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Introduction Glaucoma, a leading cause of irreversible blindness in the world, is a chronic neurodegenerative disease of multifactorial origin. Extensive research is ongoing to better understand, prevent, and treat progressive degeneration of retinal ganglion cells in glaucoma. While experimental models of glaucoma and postmortem tissues of human donors are analyzed for pathophysiological comprehension and improved treatment of this blinding disease, clinical samples of intraocular biofluids and blood collected from glaucoma patients are analyzed to identify predictive, diagnostic, and prognostic biomarkers. Multiplexing techniques for protein analysis offer a valuable approach for translational glaucoma research. Areas covered This review provides an overview of the increasing applications of multiplex protein analysis for glaucoma research and also highlights current research challenges in the field and expected solutions from emerging technological advances. Expert opinion Analytical techniques for multiplex analysis of proteins can help uncover neurodegenerative processes for enhanced treatment of glaucoma and can help identify molecular biomarkers for improved clinical testing and monitoring of this complex disease. This evolving field and continuously growing availability of new technologies are expected to broaden the comprehension of this complex neurodegenerative disease and speed up the progress towards new therapeutics and personalized patient care to prevent blindness from glaucoma.

Neuroinflammation relying on the inflammatory responses of glial cells has emerged as an impactful component of the multifactorial etiology of neurodegeneration in glaucoma. It has become increasingly evident that despite early adaptive and reparative features of glial responses, prolonged reactivity of the resident glia, along with the peripheral immune cells, create widespread toxicity to retinal ganglion cell (RGC) axons, somas, and synapses. As much as the synchronized responses of astrocytes and microglia to glaucoma-related stress or neuron injury, their bi-directional interactions are critical to build and amplify neuroinflammation and to dictate the neurodegenerative outcome. Although distinct molecular programs regulate somatic and axonal degeneration in glaucoma, inhibition of neurodegenerative inflammation can provide a broadly beneficial treatment strategy to rescue RGC integrity and function. Since inflammatory toxicity and mitochondrial dysfunction are converging etiological paths that can boost each other and feed into a vicious cycle, anti-inflammatory treatments may also offer a multi-target potential. This review presents an overview of the current knowledge on neuroinflammation in glaucoma with particular emphasis on the cell-intrinsic and cell-extrinsic factors involved in the reciprocal regulation of glial responses, the interdependence between inflammatory and mitochondrial routes of neurodegeneration, and the research aspects inspiring for prospective immunomodulatory treatments. With the advent of powerful technologies, ongoing research on molecular and functional characteristics of glial responses is expected to accumulate more comprehensive and complementary information and to rapidly move the field forward to safe and effective modulation of the glial pro-inflammatory activities, while restoring or augmenting the glial immune-regulatory and neurosupport functions.

This review aims to highlight the current knowledge about inflammatory mechanisms of neurodegeneration in glaucoma with emphasis on potential immunomodulation strategies. Glaucomatous retina and optic nerve present multiple evidences of inflammatory responses of astroglia, microglia, and blood-borne immune cells. Although adaptive/protective responses of resident or systemic immune cells can support neurons and promote tissue repair mechanisms after injurious insults, prolonged inflammatory processes can also produce neurotoxic mediators. Treatments targeting these neurodestructive outcomes may restore immune homeostasis and protect neurons from inflammatory injury. Due to widespread and chronic nature of neuroinflammation in glaucoma, immunomodulation offers a treatment strategy to protect different neuronal compartments of RGCs during the chronic and asynchronous course of neurodegeneration. Uncovering of distinct molecular responses and interactions of different immune cells that determine the neuroinflammatory phenotype and participate in neurodegenerative outcomes will be critical to develop effective strategies for immunomodulation in glaucoma. Neuroinflammation has increasingly been recognized to play an important role in glaucomatous neurodegeneration, and its modulation appears to be a promising treatment strategy for neuroprotection.

Glaucoma is a complex neurodegenerative disease involving RGC axons, somas, and synapses at dendrites and axon terminals. Recent research advancements in the field have revealed a bigger picture of glaucomatous neurodegeneration that encompasses multiple stressors, multiple injury sites, multiple cell types, and multiple signaling pathways for asynchronous degeneration of RGCs during a chronic disease period. Optic nerve head is commonly viewed as the critical site of injury in glaucoma, where early injurious insults initiate distal and proximal signaling for axonal and somatic degeneration. Despite compartmentalized processes for degeneration of RGC axons and somas, there are intricate interactions between the two compartments and mechanistic overlaps between the molecular pathways that mediate degeneration in axonal and somatic compartments. This review summarizes the recent progress in the molecular understanding of RGC degeneration in glaucoma and highlights various etiological paths with biomechanical, metabolic, oxidative, and inflammatory components. Through this growing body of knowledge, the glaucoma community moves closer toward causative treatment of this blinding disease.

Background Glia-driven neuroinflammation promotes neuron injury in glaucoma that is a chronic neurodegenerative disease of the optic nerve and a leading cause of irreversible blindness. Although therapeutic modulation of neuroinflammation is increasingly viewed as a logical strategy to avoid inflammatory neurotoxicity in glaucoma, current understanding of the molecular regulation of neuroinflammation is incomplete, and the molecular targets for immunomodulation remains unknown. Growing datasets pointed to nuclear factor-kappaB (NF-κB), a key transcriptional activator of inflammation, which was identified to be most affected in glaucomatous astroglia. Using a cell type-specific experimental approach, this study aimed to determine the value of astroglial NF-κB as a potential treatment target for immunomodulation in experimental mouse glaucoma. Methods Neuroinflammatory and neurodegenerative outcomes of experimental glaucoma were comparatively analyzed in mice with or without cre/lox-based conditional deletion of astroglial IκKβ, which is the main activating kinase involved in IκB degradation through the canonical pathway of NF-κB activation. Glial responses and the inflammatory status of the retina and optic nerve were analyzed by cell morphology and cytokine profiling, and neuron structure and function were analyzed by counting retinal ganglion cell (RGC) axons and somas and recording pattern electroretinography (PERG) responses. Results Analysis of glial inflammatory responses showed immunomodulatory outcomes of the conditional transgenic deletion of IκKβ in astroglia. Various pro-inflammatory cytokines known to be transcriptional targets for NF-κB exhibited decreased production in IκKβ-deleted astroglia, which included TNF-α that can induce RGC apoptosis and axon degeneration during glaucomatous neurodegeneration. Indeed, transgenic modulation of inflammatory responses by astroglial IκKβ deletion reduced neurodegeneration at different neuronal compartments, including both RGC axons and somas, and protected PERG responses. Conclusions The findings of this study support a key role for astroglial NF-κB in neuroinflammatory and neurodegenerative outcomes of experimental glaucoma and the potential of this transcriptional regulator pathway as a glial treatment target to provide neuroprotection through immunomodulation. By pointing to a potential treatment strategy targeting the astroglia, these experimental findings are promising for future clinical translation through transgenic applications to improve the treatment of this blinding disease.

Glaucoma is a complex neurodegenerative disease involving RGC axons, somas, and synapses at dendrites and axon terminals. Recent research advancements in the field have revealed a bigger picture of glaucomatous neurodegeneration that encompasses multiple stressors, multiple injury sites, multiple cell types, and multiple signaling pathways for asynchronous degeneration of RGCs during a chronic disease period. Optic nerve head is commonly viewed as the critical site of injury in glaucoma, where early injurious insults initiate distal and proximal signaling for axonal and somatic degeneration. Despite compartmentalized processes for degeneration of RGC axons and somas, there are intricate interactions between the two compartments and mechanistic overlaps between the molecular pathways that mediate degeneration in axonal and somatic compartments. This review summarizes the recent progress in the molecular understanding of RGC degeneration in glaucoma and highlights various etiological paths with biomechanical, metabolic, oxidative, and inflammatory components. Through this growing body of knowledge, the glaucoma community moves closer toward causative treatment of this blinding disease.

Preclinical imaging, especially of rodent models, plays a major role in experimental ophthalmology. Our aim was to determine if ultrasound can be used to visualize and measure flow dynamics in the retrobulbar vessels supplying and draining the eye and the potential of contrast microbubbles to provide image and measurement enhancement. To accomplish this, we used a 128-element, 18 MHz linear array ultrasound probe and performed plane-wave imaging of the eyes of Sprague Dawley rats. Compound images were acquired by emitting unfocused wavefronts at multiple angles and combining echo data from all angles to form individual B-scans. Multiple imaging sequences were utilized, compounding up to six angles, with imaging rate of up to 3,000 compound B-scans per second and sequence durations from 1.5 to 180 seconds. Data were acquired before and after intravenous introduction of contrast microbubbles. We found the total power of the Doppler signal in the image plane to increase approximately 20 fold after injection of contrast, followed by an exponential decay to baseline in about 90 seconds, The best-fit time constant of the decay averaged 41 sec. While major vessels and the retinal/choroidal complex were evident pre-contrast, they were dramatically enhanced with contrast present, with details such as choroidal arterioles seen only with contrast. Ocular arteriovenous transit time determined from comparative enhancement curves in arteries and veins was approximately 0.2 sec. In conclusion, plane wave ultrasound, especially with enhancement by contrast microbubbles, offers a means for the study of ocular hemodynamics using the rat eye as a model.

PURPOSE OF REVIEW: This review aims to highlight the current knowledge about inflammatory mechanisms of neurodegeneration in glaucoma with emphasis on potential immunomodulation strategies. RECENT FINDINGS: Glaucomatous retina and optic nerve present multiple evidences of inflammatory responses of astroglia, microglia, and blood-born immune cells. Although adaptive/protective responses of resident or systemic immune cells can support neurons and promote tissue repair mechanisms after injurious insults, prolonged inflammatory processes can also produce neurotoxic mediators. Treatments targeting these neurodestructive outcomes may restore immune homeostasis and protect neurons from inflammatory injury. Due to widespread and chronic nature of neuroinflammation in glaucoma, immunomodulation offers a treatment strategy to protect different neuronal compartments of RGCs during the chronic and asynchronous course of neurodegeneration. Uncovering of distinct molecular responses and interactions of different immune cells that determine the neuroinflammatory phenotype and participate in neurodegenerative outcomes will be critical to develop effective strategies for immunomodulation in glaucoma. SUMMARY: Neuroinflammation has increasingly been recognized to play an important role in glaucomatous neurodegeneration, and its modulation appears to be a promising treatment strategy for neuroprotection.

Most studies of the effect of acute elevation of intraocular pressure (IOP) on ocular blood-flow have utilized optical coherence tomography (OCT) to characterize retinal and choroidal flow and vascular density. This study investigates the effect of acute IOP elevation on blood flow velocity in the retrobulbar arteries and veins supplying and draining the eye, which, unlike the retinal and choroidal vasculature, are not directly compressed as IOP is increased. By cannulation of the anterior chamber of 20 Sprague-Dawley rats, we increased IOP in 10 mmHg steps from 10 to 60 mmHg and returned to 10 mmHg. After 1 minute at each IOP (and 3 minutes after return to 10 mmHg), we acquired 18 MHz plane-wave ultrasound data at 3000 compound images/sec for 1.5 sec. We produced color-flow Doppler images by digital signal processing of the ultrasound data, identified retrobulbar arteries and veins, generated spectrograms depicting flow velocity over the cardiac cycle and characterized changes of vascular density and perfusion in the orbit overall. Systolic, diastolic and mean velocities and resistive and pulsatile indices were determined from arterial spectrograms at each IOP level. Baseline mean arterial and mean venous velocities averaged 30.9±10.8 and 8.5 ±3.3 mm/sec, respectively. Arterial velocity progressively decreased and resistance indices increased at and above an IOP of 30 mmHg. Mean arterial velocity at 60 mmHg dropped by 55% with respect to baseline, while venous velocity decreased by 20%. Arterial and venous velocities and resistance returned to near baseline after IOP was restored to 10 mmHg. Both vascular density and orbital perfusion decreased with IOP, but while perfusion returned to near normal when IOP returned to 10 mmHg, density remained reduced. Our findings are consistent with OCT-based studies showing reduced perfusion of the retina at levels comparable to retrobulbar arterial flow velocity change with increased IOP. The lesser effect on venous flow is possibly attributable to partial collapse of the venous lumen as volumetric venous outflow decreased at high IOP. The continued reduction in orbital vascular density 3 minutes after restoration of IOP to 10 mmHg might be attributable to persisting narrowing of capillaries, but this needs to be verified in future studies.

Retinal ganglion cells (RGCs) expanding from the retina to the brain are primary victims of neurodegeneration in glaucoma, a leading cause of blindness; however, the neighboring astroglia survive the glaucoma-related stress and promote neuroinflammation. In light of diverse functions of caspase-8 in apoptosis, cell survival, and inflammation, this study investigated the importance of caspase-8 in different fates of glaucomatous RGCs and astroglia using two experimental approaches in parallel. In the first approach, cell type-specific responses of RGCs and astroglia to a caspase-8 cleavage-inhibiting pharmacological treatment were studied in rat eyes with or without experimentally induced glaucoma. The second approach utilized an experimental model of glaucoma in mice in which astroglial caspase-8 was conditionally deleted by cre/lox. Findings of these experiments revealed cell type-specific distinct processes that regulate caspase-8 functions in experimental glaucoma, which are involved in inducing the apoptosis of RGCs and promoting the survival and inflammatory responses of astroglia. Deletion of caspase-8 in astroglia protected RGCs against glia-driven inflammatory injury, while the inhibition of caspase-8 cleavage inhibited apoptosis in RGCs themselves. Various caspase-8 functions impacting both RGC apoptosis and astroglia-driven neuroinflammation may suggest the multi-target potential of caspase-8 regulation to provide neuroprotection and immunomodulation in glaucoma.

Glaucoma is a chronic neurodegenerative disease of the optic nerve and a leading cause of irreversible blindness, worldwide. While the experimental research using animal models provides growing information about cellular and molecular processes, parallel analysis of the clinical presentation of glaucoma accelerates the translational progress towards improved understanding, treatment, and clinical testing of glaucoma. Optic nerve axon injury triggers early alterations of retinal ganglion cell (RGC) synapses with function deficits prior to manifest RGC loss in animal models of glaucoma. For testing the clinical relevance of experimental observations, this study analyzed the functional correlation of localized alterations in the inner plexiform layer (IPL), where RGCs establish synaptic connections with retinal bipolar and amacrine cells. Participants of the study included a retrospective cohort of 36 eyes with glaucoma and a control group of 18 non-glaucomatous subjects followed for two-years. The IPL was analyzed on consecutively collected macular SD-OCT scans, and functional correlations with corresponding 10–2 visual field scores were tested using generalized estimating equations (GEE) models. The GEE-estimated rate of decrease in IPL thickness (R = 0.36, P0.05). These findings support early localized IPL alterations in correlation with progressing visual field defects in glaucomatous eyes. Considering the experimental data, glaucoma-related increase in IPL thickness/density might reflect dendritic remodeling, mitochondrial redistribution, and glial responses for synapse maintenance, but decreased IPL thickness/density might correspond to dendrite atrophy. The bridging of experimental data with clinical findings encourages further research along the translational path.

Gözde Hondur,1,2 Emre Göktaş,1 Lama Al-Aswad,1 Gülgün Tezel1 1Department of Ophthalmology, Columbia University, College of Physicians and Surgeons, New York, NY, USA; 2Department of Ophthalmology, Ulucanlar Training and Research Hospital, Ankara, Turkey Purpose: This pilot cross-sectional study aimed to determine age-related changes of the retinal nerve fiber layer (RNFL) thickness in retinal periphery by swept-source optical coherence tomography-based analysis.Methods: Forty eyes of 40 healthy subjects were studied in three age groups, group 1 (20–40 years, n=15), group 2 (41–60 years, n=14), and group 3 (≥61 years, n=11). Wide-angle swept-source optical coherence tomography scans, including the optic disc and macula, were montaged with the nasal peripheral optical coherence tomography images acquired with a contralateral gaze. The peripapillary and peripheral RNFL thickness values were obtained for nasal and temporal sides. The ratio of peripheral-to-peripapillary RNFL thickness was also calculated for these sectors.Results: We detected a significantly thinner RNFL in older than younger subjects at a distance of 6 mm from the optic disc on nasal and temporal sides (P

Glaucoma, a leading cause of blindness, is a progressive neurodegenerative disease affecting the optic nerve. Multiple pathogenic mechanisms for glaucomatous neurodegeneration have been linked to o...

Purpose Glaucoma-related molecular biomarkers can improve clinical testing to diagnose the disease early, predict its prognosis, and monitor treatment responses. Based on the evidence of increased oxidative stress in glaucomatous tissues, this study analyzed oxidative stress-related biomarker candidates in blood and aqueous humor samples with or without glaucoma. Methods The blood and aqueous humor samples collected from carefully selected groups of 96 patients with glaucoma and 64 healthy subjects without glaucoma were included in the study. The samples were analyzed for protein carbonyls and advanced glycation end products (AGEs) through ELISA-based quantification assays. To allow proper comparisons, the Goldmann-Witmer coefficient that reflects the ratio of aqueous humor to blood values corrected to total protein concentration in individual samples was calculated. Results Blood and aqueous humor levels of protein carbonyls and AGEs were found significantly higher in glaucomatous samples compared with age-matched nonglaucomatous controls (P < 0.001). The glaucoma-related increase in protein carbonyls and AGEs was more prominent in aqueous humor samples than blood samples (2.6-fold versus 1.9-fold for protein carbonyls, and 3.1-fold versus 1.9-fold for AGEs; P < 0.001). Comparison of the Goldmann-Witmer coefficients indicated greater values for protein carbonyls (1.37 ± 0.3 vs. 3.07 ± 0.8) and AGEs (1.2 ± 0.3 vs. 3.2 ± 1.1) in the glaucoma group (P < 0.001). Conclusions Findings of this study encourage further validation studies of oxidative stress-related biomarkers in glaucoma. Analysis of protein carbonyls and AGEs in longitudinal studies of larger and heterogeneous patient cohorts should better assess the value of these promising candidates as molecular biomarkers of glaucoma for clinical predictions.

Glaucoma is a leading cause of blindness; however, limited understanding of the molecular mechanisms involved in optic nerve degeneration hinders the development of improved treatment strategies. Proteomics techniques that combine the protein chemistry, mass spectrometry, and bioinformatics offer the opportunity to shed light on molecular mechanisms so that new treatment strategies can be developed for immunomodulation, neuroprotection, neurorescue, neuroregeneration, and function gain in glaucoma. The proteomics technologies also hold great promise for biomarker discovery, another important goal of glaucoma research. As much as developing new treatment strategies, molecular biomarkers are strongly needed for early diagnosis of glaucoma, prediction of its prognosis, and monitoring the responses to new treatments. It is now a decade that the proteomics analysis techniques have been using to move glaucoma research forward. This review will focus on valuable applications of proteomics in the field of glaucoma research and highlight the power of this analytical toolbox in translational and clinical research towards better characterization and improved treatment of glaucomatous neurodegeneration and discovery of glaucoma-related molecular biomarkers.

Many blinding diseases of the inner retina are associated with degeneration and loss of retinal ganglion cells (RGCs). Recent evidence implicates several new signaling mechanisms as causal agents associated with RGC injury and remodeling of the optic nerve head. Ion channels such as Transient receptor potential vanilloid isoform 4 (TRPV4), pannexin-1 (Panx1) and P2X7 receptor are localized to RGCs and act as potential sensors and effectors of mechanical strain, ischemia and inflammatory responses. Under normal conditions, TRPV4 may function as an osmosensor and a polymodal molecular integrator of diverse mechanical and chemical stimuli, whereas P2X7R and Panx1 respond to stretch- and/or swelling-induced adenosine triphosphate release from neurons and glia. Ca(2+) influx, induced by stimulation of mechanosensitive ion channels in glaucoma, is proposed to influence dendritic and axonal remodeling that may lead to RGC death while (at least initially) sparing other classes of retinal neuron. The secondary phase of the retinal glaucoma response is associated with microglial activation and an inflammatory response involving Toll-like receptors (TLRs), cluster of differentiation 3 (CD3) immune recognition molecules associated with the T-cell antigen receptor, complement molecules and cell type-specific release of neuroactive cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). The retinal response to mechanical stress thus involves a diversity of signaling pathways that sense and transduce mechanical strain and orchestrate both protective and destructive secondary responses.Mechanistic understanding of the interaction between pressure-dependent and independent pathways is only beginning to emerge. This review focuses on the molecular basis of mechanical strain transduction as a primary mechanism that can damage RGCs. The damage occurs through Ca(2+)-dependent cellular remodeling and is associated with parallel activation of secondary ischemic and inflammatory signaling pathways. Molecules that mediate these mechanosensory and immune responses represent plausible targets for protecting ganglion cells in glaucoma, optic neuritis and retinal ischemia.

Although the immune system functions to preserve and restore tissue homeostasis, accumulating risk factors, prolonged glial activation, and sustained release of pro-inflammatory mediators in glaucoma may lead to a failure in the regulation of stress-induced immune response, and innate immune cells, autoreactive T cells, autoantibodies, and excess complement attack may exhibit potent stimuli that harm retinal ganglion cell somas, axons, and synapses. Identification of the cellular and molecular components of immune response pathways can provide immunomodulatory treatment strategies to attenuate neuroinflammation, protect neural tissue from collateral injury, and enhance endogenous recovery processes. This review highlights the current knowledge of molecular mechanisms regulating neuroinflammation in glaucoma.

Neurodegenerative insults and glial activation during glaucomatous neurodegeneration initiate an immune response to restore tissue homeostasis and facilitate tissue cleaning and healing. However, increasing risk factors over a chronic and cumulative period may lead to a failure in the regulation of innate and adaptive immune response pathways and represent a route for conversion of the beneficial immunity into a neuroinflammatory degenerative process contributing to disease progression. Oxidative stress developing through the pathogenic cellular processes of glaucoma, along with the aging-related component of oxidative stress, likely plays a critical role in shifting the physiological equilibrium. This review aims to provide a perspective on the complex interplay of cellular events during glaucomatous neurodegeneration by proposing a unifying scheme that integrates oxidative stress-related risk factors with the altered regulation of immune response in glaucoma.

Glaucomatous optic neuropathy causes blindness through the degeneration of retinal ganglion cells (RGCs) and their axons, which comprise the optic nerve. Glaucoma traditionally is associated with elevated intraocular pressure, but often occurs or may progress with intraocular pressure in the normal range. Like other diseases of the CNS, a subset of glaucoma has been proposed to involve an autoimmune component to help explain the loss of RGCs in the absence of elevated intraocular pressure. One hypothesis involves heat shock proteins (HSPs), because increased serum levels of HSP autoantibodies are prominent in some glaucoma patients with normal pressures. In the first direct support of this hypothesis, we found that HSP27 and HSP60 immunization in the Lewis rat induced RGC degeneration and axon loss 1–4 months laterin vivoin a pattern with similarities to human glaucoma, including topographic specificity of cell loss. Infiltration of increased numbers of T-cells in the retina occurred much earlier, 14–21 d after HSP immunization, and appeared to be transient.In vitrostudies found that T-cells activated by HSP immunization induced RGC apoptosis via the release of the inflammatory cytokine FasL, whereas HSP immunization induced activation of microglia cells and upregulation of the FasL receptor in RGCs. In summary, our results suggest that RGC degeneration in glaucoma for selected individuals likely involves failed immunoregulation of the T-cell-RGC axis and is thus a disturbance of both proapoptotic and protective pathways.

Glaucoma, a leading cause of blindness, is a multifactorial neurodegenerative disease damaging the optic nerve, including retinal ganglion cell (RGC) somas, axons, and synapses. The optic nerve degeneration in glaucoma has been linked to intraocular pressure–generated mechanical and/or vascular stress,1,2 aging,3 genetic predispositions,4 epigenetic risk factors,5 compartmentalized subcellular processes,6 and secondary neurodegenerative events due to oxidative stress,7,8 glial activation/dysfunction,8–10 and glia-mediated inflammation.10–13 A major goal of glaucoma research has been uncovering the molecular pathways of neurodegeneration to thereby develop new and improved treatment strategies for neuroprotection/rescue, neuroregeneration, and immunomodulation in patients with glaucoma. Proteomics analysis techniques offer an important toolbox for accomplishing this research aim. Indeed, many previous studies analyzing the proteomic alterations in human glaucoma and animal models have provided important insights into molecular pathways of glaucomatous neurodegeneration.14,15 While glaucoma refers to patients with clinical characteristics of optic nerve injury that can be assessed by structural and functional analysis techniques, in a group of patients, intraocular pressure is found elevated (>21 mm Hg) with no detectable damage to the optic nerve. These individuals who are at increased risk for developing glaucoma are referred to as ocular hypertensives. Based on the multicenter clinical trial by the Ocular Hypertension Treatment Study group, 9.5% of these patients with ocular hypertension convert to glaucoma over 5 years if their high intraocular pressures are untreated, while 4.4% among the patients treated to lower intraocular pressure develop glaucoma.16,17 Clinical risk factors for glaucoma development are well studied; however, molecular understanding of human glaucoma is limited. The complexity of glaucomatous neurodegeneration that involves multiple molecular pathways for primary injury increases even further with a range of secondary injury processes. Undoubtedly, early molecular alterations, as opposed to secondary molecular responses, are more critical to determine the molecular mechanisms underlying the initiation of glaucomatous neurodegeneration. To gain information about ocular hypertension–related early molecular alterations, this study analyzed retinal protein samples from ocular hypertensive human donors. Herein, we present our retinal proteomics data from six human donors with ocular hypertension in comparison to data from age- and sex-matched ocular normotensive controls (as well as by considering the previous retinal proteomics data from glaucomatous human donors18–21). The presented data reflect ocular hypertension–related “molecular risk factors,” the accumulation of which has potential to distress the physiological equilibrium toward glaucoma development.

Reactive oxygen species (ROS) are generated as by-products of cellular metabolism, primarily in the mitochondria. Although ROS are essential participants in cell signaling and regulation, when their cellular production overwhelms the intrinsic antioxidant capacity, damage to cellular macromolecules such as DNA, proteins, and lipids ensues. Such a state of “oxidative stress” is thought to contribute to the pathogenesis of a number of neurodegenerative diseases. Growing evidence supports the involvement of oxidative stress as a common component of glaucomatous neurodegeneration in different subcellular compartments of retinal ganglion cells (RGCs). Besides the evidence of direct cytotoxic consequences leading to RGC death, it also seems highly possible that ROS are involved in signaling RGC death by acting as a second messenger and/or modulating protein function by redox modifications of downstream effectors through enzymatic oxidation of specific amino acid residues. Different studies provide cumulating evidence, which supports the association of ROS with different aspects of the neurodegenerative process. Oxidative protein modifications during glaucomatous neurodegeneration increase neuronal susceptibility to damage and also lead to glial dysfunction. Oxidative stress-induced dysfunction of glial cells may contribute to spreading neuronal damage by secondary degeneration. Oxidative stress also promotes the accumulation of advanced glycation end products in glaucomatous tissues. It is also evident that oxidative stress takes part in the activation of immune response during glaucomatous neurodegeneration, as ROS stimulate the antigen presenting ability of glial cells and also function as co-stimulatory molecules during antigen presentation. By discussing current evidence, this review provides a broad perspective on cellular mechanisms and potential consequences of oxidative stress in glaucoma.

Glaucoma is no longer viewed simply as elevated intraocular pressure (IOP) that damages the optic nerve. In addition to high IOP, evidence is rapidly accumulating that suggests damage to the optic nerve may be initiated or sustained by any number of factors including ischemia, excitotoxicity, neurotrophin insufficiency, peroxynitrite damage or others not yet defined. These different harmful influences then likely act through common final pathways that eventually activate the cellular proteases that accompany neuronal programmed cell death. We believe aberrant immune signal processing may also result in retinal ganglion cell death. We hypothesized that one form of glaucoma may be an autoimmune neuropathy in which an individual's immune system is not only inappropriately regulated, but a cytotoxic effect is rendered by the very system which normally serves to protect it against stress. We propose that the family of proteins termed "heat shock proteins" are critical modulators of both the homeostatic/cytoprotective as well as pathogenic/neurodegenerative arms of the immune system in retinal ganglion cells or glial cells and are thus integral to glaucomatous neurodegeneration.

To assess the specific role of tumor necrosis factor (TNF) death receptor signaling in the induction of retinal ganglion cell (RGC) death, optic nerves of mice deficient for TNF receptor-1 (TNF-R1-/-) and control mice (C57BL/6J) were unilaterally subjected to crush injury. Counts of RGCs and their axons 6 weeks after the injury demonstrated that their loss was significantly less in TNF-R1-/- mice compared to controls. The most prominent decrease in neuronal loss detected in TNF-R1-/- mice was beyond the initial 2-week period after the injury. This time period was correlated with the period of glial activation and increased glial immunolabeling for TNF-alpha in these eyes. No further protection against neuronal loss was detectable in TNF-R1-/- mice treated with D-JNKI1, a specific inhibitor of c-Jun N-terminal protein kinase (JNK). However, anti-JNK treatment of control animals provided a significant protection against neuronal loss during the same secondary degeneration period. Phospho-JNK immunolabeling of RGCs in control mice subjected to optic nerve crush significantly decreased following their treatment with D-JNKI1, and anti-JNK treatment protected RGCs from degeneration in these animals, similar to the lack of TNF-R1. These findings provide evidence that TNF death receptor signaling is involved in the secondary degeneration of RGCs following optic nerve injury, and is associated with JNK signaling. Since secondarily degenerating neurons are viable targets for neuroprotection, inhibition of TNF death receptor signaling may be an effective strategy to protect RGCs in several neurodegenerative injuries.

Retinal ganglion cell (RGC) neurons are believed to die via apoptosis in human primary and secondary open-angle glaucoma. Previous studies have relied solely on the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate [UTP]-biotin nick end-labeling) method of detecting DNA fragmentation to identify apoptotic nuclei. However, it is now clear that the TUNEL method cannot distinguish between the single- and double-strand DNA breaks that can be a feature of both apoptosis and necrosis. We have developed a double fluorescent labeling method that simultaneously combines in situ end-labeling (ISEL) to detect DNA fragmentation followed by staining with a cyanine dye, YOYO-1, to visualize apoptotic chromatin condensation. This allows for the unequivocal identification of an apoptotic nucleus. Our preliminary data obtained from one case of normal pressure glaucoma suggests that RGC neurons may die via apoptosis when intraocular pressure is not elevated.

In order to improve understanding of remodeling events in the glaucomatous optic nerve head, the migration of optic nerve head astrocytes was studied in vitro. Since elevated intraocular pressure is an important stress factor identified in glaucomatous eyes, optic nerve head astrocytes were incubated under physical stress created by elevated hydrostatic pressure. In addition, they were incubated in the presence of a chemical stimulus, lipolysaccharide (LPS). Migration of reactivated astrocytes in the presence of these stressors was examined using chambers in which cell migration through extracellular matrix-coated pores is only possible following proteolytic digestion of the matrix. We observed that the migratory ability of optic nerve head astrocytes was approximately 4-6 times greater following exposure to elevated hydrostatic pressure or LPS for up to 48 h. Phosphoinositide 3-kinase, protein kinase C, and tyrosine kinase were found to be involved in the signal transduction for activated migration of optic nerve head astrocytes in response to elevated hydrostatic pressure or LPS. In addition, we observed that the stress-induced migration of optic nerve head astrocytes, which is accompanied by proteolytic degradation, resulted in the formation of culture cavities containing mucopolysaccharides. These in vitro findings provide a clearer understanding of the pathophysiologic mechanisms of characteristic tissue remodeling events that occur, in vivo, in the glaucomatous optic nerve head.

Although glial cells in the optic nerve head undergo a reactivation process in glaucoma, the role of glial cells during glaucomatous neurodegeneration of retinal ganglion cells is unknown. Using a coculture system in which retinal ganglion cells and glial cells are grown on different layers but share the same culture medium, we studied the influences of glial cells on survival of retinal ganglion cells after exposure to different stress conditions typified by simulated ischemia and elevated hydrostatic pressure. After the exposure to these stressors, we observed that glial cells secreted tumor necrosis factor-alpha (TNF-alpha) as well as other noxious agents such as nitric oxide into the coculture media and facilitated the apoptotic death of retinal ganglion cells as assessed by morphology, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and caspase activity. The glial origin of these noxious effects was confirmed by passive transfer experiments. Furthermore, retinal ganglion cell apoptosis was attenuated approximately 66% by a neutralizing antibody against TNF-alpha and 50% by a selective inhibitor of inducible nitric oxide synthase (1400W). Because elevated intraocular pressure and ischemia are two prominent stress factors identified in the eyes of patients with glaucoma, these findings reveal a novel glia-initiated pathogenic mechanism for retinal ganglion cell death in glaucoma. In addition, these findings suggest that the inhibition of TNF-alpha that is released by reactivated glial cells may provide a novel therapeutic target for neuroprotection in the treatment of glaucomatous optic neuropathy.

Objective To determine whether the extent and location of progressive parapapillary chorioretinal atrophy noted in some patients with ocular hypertension are correlated with the extent and location of visual field defects that occur with progression to glaucoma. Study design Retrospective cohort study. Participants Thirty patients with ocular hypertension who had progressive changes of parapapillary atrophy develop before clinically detectable optic disc or visual field damage. Main outcome measures Assessment of changes in the parapapillary atrophy and visual field parameters. Methods Baseline and follow-up optic disc photographs and visual field test results were retrospectively analyzed. The relationship between the extent of parapapillary atrophy observed during the ocular hypertension period and initial visual field abnormalities detected after glaucoma development, as well as their spatial relationship, was statistically analyzed. Results The extent of progressive changes of the parapapillary atrophy detected during the ocular hypertension period was correlated with the extent of changes in the visual field parameters, including corrected pattern standard deviation and mean deviation measured after glaucoma development (Mantel-Haenszel chi-square test, P = 0.026, P = 0.037, respectively). In addition, the visual field abnormalities occurred in the corresponding quadrants of the progressive parapapillary atrophy. Analysis of the spatial relationship revealed that the location of progressive changes of the parapapillary atrophy was concordant with the location of visual field abnormalities in 78% of the quadrants (94 of 120 quadrants) (chi-square test, P = 0.001). Conclusions The extent and location of visual field abnormalities that develop in ocular hypertensive eyes with progression to glaucoma exhibit a concordance with the extent and location of progressive parapapillary atrophy noted in the ocular hypertension period. This suggests the importance of detailed examination of the parapapillary area in ocular hypertensive eyes.

Although elevated titers of serum antibodies to hsp27 accompany human diseases such as cancer and glaucoma, evidence of their pathogenic effects is lacking. Here we present novel evidence that exogenously applied hsp27 antibody enters neuronal cells in human retina by an endocytic mechanism. Subsequent to internalization, hsp27 antibody facilitates apoptotic cell death as characterized by morphological assessment, DNA fragmentation, and the activation of cysteine aspartic acid proteases. In addition, we demonstrate that after internalization, hsp27 antibody is detected in discrete cytoplasmic and nuclear structures and colocalizes to actin cytoskeleton. Hsp27 antibody binding to actin results in depolymerization and proteolytic cleavage of actin in a dose-dependent manner. These results suggest that exogenous hsp27 antibody may induce neuronal apoptosis by inactivating or attenuating the ability of native hsp27 to stabilize actin cytoskeleton, thereby providing a novel mechanism by which autoantibodies to hsp27 may impair cell survival in selective human diseases.

Purpose. To count retrogradely labeled retinal ganglion cells by Fluoro-Gold. Methods. Retinal ganglion cells were retrogradely labeled using bilateral injections of Fluoro-Gold into the superior colliculus. One week after injections, retinas were dissociated and immunolabeled using specific antibody against Fluoro-Gold. The Fluoro-Gold labeled cells were then counted using flow cytometry. Results. Flow cytometry revealed that approximately 7% of the dissociated retinal cells were ganglion cells retrogradely labeled by Fluoro-Gold (Fig. 1B). Based on the total count of retinal cells per eye, the total number of retinal ganglion cells was estimated at approximately 131,250 ± 2,542 per rat eye. The coefficient of variation of counts was calculated as 1.98%. Conclusions. The use of flow cytometry facilitates simple, reproducible and rapid quantification of virtually all of the retinal ganglion cells labeled by Fluoro-Gold in a single rat eye.

To study effects of cell density on retinal cell survival.Apoptotic cell death was induced in cultured retinal cells seeded at higher or lower density by various stimuli including simulated ischemia, excitotoxicity and antibody against heat shock protein 27 (hsp27). Quantitative analysis of apoptotic cells was performed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling technique and flow cytometry. Cytoskeleton was examined using immunocytochemistry and specific staining of actin by phalloidin and DNase I. In addition, alterations in the cytoskeletal proteins, bcl-2 family of proteins and hsp27 were studied using western blotting.Incubation of the cells under apoptotic stimuli caused higher rates of apoptosis in lower density cultures as determined by TUNEL technique and flow cytometric analysis. Both morphologic examination of cytoskeleton and western blotting revealed that after incubation with various stressors, degradation of actin and tubulin was more prominent in lower density cultures compared to higher density cultures. The expression of bcl-2 and bcl-xL was higher and the expression of bax was lower in lower density cultures compared to higher density cultures at basal condition. After incubation with stressors, bcl-2 and bcl-xL expressions decreased and bax expression increased in both lower and higher density cultures. However, we observed that the expression of hsp27 was higher in higher density cultures than in lower density cultures in the presence or absence of apoptotic stimuli.These findings demonstrate that retinal cells are more resistant to apoptosis in higher density cultures, independent of the inducer. This might be partly due to protective activity of endogenous hsp27 in the cells at higher density, which contributes to cytoskeletal integrity in response to apoptotic stimuli.

Purpose To describe the clinical and laboratory findings in 10 patients with normal-pressure glaucoma and anti-Ro/SS-A positivity by enzymelinked immunosorbent assay (ELISA) and to determine whether that positivity may be related to an autoimmune mechanism for the optic neuropathy. Methods In this prospective study, we evaluated ocular and systemic clinical findings of 10 patients with normal-pressure glaucoma and anti-Ro/SS-A positivity by ELISA, including sicca complex features. Ouchterlony immunodiffusion was performed to confirm the presence of antibodies for Ro/SS-A, and the presence of other serum antibodies and their possible cross-reactivities with Ro/SS-A were investigated. Results None of the 10 patients with normalpressure glaucoma and anti-Ro/SS-A positivity (by ELISA) had clinical or laboratory signs of Sjogren syndrome or other connective tissue diseases. Only one of 10 patients had evidence of anti-Ro/SS-A antibodies by Ouchterlony immunodiffusion. All patients demonstrated serum immunoreactivity to bacterial heat shock protein 60 (hsp60) by Western blotting. Cross-reactivity between bacterial hsp60 and Ro/SS-A was demonstrated by Western blotting. Immunoreactivity to bacterial hsp60 by ELISA was significantly elevated in the sera of patients with normal-pressure glaucoma. Furthermore, patients with either normal-pressure or primary open-angle glaucoma had increased serum immunoreactivity to human hsp60. Conclusions Anti-Ro/SS-A positivity by ELISA in 10 patients with normal-pressure glaucoma was associated with a high level of serum immunoreactivity to bacterial hsp60, which may indicate that their glaucomatous optic neuropathy involves an as yet unidentified autoimmune mechanism. The identification of autoantibodies that react with human hsp60 in patients with normalpressure and primary open-angle glaucoma may signify a common finding associated with the glaucomatous optic neuropathy process in some patients and appears to be unrelated to intraocular pressure levels.

* BACKGROUND AND OBJECTIVES: The refinements of small-incision cataract surgery by phacoemulsification with foldable intraocular lens (IOL) implantation have recently permitted new options for combined trabeculectomy with cataract extraction. The objectives of this study were to compare the longterm intraocular pressure (IOP) control and visual outcome between trabeculectomies combined with extracapsular cataract extraction (ECCE) versus those with phacoemulsification, and to analyze these same factors for foldable versus rigid IOLs. * PATIENTS AND METHODS: The authors retrospectively reviewed the charts of 311 patients (397 eyes) who underwent combined trabeculectomy with cataract extraction and posterior chamber IOL implantation. In all of the surgeries, releasable scleral flap sutures were used. The mean follow-up was 22.9 ± 15.1 months, with a minimum follow-up of 12 months. * RESULTS: Trabeculectomy combined with phacoemulsification had a lower postoperative complication rate and was more effective than trabeculectomy combined with ECCE in reducing IOP to less than 20 mm Hg with or without medication (95% vs 82%) and in improving vision to levels of 20/40 or better (71% vs 52%) (P < .001). Regarding IOLs, foldable silicone lenses were found to be an effective alternative to polymethylmethacrylate lenses in combined surgeries in terms of a controlled IOP of less than 20 mm Hg (97% vs 97%) and visual recovery to 20/40 or better (78% vs 63%). * CONCLUSION: The combination of trabeculectomy with releasable scleral flap sutures and small incision cataract surgery with foldable IOL implantation has improved postoperative IOP control and visual rehabilitation. [Ophthalmic Surg Lasers 1997;28:539-550.]

* BACKGROUND AND OBJECTIVE: One of the variables to be considered in a combined procedure for glaucoma and cataract is the type of conjunctival flap to be used. The objective of this study was to compare the effects of limbus-based and fornix-based conjunctival flaps on postoperative long-term intraocular pressure (IOP) control and visual acuity after combined trabeculectomy with phacoemulsification. * PATIENTS AND METHODS: The authors retrospectively reviewed the charts of 189 patients (215 eyes) who underwent combined trabeculectomy with phacoemulsification, posterior chamber intraocular lens (IOL) implantation, and intraoperative mitomycin-C administration and who had a minimum follow-up of 12 months. The results of the limbus-based (151 eyes) versus fornix-based (64 eyes) conjunctival incisions used in these combined procedures were compared. * RESULTS: In the limbus-based conjunctival flap group, 146 eyes (97%) achieved an IOP of less than 20 mm Hg, with or without medication; 62 eyes (97%) of the fornix-based conjunctival flap group (P > .05) achieved this result. A visual acuity of 20/40 or better was noted in 106 eyes (70%) in the limbusbased conjunctival flap group and in 45 eyes (70%) in the fornix-based conjunctival flap group (P > .05) at the last examination. Early wound leakage was observed more frequently in the fornix-based conjunctival flap group (8% vs 1%) (P= .014); however, it was not a serious clinical problem, as only 1 eye required surgical repair. Posterior capsular opacification was found more often in the limbus-based conjunctival flap group (25% vs 14%) (P = .072) and required more frequent laser capsulotomy (22% vs 9%) (P= 0.03). * CONCLUSION: Limbus-based and fornix-based conjunctival flaps appear to be comparable with respect to postoperative IOP control and visual acuity after a combined trabeculectomy with phacoemulsification and posterior chamber IOL implantation in cases supplemented by intraoperative mitomycin-C. [Ophthalmic Surg Lasers 1997;28:551-557.]

Despite improving understanding of glaucoma, key molecular players of neurodegeneration that can be targeted for treatment of glaucoma, or molecular biomarkers that can be useful for clinical testing, remain unclear. Proteomics technology offers a powerful toolbox to accomplish these important goals of the glaucoma research and is increasingly being applied to identify molecular mechanisms and biomarkers of glaucoma. Recent studies of glaucoma using proteomics analysis techniques have resulted in the lists of differentially expressed proteins in human glaucoma and animal models. The global analysis of protein expression in glaucoma has been followed by cell-specific proteome analysis of retinal ganglion cells and astrocytes. The proteomics data have also guided targeted studies to identify post-translational modifications and protein–protein interactions during glaucomatous neurodegeneration. In addition, recent applications of proteomics have provided a number of potential biomarker candidates. Proteomics technology holds great promise to move glaucoma research forward toward new treatment strategies and biomarker discovery. By reviewing the major proteomics approaches and their applications in the field of glaucoma, this article highlights the power of proteomics in translational and clinical research related to glaucoma and also provides a framework for future research to functionally test the importance of specific molecular pathways and validate candidate biomarkers.

Purpose Besides primary neurotoxicity, oxidative stress may compromise the glial immune regulation and shift the immune homeostasis toward neurodegenerative inflammation in glaucoma. We tested this hypothesis through the analysis of neuroinflammatory and neurodegenerative outcomes in mouse glaucoma using two experimental paradigms of decreased or increased oxidative stress. Methods The first experimental paradigm tested the effects of Tempol, a multifunctional antioxidant, given through osmotic mini-pumps for drug delivery by constant infusion. Following a 6-week treatment period after microbead/viscoelastic injection-induced ocular hypertension, retina and optic nerve samples were analyzed for markers of oxidative stress and cytokine profiles using specific bioassays. We also analyzed a redox-sensitive transcriptional regulator of neuroinflammation, namely NF-κB. The second paradigm included a similar analysis of the effects of overloaded oxidative stress on retina and optic nerve inflammation in mice knockout for a major antioxidant enzyme (SOD1(-/-)). Results Increased antioxidant capacity and decreased protein carbonyls and HNE adducts with Tempol treatment verified the drug delivery and biological function. Among a range of cytokines measured, proinflammatory cytokines, including IL-1, IL-2, IFN-γ, and TNF-α, exhibited more than 2-fold decreased titers in Tempol-treated ocular hypertensive eyes. Antioxidant treatment also resulted in a prominent decrease in NF-κB activation in the ocular hypertensive retina and optic nerve. Although pharmacological treatment limiting the oxidative stress resulted in decreased neuroinflammation, ocular hypertension-induced neuroinflammatory responses were increased in SOD1(-/-) mice with defective antioxidant response. Conclusions These findings support the oxidative stress-related mechanisms of neuroinflammation and the potential of antioxidant treatment as an immunomodulation strategy for neuroprotection in glaucoma.

Purpose Evidence supporting the immune system involvement in glaucoma includes increased titers of serum antibodies to retina and optic nerve proteins, although their pathogenic importance remains unclear. This study using an antibody-based proteomics approach aimed to identify disease-related antigens as candidate biomarkers of glaucoma. Methods Serum samples were collected from 111 patients with primary open-angle glaucoma and an age-matched control group of 49 healthy subjects without glaucoma. For high-throughput characterization of antigens, serum IgG was eluted from five randomly selected glaucomatous samples and analyzed by linear ion trap mass spectrometry (LC-MS/MS). Serum titers of selected biomarker candidates were then measured by specific ELISAs in the whole sample pool (including an additional control group of diabetic retinopathy). Results LC-MS/MS analysis of IgG elutes revealed a complex panel of proteins, including those detectable only in glaucomatous samples. Interestingly, many of these antigens corresponded to upregulated retinal proteins previously identified in glaucomatous donors (or that exhibited increased methionine oxidation). Moreover, additional analysis detected a greater immunoreactivity of the patient sera to glaucomatous retinal proteins (or to oxidatively stressed cell culture proteins), thereby suggesting the importance of disease-related protein modifications in autoantibody production/reactivity. As a narrowing-down strategy for selection of initial biomarker candidates, we determined the serum proteins overlapping with the retinal proteins known to be up-regulated in glaucoma. Four of the selected 10 candidates (AIF, cyclic AMP-responsive element binding protein, ephrin type-A receptor, and huntingtin) exhibited higher ELISA titers in the glaucomatous sera. Conclusions A number of serum proteins identified by this immunoproteomic study of human glaucoma may represent diseased tissue-related antigens and serve as candidate biomarkers of glaucoma.

This study aimed to determine retinal proteomic alterations in human glaucoma, with particular focus on links to TNF-α/TNFR1 signaling.Human retinal protein samples were obtained from 20 donors with (n = 10) or without (n = 10) glaucoma. Alterations in protein expression were individually analyzed by quantitative LC-MS/MS. Quantitative Western blot analysis with cleavage or phosphorylation site-specific antibodies was used for data validation, and cellular localization of selected proteins was determined by immunohistochemical analysis of the retina in an additional group of glaucomatous human donor eyes (n = 38) and nonglaucomatous controls (n = 30).Upregulated retinal proteins in human glaucoma included a number of downstream adaptor/interacting proteins and protein kinases involved in TNF-α/TNFR1 signaling. Bioinformatic analysis of the high-throughput data established extended networks of diverse functional interactions with death-promoting and survival-promoting pathways and mediation of immune response. Upregulated pathways included death receptor-mediated caspase cascade, mitochondrial dysfunction, endoplasmic reticulum stress, calpains leading to apoptotic cell death, NF-κB and JAK/STAT pathways, and inflammasome-assembly mediating inflammation. Interestingly, retinal expression pattern of a regulator molecule, TNFAIP3, exhibited prominent variability between individual samples, and methylation of cytosine nucleotides in the TNFAIP3 promoter was found to be correlated with this variability among glaucomatous donors.Findings of this study reveal a number of proteins upregulated in the glaucomatous human retina that exhibit many links to TNF-α/TNFR1 signaling. By highlighting various signaling molecules and regulators involved in cell death and immune response pathways and by correlating proteomic findings with epigenetic alterations, these findings provide a framework motivating further research.

To determine the regulation of immune system activity associated with Toll-like receptor (TLR) signaling in glaucoma.Retinal protein samples obtained from human donor eyes with (n = 10) or without (n = 10) glaucoma were analyzed by a quantitative proteomic approach involving mass spectrometry. Cellular localization of TLR2, -3, and -4 was also determined by immunohistochemical analysis of an additional group of human donor eyes with glaucoma (n = 34) and control eyes (n = 20). In addition, in vitro experiments were performed in rat retinal microglia and astrocytes to determine glial TLR expression and immunoregulatory function after exposure to exogenous heat shock proteins (HSPs) and H(2)O(2)-induced oxidative stress.Proteomic analyses of the human retina detected expression and differential regulation of different TLRs in glaucomatous samples. Parallel to the upregulation of TLR signaling, proteomic findings were also consistent with a prominent increase in the expression of HSPs in glaucoma. Immunohistochemical analysis supported upregulated expression of TLRs on both microglia and astrocytes in the glaucomatous retina. In vitro experiments provided additional evidence that HSPs and oxidative stress upregulate glial TLR and MHC class II expression and cytokine production through TLR signaling and stimulate proliferation and cytokine secretion of co-cultured T cells during antigen presentation.The findings of this study support the upregulation of TLR signaling in human glaucoma, which may be associated with innate and adaptive immune responses. In vitro findings showed that components of glaucomatous tissue stress, including upregulated HSPs and oxidative stress, may initiate the immunostimulatory signaling through glial TLRs.

Clinical and experimental studies over the past decade highlight the involvement of the immune system in glaucomatous neurodegeneration. Different components, including both innate and adaptive immunity, exhibit prominent activity in glaucoma.1–3 Despite the fact that immune activity is a necessary intrinsic response to promote the tissue cleaning, healing, and regeneration process, if there is a failure in the immune system regulation because of increasing risk factors, initially beneficial immune activity may turn into an autoimmune injury process. In addition to the potential cytotoxicity of autoreactive T cells4 and autoantibodies,5 present evidence suggests that uncontrolled complement activation may also contribute to the progression of degenerative injury to retinal ganglion cells (RGCs), their synapses, and axons in glaucoma. Recent histopathologic studies of human tissues and in vivo studies using different animal models have demonstrated that complement components, including C1q and C3, are synthesized and terminal complement complex is formed in the glaucomatous retina.6,7 Findings of another study using mice deficient in complement components C1q and C3 have also provided evidence to suggest that the classical complement cascade may be involved in synapse elimination during neurodegenerative injury.8 These findings support that injured RGCs in glaucoma may be similarly targeted and destroyed through complement-mediated processes involving reactive glia. This study aimed to further explore complement activation in glaucoma by focusing particularly on proteomic and immunohistochemical findings in human donor eyes. In addition, based on potential immunostimulatory consequences of oxidative stress in glaucoma,3 including the recently identified regulatory roles of oxidative stress in T-cell–mediated immunity,9 this study aimed to determine whether oxidative stress may be involved in the regulation of complement activation in glaucoma. Therefore, we also performed in vitro experiments using primary cultures of retinal cells in the presence and absence of oxidative stress. Findings of these studies collectively support complement activation in the glaucomatous human retina. In addition to the classical pathway, the lectin pathway is likely involved in complement activation during glaucomatous neurodegeneration. By targeting and removing the toxic debris from dying neurons in glaucoma, complement activation may participate in tissue healing and may minimize inflammatory insults. However, a potential deficiency in the intrinsic regulation of complement activation, as is evident in the presence of oxidative stress, may facilitate the progression of neurodegenerative injury by collateral cell lysis, inflammation, and autoimmunity.

Author(s): Tezel, Gulgun; Fourth ARVO/Pfizer Ophthalmics Research Institute Conference Working Group

Glaucomatous neurodegeneration has been associated with the activation of multiple pathogenic mechanisms that can result in RGC death and axonal degeneration. Growing evidence obtained from clinical and experimental studies over the last decade also strongly suggests the involvement of the immune system in the neurodegenerative process of glaucoma. The roles of the immune system in glaucoma have been described as either neuroprotective or neurodestructive. It has been proposed that a critical balance between beneficial protective immunity and harmful sequelae of autoimmune neurodegenerative injury determines the ultimate fate of RGCs in response to various stressors in patients with glaucoma. Here, we review the key role for immunoregulation in cell fate decisions regarding RGC survival in response to glaucomatous tissue stress. Furthermore, we review the mechanisms by which autoimmunity to specific antigens such as heat shock proteins may result in RGC demise in some patients with glaucoma. In these patients, we hypothesized that one form of glaucoma may be an autoimmune optic neuropathy in which an individual's immune system facilitates a somatic or axonal degeneration of RGCs by the very system which normally serves to protect it against stress.

Glaucoma is a chronic neurodegenerative disease of the optic nerve, in which apoptosis of retinal ganglion cells (RGCs) and progressive loss of optic nerve axons result in structural and functional deficits in glaucoma patients. This neurodegenerative disease is indeed a leading cause of blindness in the world. The glaucomatous neurodegenerative environment has been associated with the activation of multiple pathogenic mechanisms for RGC death and axon degeneration. Growing evidence obtained from clinical and experimental studies over the last decade also strongly suggests the involvement of the immune system in this neurodegenerative process. Paradoxically, the roles of the immune system in glaucoma have been described as either neuroprotective or neurodestructive. A balance between beneficial immunity and harmful autoimmune neurodegeneration may ultimately determine the fate of RGCs in response to various stressors in glaucomatous eyes. Based on clinical data in humans, it has been proposed that one form of glaucoma may be an autoimmune neuropathy, in which an individual's immune response facilitates a somatic and/or axonal degeneration of RGCs by the very system which normally serves to protect it against tissue stress.

TNF-alpha has recently been identified to be a mediator of retinal ganglion cell (RGC) death, while glial cells are relatively protected against this death stimulus. To identify molecular mechanisms that control diverse responses of RGCs and glial cells to TNF-alpha, we studied differential gene expression between primary cultures of RGCs and glial cells exposed to TNF-alpha using cDNA array analysis of MAPK and NF-kappaB signaling pathways. Findings of this comparative analysis demonstrated differential regulation of various genes between RGCs and glial cells exposed to TNF-alpha. RT-PCR confirmed the differential expression of selected genes, and immunocytochemistry demonstrated gene products in cultured cells. Immunolabeling with phosphorylation site-specific antibodies also revealed differential post-translational modifications of selected proteins between cell types. Identification of signaling molecules differentially regulated in RGCs and glial cells can improve our understanding of the diverse cellular responses and provide targets for neuroprotective interventions in several neurodegenerative conditions.

Compelling evidence obtained from studies over the last decade strongly suggests the involvement of immune system regulation in cell fate decisions in glia and retinal ganglion cells that lead to glaucomatous optic nerve degeneration.Recent studies reveal seemingly conflicting roles of the immune system in glaucomatous optic nerve degeneration. T cells directed against specific antigens may have a beneficial effect to protect neurons from the consequences of axonal injury. However, the immune response in glaucoma also has the capacity to cause neuronal injury. The balance between the benefit of protective immunity and the risk of inducing an autoimmune neurodegenerative disease is critical. The immunoregulatory function of glial cells and the presence of tissue stress seem to be important factors that determine the balance between diverse roles of the immune system in glaucomatous optic nerve degeneration. Thus, the net effect of immunoregulation may be either neuroprotective or neurodestructive.Despite the neuroprotective features of the immune system, an autoimmune component, resulting from a failure to properly control aberrant, stress-induced immune response, likely accompanies the progression of neurodegeneration in glaucoma in some patients. A better understanding of the diverse roles of the immune system in all forms of glaucomatous optic nerve degeneration will facilitate the development of effective neuroprotective strategies in glaucoma. The basis of a sustainable neuroprotective strategy is to harness immunoregulation of glial and retinal ganglion cell fate to maximize beneficial effects, while minimizing negative sequelae, for therapeutic gain.