巩膜生物力学改变与青光眼性视神经损伤

青光眼的主要病理特征是视网膜神经节细胞(RGC)的丧失,从而导致进行性、不可逆性的视力丧失。目前已有研究表明,巩膜的生物力学性质会影响视神经的生物力学变化,并且在RGC损伤和视力丧失的病理过程中起重要作用。因此,巩膜生物力学与青光眼关系的研究对深入了解青光眼的发病机制有着重要的意义。本文就巩膜的生物力学特性、巩膜胶原纤维结构、巩膜重塑、巩膜刚度及通透性与青光眼性视神经损伤的关系进行综述,以利于更深入地了解青光眼性视神经损害的机制,为青光眼的预防和治疗提供新思路。     

Obstructed axonal transport of BDNF and its receptor TrkB in experimental glaucoma

PURPOSE: In both animal model system and in human glaucoma, retinal ganglion cells (RGCs) die by apoptosis. To understand how RGC apoptosis is initiated in these systems, the authors studied RGC neurotrophin transport in experimental glaucoma using acute intraocular pressure (IOP) elevations in rats and chronic IOP elevation and unilateral optic nerve transections in monkeys. METHODS: Eyes were studied in masked fashion by light and electron microscopy and by immunohistochemistry with antibodies directed against the tyrosine kinase receptors (TrkA, B, and C) and against brain-derived neurotrophic factor (BDNF), as well as by autoradiography to identify retrograde axonal transport of 125I-BDNF injected into the superior colliculus. RESULTS: With acute glaucoma in the rat, RGC axons became abnormally dilated, accumulating vesicles presumed to be moving in axonal transport at the optic nerve head. Label for TrkB, but not TrkA, was relatively increased at and behind the optic nerve head with IOP elevation. Abnormal, focal labeling for TrkB and BDNF was identified in axons of monkey optic nerve heads with chronic glaucoma. With acute IOP elevation in rats, radiolabeled BDNF arrived at cells in the RGC layer at less than half the level of control eyes. CONCLUSIONS: Interruption of BDNF retrograde transport and accumulation of TrkB at the optic nerve head in acute and chronic glaucoma models suggest a role for neurotrophin deprivation in the pathogenesis of RGC death in glaucoma.     

玻璃体腔注射大麻素HU-211治疗鼠青光眼视神经损伤的研究

目的：评价玻璃体腔内注射大麻素HU-211对大鼠青光眼模型视神经的保护作用,为青光眼视神经损伤治疗提供实验依据。

方法：采用电凝巩膜表面静脉法制作大鼠青光眼模型18眼,随机分为3组：A组分别隔日一次玻璃体腔注射1mg/0.1mL大麻素HU-211,B组隔日一次玻璃体腔注射0.1mL生理盐水,C组为高眼压组。随机选6只对侧眼为空白对照组,每日观察眼压变化情况,用药4wk后处死大鼠,视网膜冰冻切片,HE染色通过视网膜神经元的密度变化评估大鼠慢性高眼压模型视网膜神经元的损伤程度。

结果： B组的凋亡程度及RGC的损伤程度明显高于A组,差异有统计学意义(P<0.05), B组与C组比较差异无统计学意义(P>0.05)。

结论： 玻璃体腔注射大麻素(HU-211)对大鼠青光眼模型视神经视网膜有明显的保护作用。     

Quantitative Mapping of Collagen Fiber Orientation in Non-glaucoma and Glaucoma Posterior Human Sclerae

Purpose. The posterior sclera has a major biomechanical influence on the optic nerve head, and may therefore be important in glaucoma. Scleral material properties are influenced significantly by collagen fiber architecture. Here we quantitatively map fiber orientation in non-glaucoma and glaucoma posterior human sclerae. Methods. Wide-angle x-ray scattering quantified fiber orientation at 0.5-mm intervals across seven non-glaucoma post-mortem human sclerae, and five sclerae with glaucoma history and confirmed axon loss. Multiphoton microscopy provided semiquantitative depth-profiling in the peripapillary sclera. Results. Midposterior fiber orientation was either uniaxial (one preferred direction) or biaxial (two directions). The peripapillary sclera was characterized by a ring of fibers located mainly in the mid-/outer stromal depth and encompassing ～50% of the total tissue thickness. Fiber anisotropy was 37% higher in the peripapillary sclera compared with midposterior, varied up to 4-fold with position around the scleral canal, and was consistently lowest in the superior-nasal quadrant. Mean fiber anisotropy was significantly lower in the superior-temporal (P < 0.01) and inferior-nasal (P < 0.05) peripapillary scleral quadrants in glaucoma compared with non-glaucoma eyes. Conclusions. The collagen fiber architecture of the posterior human sclera is highly anisotropic and inhomogeneous. Regional differences in peripapillary fiber anisotropy between non-glaucoma and glaucoma eyes may represent adaptive changes in response to elevated IOP and/or glaucoma, or baseline structural properties that associate with predisposition to glaucomatous axon damage. Quantitative fiber orientation data will benefit numerical eye models aimed at predicting the sclera's influence on nerve head biomechanics, and thereby its possible role in glaucoma.     

Factors influencing optic nerve head biomechanics

PURPOSE: The biomechanical environment within the optic nerve head (ONH) may play a role in retinal ganglion cell loss in glaucomatous optic neuropathy. This was a systematic analysis in which finite element methods were used to determine which anatomic and biomechanical factors most influenced the biomechanical response of the ONH to acute changes in IOP. METHODS: Based on a previously described computational model of the eye, each of 21 input factors, representing the biomechanical properties of relevant ocular tissues, the IOP, and 14 geometric factors were independently varied. The biomechanical response of the ONH tissues was quantified through a set of 29 outcome measures, including peak and mean stress and strain within each tissue, and measures of geometric changes in ONH tissues. Input factors were ranked according to their aggregated influence on groups of outcome measures. RESULTS: The five input factors that had the largest influence across all outcome measures were, in ranked order: stiffness of the sclera, radius of the eye, stiffness of the lamina cribrosa, IOP, and thickness of the scleral shell. The five least influential factors were, in reverse ranked order: retinal thickness, peripapillary rim height, cup depth, cup-to-disc ratio, and pial thickness. Factor ranks were similar for various outcome measure groups and factor ranges. CONCLUSIONS: The model predicts that ONH biomechanics are strongly dependent on scleral biomechanical properties. Acute deformations of ONH tissues, and the consequent high levels of neural tissue strain, were less strongly dependent on the action of IOP directly on the internal surface of the ONH than on the indirect effects of IOP on the sclera. This suggests that interindividual variations in scleral properties could be a risk factor for the development of glaucoma. Eye size and lamina cribrosa biomechanical properties also have a strong influence on ONH biomechanics.     

Lack of neuroprotection against experimental glaucoma in c-Jun N-terminal kinase 3 knockout mice

To determine if the absence of c-Jun N-terminal kinase 3 (JNK3) in the mouse retina would reduce retinal ganglion cell (RGC) loss in mice with experimental glaucoma. C57BL/6 mice underwent experimental intraocular pressure (IOP) elevation with a bead/viscoelastic injection into one eye. One-half of the mice were Jnk3 homozygous knockouts (KO) and were compared to wild type (WT) mice. IOP was measured under anesthesia with the TonoLab, axial length was measured post-mortem with calipers after inflation to 15 mmHg, and RGC layer counts were performed on retinal whole mount images stained with DAPI, imaged by confocal microscopy, and counted by masked observers in an image analysis system. Axon counts were performed in optic nerve cross-sections by semi-automated image analysis. Both WT and Jnk3^−/− mice had mean elevations of IOP of more than 50% after bead injection. Both groups underwent the expected axial globe elongation due to chronic IOP elevation. The absence of JNK3 in KO retina was demonstrated by Western blots. RGC layer neuron counts showed modest loss in both WT and Jnk3^−/− animals; local differences by retinal eccentricity were detected, in each case indicating greater loss in KO animals than in WT. The baseline number of RGC layer cells in KO animals was 10% higher than in WT, but the number of optic nerve axons was identical in KO and WT controls. A slightly greater loss of RGC in Jnk3^−/− mice compared to controls was detected in experimental mouse glaucoma by RGC layer counting and there was no protective effect shown in axon counts. Counts of RGC layer cells and optic nerve axons indicate that Jnk3^−/− mice have an increased number of amacrine cells compared to WT controls.     

Glaucoma 2.0: neuroprotection, neuroregeneration, neuroenhancement

Glaucoma is a progressive neurodegenerative disease of retinal ganglion cells (RGCs) associated with characteristic axon degeneration in the optic nerve. Clinically, our only method of slowing glaucomatous loss of vision is to reduce intraocular pressure (IOP), but lowering IOP is only partially effective and does not address the underlying susceptibility of RGCs to degeneration. We review the recent steps forward in our understanding of the pathophysiology of glaucoma and discuss how this understanding has given us a next generation of therapeutic targets by which to maintain RGC survival, protect or rebuild RGC connections in the retina and brain, and enhance RGC function.     

Is open‐angle glaucoma caused by impaired cerebrospinal fluid circulation: around the optic nerve?

Chronic open-angle glaucoma is the most frequent type of glaucoma and a leading cause for blindness. The role of intraocular pressure (IOP) in the pathogenesis of open-angle glaucoma has been challenged by patients with typical glaucomatous optic disc changes and visual field loss in whom the IOP never exceeded normal values (normal-tension glaucoma), as well as by patients with persistently elevated IOP who do not develop glaucomatous disc or field changes. Recent research has demonstrated that the cerebrospinal fluid (CSF) is not evenly distributed in all CSF spaces and that the subarachnoid space of the optic nerve can turn into a CSF compartment on its own. The biochemical components in this optic nerve compartment can differ markedly from normal CSF and some of its components (such as L-PGDS) may produce a toxic effect on the optic nerve and may therefore play an important role in the pathophysiology of open-angle glaucoma.     

Activation of retinal glial cells contributes to the degeneration of ganglion cells in experimental glaucoma

Elevation of intraocular pressure (IOP) is a major risk factor for neurodegeneration in glaucoma. Glial cells, which play an important role in normal functioning of retinal neurons, are well involved into retinal ganglion cell (RGC) degeneration in experimental glaucoma animal models generated by elevated IOP. In response to elevated IOP, mGluR I is first activated and Kir4.1 channels are subsequently inhibited, which leads to the activation of Müller cells. Müller cell activation is followed by a complex process, including proliferation, release of inflammatory and growth factors (gliosis). Gliosis is further regulated by several factors. Activated Müller cells contribute to RGC degeneration through generating glutamate receptor-mediated excitotoxicity, releasing cytotoxic factors and inducing microglia activation. Elevated IOP activates microglia, and following morphological and functional changes, these cells, as resident immune cells in the retina, show adaptive immune responses, including an enhanced release of pro-inflammatory factors (tumor neurosis factor-α, interleukins, etc.). These ATP and Toll-like receptor-mediated responses are further regulated by heat shock proteins, CD200R, chemokine receptors, and metabotropic purinergic receptors, may aggravate RGC loss. In the optic nerve head, astrogliosis is initiated and regulated by a complex reaction process, including purines, transmitters, chemokines, growth factors and cytokines, which contributes to RGC axon injury through releasing pro-inflammatory factors and changing extracellular matrix in glaucoma. The effects of activated glial cells on RGCs are further modified by the interplay among different types of glial cells. This review is concluded by presenting an in-depth discussion of possible research directions in this field in the future.     

Erythropoietin promotes survival of retinal ganglion cells in DBA/2J glaucoma mice

PURPOSE: Retinal ganglion cell (RGC) loss occurs in response to increased intraocular pressure (IOP) and/or retinal ischemia in glaucoma and leads to impairment of vision. This study was undertaken to test the efficacy of erythropoietin (EPO) in providing neuroprotection to RGCs in vivo. METHODS: The neuroprotective effects of EPO were studied in the DBA/2J mouse model of glaucoma. Mice were intraperitoneally injected with control substances or various doses of EPO, starting at the age of 6 months and continuing for an additional 2, 4, or 6 months. RGCs were labeled retrogradely by a gold tracer. IOP was measured with a microelectric-mechanical system, and EPO receptor (EPOR) expression was detected by immunohistochemistry. Axonal death in the optic nerve was quantified by para-phenylenediamine staining, and a complete blood count system was used to measure the number of erythrocytes. RESULTS: In DBA/2J mice, the average number of viable RGCs significantly decreased from 4 months to 10 months, with an inverse correlation between the number of dead optic nerve axons and viable RGCs. Treatment with EPO at doses of 3000, 6000, and 12,000 U/kg body weight per week all prevented significant RGC loss, compared with untreated DBA/2J control animals. EPO effects were similar to those of memantine, a known neuroprotective agent. IOP, in contrast, was unchanged by both EPO and memantine. Finally, EPOR was expressed in the RGC layer in both DBA/2J and C57BL/6J mice. CONCLUSIONS: EPO promoted RGC survival in DBA/2J glaucomatous mice without affecting IOP. These results suggest that EPO may be a potential therapeutic neuroprotectant in glaucoma.     

【In Press】 The role of microglia in the progression of glaucomatous neurodegeneration- a review

Glaucoma is the most serious leading cause of irreversible blindness worldwide. Reducing intraocular pressure (IOP) does not always stop glaucomatous neurodegeneration and the optic nerve may continue to be damaged in the normal IOP. Microglial activity has been recognized to play essential roles in pathologenesis of the central nervous system (CNS) as well as retinal ganglion cell (RGC) survival. The relationship between the neurodegeneration and the microglia cells in glaucoma is very complicated and still remains unclear. In the present review, we summarize the recent studies of mechanisms of microglia in glaucoma neurodegeneration, which might provide a new therapeutic target for the treatment of glaucoma.     

The role of microglia in the progression of glaucomatous neurodegeneration- a review

Glaucoma is a serious leading cause of irreversible blindness worldwide. Reducing intraocular pressure (IOP) does not always stop glaucomatous neurodegeneration and the optic nerve may continue to be damaged in the normal IOP. Microglial activity has been recognized to play essential roles in pathogenesis of the central nervous system (CNS) as well as retinal ganglion cell (RGC) survival. The relationship between the neurodegeneration and the microglia cells in glaucoma is very complicated and still remains unclear. In the present review, we summarize the recent studies of mechanisms of microglia in glaucoma neurodegeneration, which might provide new ways to treat glaucoma.     

Glaucoma update: epidemiology and new approaches to medical management

Glaucoma describes a group of ocular conditions which involve progressive optic nerve damage associated with loss of visual function and, frequently, with elevated intraocular pressure. Recent estimates of worldwide prevalence predict that 67 million people will suffer from glaucoma by the year 2000. Although the clinical features of glaucoma are reasonably well described, the pathogenesis of optic nerve damage remains unclear. Intraocular pressure (IOP) is accepted as an important risk factor; however, it is clear that other factors play a role in the pathogenesis of the disease, and such factors may interact with IOP to greatly enhance its harmful effects. Many of the new therapeutic approaches to the stabilisation and potential cure of glaucoma attempt to address these non-IOP factors. The aim of the following paper is to consider the implications of new estimates of disease prevalence, discuss theories related to optic nerve damage and outline new and future approaches to the medical management of the disease.     

Use of a mathematical model to estimate stress and strain during elevated pressure induced lamina cribrosa deformation.

BACKGROUND: High intraocular pressure (IOP), which is generally associated with glaucoma, causes lamina cribrosa retrodisplacement and deformation. Shear stress and strain resulting from lamina cribrosa deformation have been implicated in tissue remodeling, changes in retinal astrocyte function and retinal ganglion cell (RGC) death observed in vivo during glaucoma. METHODS: A mathematical model was developed to describe the lamina cribrosa exposed to elevated intraocular pressure (IOP). The model is based on the bending theory of plates, incorporates anatomical properties of the lamina cribrosa, and provides estimates of its biomechanical properties. The model relates IOP, the parameter normally correlated with glaucoma, and lamina cribrosa retrodisplacement to stress and strain experienced by cells, parameters that may be more closely associated with cell injury. RESULTS: We estimate that shear strains of 0.05 occur at the edge of a 200 microm thick lamina cribrosa at an IOP of 25 mm Hg. We estimate greater lamina cribrosa deformation and higher shear stress and strain for thinner lamina cribrosa and lamina cribrosa of larger radii. CONCLUSION: These results may provide better estimates of the stress and strain experienced by cells in the lamina cribrosa and may further our understanding of the forces that contribute to optic nerve degeneration during glaucoma.     

Head-up tilt lowers IOP and improves RGC dysfunction in glaucomatous DBA/2J mice

The inbred DBA/2J (D2) mouse strain is a well established model of spontaneously elevated intraocular pressure (IOP), progressive glaucomatous loss of retinal ganglion cells (RGCs), and early damage of RGC axons at the level of optic nerve head. Pattern electroretinogram (PERG) studies have shown that surviving RGCs in mice 6-12-month-old may be dysfunctional. RGC dysfunction seems to be IOP-dependent, since it may be exacerbated by means of acute IOP elevation with head-down body tilt. Here we test the hypothesis that head-up body posture lowers IOP, resulting in improvement of PERG amplitude in aged D2 mice with glaucoma. We show that head-up body tilt induces age-independent IOP lowering whose magnitude increases with the angle of tilt. For a fixed angle (−60°) of head-up tilt, IOP progressively decreases with a time constant of about 5 min and stabilizes at a value lower by about 5-6 mm Hg compared to the baseline. Head-up tilt also results in an improvement of PERG amplitude in older D2 mice with glaucoma but not in younger D2 mice without glaucoma. Improvement of PERG amplitude in aged D2 mice upon head-up-induced IOP lowering is consistent with the idea that RGCs undergo a stage of IOP-dependent, reversible dysfunction before death. The head-up IOP/PERG protocol may represent a non-invasive way to probe the potential for recovery of RGC dysfunction in D2 mice.     

Diabetes Exacerbates the Intraocular Pressure-Independent Retinal Ganglion Cells Degeneration in the DBA/2J Model of Glaucoma

PurposeGlaucoma is a multifactorial disease, causing retinal ganglion cells (RGCs) and optic nerve degeneration. The role of diabetes as a risk factor for glaucoma has been postulated but still not unequivocally demonstrated. The purpose of this study is to clarify the effect of diabetes in the early progression of glaucomatous RGC dysfunction preceding intraocular pressure (IOP) elevation, using the DBA/2J mouse (D2) model of glaucoma.MethodsD2 mice were injected with streptozotocin (STZ) obtaining a combined model of diabetes and glaucoma (D2 + STZ). D2 and D2 + STZ mice were monitored for weight, glycemia, and IOP from 3.5 to 6 months of age. In addition, the activity of RGC and outer retina were assessed using pattern electroretinogram (PERG) and flash electroretinogram (FERG), respectively. At the end point, RGC density and astrogliosis were evaluated in flat mounted retinas. In addition, Müller cell reactivity was evaluated in retinal cross-sections. Finally, the expression of inflammation and oxidative stress markers were analyzed.ResultsIOP was not influenced by time or diabetes. In contrast, RGC activity resulted progressively decreased in the D2 group independently from IOP elevation and outer retinal dysfunction. Diabetes exacerbated RGC dysfunction, which resulted independent from variation in IOP and outer retinal activity. Diabetic retinas displayed decreased RGC density and increased glial reactivity given by an increment in oxidative stress and inflammation.ConclusionsDiabetes can act as an IOP-independent risk factor for the early progression of glaucoma promoting oxidative stress and inflammation-mediated RGC dysfunction, glial reactivity, and cellular death.     

Neuroprotection of retinal ganglion cells with GDNF-Loaded biodegradable microspheres in experimental glaucoma

Glaucoma is the second leading cause of blindness worldwide, and also the most common optic neuropathy. The ultimate cause of vision loss in glaucoma is thought to be retinal ganglion cell (RGC) death. Neuroprotection of RGC is therefore an important goal of glaucoma therapy. Currently, glaucoma treatment relies on pharmacologic or surgical reduction of intraocular pressure (IOP). It is critical to develop treatment approaches that actively prevent the death of RGCs at risk in glaucoma. Neurotrophic factors have the ability to promote the survival and influence the growth of neurons. Neurotrophic factor deprivation has been proposed as one mechanism leading to RGC death in glaucoma. Effective neuroprotection in glaucoma likely requires the consistent availability of the active agent for prolonged periods of time. Biodegradable microspheres are especially attractive as drug delivery vehicles for a number of reasons. Sustained GDNF delivery by biodegradable microspheres offers significant neuroprotection to injured RGC in experimental glaucoma. PLGA microsphere-delivered GDNF represents an important neuroprotective strategy in the treatment of glaucomatous optic neuropathy and provides direction for further investigations of this hypothesis.     

Different responses of macrophages in retinal ganglion cell survival after acute ocular hypertension in rats with different autoimmune backgrounds

Recently macrophages were shown to play a protective role in retinal ganglion cells (RGCs) after optic nerve (ON) injury. In the present study, we investigated how macrophages responded after acute intraocular pressure (IOP) elevation in experimental autoimmune encephalomyelitis (EAE)-resistant Fischer 344 (F344) and Sprague Dawley (SD) rats and EAE-vulnerable Lewis rats. Acute IOP elevation was performed at 110mmHg for 2h to mimic acute glaucoma. Phagocytic cells in the eye were removed by intravitreal application of clodronate liposomes whereas macrophage activation was achieved by intravitreal injection of zymosan, a yeast wall preparation. Fluorescence dye, FluoroGold, was applied behind the eyeballs to retrogradely label surviving RGCs 40h before animal sacrifice. Macrophages in the retina were identified by ED1 immunostaining. Loss of 25% RGCs in F344 but over 90% in Lewis rats was seen 2 weeks after acute IOP elevation. Significant increase in the number of macrophages in the retina was seen to accompany the great RGC loss in Lewis rats; removal of these macrophages reduced the extent of RGC loss, suggesting the involvement of macrophages in RGC death in Lewis strain. Low numbers of macrophages were seen in F344 retinas after acute IOP elevation, and removal of macrophages did not show clear effect on RGC viability. Whereas macrophage activation by zymosan protected RGCs after ON axotomy in F344 rats, the same macrophage activation became detrimental to RGCs after acute IOP elevation. The extent of RGC loss 3 weeks after acute IOP elevation or after macrophage activation by zymosan in EAE-resistant SD rats was similar to that in F344 rats. We thus demonstrate that macrophages in rats with different autoimmune backgrounds react differently to acute IOP elevation and differentially modulate RGC loss, a phenomenon contrary to the protective action in RGCs after ON axotomy. These data suggest that autoimmune background plays a role in modulating vulnerability of RGCs to acute IOP elevation.     

青光眼视神经节细胞凋亡的作用机制研究进展

青光眼是一组以视神经退行性病变、视野缺损和视力下降为特征的不可逆性致盲性眼病。青光眼的发生主要涉及视网膜神经节细胞（retinal ganglion cells，RGC）的变性、凋亡、进行性丧失。目前，青光眼RGC凋亡的具体机制尚未明确，其中涉及多种凋亡途径和凋亡相关因子，包括死亡受体途径、线粒体途径、内质网途径等凋亡机制及caspases等凋亡因子参与。本文就RGC几种常见凋亡途径及凋亡相关因素做一简要综述，对青光眼的靶向治疗提供一定的理论依据，进而为临床治疗青光眼提供可靠的分子依据。     

Retinal ganglion cell apoptosis in glaucoma is related to intraocular pressure and IOP-induced effects on extracellular matrix

PURPOSE: To investigate the effect of IOP on retinal ganglion cell (RGC) apoptosis and correlate the effects with IOP-induced changes in extracellular matrix (ECM) in the retina and optic nerve head (ONH) in glaucomatous rat eyes. METHODS: Thirty-seven Dark Agouti rats had elevated IOP induced in the left eye by hypertonic saline episcleral vein injections. Eyes were examined at 3 months histologically for RGC apoptosis and expression of specific ECM components. RESULTS: RGC apoptosis was significantly related to IOP exposure (integral DeltaIOP P <0.001; peak IOP P <0.01). In the RGC layer, elevated IOP correlated positively to a significant increase in MMP-9 activity (P <0.001), tissue inhibitor of matrix metalloproteinase (TIMP-1) (P <0.05), and collagen I (P <0.01), and negatively correlated to deposition of laminin (P <0.05) and TGF-beta2 (P <0.05). There was a significant correlation between MMP-9 activity and both RGC apoptosis (P <0.001) and loss of laminin (P <0.01). IOP exposure was also associated with increased deposition of TGF-beta2 and collagen I at the ONH (P <0.01). CONCLUSIONS: The results demonstrated that RGC apoptosis in glaucoma correlates strongly with elevated IOP and is significantly associated with IOP-induced changes in specific ECM components in the RGC layer. The study shows for the first time a link between MMP-9, laminin degradation, RGC apoptosis, and IOP exposure in glaucoma. The findings suggest that abnormal ECM remodeling in the glaucomatous retina may relate to RGC death and support the notion that the retina is a primary site of injury in glaucoma.