Optic Nerve Head Astrocytes Research Articles

Therapeutic Retrobulbar Inhibition of STAT3 Protects Ischemic Retina Ganglion Cells.

Astrocytes play an important role in the pathogenesis of glaucoma. Abnormal activation and/or proliferation of astrocytes, termed astrogliosis, have been observed during optic nerve degeneration. Our previous study identified signal transducer and activator of transcription 3 (STAT3) signaling as an important regulator of astrogliosis in the optic nerve in a rat transient ischemia/reperfusion model. In this study, we used pharmacological inhibition of STAT3 activation in the same model to assess whether it could attenuate reactive astrogliosis and to observe its influence on optic nerve damage and retinal ganglion cell (RGC) damage. Our findings show that retrobulbar inhibition of STAT3 in optic nerve head astrocytes leads to (a) increased nerve fiber bundle survival in the optic nerve, (b) increased nerve fiber bundle and RGC survival in the retina, (c) decreased astrocyte reactivation in the optic nerve (d) decreased remodeling of astrocytes in the optic nerve, and (e) no influence of astrocyte reactivation inside the retina. Taken together, the Janus kinase/STAT3 pathway contributes to astrocyte reactivation in the optic nerve, which plays a pivotal role in neurodegeneration after transient ischemia/reperfusion in vivo. Inhibition of this pathway provides a potential therapeutic strategy for the treatment of glaucomatous neuropathy, and could extend to other neurodegenerative diseases.

Astrocyte processes label for filamentous actin and reorient early within the optic nerve head in a rat glaucoma model.

To determine if astrocyte processes label for actin and to quantify the orientation of astrocytic processes within the optic nerve head (ONH) in a rat glaucoma model. Chronic intraocular pressure (IOP) elevation was produced by episcleral hypertonic saline injection and tissues were collected after 5 weeks. For comparison, eyes with optic nerve transection were collected at 2 weeks. Fellow eyes served as controls. Axonal degeneration in retrobulbar optic nerves was graded on a scale of 1 to 5. Optic nerve head sections (n ≥ 4 eyes per group) were colabeled with phalloidin (actin marker) and antibodies to astrocytic glial fibrillary acidic protein and aquaporin 4, or axonal tubulin βIII. Confocal microscopy and FIJI software were used to quantify the orientation of actin bundles. Control ONHs showed stereotypically arranged actin bundles within astrocyte processes. Optic nerve head actin bundle orientation was nearly perpendicular to axons (82.9° ± 6.3° relative to axonal axis), unlike the retrobulbar optic nerve (45.4° ± 28.7°, P < 0.05). With IOP elevation, ONH actin bundle orientation became less perpendicular to axons, even in eyes with no perceivable axonal injury (i.e., 38.8° ± 15.1° in grade 1, P < 0.05 in comparison to control ONHs). With severe injury, ONH actin bundle orientation became more parallel to the axonal axis (24.1° ± 28.4°, P < 0.05 in comparison to control ONHs). Optic nerve head actin bundle orientation in transected optic nerves was unchanged. Actin labeling identifies fine astrocyte processes within the ONH. Optic nerve head astrocyte process reorientation occurs early in response to elevated IOP.

Opioid receptors: methods for detection and their modes of actions in the eye.

This study provides evidence for the presence of opioid-receptors in the retina, optic nerve, and optic nerve head astrocytes. These receptors were measured by more than one technique including Western blotting, immunohistochemistry, and functional assays such as scotopic electroretinogram (ERG) and Pattern ERG. I also have provided evidence in recently published work from my laboratory that opioid receptors, more specifically δ-opioid receptors, play crucial roles in retina neuroprotection against ischemic and glaucomatous injuries. This chapter provides detailed procedures to measure opioid receptor activation and their roles in retina neuroprotection using functional assays such as scotopic ERG and pattern ERG.

Combinatorial targeting of early pathways profoundly inhibits neurodegeneration in a mouse model of glaucoma

The endothelin system is implicated in various human and animal glaucomas. Targeting the endothelin system has great promise as a treatment for human glaucoma, but the cell types involved and the exact mechanisms of action are not clearly elucidated. Here, we report a detailed characterization of the endothelin system in specific cell types of the optic nerve head (ONH) during glaucoma in DBA/2J mice. First, we show that key components of the endothelin system are expressed in multiple cell types. We discover that endothelin 2 (EDN2) is expressed in astrocytes as well as microglia/monocytes in the ONH. The endothelin receptor type A (Ednra) is expressed in vascular endothelial cells, while the endothelin receptor type B (Ednrb) receptor is expressed in ONH astrocytes. Second, we show that Macitentan treatment protects from glaucoma. Macitentan is a novel, orally administered, dual endothelin receptor antagonist with greater affinity, efficacy and safety than previous antagonists. Finally, we test the combinatorial effect of targeting both the endothelin and complement systems as a treatment for glaucoma. Similar to endothelin, the complement system is implicated in a variety of human and animal glaucomas, and has great promise as a treatment target. We discovered that combined targeting of the endothelin (Bosentan) and complement (C1qa mutation) systems is profoundly protective. Remarkably, 80% of DBA/2J eyes subjected to this combined inhibition developed no detectable glaucoma. This opens an exciting new avenue for neuroprotection in glaucoma.

Mechanosensitive release of adenosine 5'-triphosphate through pannexin channels and mechanosensitive upregulation of pannexin channels in optic nerve head astrocytes: a mechanism for purinergic involvement in chronic strain.

As adenosine 5'-triphosphate (ATP) released from astrocytes can modulate many neural signaling systems, the triggers and pathways for this ATP release are important. Here, the ability of mechanical strain to trigger ATP release through pannexin channels and the effects of sustained strain on pannexin expression were examined in rat optic nerve head astrocytes. Astrocytes released ATP when subjected to 5% of equibiaxial strain or to hypotonic swelling. Although astrocytes expressed mRNA for pannexins 1-3, connexin 43, and VNUT, pharmacological analysis suggested a predominant role for pannexins in mechanosensitive ATP release, with Rho kinase contribution. Astrocytes from panx1(-/-) mice had reduced baseline and stimulated levels of extracellular ATP, confirming the role for pannexins. Swelling astrocytes triggered a regulatory volume decrease that was inhibited by apyrase or probenecid. The swelling-induced rise in calcium was inhibited by P2X7 receptor antagonists A438079 and AZ10606120, in addition to apyrase and carbenoxolone. Extended stretch of astrocytes in vitro upregulated expression of panx1 and panx2 mRNA. A similar upregulation was observed in vivo in optic nerve head tissue from the Tg-MYOC(Y437H) mouse model of chronic glaucoma; genes for panx1, panx2, and panx3 were increased, whereas immunohistochemistry confirmed increased expression of pannexin 1 protein. In summary, astrocytes released ATP in response to mechanical strain, with pannexin 1 the predominant efflux pathway. Sustained strain upregulated pannexins in vitro and in vivo. Together, these findings provide a mechanism by which extracellular ATP remains elevated under chronic mechanical strain, as found in the optic nerve head of patients with glaucoma.

Osteopontin Is Induced by TGF-β2 and Regulates Metabolic Cell Activity in Cultured Human Optic Nerve Head Astrocytes

The aqueous humor (AH) component transforming growth factor (TGF)-β2 is strongly correlated to primary open-angle glaucoma (POAG), and was shown to up-regulate glaucoma-associated extracellular matrix (ECM) components, members of the ECM degradation system and heat shock proteins (HSP) in primary ocular cells. Here we present osteopontin (OPN) as a new TGF-β2 responsive factor in cultured human optic nerve head (ONH) astrocytes. Activation was initially demonstrated by Oligo GEArray microarray and confirmed by semiquantitative (sq) RT-PCR, realtime RT-PCR and western blot. Expressions of most prevalent OPN receptors CD44 and integrin receptor subunits αV, α4, α 5, α6, α9, β1, β3 and β5 by ONH astrocytes were shown by sqRT-PCR and immunofluorescence labeling. TGF-β2 treatment did not affect their expression levels. OPN did not regulate gene expression of described TGF-β2 targets shown by sqRT-PCR. In MTS-assays, OPN had a time- and dose-dependent stimulating effect on the metabolic activity of ONH astrocytes, whereas TGF-β2 significantly reduced metabolism. OPN signaling via CD44 mediated a repressive outcome on metabolic activity, whereas signaling via integrin receptors resulted in a pro-metabolic effect. In summary, our findings characterize OPN as a TGF-β2 responsive factor that is not involved in TGF-β2 mediated ECM and HSP modulation, but affects the metabolic activity of astrocytes. A potential involvement in a protective response to TGF-β2 triggered damage is indicated, but requires further investigation.

Comparative quantitative study of astrocytes and capillary distribution in optic nerve laminar regions

Retinal ganglion cell (RGC) axonal structure and function in the optic nerve head (ONH) is predominantly supported by astrocytes and capillaries. There is good experimental evidence to demonstrate that RGC axons are perturbed in a non-uniform manner following ONH injury and it is likely that the pattern of RGC axonal modification bears some correlation with the quantitative properties of astrocytes and capillaries within laminar compartments. Although there have been some excellent topographic studies concerning glial and microvascular networks in the ONH our knowledge regarding the quantitative properties of these structures are limited. This report is an in-depth quantitative, structural analysis of astrocytes and capillaries in the pre laminar, lamina cribrosa and post laminar compartments of the ONH. 49 optic nerves from human (n = 10), pig (n = 12), horse (n = 6), rat (n = 11) and rabbit (n = 10) eyes are studied. Immunohistochemical and high-magnification confocal microscopy techniques are used to co-localise astrocytes, capillaries and nuclei in the mid-portion of the optic nerve. Quantitative methodology is used to determine the area occupied by astrocyte processes, microglia processes, nuclei density and the area occupied by capillaries in each laminar compartment. Comparisons are made within and between species. Relationships between ONH histomorphometry and astrocyte-capillary constitution are also explored. This study demonstrates that there are significant differences in the quantitative properties of capillaries and astrocytes between the laminar compartments of the human ONH. Astrocyte processes occupied the greatest area in the lamina cribrosa compartment of the human ONH implicating it as an area of great metabolic demands. Microglia were found to occupy only a small proportion of tissue in the rat, rabbit and pig optic nerve suggesting that the astrocyte is the predominant glia cell type in the optic nerve. This study also demonstrates that there is significant uniformity, with respect to astrocyte and capillary constitution, in the post laminar region of species with an unmyelinated anterior optic nerve. This implicates an important role served by oligodendrocytes and myelin in governing the structural characteristics of the post laminar optic nerve. Finally, this study demonstrates that eyes with similar lamina cribrosa structure do not necessarily share an identical cellular constitution with respect to astrocytes. The quantitative properties of astrocytes in the pre laminar and lamina cribrosa regions of the rat, which has a rudimentary lamina cribrosa with only a few collagenous beams, shared more similarities to the human eye than the pig or horse. The quantitative properties of astrocytes and capillaries in the laminar compartments of the ONH provide a basis for understanding the pathogenic mechanisms that are involved in diseases such as glaucoma and ischemic optic neuropathy. The findings in this study also provide valuable information about the distinct advantages of different animal models for studying human optic nerve diseases. Utilisation of structural data provided in this report together with emerging in vivo technology may potentially permit the early identification of RGC axonal injury by quantifying changes in ONH capillaries and astrocytes.

Delta-Opioid Receptors Attenuate TNF-α–Induced MMP-2 Secretion From Human ONH Astrocytes

We examined the signaling mechanisms involved in δ-opioid-receptor agonist, SNC-121-mediated attenuation of TNF-α-induced matrix metalloproteinase-2 (MMP-2) secretion from human optic nerve head (ONH) astrocytes. Human ONH astrocytes were treated with SNC-121 (1 μmol/L) for 15 minutes followed by TNF-α (25 ng/mL) treatment for 6 or 24 hours. Cells were pretreated with inhibitors of p38 mitogen-activated protein (MAP) kinase (SB-203580) or NF-κB (Helenalin) prior to TNF-α treatment. Changes in phosphorylation and expression of p38 MAP kinase, IκBα, NF-κB, and MMP-2 were measured by Western blotting. Translocation of NF-κB was determined by immunocytochemistry. TNF-α treatment increased MMP-2 secretion from ONH astrocytes to 236% ± 17% and 142% ± 8% at 6 and 24 hours, respectively; while SNC-121 treatment reduced MMP-2 secretion to 149% ± 11% and 108% ± 7% at 6 and 24 hours, respectively. The SNC-121-mediated inhibitory response was blocked by the δ-opioid-receptor antagonist naltrindole. TNF-α treatment resulted in a sustained phosphorylation of p38 MAP kinase up to 24 hours (226% ± 15% over control levels), which was reduced to 150% ± 20% by SNC-121 treatment. TNF-α treatment increased the expression of NF-κB to 179% ± 21% and 139% ± 6% at 6 and 24 hours, respectively, which was significantly blocked by SNC-121 treatment. Furthermore, TNF-α-induced MMP-2 secretion was blocked by 100% and 78% in the presence of SB-203580 and Helenalin, respectively. Evidence is provided that SNC-121 attenuated TNF-α-induced MMP-2 secretion from ONH astrocytes. Data also supported the idea that p38 MAP kinase and NF-κB played central roles in TNF-α-induced MMP-2 secretion, and both were negatively regulated by SNC-121.

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.

Aqueous humour levels of placental growth factor in diabetic retinopathy

Placental growth factor (PlGF), a member of the vascular endothelial growth factor (VEGF) family, is a proangiogenic factor, originally isolated from human placenta (Maglione et al. 1991). Lines of evidence have elucidated that PlGF participates in angiogenic process not only directly by signalling via VEGF receptor (VEGFR)-1 but indirectly by amplifying VEGF-driven angiogenesis through regulation of inter- and intramolecular cross-talk between VEGFR-1 and VEGFR-2 (Autiero et al. 2003). Therefore, it is plausible that PlGF, synergistically with VEGF, plays a role in the development of ocular angiogenic disorders. So far, it has been reported that vitreous level of PlGF is increased in patients with proliferative diabetic retinopathy (PDR) compared with nondiabetic patients (Yamashita et al. 1999). Herein, we report the elevation of PlGF levels in aqueous humour from patients with diabetic retinopathy (DR), particularly in patients with neovascular glaucoma (NVG) due to DR. Aqueous humour samples were collected from 20 eyes of 20 patients with DR, including six with diabetic macular edema (DME) (mean age, 58.3 ± 3.5 years old), eight with PDR (50.5 ± 6.6 years old) and six with NVG (64.8 ± 2.3 years old). For control, samples were obtained from eight eyes of eight age-matched patients without diabetes, that is, epiretinal membrane or senile cataract (68.0 ± 3.3 years old). Protein level of PlGF was measured using ELISA kits for human PlGF (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's protocol. Aqueous levels of PlGF were significantly elevated in patients with DME (24.5 ± 4.8 pg/ml) and PDR (52.8 ± 27.1 pg/ml), whereas PlGF was undetectable in patients without diabetic ocular complication (Fig. 1A). Notably, PlGF levels in aqueous samples of NVG group showed approximately 10-fold increase (577.4 ± 277.2 pg/ml) compared with those of PDR group (p < 0.05, Mann–Whitney U-test) (Fig. 1B). Yamashita et al. (1999) have reported intravitreal concentrations of PlGF to be significantly elevated in patients with PDR (360 ± 272 pg/ml) than those with nonischaemic retinal diseases. The results of the present study are in accordance with previous data showing that PlGF was not detectable in aqueous humour samples from non-DR patients; however, in patients with DME and PDR, PlGF level was elevated. Furthermore, the aqueous level of PlGF was markedly elevated in NVG group. The current data indicate that aqueous level of PlGF is related to the severity of diabetic ocular complication and accumulation of proangiogenic factor PlGF in anterior chamber aggravates iris neovascularization in DR. The previous and current data have shown that aqueous level of PlGF is lower in comparison with the vitreous levels of PlGF, suggesting that PlGF is secreted in the posterior segment of the eye and diffuses through the vitreous cavity to the anterior chamber during DR progression. It has been shown that retinal pigment epithelial (RPE) cells can produce PlGF in response to growth factors such as bone morphogenetic protein-4, known as a participant in ocular angiogenesis (Hollborn et al. 2006). In addition, it was reported that cultured optic nerve head astrocytes secreted PlGF (Kernt et al. 2010). Therefore, it is possible that RPE and glial cells are cellular sources of PlGF in the diabetic retina. Further investigation is necessary to elucidate the mechanism(s) by which PlGF is elevated in the anterior chamber in case with DR. Therefore, PlGF may be an attractive molecular target in the prevention of pathological angiogenesis, and the current data suggest that targeting PlGF may prove beneficial in the treatment of PDR, particularly in case with NVG.

Multikinase-Inhibitoren als therapeutischer Ansatz bei neovaskulärer AMD: In-vitro-Evaluation der Sicherheit von Axitinib, Pazopanib und Sorafenib zur intraokularen Anwendung

Multikinase inhibitors (MKI) interfere effectively at different levels of the neovascularisation cascade. Early clinical and experimental data suggest that MKIs represent a promising novel approach for the treatment of neovascular age-related macular degeneration (AMD). However, so far little is known about the biocompatibility of MKIs regarding human ocular cells. This in vitro study investigates and compares the biocompatibility of three MKIs, axitinib, pazopanib, and sorafenib regarding ocular cells of the anterior and posterior segments, as well as organ-cultured donor corneas. Primary human optic nerve head astrocytes (ONHA), trabecular meshwork cells (TMC), and retinal pigment epithelium (RPE), human corneal endothelial and lens epithelial cells (CEC and LEC) were treated with different concentrations of axitinib, pazopanib, or sorafenib (0.1 to 100 µg/mL). To simulate oxidative stress, the cells were additionally co-incubated with 400 µM hydrogen peroxide. Induction of cell death and cellular viability were examined by live-dead assay and tetrazolium dye reduction assay (MTT). In addition, the influence of the three substances on human corneal endothelium was evaluated in seropositive donor corneas in organ culture by phase contrast microscopy. Up to a concentration of 7.5 mg/mL of the substances tested in any cell type examined, no toxic effects were found. Even after 10 days of incubation of organ-cultured donor corneas with 7.5 µg/mL, axitinib, pazopanib, or sorafenib, no evidence for endothelial toxicity was found. All three MKIs tested, axitinib, pazopanib, and sorafenib showed a good biocompatibility on the investigated ocular cells. Even under conditions of oxidative stress, there were no toxic effects up to a concentration of 7.5 µg/mL. Only at higher concentrations, there was a dose-dependent decrease in cellular viability and pronounced induction of cell death. These effects on cellular viability and induction of cell death appeared to be stronger with pazopanib, followed by sorafenib, than with axitinib.

Idebenone Prevents Human Optic Nerve Head Astrocytes From Oxidative Stress, Apoptosis, and Senescence by Stabilizing BAX/Bcl-2 Ratio

Oxidative stress plays an important role in the pathogenesis of several neurodegenerative diseases including glaucoma. Astrocytes are supposed to play a role in glaucoma pathogenesis. This study investigates the antiapoptotic and cytoprotective effects of idebenone on optic nerve head astrocytes (ONHA) under oxidative stress. ONHA were treated with 1 to 150 µM idebenone. Cell viability (MTT assay and live-dead assay), induction of intracellular reactive oxygen species, senescence-associated β-galactosidase activity were investigated. In addition, apoptosis (detection of histone-associated DNA fragmentation), and expression of BAX and Bcl-2, and their mRNA were determined after 48 hours and after hydrogen peroxide (H2O2) treatment. Idebenone concentrations from 1 to 50 µM showed no effects on ONHA viability. Pretreatment with 10 µM idebenone led to an increase in viability of ONHA after H2O2 treatment. In addition, idebenone pretreatment significantly attenuated the increase of histone-associated DNA fragmentation, induction of senescence-associated β-galactosidase, and intracellular reactive oxygen species after treatment with H2O2. When ONHA cells were treated with idebenone and H2O2, real-time polymerase chain reaction and Western blot analysis yielded an increased expression of Bcl-2 and a decrease of BAX compared with those cells that were treated with H2O2 only. Idebenone reduced senescence, oxidative stress, and apoptotic cell death in cultured ONHA in vitro. Our results suggest that idebenone may help to protect ONHA in vivo, and therefore might be helpful in preventing the progression of glaucomatous degeneration.

Antioxidants reduce TGF-beta2-induced gene expressions in human optic nerve head astrocytes

The goal of the present study was to investigate whether the antioxidants vitamin E, vitamin C and vitamin B1 can reduce the transforming growth factor-beta2 (TGF-β2)-induced gene expressions in cultured human optic nerve head (ONH) astrocytes. Cultured human ONH astrocytes were pretreated with different concentrations of vitamin E, vitamin C and vitamin B1 and then exposed to 1.0 ng/ml TGF-β2 for 24 hr. Expression of the heat shock proteins Hsp27 and αB-crystallin, the extracellular matrix (ECM) component fibronectin and the ECM-modulating protein connective tissue growth factor (CTGF) was detected by immunohistochemistry or real-time PCR analysis. TGF-β2 increased the expression of Hsp27, αB-crystallin, fibronectin and CTGF in human ONH astrocytes. Pretreatment with different concentrations of vitamin E, vitamin C and vitamin B1 reduced the TGF-β2-stimulated gene expressions. In cultured human ONH astrocytes, the TGF-β2-stimulated gene expressions could be reduced by pretreatment with vitamin E, vitamin C and vitamin B1. Therefore, the use of antioxidants in glaucomatous optic neuropathy might be a promising approach to prevent TGF-β2-induced cellular changes in ONH astrocytes.

Pax2+ astrocytes in the fish optic nerve head after optic nerve crush

The transcription factor Pax2 actively participates in the development of the vertebrate visual system. In adults, Pax2 expression persists in a subpopulation of Müller cells and/or astrocytes in the retina and optic nerve head (ONH), although its function remains elusive. In a previous work we showed that the pax2 gene expression is modified and the Pax2+ astrocyte population in the ONH strongly reacted during the regeneration of the retina after a lesion in goldfish. In the present work we have analyzed Pax2 expression in the goldfish ONH after optic nerve (ON) crush. At one week post-injury, when the regenerating axons arrive at the ONH, the pax2 gene expression level increases as well as the number of Pax2+ astrocytes in this region. These Pax2+ astrocytes show a higher number of Cytokeratin (Ck)+/GFAP+ processes compared with control animals. In contrast, a different S100+ astrocyte population is not modified and persists similar to that of controls. Furthermore, we find a ring that surrounds the posterior ONH that is formed by highly reactive astrocytes, positive to Pax2, GFAP, Ck, S100, GS and ZO1. In this region we also find a source of new astrocytes Pax2+/PCNA+ that is activated after the injury. We conclude that Pax2+ astrocytes constitute a subpopulation of ONH astrocytes that strongly reacts after ON crush and supports our previous results obtained after retina regeneration. Altogether, this suggests that pax2 gene expression and Pax2+ astrocytes are probably directly involved in the process of axonal regeneration.

Olfactory Ensheathing Cells Rescue Optic Nerve Fibers in a Rat Glaucoma Model

To determine if transplantation of olfactory ensheathing cells (OECs) can reduce loss of optic nerve axons after raised intraocular pressure (IOP) in the rat. OECs cultured from the adult olfactory mucosa were transplanted into the region of the optic disc. The IOP was raised by injection of magnetic microspheres into the anterior chamber. At 4 weeks after raising the IOP, the transplanted OECs had migrated into the dorsal area of the optic nerve head (ONH) where they surrounded the optic nerve fibers with a non-myelinated ensheathment. The mean amount of damage to the ONH astrocytic area in rats was 51.0% compared with 85.8% in those without OEC transplants (P < 0.02) and the mean loss of axons in the optic nerve was 51.0% compared with 80.3% in the absence of OECs (P < 0.01). OECs transplanted into the region of the ONH of the rat can reduce the loss of axons and the damage to ONH astrocytes caused by raised IOP. Confirmation of these preliminary experimental data, further understanding of possible mechanisms of axonal protection by OECs, and the longer-term time course of protection could provide a basis for future human clinical trials of autografted OECs, which would be available from autologous nasal epithelial biopsies.

Pathogenesis of glaucoma: does light play a part in the process?

AbstractPathogenesis of glaucoma: does light play a part in the process? By: Neville N. Osborne Fundación de Investigación Oftalmológica, Instituto Oftalmológico Fernández‐Vega, Oviedo 33012, Spain. Neurones are absolutely dependent on their mitochondria for their energy needs unlike dividing non‐neuronal glial cells. Retinal ganglion neurones are particularly dependent on their mitochondria because a lot of energy is required to propagate the action potential along the non‐mylinated axons within the globe. We have propose that glaucoma is initiated by an alteration in the blood flow dynamics in the optic nerve head causing a defined “ischemic‐like” insult to the retinal ganglion neurone axon mitochondria and also surrounding astrocytes and microglia. While this results in ganglion cells being metabolically weaker, but still functionally normally, they are now susceptible to secondary insults that they would have normally tolerated. The major secondary insults to subsequently result in ganglion cells dying at different times probably arise from chemicals released from activated optic nerve head astrocytes and microglia. Yet another factor may be the very same light that is involved in vision. This is because basic science studies have shown that light as impinging on ganglion cell axons can affect mitochondrial function. Thus while it is not suggested that light causes glaucoma it is proposed that particularly the blue light component impinging on the retina can exacerbate already metabolically effected ganglion cell mitochondria.

Purification and characterization of adult optic nerve head astrocytes

To establish a method of purifying and characterizing adult astrocytes from optic nerve head (ONH). Experimental study. The lamina cribrosa tissue from ONH of human eye was isolated under anatomic microscopy, and then 4 to 6 little explants were incubated in each culture plate containing culture medium DMEM/F12. After 8 to 10 weeks, the cells were removed by digesting cells with 0.25% trypsogen. Selective astrocyte culture medium is subsequently used. After two passages, astrocytes were identified by the observation of cell morphology and immunofluorescent staining of GFAP and NCAM. After 2 to 3 weeks of explants planting, cells showed an obvious migration procession by crawling in succession from the verge of the explants and rapidly splitting. Most cells displayed a flat star shape or polygon after digested with trypsogen. Several cells are long fusiformis. Almost all cells presented a flat star shape and simultaneously expressed GFAP and NCAM when the cells cultured with selective astrocyte culture medium. Cultured human ONH astrocytes can be obtained by precisely separating lamina cribrosa and placing the explants on the margin of culture medium, a method that promotes cell adherence. Using selective astrocyte culture medium is very effective and convenient in purifying primary astrocytes.

Involvement of Glial Cells in the Autoregulation of Optic Nerve Head Blood Flow in Rabbits

To investigate the involvement of glial cells in the autoregulation of optic nerve head (ONH) blood flow in response to elevated intraocular pressure (IOP). Rabbit eyes were treated with an intravitreal injection of l-2-aminoadipic acid (LAA), a gliotoxic compound. Twenty-four hours after the injection IOP was artificially elevated from a baseline of 20 to 50 or 70 mm Hg and maintained at each IOP level for 30 minutes. ONH blood flow was measured by laser speckle flowgraphy every 10 minutes. Ocular perfusion pressure (OPP) was calculated to investigate the relationship between ONH blood flow and OPP. To evaluate the effects of LAA on the function and morphology of retinal neurons and glial cells, electroretinogram (ERG) was monitored after injections of LAA (2.0 and 6.0 mM) or saline as a control. Histologic and immunohistochemical examinations were then performed. In the LAA-treated eyes, histologic changes selectively occurred in the retinal Müller cells and ONH astrocytes. There was not any significant reduction of amplitude or elongation of implicit time of each parameter in the ERG after LAA injection compared with control. ONH blood flow in LAA-treated eyes was significantly decreased with a reduction of OPP during IOP elevation to 50 and 70 mm Hg, whereas blood flow was maintained in control eyes during IOP elevation to 50 mm Hg. These results indicate the involvement of glial ells in the autoregulation of ONH blood flow during IOP elevation.

LOXL1 Deficiency in the Lamina Cribrosa as Candidate Susceptibility Factor for a Pseudoexfoliation-Specific Risk of Glaucoma

To test the hypothesis that a primary disturbance in lysyl oxidase-like 1 (LOXL1) and elastin metabolism in the lamina cribrosa of eyes with pseudoexfoliation syndrome constitutes an independent risk factor for glaucoma development and progression. Observational, consecutive case series. Posterior segment tissues obtained from 37 donors with early and late stages of pseudoexfoliation syndrome without glaucoma, 37 normal age-matched control subjects, 5 eyes with pseudoexfoliation-associated open-angle glaucoma, and 5 eyes with primary open-angle glaucoma (POAG). Protein and mRNA expression of major elastic fiber components (elastin, fibrillin-1, fibulin-4), collagens (types I, III, and IV), and lysyl oxidase crosslinking enzymes (LOX, LOXL1, LOXL2) were assessed in situ by quantitative real-time polymerase chain reaction, (immuno)histochemistry, and light and electron microscopy. Lysyl oxidase-dependent elastin fiber assembly was assessed by primary optic nerve head astrocytes in vitro. Expression levels of elastic proteins, collagens, and lysyl oxidases in the lamina cribrosa. Lysyl oxidase-like 1 proved to be the major lysyl oxidase isoform in the normal lamina cribrosa in association with a complex elastic fiber network. Compared with normal and POAG specimens, lamina cribrosa tissues obtained from early and late stages of pseudoexfoliation syndrome without and with glaucoma consistently revealed a significant coordinated downregulation of LOXL1 and elastic fiber constituents on mRNA and protein level. In contrast, expression levels of collagens and other lysyl oxidase isoforms were not affected. Dysregulated expression of LOXL1 and elastic proteins was associated with pronounced (ultra)structural alterations of the elastic fiber network in the laminar beams of pseudoexfoliation syndrome eyes. Inhibition of LOXL1 interfered with elastic fiber assembly by optic nerve head astrocytes in vitro. The findings provide evidence for a pseudoexfoliation-specific elastinopathy of the lamina cribrosa resulting from a primary disturbance in LOXL1 regulation and elastic fiber homeostasis, possibly rendering pseudoexfoliation syndrome eyes more vulnerable to pressure-induced optic nerve damage and glaucoma development and progression.

The Cell and Molecular Biology of Glaucoma: Biomechanical Factors in Glaucoma

Glaucoma is a heterogeneous group of diseases that progressively damage the optic nerve, leading to visual impairment and blindness. One major risk factor for most forms of glaucoma is the pressure inside the eye (intraocular pressure [IOP]). IOP is involved in both the development and progression of glaucoma. It can directly affect pressure-sensitive cells and tissues in the eye, especially those cells involved in glaucomatous damage to the eye. These pressure-sensitive cells and tissues are (1) the trabecular meshwork (TM); (2) cells in the optic nerve head (ONH), including lamina cribrosa cells and optic nerve head astrocytes (ONHAs); (3) the peripapillary sclera around the ONH; (4) retinal ganglion cells (RGCs); and (5) RGC axons in the retinal nerve fiber layer (RNFL) and in the ONH (Fig. 1A). These biological cells and tissues experience and respond to pressure-induced mechanical deformation, such as stretching and compression (i.e., biomechanical stress). In addition, cells in certain regions of the TM are subjected to sheer stress from the flow of the aqueous humor, the nutritive fluid in the anterior portion of the eye, through the outflow pathway. The eye experiences normal physiological fluctuations in IOP caused by eye blinking and movement, eye rubbing, and the daily variations in the rate of aqueous humor production that do not damage the eye. However, the pressure-sensitive tissues of the eye are pathologically affected by the chronically elevated IOP and abnormal IOP spikes associated with glaucoma. In addition, there are molecular, morphologic, and physiological changes in the eye that occur with aging and may make the eye more susceptible to the biomechanical insults associated with glaucoma.