Proteomics analyses of human optic nerve head astrocytes following biomechanical strain

We investigate the role of glial cell activation in the human optic nerve caused by raised intraocular pressure, and their potential role in the development of glaucomatous optic neuropathy. To do this we present a proteomics study of the response of cultured, optic nerve head astrocytes to biomechanical strain, the magnitude and mode of strain based on previously published quantitative models. In this case, astrocytes were subjected to 3 and 12% stretches for either 2 h or 24 h. Proteomic methods included nano-liquid chromatography, tandem mass spectrometry, and iTRAQ labeling. Using controls for both stretch and time, a six-plex iTRAQ liquid chromatography- tandem MS (LC/MS/MS) experiment yielded 573 proteins discovered at a 95% confidence limit. The pathways included transforming growth factor β1, tumor necrosis factor, caspase 3, and tumor protein p53, which have all been implicated in the activation of astrocytes and are believed to play a role in the development of glaucomatous optic neuropathy. Confirmation of the iTRAQ analysis was performed by Western blotting of various proteins of interest including ANXA 4, GOLGA2, and αB-Crystallin.     

Myocilin expression in the astrocytes of the optic nerve head

We investigated the expression of myocilin in the optic nerve head of porcine eyes by Western blotting and immunohistochemical staining. Myocilin was localized in the nucleus, centrosome, glial filament, mitochondria, and some parts of the cell membranes of the astrocytes. Myocilin was also detected at the edge-feet portion of the processes of astrocytes adjacent to the inner limiting membrane and blood vessel wall. The astrocytes are the major cell population in the optic nerve head, contributing to the architecture of the nerve axon and blood vessels. Therefore, myocilin gene mutation and change of myocilin protein are likely to affect the architecture of the optic nerve head and induce various forms of glaucomatous optic nerve damage.     

Hypoxia augments TNF-alpha-mediated endothelin-1 release and cell proliferation in human optic nerve head astrocytes

The effect of hypoxia (24 h) on TNF-alpha-mediated release of endothelin-1 (ET-1) from human optic nerve head astrocytes (hONAs) and TNF-alpha- and ET-1-induced hONA proliferation was determined. ET-1 synthesis and release was quantitated using ELISA while TNF-alpha (10 nM)- and ET-1 (100 nM)-mediated hONA proliferation was assessed by CellTiter 96 aqueous one-solution cell proliferation assay, respectively. hONAs appeared to be more rounded with fewer processes following 24 h hypoxia compared to thodr seen in normoxia. Hypoxia enhanced TNF-alpha-mediated ET-1 synthesis and release (by 5-fold) and also significantly increased TNF-alpha- and ET-1-mediated hONA proliferation. PD142893 (1 microM), an ET(A/B) receptor antagonist, blocked ET-1-mediated hONA proliferation both under normoxia and hypoxia, while doing so only under normoxia following TNF-alpha treatment. Also, U0126 (10 microM; an upstream ERK1/2 inhibitor) completely blocked agonist-induced hONA proliferation in normoxia and partially blocked the same in hypoxia. These results demonstrate for the first time that hONAs secrete ET-1 and that TNF-alpha and hypoxia can regulate its levels. Moreover, hypoxia augments the proliferative responses of hONAs to TNF-alpha and ET-1. These agonist-mediated effects following hypoxia could contribute to astroglial activation as seen in glaucomatous optic nerve heads.     

Accumulation of lipid inclusions in astrocytes of aging human optic nerve

We examined age-related changes in the human optic nerve (ON) from 10 postmortem donor eye samples (age: 21- to 94-year-old). In aged ON, many axons showed paucity of cytoskeleton, and possessed disorganized myelin that remained in the extracellular space. Lipid inclusions were detected in glia, as stained by oil red O, and these accumulated with aging. To identify and confirm which glial cell type possessed lipid inclusions, we performed immunohistochemistry (IHC) and transmission electron microscopy (TEM). Comparisons were made from TEM features and size of the glia immunolabeled with glial fibrillary acidic protein and glutamine synthetase (markers for astrocytes) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (a marker for oligodendrocytes). It was found that lipid inclusions were restricted to the astrocytes having larger perikarya than the oligodendrocytes (IHC) and possessing filaments in cytoplasm (TEM). These astrocytes also possessed myelin debris and it is thus likely that those inclusions originated from degenerated myelin of the ON axons. These data indicate that astrocytes play a role in phagocytosis and clearance of disorganized myelin in aging human ON.     

Ouabain-induced stimulation of sodium-hydrogen exchange in rat optic nerve astrocytes

Sodium-dependent transporters are inhibited indirectly by the Na-K-ATPase inhibitor ouabain. Here we report stimulation of sodium-hydrogen exchange (NHE) in ouabain-treated cells. BCECF was used to measure cytoplasmic pH in cultured rat optic nerve astrocytes. Ammonium chloride was applied to acid load the cells. On removal of ammonium chloride, cytoplasmic pH fell abruptly, then gradually recovered toward baseline. Ouabain (1 microM) did not change cell sodium content, but the rate of pH recovery increased by 68%. Ouabain speeded pH recovery both in the presence and absence of bicarbonate. In bicarbonate-free medium, dimethylamiloride, an NHE inhibitor, eliminated the effect of 1 microM ouabain on pH recovery. Western blot analysis showed an NHE1 immunoreactive band but not NHE2, NHE3, or NHE4. Immunoprecipitation studies showed phosphorylation of NHE1 in cells treated with 1 microM ouabain. Ouabain evoked an increase of cAMP, and the effect of 1 microM ouabain on pH recovery was abolished by H-89, a protein kinase A inhibitor. 8-Bromoadenosine-cAMP increased the pH recovery rate, and this recovery was not further increased by ouabain. Although 1 microM ouabain did not alter cytoplasmic calcium concentration, it stimulated calcium entry after store depletion, a response abolished by 2-APB. Ouabain-induced stimulation of pH recovery was suppressed by inhibitors of capacitative calcium entry, SKF-96365, and 2-APB, as well as the cytoplasmic calcium chelator BAPTA. The cAMP increase in ouabain-treated cells was abolished by BAPTA and 2-APB. Taken together, the results are consistent with increased capacitative calcium entry and subsequent cAMP-PKA-dependent stimulation of NHE1 in ouabain-treated cells.     

Choroidal melanoma invading the optic nerve head

Choroidal melanoma is the most common primary intraocular tumor, however, involvement of the optic nerve is rare. This case report presents a patient with an amelanotic juxtapapillary malignant choroidal melanoma with secondary optic disc invasion. The clinical signs and symptoms, differential diagnosis, management and prognosis for survival are discussed.     

The astrocytes in the retina and optic nerve head of mammals: A special glia for the ganglion cell axons

Summary The neuroglia in the retina and the intraocular portion of the optic nerve of the monkey and cat has been examined by light and electron microscopy. In the retina two types of macroglial cells can be distinguished: 1) Müller cells, and 2) astrocytes. The bipolar radial glial cells of Müller penetrate the entire thickness of the retina and their basal processes align in the nerve fibre layer to form septa that fasciculate the axons of the ganglion cells. In contrast to the Müller cells, the retinal astrocytes are not homogeneously distributed throughout the retina; their number correlates with the thickness of the nerve fibre layer. The processes of the astrocytes are confined to the ganglion cell layer and to the nerve fibre layer. In the latter, the astrocytic processes run parallel to and between the axons of a given nerve fibre bundle. According to cytological criteria, the retinal astrocytes are protoplasmic. In the intraocular portion of the optic nerve, however, the astrocytes are fibrous and their processes run perpendicular to the axon bundles of the prelaminar portion of the optic nerve. Thus, because of their intimate morphological relationship to axons of the nerve fibre layer and the intraocular portion of the optic nerve, the astrocytes in the eye of the monkey and the cat may be considered as a special glia for the axons of ganglion cells.     

Inhibition of oxidative stress by coenzyme Q10 increases mitochondrial mass and improves bioenergetic function in optic nerve head astrocytes

Oxidative stress contributes to dysfunction of glial cells in the optic nerve head (ONH). However, the biological basis of the precise functional role of mitochondria in this dysfunction is not fully understood. Coenzyme Q10 (CoQ₁₀), an essential cofactor of the electron transport chain and a potent antioxidant, acts by scavenging reactive oxygen species (ROS) for protecting neuronal cells against oxidative stress in many neurodegenerative diseases. Here, we tested whether hydrogen peroxide (100 μM H₂O₂)-induced oxidative stress alters the mitochondrial network, oxidative phosphorylation (OXPHOS) complex (Cx) expression and bioenergetics, as well as whether CoQ₁₀ can ameliorate oxidative stress-mediated alterations in mitochondria of the ONH astrocytes in vitro. Oxidative stress triggered the activation of ONH astrocytes and the upregulation of superoxide dismutase 2 (SOD2) and heme oxygenase-1 (HO-1) protein expression in the ONH astrocytes. In contrast, CoQ₁₀ not only prevented activation of ONH astrocytes but also significantly decreased SOD2 and HO-1 protein expression in the ONH astrocytes against oxidative stress. Further, CoQ₁₀ prevented a significant loss of mitochondrial mass by increasing mitochondrial number and volume density and by preserving mitochondrial cristae structure, as well as promoted mitofilin and peroxisome-proliferator-activated receptor-γ coactivator-1 protein expression in the ONH astrocyte, suggesting an induction of mitochondrial biogenesis. Finally, oxidative stress triggered the upregulation of OXPHOS Cx protein expression, as well as reduction of cellular adeonsine triphosphate (ATP) production and increase of ROS generation in the ONH astocytes. However, CoQ₁₀ preserved OXPHOS protein expression and cellular ATP production, as well as decreased ROS generation in the ONH astrocytes. On the basis of these observations, we suggest that oxidative stress-mediated mitochondrial dysfunction or alteration may be an important pathophysiological mechanism in the dysfunction of ONH astrocytes. CoQ₁₀ may provide new therapeutic potentials and strategies for protecting ONH astrocytes against oxidative stress-mediated mitochondrial dysfunction or alteration in glaucoma and other optic neuropathies.     

Isolation of 10-nm filaments from astrocytes in the mouse optic nerve

Astroglial filaments approximately 10 nm in diameter were isolated from degenerated mouse optic nerves by Triton X-100 and DNase I treatments followed by sucrose density gradient centrifugation. 2-4 wk after bilateral enucleation, optic nerves contained virtually a single population of 10-nm filaments (astroglial filaments), free from neurofilaments. In negative-staining and thin-section electron microscopy, the isolated filaments were seen as nonbranching linear structures with smooth contour, and were morphologically identical to those in situ. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed the isolated filaments to be composed of two major polypeptides with molecular weights of 45,000 and 55,000, present in an approximate molar ratio of 1:1. These findings, together with the results of one-dimensional peptide mapping and solubility study, indicate that the astroglial filaments in the mouse optic nerve are primarily composed of these two polypeptides.     

Compressive mechanical properties of rat and pig optic nerve head

Glaucoma is the leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP), the primary risk factor for glaucoma, is thought to induce abnormally high strains in optic nerve head (ONH) tissues, which ultimately result in retinal ganglion cell damage and vision loss. The mechanisms by which excessive deformations result in vision loss remain incompletely understood. The ability of computational and in vitro models of the ONH to provide insight into these mechanisms, in many cases, depends on our ability to replicate the physiological environment, which in turn requires knowledge of tissue biomechanical properties. The majority of mechanical data published to date regarding the ONH has been obtained from tensile testing, yet compression has been shown to be the main mode of deformation in the ONH under elevated IOP. We have thus tested pig and rat ONH tissue using unconfined cyclic compression. The material constants C₁, obtained from fitting the stress vs. strain data with a neo-Hookean material model, were 428 [367, 488] Pa and 64 [53, 76] Pa (mean [95% Confidence Interval]) for pig and rat optic nerve head, respectively. Additionally, we investigated the effects of strain rate and tissue storage on C1 values. These data will inform future efforts to understand and replicate the in vivo biomechanical environment of the ONH.     

Filaments in astrocytes and axons of mice optic nerve. A freeze-etching study

Fixed and nonfixed tissues from optic nerves of 20-day-old mice were examined with the electron microscope using the freeze-etching method. In this study the filaments of fibrious astrocytes are compared with those of axons. Both the astrocytic perikaron and the processes show a characteristic aspect in view of the arrangement and density of filaments. The most reliable criterion to distinguish them from nonmyelinated axons is the presence of areas with packed filaments. Furthermore, we demonstrated morphometrically that the filaments of axons and astrocytes from prefixed specimens had a statistically significant (p less than 0.001) smaller diameter (9.5 +/- 0.3 nm) than those from nonprefixed ones (10.5 +/- 0.3 nm). The diameters of filaments in axons and astrocytes are identical in fixed as well as in nonfixed material. The fine structure of filaments displays in addition to a helical form also a certain periodicity.