Astrocytes in the Optic Nerve Are Heterogeneous in Their Reactivity to Glaucomatous Injury.

The optic nerve head is thought to be the site of initial injury to retinal ganglion cell injury in glaucoma. In the initial segment of the optic nerve directly behind the globe, the ganglion cell axons are unmyelinated and come into direct contact to astrocytes, suggesting that astrocytes may play a role in the pathology of glaucoma. As in other parts of the CNS, optic nerve head astrocytes respond to injury by characteristic changes in cell morphology and gene expression profile. Using RNA-sequencing of glaucomatous optic nerve heads, single-cell PCR, and an in-vivo assay, we demonstrate that an up-regulation of astrocytic phagocytosis is an early event after the onset of increased intraocular pressure. We also show that astrocytes in the glial lamina of the optic nerve are apparently functionally heterogeneous. At any time, even in naïve nerves, some of the cells show signs of reactivity-process hypertrophy, high phagocytic activity, and expression of genetic markers of reactivity whereas neighboring cells apparently are inactive. A period of increased intraocular pressure moves more astrocytes towards the reactive phenotype; however, some cells remain unreactive even in glaucomatous nerves.

Vitamin C protects retinal ganglion cells via SPP1 in glaucoma and after optic nerve damage.

Glaucoma is a common neurodegenerative disorder characterized by retinal ganglion cell death, astrocyte reactivity in the optic nerve, and vision loss. Currently, lowering the intraocular pressure (IOP) is the first-line treatment, but adjuvant neuroprotective approaches would be welcome. Vitamin C possesses neuroprotective activities that are thought to be related to its properties as a co-factor of enzymes and its antioxidant effects. Here, we show that vitamin C promotes a neuroprotective phenotype and increases gene expression related to neurotropic factors, phagocytosis, and mitochondrial ATP production. This effect is dependent on the up-regulation of secreted phosphoprotein 1 (SPP1) in reactive astrocytes via the transcription factor E2F1. SPP1+ astrocytes in turn promote retinal ganglion cell survival in a mouse model of glaucoma. In addition, oral administration of vitamin C lowers the IOP in mice. This study identifies an additional neuroprotective pathway for vitamin C and suggests a potential therapeutic role of vitamin C in neurodegenerative diseases such as glaucoma.

Individual astrocyte morphology in the collagenous lamina cribrosa revealed by multicolor DiOlistic labeling.

Astrocytes in the lamina region of the optic nerve head play vital roles in supporting retinal ganglion cell axon health. In glaucoma, these astrocytes are implicated as early responders to stressors, undergoing characteristic changes in cell function as well as cell morphology. Much of what is currently known about individual lamina astrocyte morphology has been learned from rodent models which lack a defining feature of the human optic nerve head, the collagenous lamina cribrosa (LC). Current methods available for evaluation of collagenous LC astrocyte morphology have significant shortcomings. We aimed to evaluate Multicolor DiOlistic labeling (MuDi) as an approach to reveal individual astrocyte morphologies across the collagenous LC. Gold microcarriers were coated with all combinations of three fluorescent cell membrane dyes, DiI, DiD, and DiO, for a total of seven dye combinations. Microcarriers were delivered to 150 µm-thick coronal vibratome slices through the LC of pig, sheep, goat, and monkey eyes via MuDi. Labeled tissues were imaged with confocal and second harmonic generation microscopy to visualize dyed cells and LC collagenous beams, respectively. GFAP labeling of DiOlistically-labeled cells with astrocyte morphologies was used to investigate cell identity. 3D models of astrocytes were created from confocal image stacks for quantification of morphological features. DiOlistic labeling revealed fine details of LC astrocyte morphologies including somas, primary branches, higher-order branches, and end-feet. Labeled cells with astrocyte morphologies were GFAP+. Astrocytes were visible across seven distinct color channels, allowing high labeling density while still distinguishing individual cells from their neighbors. MuDi was capable of revealing tens to hundreds of collagenous LC astrocytes, in situ, with a single application. 3D astrocyte models allowed automated quantification of morphological features including branch number, length, thickness, hierarchy, and straightness as well as Sholl analysis. MuDi labeling provides an opportunity to investigate morphologies of collagenous LC astrocytes, providing both qualitative and quantitative detail, in healthy tissues. This approach may open doors for research of glaucoma, where astrocyte morphological alterations are thought to coincide with key functional changes related to disease progression.

Loss of epigenetic information as a cause of mammalian aging.

All living things experience an increase in entropy, manifested as a loss of genetic and epigenetic information. In yeast, epigenetic information is lost over time due to the relocalization of chromatin-modifying proteins to DNA breaks, causing cells to lose their identity, a hallmark of yeast aging. Using a system called "ICE" (inducible changes to the epigenome), we find that the act of faithful DNA repair advances aging at physiological, cognitive, and molecular levels, including erosion of the epigenetic landscape, cellular exdifferentiation, senescence, and advancement of the DNA methylation clock, which can be reversed by OSK-mediated rejuvenation. These data are consistent with the information theory of aging, which states that a loss of epigenetic information is a reversible cause of aging.

Viability of mitochondria-labeled retinal ganglion cells in organotypic retinal explant cultures by two methods.

Retinal explant cultures provide a valuable system to study retinal function in vitro. This study established a new retinal explant culture method to prolong the survival of retinal ganglion cells (RGCs). Explants were prepared in two different ways: with or without optic nerve. Retinas from newborn mice that had received an injection of MitoTracker Red into the contralateral superior colliculus to label axonal mitochondria were cultured as organotypic culture for 7 days in vitro. At several time points during the culture, viability of RGCs was assessed by multi-electrode array recording, and morphology by immunohistochemical methods. During the culture, the thickness of the retinal tissue in both groups gradually decreased, however, the structure of the layers of the retina could be identified. Massive apoptosis in the retinal ganglion cell layer (GCL) appeared on the first day of culture, thereafter the number of apoptotic cells decreased. Glial activation was observed throughout the culture, and there was no difference in morphology between the two groups. RGCs loss was exacerbated on 3rdday of culture, and RGCs loss in retinal explants with preserved optic nerve was significantly lower than in retinas that did not preserve the optic nerve. More and longer-lasting mitochondrial signals were observed in the injured area of the optic nerve-preserving explants. Retinal explants provide an invaluable tool for studying retinal function and developing treatments for ocular diseases. The optic nerve-preserving culture helps preserve the integrity of RGCs. The higher number of mitochondria in the nerve-preserving cultures may help maintain viability of RGCs.

Secreted phosphoprotein 1 slows neurodegeneration and rescues visual function in mouse models of aging and glaucoma.

Aging causes an irreversible, cumulative decline in neuronal function. Using the visual system as a model, we show that astrocytes play a critical role in maintaining retinal ganglion cell health and that deletion of SPP1 (secreted phosphoprotein 1, or osteopontin) from astrocytes leads to increased vulnerability of ganglion cells to age, elevated intraocular pressure, and traumatic optic nerve damage. Overexpression of SPP1 slows the age-related decline in ganglion cell numbers and is highly protective of visual function in a mouse model of glaucoma. SPP1 acts by promoting phagocytosis and secretion of neurotrophic factors while inhibiting production of neurotoxic and pro-inflammatory factors. SPP1 up-regulates transcription of genes related to oxidative phosphorylation, functionally enhances mitochondrial respiration, and promotes the integrity of mitochondrial microstructure. SPP1 increases intracellular ATP concentration via up-regulation of VDAC1.

Microglia depletion exacerbates retinal ganglion cell loss in a mouse model of glaucoma.

To test whether depletion of microglia in the optic nerve head has a beneficial effect on retinal ganglion cell numbers and function, we depleted microglia by oral administration of the CSF1R antagonist PLX5622. Then, ocular hypertension was induced by unilateral injection of magnetic microbeads into the anterior chamber. Visual function was assessed with pattern electroretinography and measurement of the optomotor reflex. Retinal ganglion cell bodies and axons were counted and gene expression patterns in optic nerve head astrocytes were tested on freshly dissociated astrocytes. PLX5622 efficiently depleted microglia in the retina and the optic nerve head, but about 20% of microglia persisted in the myelinated optic nerve proper even after prolonged exposure to the drug. PLX5622 did not affect ganglion cell function by itself. Elevation of the IOP for four weeks led to the expected decrease in visual acuity and pattern ERG amplitude. Microglia ablation did not affect these parameters. Ganglion cell and axon numbers were counted histologically post mortem. Mice in the microglia depletion group showed a moderate but significantly greater loss of ganglion cells than the control group. At four weeks post microbead injection, gene expression patterns in optic nerve head astrocytes are consistent with an A2 (or neuroprotective) pattern. Microglia depletion blunted the up-regulation of A2 genes in astrocytes. In conclusion, microglia depletion is unlikely to protect retinal ganglion cells in early glaucoma.

Lamina cribrosa vessel and collagen beam networks are distinct.

Our goal was to analyze the spatial interrelation between vascular and collagen networks in the lamina cribrosa (LC). Specifically, we quantified the percentages of collagen beams with/without vessels and of vessels inside/outside of collagen beams. To do this, the vasculature of six normal monkey eyes was labeled by perfusion post-mortem. After enucleation, coronal cryosections through the LC were imaged using fluorescence and polarized light microscopy to visualize the blood vessels and collagen beams, respectively. The images were registered to form 3D volumes. Beams and vessels were segmented, and their spatial interrelationship was quantified in 3D. We found that 22% of the beams contained a vessel (range 14%-32%), and 21% of vessels were outside beams (13%-36%). Stated differently, 78% of beams did not contain a vessel (68%-86%), and 79% of vessels were inside a beam (64%-87%). Individual monkeys differed significantly in the fraction of vessels outside beams (p < 0.01 by linear mixed effect analysis), but not in the fraction of beams with vessels (p > 0.05). There were no significant differences between contralateral eyes in the percent of beams with vessels and of vessels outside beams (p > 0.05). Our results show that the vascular and collagenous networks of the LC in monkey are clearly distinct, and the historical notions that each LC beam contains a vessel and all vessels are within beams are inaccurate. We postulate that vessels outside beams may be relatively more vulnerable to mechanical compression by elevated IOP than are vessels shielded inside of beams.

Ultrastructural Morphology of the Optic Nerve Head in Aged and Glaucomatous Mice.

Purpose: To study age- and intraocular pressure-induced changes in the glial lamina of the murine optic nerve on the ultrastructural level. Methods: Naïve C57bl/6 mice at various ages spanning the time between early adulthood (3 months) and senescence (30 months) were used in this study. In addition, the intraocular pressure (IOP) was increased in a group of young mice by injection of microbeads into the anterior chamber. The unmyelinated segments of the optic nerve containing the glial lamina were prepared for transmission electron microscopy and imaged at high resolution. Results: Axon packing density decreased slightly with age. Aging nerves contained higher numbers of enlarged and degenerating axons. Mean axonal diameter and in particular the variance of axonal diameter correlated well with age. Axonal mitochondria also showed age-dependent signs of pathology. The mean diameter of axonal mitochondria increased, and aged axons often contained profiles of mitochondria with very few or no cristae. Astrocytic mitochondria remained normal even in very old nerves. Changes to axons and axonal mitochondria in young glaucomatous nerves were comparable with those of 18- to 30-month-old naïve mice. In addition to axons and mitochondria, aged and glaucomatous nerves showed thickening of the blood vessel basement membranes and increased deposition of basement membrane collagen. Conclusions: On the ultrastructural level, the effects of age and elevated IOP are quite similar. One month of elevated IOP seems to have as strongly detrimental effects on the nerve as at least 18 months of normal aging.

Single-Cell Dissociation and Characterization in the Murine Retina and Optic Nerve.

Recent technological advances have extended the range of analytic tools to very small samples. It is now possible to assay the transcriptome, and in some cases even the proteome, of single cells reliably. This allows addressing novel questions, such as the genotype/phenotype relationships of single neurons, heterogeneity within individual cells of the same type, or the basis of differential vulnerability to injury. An important prerequisite for these kinds of studies is the ability to isolate well-defined individual cells without contamination by adjacent tissue. In the retina and optic nerve, cells of different types and functions are closely intermingled, limiting the use of standard methods such as laser capture microdissection. Here, we describe a simple method to isolate morphologically intact cells from the retina and the optic nerve and discuss considerations in recognizing and isolating different cell types after dissociation.

Optic nerve astrocyte reactivity protects function in experimental glaucoma and other nerve injuries.

Reactive remodeling of optic nerve head astrocytes is consistently observed in glaucoma and other optic nerve injuries. However, it is unknown whether this reactivity is beneficial or harmful for visual function. In this study, we used the Cre recombinase (Cre)-loxP system under regulation of the mouse glial fibrillary acidic protein promoter to knock out the transcription factor signal transducer and activator of transcription 3 (STAT3) from astrocytes and test the effect this has on reactive remodeling, ganglion cell survival, and visual function after experimental glaucoma and nerve crush. After injury, STAT3 knockout mice displayed attenuated astrocyte hypertrophy and reactive remodeling; astrocytes largely maintained their honeycomb organization and glial tubes. These changes were associated with increased loss of ganglion cells and visual function over a 30-day period. Thus, reactive astrocytes play a protective role, preserving visual function. STAT3 signaling is an important mediator of various aspects of the reactive phenotype within optic nerve astrocytes.

Astrocytes in the Optic Nerve Head of Glaucomatous Mice Display a Characteristic Reactive Phenotype.

Purpose: Optic nerve head astrocytes, a subtype of white-matter astrocytes, become reactive early in the course of glaucoma. It was shown recently that in the DBA/2J mouse model of inherited glaucoma optic nerve astrocytes extend new longitudinal processes into the axon bundles before ganglion cell loss becomes apparent. The present study aims at testing whether this behavior of astrocytes is typical of early glaucomatous damage. Methods: Mice expressing green fluorescent protein in individual astrocytes were used to evaluate the early response of astrocytes in the glial lamina of the optic nerve head after increasing the IOP using the microbead occlusion method. Tissue sections from the glial lamina were imaged consecutively by confocal and electron microscopy. Results: Confocal and electron microscope images show that astrocytes close to the myelination transition zone in the hypertensive nerve heads extend new processes that follow the longitudinal axis of the optic nerve and invade axon bundles in the nerve head. Ultrastructurally, the longitudinal processes were largely devoid of subcellular organelles except for degenerating mitochondria. Conclusions: The longitudinal processes are a common feature of glaucomatous optic nerve astrocytes, whereas they are not observed after traumatic nerve injury. Thus, astrocytes appear to fine-tune their responses to the nature and/or timing of the injury to the neurons that they surround.

Mice Homozygous for a Deletion in the Glaucoma Susceptibility Locus INK4 Show Increased Vulnerability of Retinal Ganglion Cells to Elevated Intraocular Pressure.

A genomic region located on chromosome 9p21 is associated with primary open-angle glaucoma and normal tension glaucoma in genome-wide association studies. The genomic region contains the gene for a long noncoding RNA called CDKN2B-AS, two genes that code for cyclin-dependent kinase inhibitors 2A and 2B (CDKN2A/p16(INK4A) and CDKN2B/p15(INK4B)) and an additional protein (p14(ARF)). We used a transgenic mouse model in which 70 kb of murine chromosome 4, syntenic to human chromosome 9p21, are deleted to study whether this deletion leads to a discernible phenotype in ocular structures implicated in glaucoma. Homozygous mice of this strain were previously reported to show persistent hyperplastic primary vitreous. Fundus photography and optical coherence tomography confirmed that finding but showed no abnormalities for heterozygous mice. Optokinetic response, eletroretinogram, and histology indicated that the heterozygous and mutant retinas were normal functionally and morphologically, whereas glial cells were activated in the retina and optic nerve head of mutant eyes. In quantitative PCR, CDKN2B expression was reduced by approximately 50% in the heterozygous mice and by 90% in the homozygous mice, which suggested that the CDKN2B knock down had no deleterious consequences for the retina under normal conditions. However, compared with wild-type and heterozygous animals, the homozygous mice are more vulnerable to retinal ganglion cell loss in response to elevated intraocular pressure.

Synapse Loss and Dendrite Remodeling in a Mouse Model of Glaucoma.

It has been hypothesized that synaptic pruning precedes retinal ganglion cell degeneration in glaucoma, causing early dysfunction to retinal ganglion cells. To begin to assess this, we studied the excitatory synaptic inputs to individual ganglion cells in normal mouse retinas and in retinas with ganglion cell degeneration from glaucoma (DBA/2J), or following an optic nerve crush. Excitatory synapses were labeled by AAV2-mediated transfection of ganglion cells with PSD-95-GFP. After both insults the linear density of synaptic inputs to ganglion cells decreased. In parallel, the dendritic arbors lost complexity. We did not observe any cells that had lost dendritic synaptic input while preserving a normal or near-normal morphology. Within the temporal limits of these observations, dendritic remodeling and synapse pruning thus appear to occur near-simultaneously.

Astrocytes in the optic nerve head express putative mechanosensitive channels.

PURPOSE: To establish whether optic nerve head astrocytes express candidate molecules to sense tissue stretch. METHODS: We used conventional PCR, quantitative PCR, and single-cell reverse transcription PCR (RT-PCR) to assess the expression of various members of the transient receptor potential (TRP) channel family and of the recently characterized mechanosensitive channels Piezo1 and 2 in optic nerve head tissue and in single, isolated astrocytes. RESULTS: Most TRP subfamilies (TRPC, TRPM, TRPV, TRPA, and TRPP) and Piezo1 and 2 were expressed in the optic nerve head of the mouse. Quantitative real-time PCR analysis showed that TRPC1, TRPM7, TRPV2, TRPP2, and Piezo1 are the dominant isoforms in each subfamily. Single-cell RT-PCR revealed that many TRP isoforms, TRPC1-2, TRPC6, TRPV2, TRPV4, TRPM2, TRPM4, TRPM6-7, TRPP1-2, and Piezo1-2, are expressed in astrocytes of the optic nerve head, and that most astrocytes express TRPC1 and TRPP1-2. Comparisons of the TRPP and Piezo expression levels between different tissue regions showed that Piezo2 expression was higher in the optic nerve head and the optic nerve proper than in the brain and the corpus callosum. TRPP2 also showed higher expression in the optic nerve head. CONCLUSIONS: Astrocytes in the optic nerve head express multiple putative mechanosensitive channels, in particular the recently identified channels Piezo1 and 2. The expression of putative mechanosensitive channels in these cells may contribute to their responsiveness to traumatic or glaucomatous injury.

Isolation of intact astrocytes from the optic nerve head of adult mice.

The astrocytes of the optic nerve head are a specialized subtype of white matter astrocytes that form the direct cellular environment of the unmyelinated ganglion cell axons. Due to their potential involvement in glaucoma, these astrocytes have become a target of research. Due to the heterogeneity of the optic nerve tissue, which also contains other cell types, in some cases it may be desirable to conduct gene expression studies on small numbers of well-characterized astrocytes or even individual cells. Here, we describe a simple method to isolate individual astrocytes. This method permits obtaining astrocytes with intact morphology from the adult mouse optic nerve and reduces contamination of the isolated astrocytes by other cell types. Individual astrocytes can be recognized by their morphology and collected under microscopic control. The whole procedure can be completed in 2-3 h. We also discuss downstream applications like multiplex single-cell PCR and quantitative PCR (qPCR).

Differential gene expression in glaucoma.

In glaucoma, regardless of its etiology, retinal ganglion cells degenerate and eventually die. Although age and elevated intraocular pressure (IOP) are the main risk factors, there are still many mysteries in the pathogenesis of glaucoma. The advent of genome-wide microarray expression screening together with the availability of animal models of the disease has allowed analysis of differential gene expression in all parts of the eye in glaucoma. This review will outline the findings of recent genome-wide expression studies and discuss their commonalities and differences. A common finding was the differential regulation of genes involved in inflammation and immunity, including the complement system and the cytokines transforming growth factor ß (TGFß) and tumor necrosis factor α (TNFα). Other genes of interest have roles in the extracellular matrix, cell-matrix interactions and adhesion, the cell cycle, and the endothelin system.

The Time Course of Gene Expression during Reactive Gliosis in the Optic Nerve.

Reactive gliosis is a complex process that involves changes in gene expression and morphological remodeling. The mouse optic nerve, where astrocytes, microglia and oligodendrocytes interact with retinal ganglion cell axons and each other, is a particularly suitable model for studying the molecular mechanisms of reactive gliosis. We triggered gliosis at the mouse optic nerve head by retro orbital nerve crush. We followed the expression profiles of 14,000 genes from 1 day to 3 months, as the optic nerve formed a glial scar. The transcriptome showed profound changes. These were greatest shortly after injury; the numbers of differentially regulated genes then dropped, returning nearly to resting levels by 3 months. Different genes were modulated with very different time courses, and functionally distinct groups of genes responded in partially overlapping waves. These correspond roughly to two quick waves of inflammation and cell proliferation, a slow wave of tissue remodeling and debris removal, and a final stationary phase that primarily reflects permanent structural changes in the axons. Responses from astrocytes, microglia and oligodendrocytes were distinctively different, both molecularly and morphologically. Comparisons to other models of brain injury and to glaucoma indicated that the glial responses depended on both the tissue and the injury. Attempts to modulate glial function after axonal injuries should consider different mechanistic targets at different times following the insult.