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A major risk factor for glaucomatous optic neuropathy is the level of intraocular pressure (IOP), which can lead to retinal ganglion cell axon injury and cell death. The optic nerve has a rostral unmyelinated portion at the optic nerve head followed by a caudal myelinated region. The unmyelinated region is differentially susceptible to IOP-induced damage in rodent models and human glaucoma. While several studies have analyzed gene expression changes in the mouse optic nerve following optic nerve injury, few were designed to consider the regional gene expression differences that exist between these distinct areas. We performed bulk RNA-sequencing on the retina and separately micro-dissected unmyelinated and myelinated optic nerve regions from naïve C57BL/6 mice, mice after optic nerve crush, and mice with microbead-induced experimental glaucoma (total = 36). Gene expression patterns in the naïve unmyelinated optic nerve showed significant enrichment of the Wnt, Hippo, PI3K-Akt, and transforming growth factor β pathways, as well as extracellular matrix-receptor and cell membrane signaling pathways, compared to the myelinated optic nerve and retina. Gene expression changes induced by both injuries were more extensive in the myelinated optic nerve than the unmyelinated region, and greater after nerve crush than glaucoma. Changes present three and fourteen days after injury largely subsided by six weeks. Gene markers of reactive astrocytes did not consistently differ between injury states. Overall, the transcriptomic phenotype of the mouse unmyelinated optic nerve was significantly different from immediately adjacent tissues, likely dominated by expression in astrocytes, whose junctional complexes are inherently important in responding to IOP elevation.

The responses of astrocytes in the optic nerve head (ONH) to mechanical and biochemical stimuli are important to understanding the degeneration of retinal ganglion cell axons in glaucoma. The ONH in glaucoma is vulnerable to stress produced by the intraocular pressure (IOP). Notably, after three days of elevated IOP in a mouse model, the junctions between the astrocytic processes and the peripapillary sclera were altered and the structural compliance of the ONH increased. In order to simulate this aspect of glaucomatous remodeling, explanted mouse eyes were treated with TrypLE, a recombinant trypsin enzyme. Treatment with TrypLE caused the periphery of the astrocytic lamina to contract radially by 0.044 ± 0.038. Transmission electron microscopy showed that TrypLE caused a separation of the end-feet of the astrocyte processes from the basement membrane at the junction with the sclera. Inflation testing after treatment with TrypLE caused an increased strain response in the astrocytic lamina compared to the strain response before treatment. The greatest increase was in the radial Green-Lagrange strain, E rr = 0.028 ± 0.009, which increased by 340%. The alterations in the microstructure and in the strain response of the astrocytic lamina reported in mouse experimental glaucoma were partially reproduced by experimental treatment of mouse eyes with TrypLE. The results herein suggest that separation of junctions between the astrocyte processes and the sclera may be instrumental in increasing the structural compliance of the ONH after a period of elevated IOP. STATEMENT OF SIGNIFICANCE: Astrocytes of the optic nerve of the eye spread out from edge to edge across the optic nerve in a region referred to as the astrocytic lamina. In an experimental model of glaucoma caused by elevated eye-pressure, there is disruption of the connections between astrocytes and the edge of the astrocytic lamina. We caused a similar event in the lamina by incubating explanted mouse eyes with an enzyme. Disruption of the astrocyte connections to the edge of their tissue caused the tissue to stretch more when we increased the eye-pressure, compared to the control tissue. This work is the first on the tissue of the optic nerve to demonstrate the importance of cell connections in preventing the over-stretching of the astrocytic lamina., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Purpose: To measure quantitatively changes in lamina cribrosa (LC) cell and connective tissue structure in human glaucoma eyes., Methods: We studied 27 glaucoma and 19 age-matched non-glaucoma postmortem eyes. In 25 eyes, LC cross-sections were examined by confocal and multiphoton microscopy to quantify structures identified by anti-glial fibrillary acidic protein (GFAP), phalloidin-labeled F-actin, nuclear 4',6-diamidino-2-phenylindole (DAPI), and by second harmonic generation imaging of LC beams. Additional light and transmission electron microscopy were performed in 21 eyes to confirm features of LC remodeling, including immunolabeling by anti-SOX9 and anti-collagen IV. All glaucoma eyes had detailed clinical histories of open-angle glaucoma status, and degree of axon loss was quantified in retrolaminar optic nerve cross-sections., Results: Within LC pores, the proportionate area of both GFAP and F-actin processes was significantly lower in glaucoma eyes than in controls (P = 0.01). Nuclei were rounder (lower median aspect ratio) in glaucoma specimens (P = 0.02). In models assessing degree of glaucoma damage, F-actin process width was significantly wider in glaucoma eyes with more damage (P = 0.024), average LC beam width decreased with worse glaucoma damage (P = 0.042), and nuclear count per square millimeter rose with worse damage (P = 0.019). The greater cell count in LC pores represented 92.3% astrocytes by SOX9 labeling. The results are consistent with replacement of axons in LC pores by basement membrane labeled by anti-collagen IV and in-migrating astrocytes., Conclusions: Alteration in LC structure in glaucoma involves migration of astrocytes into axonal bundles, change in astrocyte orientation and processes, production of basement membrane material, and thinning of connective tissue beams.

Aquaporin 4 is absent from astrocytes in the rodent optic nerve head, despite high expression in the retina and myelinated optic nerve. The purpose of this study was to quantify regional aquaporin channel expression in astrocytes of the porcine and human mouse optic nerve (ON). Ocular tissue sections were immunolabeled for aquaporins 1(AQP1), 4(AQP4), and 9(AQP9), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP) and alpha-dystroglycan (αDG) for their presence in retina, lamina, myelin transition zone (MTZ, region just posterior to lamina) and myelinated ON (MON). Semi- quantification of AQP4 labeling & real-time quantitative PCR (qPCR) data were analyzed in retina and ON tissue. Porcine and control human eyes had abundant AQP4 in Müller cells, retinal astrocytes, and myelinated ON (MON), but minimal expression in the lamina cribrosa. AQP1 and AQP9 were present in retina, but not in the lamina. Immunolabeling of GFAP and αDG was similar in lamina, myelin transition zone (MTZ) and MON regions. Semi-quantitative AQP4 labeling was at background level in lamina, increasing in the MTZ, and highest in the MON (lamina vs MTZ, MON; p≤0.05, p≤0.01, respectively). Expression of AQP4 mRNA was minimal in lamina and substantial in MTZ and MON, while GFAP mRNA expression was uniform among the lamina, MTZ, and MON regions. Western blot assay showed AQP4 protein expression in the MON samples, but none was detected in the lamina tissue. The minimal presence of AQP4 in the lamina is a specific regional phenotype of astrocytes in the mammalian optic nerve head., Competing Interests: The authors have declared that no competing interests exist.

Glaucoma is the leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP) is one of the major risk factors for glaucoma onset and progression, and available pharmaceutical interventions are exclusively targeted at IOP lowering. However, degeneration of retinal ganglion cells (RGCs) may continue to progress despite extensive lowering of IOP. A complementary strategy to IOP reduction is the use of neuroprotective agents that interrupt the process of cell death by mechanisms independent of IOP. Here, we describe an ion complexation approach for formulating microcrystals containing ~50% loading of a protein kinase inhibitor, sunitinib, to enhance survival of RGCs with subconjunctival injection. A single subconjunctival injection of sunitinib-pamoate complex (SPC) microcrystals provided 20 weeks of sustained retina drug levels, leading to neuroprotection in a rat model of optic nerve injury. Furthermore, subconjunctival injection of SPC microcrystals also led to therapeutic effects in a rat model of corneal neovascularization. Importantly, therapeutically relevant retina drug concentrations were achieved with subconjunctival injection of SPC microcrystals in pigs. For a chronic disease such as glaucoma, a formulation that provides sustained therapeutic effects to complement IOP lowering therapies could provide improved disease management and promote patient quality of life.

Purpose: To study aquaporin channel expression in astrocytes of the mouse optic nerve (ON) and the response to IOP elevation in mice lacking aquaporin 4 (AQP4 null)., Methods: C57BL/6 (B6) and AQP4 null mice were exposed to bead-induced IOP elevation for 3 days (3D-IOP), 1 and 6 weeks. Mouse ocular tissue sections were immunolabeled against aquaporins 1(AQP1), 4(AQP4), and 9(AQP9). Ocular tissue was imaged to identify normal AQP distribution, ON changes, and axon loss after IOP elevation. Ultrastructure examination, cell proliferation, gene expression, and transport block were also analyzed., Results: B6 mice had abundant AQP4 expression in Müller cells, astrocytes of retina and myelinated ON (MON), but minimal AQP4in prelaminar and unmyelinated ON (UON). MON of AQP4 nulls had smaller ON area, smaller axon diameter, higher axon density, and larger proportionate axon area than B6 (all p≤0.05). Bead-injection led to comparable 3D-IOP elevation (p = 0.42) and axonal transport blockade in both strains. In B6, AQP4 distribution was unchanged after 3D-IOP. At baseline, AQP1 and AQP9 were present in retina, but not in UON and this was unaffected after IOP elevation in both strains. In 3D-IOP mice, ON astrocytes and microglia proliferated, more in B6 than AQP4 null. After 6 week IOP elevation, axon loss occurred equally in the two mouse types (24.6%, AQP4 null vs. 23.3%, B6)., Conclusion: Lack of AQP4 was neither protective nor detrimental to the effects of IOP elevation. The minimal presence of AQP4 in UON may be a vital aspect of the regionally specific phenotype of astrocytes in the mouse optic nerve head., Competing Interests: The authors have declared that no competing interests exist.

Retinal ganglion cell (RGC) replacement holds potential for restoring vision lost to optic neuropathy. Transplanted RGCs must undergo neuroretinal integration to receive afferent visual signals for processing and efferent transmission. To date, retinal integration following RGC transplantation has been limited. We sought to overcome key barriers to transplanted human stem cell-derived RGC integration. Following co-culture ex vivo on organotypic mouse retinal explants, human RGCs cluster and extend bundled neurites that remain superficial to the neuroretina, hindering afferent synaptogenesis. To enhance integration, we increased the cellular permeability of the internal limiting membrane (ILM). Extracellular matrix digestion using proteolytic enzymes achieved ILM disruption while minimizing retinal toxicity and preserving glial reactivity. ILM disruption is associated with dispersion rather than clustering of co-cultured RGC bodies and neurites, and increased parenchymal neurite ingrowth. The ILM represents a significant obstacle to transplanted RGC connectivity and its circumvention may be necessary for functional RGC replacement., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: To delineate responses of optic nerve head astrocytes to sustained intraocular pressure (IOP) elevation in mice., Methods: We elevated IOP for 1 day to 6 weeks by intracameral microbead injection in 4 strains of mice. Astrocyte alterations were studied by transmission electron microscopy (TEM) including immunogold molecular localization, and by laser scanning microscopy (LSM) with immunofluorescence for integrin β1, α-dystroglycan, and glial fibrillary acidic protein (GFAP). Astrocyte proliferation and apoptosis were quantified by Ki67 and TUNEL labeling, respectively., Results: Astrocytes in normal optic nerve head expressed integrin β1 and α-dystroglycan by LSM and TEM immunogold labeling at electron dense junctional complexes that were found only on cell membrane zones bordering their basement membranes (BM) at the peripapillary sclera (PPS) and optic nerve head capillaries. At 1-3 days after IOP elevation, abnormal extracellular spaces appeared between astrocytes near PPS, and axonal vesical and mitochondrial accumulation indicated axonal transport blockade. By 1 week, abnormal spaces increased, new collagen formation occurred, and astrocytes separated from their BM, leaving cell membrane fragments. Electron dense junctional complexes separated or were absent at the BM. Astrocyte proliferation was modest during the first week, while only occasional apoptotic astrocytes were observed by TEM and TUNEL., Conclusions: Astrocytes normally exhibit junctions with their BM which are disrupted by extended IOP elevation. Responses include reorientation of cell processes, new collagen formation, and cell proliferation., Competing Interests: The authors have declared that no competing interests exist.