang-(1-12) expression and internalization in adult retinal pigment epithelial-19 (ARPE-19) cultured cells. We performed the internalization of Ang-(1-12) in ARPE-19 cells in the presence of a highly specific monoclonal antibody (mAb) developed against the C-terminal end of the Ang-(1-12) sequence. Methods: All experiments were performed in confluent ARPE-19 cells (passage 28-35). We employed high-performance liquid chromatography to purify radiolabeled, 125I-Ang-(1-12) and immuno-neutralization with Ang-(1-12) mAb to demonstrate Ang-(1-12)'s internalization in ARPE-19 cells. Internalization was also demonstrated by immunofluorescence (IF) method. Results: These procedures revealed internalization of an intact 125I-Ang-(1-12) in ARPE-19 cells. A significant reduction (∼53%, P < 0.0001) in 125I-Ang-(1-12) internalization was detected in APRE-19 cells in the presence of the mAb. IF staining experiments further confirms internalization of Ang-(1-12) into the cells from the extracellular culture medium. No endogenous expression was detected in the ARPE-19 cells. An increased intensity of IF staining was detected in cells exposed to 1.0 µM Ang-(1-12) compared with 0.1 µM. Furthermore, we found hydrolysis of Ang-(1-12) into Ang II by ARPE-19 cells' plasma membranes. Conclusions: Intact Ang-(1-12) peptide is internalized from the extracellular spaces in ARPE-19 cells and metabolized into Ang II. The finding that a selective mAb blocks cellular internalization of Ang-(1-12) suggests alternate therapeutic approaches to prevent/reduce the RPE cells Ang II burden.

A novel approach to sclerotomy closure in pars plana vitrectomy: a pilot study.

BACKGROUND: Methods of sclerotomy closure following a vitrectomy, including the use of sutures, have been associated with complications such as inflammation, foreign body sensation, and infection. Here, we test an innovative approach to scleral wound closure following pars plana vitrectomy that involves plugging the wound. We investigated several materials with the intent of using products that were either already approved by the FDA for other types of procedures or were biocompatible patient-derived materials. METHODS: We examined whether scleral wounds could be sealed by a clot or internal "plug" rather than a suture or an external adhesive. We tested patient-derived materials (platelet-rich plasma (PRP) and whole blood) as well as polyethylene glycol (PEG) sealant. Whole blood and PRP were prevented from clotting prematurely using sodium citrate, and were clotted for the study with thrombin. Polyethylene glycol (PEG) sealant was prepared according to manufacturer's recommendations. We used fresh-frozen cadaveric porcine eyes. We tested several methods to form plugs using the above materials, as well as various methods to deliver the plugs into the sclerotomy incisions. We used a novel technique of manual vitrectomy. Successfully generated and implanted clots were tested for efficacy with the Seidel test. RESULTS: Polyethylene glycol (PEG) sealant fractured during our attempts at molding and inserting the plug. In contrast, both whole blood and PRP yielded successful plugs for insertion. We molded a whole blood clot plug by allowing it to clot inside a 20-gauge angiocath catheter and we successfully delivered it through a 23G trocar. At baseline, no wound leakage was apparent. However, the whole blood clot dislodged during the Seidel test. We successfully molded and delivered a PRP clot plug using a tapered 2-20 µl pipette tip, using MAXGrip Forceps to push it through into the wound. No scleral wound leakage was noted at our baseline physiologic infusion pressure. Furthermore, the PRP clot plug prevented scleral wound leakage up to a pressure of 60 mmHg and was confirmed with the Seidel test. CONCLUSION: Our findings suggest that insertion of a clot plug made from either whole blood or PRP may be an effective strategy for scleral wound closure following pars plana vitrectomy. Further testing in preclinical models is warranted to further refine the materials and methods, since this appears to have the potential to improve the closure of the scleral wounds after pars plana vitrectomy.

Interleukin-6 promotes microtubule stability in axons via Stat3 protein-protein interactions.

The interleukin-6 (IL-6) family of cytokines and its downstream effector, STAT3, are important mediators of neuronal health, repair, and disease throughout the CNS, including the visual system. Here, we elucidate a transcription-independent mechanism for the neuropoietic activities of IL-6 related to axon development, regeneration, and repair. We examined the outcome of IL-6 deficiency on structure and function of retinal ganglion cell (RGC) axons, which form the optic projection. We found that IL-6 deficiency substantially delays anterograde axon transport in vivo. The reduced rate of axon transport is accompanied by changes in morphology, structure, and post-translational modification of microtubules. In vivo and in vitro studies in mice and swine revealed that IL-6-dependent microtubule phenotypes arise from protein-protein interactions between STAT3 and stathmin. As in tumor cells and T cells, this STAT3-stathmin interaction stabilizes microtubules in RGCs. Thus, this IL-6-STAT3-dependent mechanism for axon architecture is likely a fundamental mechanism for microtubule stability systemically.

Impairment of Membrane Repolarization Accompanies Axon Transport Deficits in Glaucoma.

Glaucoma is a leading cause of blindness worldwide, resulting from degeneration of retinal ganglion cells (RGCs), which form the optic nerve. In glaucoma, axon transport deficits appear to precede structural degeneration of RGC axons. The period of time between the onset of axon transport deficits and the structural degeneration of RGC axons may represent a therapeutic window for the prevention of irreversible vision loss. However, it is unclear how deficits in axon transport relate to the electrophysiological capacity of RGCs to produce and maintain firing frequencies that encode visual stimuli. Here, we examined the electrophysiological signature of individual RGCs in glaucomatous retina with respect to axon transport facility. Utilizing the Microbead Occlusion Model of murine ocular hypertension, we performed electrophysiological recordings of RGCs with and without deficits in anterograde axon transport. We found that RGCs with deficits in axon transport have a reduced ability to maintain spiking frequency that arises from elongation of the repolarization phase of the action potential. This repolarization phenotype arises from reduced cation flux and K+ dyshomeostasis that accompanies pressure-induced decreases in Na/K-ATPase expression and activity. In vitro studies with purified RGCs indicate that elevated pressure induces early internalization of Na/K-ATPase that, when reversed, stabilizes cation flux and prevents K+ dyshomeostasis. Furthermore, pharmacological inhibition of the Na/K-ATPase is sufficient to replicate pressure-induced cation influx and repolarization phase phenotypes in healthy RGCs. These studies suggest that deficits in axon transport also likely reflect impaired electrophysiological function of RGCs. Our findings further identify a failure to maintain electrochemical gradients and cation dyshomeostasis as an early phenotype of glaucomatous pathology in RGCs that may have significant bearing on efforts to restore RGC health in diseased retina.

Pressure-dependent modulation of inward-rectifying K+ channels: implications for cation homeostasis and K+ dynamics in glaucoma.

Glaucoma is the leading cause of blindness worldwide, resulting from degeneration of retinal ganglion cells (RGCs), which form the optic nerve. Prior to structural degeneration, RGCs exhibit physiological deficits. Müller glia provide homeostatic regulation of ions that supports RGC physiology through a process called K+ siphoning. Recent studies suggest that several retinal conditions, including glaucoma, involve changes in the expression of K+ channels in Müller glia. To clarify whether glaucoma-related stressors directly alter expression and function of K+ channels in Müller glia, we examined changes in the expression of inwardly rectifying K+ (Kir) channels and two-pore domain (K2P) channels in response to elevated intraocular pressure (IOP) in vivo and in vitro in primary cultures of Müller glia exposed to elevated hydrostatic pressure. We then measured outcomes of cell health, cation homeostasis, and cation flux in Müller glia cultures. Transcriptome analysis in a murine model of microbead-induced glaucoma revealed pressure-dependent downregulation of Kir and K2P channels in vivo. Changes in the expression and localization of Kir and K2P channels in response to elevated pressure were also found in Müller glia in vitro. Finally, we found that elevated pressure compromises the plasma membrane of Müller glia and induces cation dyshomeostasis that involves changes in ion flux through cation channels. Pressure-induced changes in cation flux precede both cation dyshomeostasis and membrane compromise. Our findings have implications for Müller glia responses to pressure-related conditions, i.e., glaucoma, and identify cation dyshomeostasis as a potential contributor to electrophysiological impairment observed in RGCs of glaucomatous retina.

Phenotypes of primary retinal macroglia: Implications for purification and culture conditions.

Many neurodegenerations, including those of the visual system, have complex etiologies that include roles for both neurons and glia. In the retina there is evidence that retinal astrocytes play an important role in neurodegeneration. There are several approaches for isolating and growing primary retinal astrocytes, however, they often lead to different results. In this study, we examined the influence of culture conditions on phenotypic maturation of primary, purified retinal glia. We compared retinal astrocytes and Müller glia purified by immunomagnetic separation, as differentiation between these astrocyte subtypes is critical and immuno-based methods are the standard practice of purification. We found that while time in culture impacts the health and phenotype of both astrocytes and Müller glia, the phenotypic maturation of retinal astrocytes was most impacted by serum factors. These factors appeared to actively regulate intermediate filament phenotypes in a manner consistent with the induction of astrocyte-mesenchymal transition (AMT). This propensity for retinal astrocytes to shift along an AMT continuum should be considered when interpreting resulting data. Our goal is that this study will help standardize the field so that studies are replicable, comparable, and as accurate as possible for subsequent interpretation of findings.

Increased bioavailability of cyclic guanylate monophosphate prevents retinal ganglion cell degeneration.

The nitric oxide - guanylyl cyclase-1 - cyclic guanylate monophosphate (NO-GC-1-cGMP) pathway has emerged as a potential pathogenic mechanism for glaucoma, a common intraocular pressure (IOP)-related optic neuropathy characterized by the degeneration of retinal ganglion cells (RGCs) and their axons in the optic nerve. NO activates GC-1 to increase cGMP levels, which are lowered by cGMP-specific phosphodiesterase (PDE) activity. This pathway appears to play a role in both the regulation of IOP, where reduced cGMP levels in mice leads to elevated IOP and subsequent RGC degeneration. Here, we investigated whether potentiation of cGMP signaling could protect RGCs from glaucomatous degeneration. We administered the PDE5 inhibitor tadalafil orally (10 mg/kg/day) in murine models of two forms of glaucoma - primary open angle glaucoma (POAG; GC-1-/- mice) and primary angle-closure glaucoma (PACG; Microbead Occlusion Model) - and measured RGC viability at both the soma and axon level. To determine the direct effect of increased cGMP on RGCs in vitro, we treated axotomized whole retina and primary RGC cultures with the cGMP analogue 8-Br-cGMP. Tadalafil treatment increased plasma cGMP levels in both models, but did not alter IOP or mean arterial pressure. Nonetheless, tadalafil treatment prevented degeneration of RGC soma and axons in both disease models. Treatment of whole, axotomized retina and primary RGC cultures with 8-Br-cGMP markedly attenuated both necrotic and apoptotic cell death pathways in RGCs. Our findings suggest that enhancement of the NO-GC-1-cGMP pathway protects the RGC body and axon in murine models of POAG and PACG, and that enhanced signaling through this pathway may serve as a novel glaucoma treatment, acting independently of IOP.

Ccl5 Mediates Proper Wiring of Feedforward and Lateral Inhibition Pathways in the Inner Retina.

The ß-chemokine Ccl5 and its receptors are constitutively expressed in neurons of the murine inner retina. Here, we examined the functional and structural significance of this constitutive Ccl5 signaling on retinal development. We compared outcomes of electrophysiology, ocular imaging and retinal morphology in wild-type mice (WT) and mice with Ccl5 deficiency (Ccl5-/- ). Assessment of retinal structure by ocular coherence tomography and histology revealed slight thinning of the inner plexiform layer (IPL) and inner nuclear layer (INL) in Ccl5-/- mice, compared to WT (p < 0.01). Assessment of postnatal timepoints important for development of the INL (P7 and P10) revealed Ccl5-dependent alterations in the pattern and timing of apoptotic pruning. Morphological analyses of major inner retinal cell types in WT, Ccl5-/- , gustducingfp and gustducingfp/Ccl5-/- mice revealed Ccl5-dependent reduction in GNAT3 expression in rod bipolar cells as well as a displacement of their terminals from the IPL into the GCL. RGC dendritic organization and amacrine cell morphology in the IPL was similarly disorganized in Ccl5-/- mice. Examination of the intrinsic electrophysiological properties of RGCs revealed higher spontaneous activity in Ccl5-/- mice that was characterized by higher spiking frequency and a more depolarized resting potential. This hyperactive phenotype could be negated by current clamp and correlated with both membrane resistance and soma area. Overall, our findings identify Ccl5 signaling as a mediator of inner retinal circuitry during development of the murine retina. The apparent role of Ccl5 in retinal development further supports chemokines as trophic modulators of CNS development and function that extends far beyond the inflammatory contexts in which they were first characterized.

Impact of Graphene on the Efficacy of Neuron Culture Substrates.

How graphene influences the behavior of living cells or tissues remains a critical issue for its application in biomedical studies, despite the general acceptance that graphene is biocompatible. While direct contact between cells and graphene is not a requirement for all biomedical applications, it is often mandatory for biosensing. Therefore, it is important to clarify whether graphene impedes the ability of cells to interact with biological elements in their environment. Here, a systematic study is reported to determine whether applying graphene on top of matrix substrates masks interactions between these substrates and retinal ganglion cells (RGCs). Six different platforms are tested for primary RGC cultures with three platforms comprised of matrix substrates compatible with these neurons, and another three having a layer of graphene placed on top of the matrix substrates. The results demonstrate that graphene does not impede interactions between RGCs and underlying substrate matrix, such that their positive or negative effects on neuron viability and vitality are retained. However, direct contact between RGCs and graphene reduces the number, but increases basal activity, of functional cation channels. The data indicate that, when proper baselines are established, graphene is a promising biosensing material for in vitro applications in neuroscience.

The nitric oxide-guanylate cyclase pathway and glaucoma.

Glaucoma is a prevalent optic neuropathy characterized by the progressive dysfunction and loss of retinal ganglion cells (RGCs) and their optic nerve axons, which leads to irreversible visual field loss. Multiple risk factors for the disease have been identified, but elevated intraocular pressure (IOP) remains the primary risk factor amenable to treatment. Reducing IOP however does not always prevent glaucomatous neurodegeneration, and many patients progress with the disease despite having IOP in the normal range. There is increasing evidence that nitric oxide (NO) is a direct regulator of IOP and that dysfunction of the NO-Guanylate Cyclase (GC) pathway is associated with glaucoma incidence. NO has shown promise as a novel therapeutic with targeted effects that: 1) lower IOP; 2) increase ocular blood flow; and 3) confer neuroprotection. The various effects of NO in the eye appear to be mediated through the activation of the GC- guanosine 3:5'-cyclic monophosphate (cGMP) pathway and its effect on downstream targets, such as protein kinases and Ca2+ channels. Although NO-donor compounds are promising as therapeutics for IOP regulation, they may not be ideal to harness the neuroprotective potential of NO signaling. Here we review evidence that supports direct targeting of GC as a novel pleiotrophic treatment for the disease, without the need for direct NO application. The identification and targeting of other factors that contribute to glaucoma would be beneficial to patients, particularly those that do not respond well to IOP-dependent interventions.

The Microbead Occlusion Model of Ocular Hypertension in Mice.

Glaucoma is a common optic neuropathy that leads to vision loss through the degeneration of retinal ganglion cells (RGCs) and their axons. RGC degeneration in glaucoma is associated with sensitivity to intraocular pressure (IOP) and elevated IOP (also known as ocular hypertension) is the primary modifiable risk factor. Ocular hypertension is the primary characteristic of rodent models for glaucoma research. Intracameral injection of microbeads has evolved as a preferred method of IOP elevation in rodents, particularly in mice. Here, we outline the protocol and method for the Microbead Occlusion Model in mice. We highlight the importance of anesthesia choice and the utilization of glass micropipettes in combination with a micromanipulator and microsyringe pump for the successful execution of the model.

Constitutive and Stress-induced Expression of CCL5 Machinery in Rodent Retina.

Signaling by inflammatory cytokines and chemokines is associated with neurodegeneration in disease and injury. Here we examined expression of the ß-chemokine CCL5 and its receptors in the mouse retina and evaluated its relevance in glaucoma, a common optic neuropathy associated with sensitivity to intraocular pressure (IOP). Using quantitative PCR, fluorescent in situ hybridization, immunohistochemistry and quantitative image analysis, we found CCL5 mRNA and protein was constitutively expressed in the inner retina and synaptic layers. CCL5 appeared to associate with Müller cells and RGCs as well as synaptic connections between horizontal cells and bipolar cells in the OPL and amacrine cells, bipolar cells and RGCs in the IPL. Although all three high-affinity receptors (CCR5, CCR3, CCR1) for CCL5 were expressed constitutively, CCR5 expression was significantly higher than CCR3, which was also markedly greater than CCR1. Localization patterns for constitutive CCR5, CCR3 and CCR1 expression differed, particularly with respect to expression in inner retinal neurons. Stress-related expression of CCL5 was primarily altered in aged DBA/2 mice with elevated IOP. In contrast, changes in expression and localization of both CCR3 and CCR5 were evident not only in aged DBA/2 mice, but also in age-matched control mice and young DBA/2 mice. These groups do not exhibit elevated IOP, but possess either the aging stress (control mice) or the genetic predisposition to glaucoma (DBA/2 mice). Together, these data indicate that CCL5 and its high-affinity receptors are constitutively expressed in murine retina and differentially induced by stressors associated with glaucomatous optic neuropathy. Localization patterns further indicate that CCL5 signaling may be relevant for modulation of synapses in both health and disease, particularly in the inner plexiform layer.

Interleukin-6 Deficiency Attenuates Retinal Ganglion Cell Axonopathy and Glaucoma-Related Vision Loss.

The pleotropic cytokine interleukin-6 (IL-6) is implicated in retinal ganglion cell (RGC) survival and degeneration, including that associated with glaucoma. IL-6 protects RGCs from pressure-induced apoptosis in vitro. However, it is unknown how IL-6 impacts glaucomatous degeneration in vivo. To study how IL-6 influences glaucomatous RGC axonopathy, accompanying glial reactivity, and resultant deficits in visual function, we performed neural tracing, histological, and neurobehavioral assessments in wildtype (B6;129SF2/J; WT) and IL-6 knock-out mice (B6;129S2-IL6tm1kopf/J; IL-6-/-) after 8 weeks of unilateral or bilateral microbead-induced glaucoma (microbead occlusion model). IOP increased by 20% following microbead injection in both genotypes (p < 0.05). However, deficits in wound healing at the site of corneal injection were noted. In WT mice, elevated IOP produced degenerating axon profiles and decreased axon density in the optic nerve by 15% (p < 0.01). In IL-6-/- mice, axon density in the optic nerve did not differ between microbead- and saline-injected mice (p > 0.05) and degenerating axon profiles were minimal. Preservation of RGC axons was reflected in visual function, where visual acuity decreased significantly in a time-dependent manner with microbead-induced IOP elevation in WT (p < 0.001), but not IL-6-/- mice (p > 0.05). Despite this preservation of RGC axons and visual acuity, both microbead-injected WT and IL-6-/- mice exhibited a 50% decrease in anterograde CTB transport to the superior colliculus, as compared to saline-injected controls (p < 0.01). Assessment of glial reactivity revealed no genotype- or IOP-dependent changes in retinal astrocytes. IOP elevation decreased microglia density and percent retinal area covered in WT mice (p < 0.05), while IL-6-/- mice exhibited only a decrease in density (p < 0.05). Together, our findings indicate that two defining features of RGC axonopathy-axon transport deficits and structural degeneration of axons-likely occur via independent mechanisms. Our data suggest that IL-6 is part of a mechanism that specifically leads to structural degeneration of axons. Furthermore, its absence is sufficient to prevent both structural degeneration of the optic nerve and vision loss. Overall, our work supports the proposition that functional deficits in axon transport represent a therapeutic window for RGC axonopathy and identify IL-6 signaling as a strong target for such a therapeutic.

Oral Delivery of a Synthetic Sterol Reduces Axonopathy and Inflammation in a Rodent Model of Glaucoma.

Glaucoma is a group of optic neuropathies associated with aging and sensitivity to intraocular pressure (IOP). The disease is the leading cause of irreversible blindness worldwide. Early progression in glaucoma involves dysfunction of retinal ganglion cell (RGC) axons, which comprise the optic nerve. Deficits in anterograde transport along RGC axons to central visual structures precede outright degeneration, and preventing these deficits is efficacious at abating subsequent progression. HE3286 is a synthetic sterol derivative that has shown therapeutic promise in models of inflammatory disease and neurodegenerative disease. We examined the efficacy of HE3286 oral delivery in preventing loss of anterograde transport in an inducible model of glaucoma (microbead occlusion). Adult rats received HE3286 (20 or 100 mg/kg) or vehicle daily via oral gavage for 4 weeks. Microbead occlusion elevated IOP ~30% in all treatment groups, and elevation was not affected by HE3286 treatment. In the vehicle group, elevated IOP reduced anterograde axonal transport to the superior colliculus, the most distal site in the optic projection, by 43% (p = 0.003); HE3286 (100 mg/kg) prevented this reduction (p = 0.025). HE3286 increased brain-derived neurotrophic factor (BDNF) in the optic nerve head and retina, while decreasing inflammatory and pathogenic proteins associated with elevated IOP compared to vehicle treatment. Treatment with HE3286 also increased nuclear localization of the transcription factor NFκB in collicular and retinal neurons, but decreased NFκB in glial nuclei in the optic nerve head. Thus, HE3286 may have a neuroprotective influence in glaucoma, as well as other chronic neurodegenerations.

Probing electrical signals in the retina via graphene-integrated microfluidic platforms.

Graphene has attracted extensive attention in biological and biomedical fields due to its unique physical properties and excellent biocompatibility. We combine graphene field-effect transistors and scanning photocurrent microscopy with microfluidic platforms to investigate electrical signals in mouse retina. Remarkable photocurrent signals were detected from the graphene underneath the optic nerve head (ONH) of the retina, where the electrical activity from this region can modulate the carrier concentration of the graphene and induce local potential gradients. These built-in electrical potential gradients can efficiently separate photo-excited electron-hole pairs, leading to strong photocurrent responses in the graphene underneath the ONH. We also show that no significant photocurrent signal was observed in the graphene underneath either dehydrated or fixed retinal tissues, verifying that the photocurrent responses generated in the graphene underneath the ONH were indeed induced by the electrical activity in living retina. This method not only provides a way to investigate electrical processes in living retinal tissues, but also offers opportunities to study many other cellular systems involving cell-cell interactions through electrical signaling.

Interleukin-6: A Constitutive Modulator of Glycoprotein 130, Neuroinflammatory and Cell Survival Signaling in Retina.

OBJECTIVE: The interleukin-6 (IL-6) family of cytokines and their signal transducer glycoprotein (gp130) are implicated in inflammatory and cell survival functions in glaucoma. There are several avenues for interdependent modulation of IL-6 family members and gp130 signaling. Here we investigated whether IL-6 modulates gp130 and related neuroinflammatory, cell survival and regulatory signaling in both healthy and glaucomatous retina. METHODS: In naïve and glaucomatous (Microbead Occlusion Model), wildtype (WT) and IL-6 knockout (IL-6-/-) mice, we examined gp130 protein expression and localization, using western blot and immunohistochemistry. Gene targets related to IL-6 and gp130 signaling and pertinent to neuroinflammation (TNFα, IL-1ß), cell health (Bax, Bcl-xl) and STAT3 regulation (Socs3) were quantified using qRTPCR. RESULTS: In the naïve retina, IL-6-/- retina contained significantly less gp130 compared to WT retina. This IL-6-related decrease in gp130 was accompanied by a reduction in mRNA expression of TNFα, Socs3 and Bax. After 4 weeks of microbead-induced ocular hypertension, both microbead- and saline-injected (control) eyes of IL-6-/- mice exhibited higher expression of TNFα, compared to WT mice. IL-1ß expression was also reduced specifically in IL-6-/- retina with microbead-induced glaucoma. While saline and microbead injection increased Bcl-xl and Socs3 mRNA in both WT and IL-6-/- mice, IL-6-/- deficiency led to smaller increases for both Bcl-xl and Socs3. CONCLUSIONS: Our findings support a role for IL-6 in setting baseline parameters for neuroinflammatory, cell health and gp130 regulatory signaling that can impact the nature and magnitude of retinal responses to glaucoma-related stressors.