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Purpose The purpose of this study was to examine the expression of glial derived neurotrophic factor (GDNF), the GDNF receptors GFRα1 and GFRα2, ciliary neurotrophic factor (CNTF), and the CNTF receptor CNTFRα in normal and glaucomatous human tissue. Methods Human retinas were collected from 8 donors that had been clinically diagnosed and treated for glaucoma, and also from 9 healthy control donors. Immunohistochemical analysis for each trophic factor and receptor was performed. The percent of each retinal section labeled with each antibody was quantified for the total retinal thickness, and separately for the retinal ganglion cell (RGC) complex + retinal nerve fiber layer (RNFL). The expression of each protein was correlated with measures of the subject's ocular histories. Results The percentage area immunopositive for GFRα2 was significantly decreased in the total retinal thickness containing all retinal layers and in the combined RGC complex + RNFL in glaucomatous eyes in both the peripapillary region and more peripheral retinal locations. We also observed a decrease in GFRα1 expression in the peripapillary RGC Complex + RNFL in glaucoma patients compared to healthy control patients. We also observed a relationship between GDNF and its receptors with several outcomes obtained from the medical record. No differences in CNTF or CNTFR labeling were observed. Conclusion Decreases in GDNF receptor expression in glaucomatous tissue may limit the potential for neuroprotective therapy by supplementation with GDNF.

To examine the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor, tropomyosin-related kinase receptor-B (TrkB), in normal and glaucomatous human retinas.Human retinas were collected from 8 donors who had been clinically diagnosed and treated for glaucoma, and from 9 control donors. Immunohistochemical analysis for BDNF and TrkB was performed. The percent of each retina expressing BDNF and TrkB was quantified for the total retinal thickness, and separately for the retinal ganglion cell (RGC) complex + retinal nerve fiber layer (RNFL). The expression of each protein was correlated with clinical outcomes obtained from the subject's ocular histories.There was no significant difference in BDNF or TrkB expression when comparing glaucomatous and control retinas. Correlation analysis revealed a significant relationship between BDNF expression and the use of prostaglandin analogs. TrkB expression was highly correlated with the last-measured intraocular pressure (IOP), the use of carbonic anhydrase inhibitors, the use of beta blockers, and the total number of drugs used for the treatment of glaucoma.Topical drugs used to treat glaucoma were associated with an increase in retinal BDNF and TrkB expression in human retina, independent of IOP, which may represent molecular evidence of neuroprotective pathway activation.

Neuroinflammation significantly contributes to the pathophysiology of several neurodegenerative diseases. This is also the case in glaucoma and may be a reason why many patients suffer from progressive vision loss despite maximal reduction in intraocular pressure. Pioglitazone is an agonist of the peroxisome proliferator-activated receptor gamma (PPARγ) whose pleiotrophic activities include modulation of cellular energy metabolism and reduction in inflammation. In this study we employed the DBA2/J mouse model of glaucoma with chronically elevated intraocular pressure to investigate whether oral low-dose pioglitazone treatment preserves retinal ganglion cell (RGC) survival. We then used an inducible glaucoma model in C57BL/6J mice to determine visual function, pattern electroretinographs, and tracking of optokinetic reflex. Our findings demonstrate that pioglitazone treatment does significantly protect RGCs and prevents axonal degeneration in the glaucomatous retina. Furthermore, treatment preserves and partially reverses vision loss in spite of continuously elevated intraocular pressure. These data suggest that pioglitazone may provide treatment benefits for those glaucoma patients experiencing continued vision loss.

Glaucoma is a multifactorial disease resulting in progressive vision loss due to retinal ganglion cell (RGC) dysfunction and death. Early events in the pathobiology of the disease include oxidative, metabolic, or mechanical stress that acts upon RGC, causing these to rapidly release danger signals, including extracellular ATP, resulting in micro- and macroglial activation and neuroinflammation. Danger signaling also leads to the formation of inflammasomes in the retina that enable maturation of proinflammatory cytokines such IL-1β and IL-18. Chronic neuroinflammation can have directly damaging effects on RGC, but it also creates a proinflammatory environment and compromises the immune privilege of the retina. In particular, continuous synthesis of proinflammatory mediators such as TNFα, IL-1β, and anaphylatoxins weakens the blood–retina barrier and recruits or activates T-cells. Recent data have demonstrated that adaptive immune responses strongly exacerbate RGC loss in animal models of the disease as T-cells appear to target heat shock proteins displayed on the surface of stressed RGC to cause their apoptotic death. It is possible that dysregulation of these immune responses contributes to the continued loss of RGC in some patients.

Glaucoma is a leading cause of irreversible blindness worldwide, and increased intraocular pressure (IOP) is a major risk factor. We aimed to determine if early functional and molecular differences in the glaucomatous retina manifest before significant retinal ganglion cell (RGC) loss is apparent. Adenoviral vectors expressing a pathogenic form of myocilin (Ad5.MYOC) were used to induce IOP elevation in C57BL/6 mice. IOP and pattern electroretinograms (pERG) were recorded, and retinas were prepared for RNA sequencing, immunohistochemistry, or to determine RGC loss. Ocular injection of Ad5.MYOC leads to reliable IOP elevation, resulting in significant loss of RGC after nine weeks. A significant decrease in the pERG amplitude was evident in eyes three weeks after IOP elevation. Retinal gene expression analysis revealed increased expression for 291 genes related to complement cascade, inflammation, and antigen presentation in hypertensive eyes. Decreased expression was found for 378 genes associated with the γ-aminobutyric acid (GABA)ergic and glutamatergic systems and axon guidance. These data suggest that early functional changes in RGC might be due to reduced GABAA receptor signaling and neuroinflammation that precedes RGC loss in this glaucoma model. These initial changes may offer new targets for early detection of glaucoma and the development of new interventions.

The cells residing in the trabecular meshwork (TM) fulfill important roles in the maintenance of the tissue and the regulation of intraocular pressure (IOP). Here we examine (i) TM cell distribution along the circumference of the human eye, (ii) differences in TM cell density between regions of high and low outflow, and (iii) whether TM cell distribution in eyes from donors with primary open angle glaucoma (POAG) differs from that of normal eyes. Toward this end, the TM cell density from 12 radial segments around the circumference of the TM of human donor eyes (n = 6) with and without POAG was determined using histochemical methods. Areas of high, median, and low outflow were mapped in a different set of human donor eyes that were perfused in organ culture, and TM cell densities in these areas were determined in normal (n = 11) and POAG eyes (n = 6). Our analysis of 1380 tissue sections taken from the first set of six eyes shows that the average TM cell density of these six eyes ranges from 15.5 to 23.7 cells/100 μm and is negatively correlated to the maximum IOP recorded for each donor eye (R2 = 0.91). Considerable differences in TM cell density exist among sections taken from the same segment of an individual eye (average standard deviation = 2.35 cells/100 μm). Less variability is observed among the segment averages across the eye's circumference (average standard deviation = 1.03 cells/100 μm). Variations in cell density are similar between normal and POAG eyes and are not correlated with the anatomic position of examined segments (p = 0.745). The analysis of the second set of eyes shows that TM regions of high outflow display a TM cell density similar to regions of median or low outflow in both normal and POAG eyes. Together these findings demonstrate that (i) statistically significant differences in TM cell density exist along the circumference of each eye (ii) TM cellularity is not correlated with segmental flow and (iii) eyes with POAG, while displaying reduced TM cellularity, do not exhibit higher TM cell variability than normal eyes. Finally, statistical analysis of sections and segments indicates that measurements from 12 sections taken from 2 segments provide a reliable and cost-effective estimate of a human eye's TM cell density.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

OBJECTIVE: Mutations in myocilin (MYOC) may cause either juvenile open angle glaucoma (JOAG) or adult-onset primary open angle glaucoma (POAG). MYOC encodes a glycoprotein that is normally secreted from trabecular meshwork cells that regulate intraocular pressure. Prior in vitro, transgenic rodent, and organ culture experiments have suggested that abnormal accumulation of MYOC protein within trabecular meshwork cells is a key step in glaucoma pathophysiology. We investigated the pathogenesis of MYOC glaucoma by examining a donor eye from a patient with JOAG caused by a Tyr437His MYOC mutation. DESIGN: Case-control, immunohistochemical study of a donor eye from a patient with JOAG caused by a Tyr437His MYOC mutation and age-matched control donor eyes. SUBJECTS: An eye from a 59-year-old male with JOAG caused by a Tyr437His MYOC mutation and eyes from five donors (ages 51–66) with no known ocular disease were examined. METHODS: Frozen fixed sections of the iridocorneal angle were prepared from the donor eyes of the MYOC glaucoma patient and control eyes. We used antibodies directed against MYOC, collagen IV, and BiP/GRP78 as well as wheat germ agglutinin and concanavalin A lectins to localize MYOC protein in the trabecular meshwork. MAIN OUTCOME MEASURE: Qualitative comparison of MYOC protein labeling and localization in the trabecular meshwork of donor eyes from a glaucoma patient with a MYOC mutation and from control subjects. RESULTS: Using immunohistochemistry, we detected more abundant MYOC protein within the trabecular meshwork of the MYOC glaucoma patient’s eye than in control eyes. We further localized MYOC protein within the trabecular meshwork cells of the MYOC glaucoma patient’s eye by co-labeling with the endoplasmic reticulum (ER) marker GRP78 (BiP). Little to no MYOC was identified within the trabecular meshwork cells of control eyes. Minimal extracellular MYOC was detected in both MYOC glaucoma eyes and control eyes. CONCLUSIONS: This is the first histopathological analysis of an eye from a glaucoma patient with a MYOC mutation. Furthermore, this analysis supports our model of MYOC-associated glaucoma, in which MYOC mutations cause abnormal intracellular retention of MYOC within the ER of trabecular meshwork cells as a key step towards development of glaucoma.

Purpose Decreased trabecular meshwork (TM) cellularity has been implicated as a major reason for TM dysfunction and aqueous humor (AH) outflow abnormalities in primary open angle glaucoma. We previously found that transplantation of induced pluripotent stem cell (iPSC)-derived TM cells can restore TM function and stimulate endogenous TM cell division. The goal of the present study is to investigate whether signaling via gap junctions is involved in this process. Methods Differentiated iPSCs were characterized morphologically, transcriptionally, and immunohistochemically. After purification, iPSC-TM were co-cultured with mouse TM (MTM) cells to mimic the transplantation procedure. Through the pharmacological antagonists and short hairpin RNA (shRNA) technique, the gap junction function in iPSC-based therapy was determined. Results In the co-culture system, iPSC-TM increase MTM cell division as well as transfer of Ca2+ to MTM. This effect was blocked by treatment with the gap junction inhibitors carbenoxolone (CBX) or flufenamic acid (FFA). The shRNA mediated knock down of connexin 43 (Cx43) expression in iPSC-TM also results in decreased Ca2+ transfer and lower MTM proliferation rates. In vivo, Cx43 downregulation in transplanted iPSC-TM weakened their regenerative role in an Ad5.myocilinY437H mouse model of glaucoma. Mice receiving these cells exhibited lower TM cellularity and higher intraocular pressure (IOP) than those receiving unmodified iPSC-TM. Conclusions Our findings reveal a crucial role of gap junction, especially Cx43, in iPSC-based TM regeneration, and provides insights to enhance the regenerative effect of iPSCs in glaucoma therapy.

Mounting evidence suggests neuroinflammation is a key process in glaucoma, yet the precise roles are not known. Understanding these complex processes, which may also be a key in other common neurodegenerations such as Alzheimer’s disease, will lead to targeted therapeutics for a disease that affects as many as 80 million people worldwide. Here, we define neuroinflammation as any immune-relevant response by a variety of cell types including astrocytes, microglia, and peripherally derived cells occurring in the optic nerve head and/or retina. In this review article, we first discuss clinical evidence for neuroinflammation in glaucoma and define neuroinflammation in glaucoma. We then review the inflammatory pathways that have been associated with glaucoma. Finally, we set out key research directions that we believe will greatly advance our understanding of the role of neuroinflammation in glaucoma. This review arose from a discussion of neuroinflammation in glaucoma at the 2015 meeting of the The Lasker/IRRF Initiative for Innovation in Vision Science. This manuscript sets out to summarize one of these sessions; “Inflammation and Glaucomatous Neurodegeneration”, as well as to review the current state of the literature surrounding neuroinflammation in glaucoma.

Purpose We previously demonstrated that passive transfer of lymphocytes from glaucomatous mice induces retinal ganglion cell (RGC) damage in recipient animals, suggesting a role for immune responses in the multifactorial pathophysiology of glaucoma. Here we evaluate whether absence of an adaptive immune response reduces RGC loss in glaucoma. Methods Elevated intraocular pressure (IOP) was induced in one eye of C57BL/6J (B6) or T- and B-cell-deficient Rag1-/- knockout mice. After 16 weeks RGC density was determined in both the induced and the normotensive contralateral eyes. Data were compared to mice having received injections of "empty" vector (controls). The number of extravascular CD3+ cells in the retinas was determined using FACS. Results Retinas of eyes with elevated IOP contain significantly more extravasated CD3+ cells than control retinas (46.0 vs. 27.1, P = 0.025). After 16 weeks of elevated IOP the average RGC density in B6 mice decreased by 20.7% (P = 1.9 × 10-4). In contrast, RGC loss in Rag1-/- eyes with elevated IOP was significantly lower (10.3%, P = 0.006 vs. B6). RGC loss was also observed in the contralateral eyes of B6 mice, despite the absence of elevated IOP in those eyes (10.1%; P = 0.008). In RAG1-/- loss in the contralateral eyes was minimal (3.1%) and significantly below that detected in B6 (P = 0.02). Conclusions Our findings demonstrate that T Rag1-/- mice are significantly protected from glaucomatous RGC loss. In this model, lymphocyte activity contributes to approximately half of all RGC loss in eyes with elevated IOP and to essentially all loss observed in normotensive contralateral eyes.

Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

PURPOSE. While nitric oxide (NO) donors are emerging as treatments for glaucoma, the mechanism by which NO lowers intraocular pressure (IOP) is unclear. NO activates the enzyme guanylyl cyclase (GC) to produce cyclic guanosine monophosphate. We studied the ocular effects of inhaled and topically applied NO gas in mice and lambs, respectively. METHODS. IOP and aqueous humor (AqH) outflow were measured in WT and GC-1 alpha subunit null (GC-1-/-) mice. Mice breathed 40 parts per million (ppm) NO in O-2 or control gas (N-2/O-2). We also studied the effect of ocular NO gas exposure (80, 250, 500, and 1000 ppm) on IOP in anesthetized lambs. NO metabolites were measured in AqH and plasma. RESULTS. In awake WT mice, breathing NO for 40 minutes lowered IOP from 14.4 1.9 mm Hg to 10.9 1.0 mm Hg (n = 11, P < 0.001). Comparable results were obtained in anesthetized WT mice (n = 10, P < 0.001). In awake or anesthetized GC-1(-/-)mice, IOP did not change under similar experimental conditions (P >= 0.08, n = 20). Breathing NO increased in vivo outflow facility in WT but not GC-1(-/-)mice (+13.7 14.% vs. -12.1 9.4%, n = 4 each, P < 0.05). In lambs, ocular exposure to NO lowered IOP in a dose-dependent manner (-0.43 mm Hg/ppm NO; n = 5 with 40 total measurements; P = 0.04) without producing corneal pathology or altering pulmonary and systemic hemodynamics. After ocular NO exposure, NO metabolites were increased in AqH (n = 8, P < 0.001) but not in plasma. CONCLUSIONS. Breathing NO reduced IOP and increased outflow facility in a GC-dependent manner in mice. Exposure of ovine eyes to NO lowers IOP.

Medical treatment of glaucoma relies on intraocular pressure (IOP)-lowering medications, typically administered daily by the patient. While these medications are effective when applied correctly, patient adherence is a major obstacle in glaucoma treatment. We have developed a sustained-release formulation of timolol maleate that can be injected subconjunctivally to avoid patient noncompliance.A biodegradable microsphere formulation for timolol maleate was injected subconjunctivally in normal rabbits. We measured timolol levels in tears, aqueous humor, vitreous humor, and serum of study rabbits. Furthermore, IOP profiles were recorded longitudinally. Tissue compatibility and side effects were evaluated using histochemistry.The microsphere formulation led to measureable amounts of timolol in the aqueous humor and the tear film for up to 90 days. Timolol was not detectable in the serum at any time. A significant reduction of IOP was observed in treated eyes. Clinically, the subconjunctival administration of the microspheres was well tolerated with no signs of inflammation or infection. The absence of local inflammation was confirmed by histology.A single subconjunctival administration of timolol microspheres achieved delivery and IOP reduction in rabbits for up to 90 days without local or systemic inflammation or toxicity. This approach has the potential to improve the management of glaucoma in patient populations, who are challenged to adhere to a regimen of daily eye drops.

Glaucoma is a common cause of vision loss or blindness and reduction of intraocular pressure (IOP) has been proven beneficial in a large fraction of glaucoma patients. The IOP is maintained by the trabecular meshwork (TM) and the elevation of IOP in open-angle glaucoma is associated with dysfunction and loss of the postmitotic cells residing within this tissue. To determine if IOP control can be maintained by replacing lost TM cells, we transplanted TM-like cells derived from induced pluripotent stem cells into the anterior chamber of a transgenic mouse model of glaucoma. Transplantation led to significantly reduced IOP and improved aqueous humor outflow facility, which was sustained for at least 9 wk. The ability to maintain normal IOP engendered survival of retinal ganglion cells, whose loss is ultimately the cause for reduced vision in glaucoma. In vivo and in vitro analyses demonstrated higher TM cellularity in treated mice compared with littermate controls and indicated that this increase is primarily because of a proliferative response of endogenous TM cells. Thus, our study provides in vivo demonstration that regeneration of the glaucomatous TM is possible and points toward novel approaches in the treatment of this disease.

The glaucomas are a group of diseases characterized by optic nerve damage that together represent a leading cause of blindness in the human population and in domestic animals. Here we report a mutation in LTBP2 that causes primary congenital glaucoma (PCG) in domestic cats. We identified a spontaneous form of PCG in cats and established a breeding colony segregating for PCG consistent with fully penetrant, autosomal recessive inheritance of the trait. Elevated intraocular pressure, globe enlargement and elongated ciliary processes were consistently observed in all affected cats by 8 weeks of age. Varying degrees of optic nerve damage resulted by 6 months of age. Although subtle lens zonular instability was a common feature in this cohort, pronounced ectopia lentis was identified in less than 10% of cats examined. Thus, glaucoma in this pedigree is attributed to histologically confirmed arrest in the early post-natal development of the aqueous humor outflow pathways in the anterior segment of the eyes of affected animals. Using a candidate gene approach, significant linkage was established on cat chromosome B3 (LOD 18.38, θ = 0.00) using tightly linked short tandem repeat (STR) loci to the candidate gene, LTBP2. A 4 base-pair insertion was identified in exon 8 of LTBP2 in affected individuals that generates a frame shift that completely alters the downstream open reading frame and eliminates functional domains. Thus, we describe the first spontaneous and highly penetrant non-rodent model of PCG identifying a valuable animal model for primary glaucoma that closely resembles the human disease, providing valuable insights into mechanisms underlying the disease and a valuable animal model for testing therapies.

Objective To determine the extent of fluctuation in circadian intraocular pressure (IOP) and the efficacy of topical dorzolamide 2% q 8 h in lowering IOP and blunting circadian fluctuation in IOP in glaucomatous cats. Animals studied Seven adult cats with primary congenital glaucoma (PCG). Procedures Measurements of IOP and pupil diameter were obtained for both eyes (OU) of each cat q 4 h for 12 days. Cats were housed in a laboratory animal facility with a 12-h light:dark cycle. Baseline values were established for 2 days. For the next 5 days, placebo (1.4% polyvinyl alcohol) was administered OU q 8 h. Dorzolamide 2% was then administered OU q 8 h for a further 5 days. A multivariate mixed linear model was fitted to the data, with parameters estimated from a Bayesian perspective. The 4 am time point was selected as the reference for the purposes of comparisons. Results Estimated mean IOP for the reference time point pre-treatment was symmetric (about 33 mmHg OU). In all cats, IOP was significantly lower during the diurnal phase, relative to the 4 am measurements, with highest IOP observed 2–6 h after the onset of the dark phase. Circadian fluctuations in IOP were dampened during the treatment period. There was a significant decrease in IOP in all cats during the dorzolamide treatment period (estimated mean for the treatment period reference = 17.9 mmHg OU). Conclusions Topical dorzolamide 2% q 8 h is effective in reducing IOP and IOP fluctuation in cats with PCG.

Reduction of intraocular pressure (IOP) by pharmaceutical or surgical means has long been the standard treatment for glaucoma. A number of excellent drugs are available that are effective in reducing IOP. These drugs are typically applied as eye drops. However, patient adherence can be poor, thus reducing the clinical efficacy of the drugs. Several novel delivery systems designed to address the issue of adherence and to ensure consistent reduction of IOP are currently under development. These delivery systems include contact lenses-releasing glaucoma medications, injectables such as biodegradable micro- and nanoparticles, and surgically implanted systems. These new technologies are aimed at increasing clinical efficacy by offering multiple delivery options and are capable of managing IOP for several months. There is also a desire to have complementary neuroprotective approaches for those who continue to show progression, despite IOP reduction. Many potential neuroprotective agents are not suitable for traditional oral or drop formulations. Their potential is dependent on developing suitable delivery systems that can provide the drugs in a sustained, local manner to the retina and optic nerve. Drug delivery systems have the potential to improve patient adherence, reduce side effects, increase efficacy, and ultimately, preserve sight for glaucoma patients. In this review, we discuss benefits and limitations of the current systems of delivery and application, as well as those on the horizon.

Data along several lines of evidence have suggested that a systemic autoimmune response may be provoked in glaucoma and could contribute to retinal ganglion cell (RGC) loss. If such an autoimmune response exists one could predict that in cases of unilateral glaucoma, autoantibodies generated would affect both eyes, leading to damage in the unaffected, contralateral, eye in an intraocular pressure (IOP) independent fashion. However, such an effect has not yet been reported. There are currently no data to reconcile these contrasting observations but a review of the literature suggests a possible explanation.

Introduction Several studies have indicated that autoimmune and neuroinflammatory processes contribute to the neurodegeneration of retinal ganglion cells in human glaucoma patients and in animal models. To test the involvement of cellular immune processes in the pathophysiology of retinal ganglion cell degeneration in vivo, we carried out adoptive transfer experiments from two independent genetic mouse models of glaucoma into normal recipient mice. Results Our findings indicate that transfer results in a progressive loss of retinal ganglion cells and their axons despite normal intraocular pressure in recipient mice. Signs of pan-retinal inflammation were not detected. Similar findings were obtained following transfer of isolated T-lymphocytes, but not after transfer of splenocytes from immune deficient glaucomatous mice. Transferred lymphocytes were detected integrated in the spleen and in the retinal ganglion cell layer of recipient animals, albeit at very low frequencies. Furthermore, we observed cell-cell interaction between transferred T-cells and recipient microglia along with focal microglial activation in recipient eyes. Conclusion This study demonstrates that the pathophysiology of glaucomatous degeneration in the tested animal models includes T-cell mediated events that are capable of causing loss of healthy retinal ganglion cells. Electronic supplementary material The online version of this article (doi:10.1186/s40478-015-0234-y) contains supplementary material, which is available to authorized users.

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.

Primary Angle Closure Glaucoma (PACG) is one of the most common types of glaucoma affecting over 15 million individuals worldwide. Family history and ethnicity are strongly associated with the development of the disease, suggesting that one or more genetic factors contribute to PACG. Although strictly heritable disease-causing mutations have not been identified, a number of recent association studies have pointed out genetic factors that appear to contribute to an individual's risk to develop PACG. In addition, genetic factors have been identified that modify PACG endophenotypes for example, axial length. Herein we review the current literature on this important topic.

Various cellular and molecular mechanisms that may lead to apoptotic cell death of retinal ganglion cells in glaucoma are discussed. These cellular mechanisms include neurotrophic factor deprivation, ischemia, glial cell activation, glutamate excitotoxicity, and abnormal immune response. Based on experimental and clinical evidence, the rationale for various neuroprotective strategies is described.

Purpose Primary open-angle glaucoma (POAG) is particularly common in older individuals and associated with pathologic degeneration of the trabecular meshwork (TM). We have shown previously that transplantation of induced pluripotent stem cell (iPSC) derived TM cells restores aqueous humor dynamics in young transgenic mice expressing a pathogenic form of human myocilin (Tg-MYOCY437H). This study was designed to determine if this approach is feasible in older mice with more pronounced TM dysfunction. Methods Mouse iPSC were differentiated toward a TM cell phenotype (iPSC-TM) and injected into the anterior chamber of 6-month-old Tg-MYOCY437H or control mice. IOP and aqueous humor outflow facility were recorded for up to 3 months. Transmission electron microscopy, Western blot, and immunohistochemistry were performed to analyze TM morphology, quantify endoplasmic reticulum (ER) stress, and assess TM cellularity. Results A 12 weeks after transplantation, IOP in iPSC-TM recipients was statistically lower and outflow facility was significantly improved compared to untreated controls. The number of endogenous TM cells increased significantly in iPSC-TM recipients along with the appearance of TM cells immmunopositive for a marker of cellular division. Morphologically, transplantation of iPSC-TM preserves ER structure 12 weeks after transplantation. However, myocilin and calnexin expression levels remain elevated in transplanted eyes of these 9-month-old Tg-MYOCY437H mice, indicating that ER stress persists within the TM. Conclusions Transplantation of iPSC-TM can restore IOP and outflow facility in aged Tg-MYOCY437H mice. This type of stem cell-based therapy is a promising possibility for restoration of IOP control in some glaucoma patients.

Purpose Loss or dysfunction of trabecular meshwork (TM) cells has been associated with the development of pathologically elevated IOP, and it is conceivable that replacement of damaged TM cells could restore function to the TM. We propose that the use of TM-like cells derived from induced pluripotent stem cells (iPSCs) created from a patient's own dermal fibroblasts offers the best solution to this challenge. Here we demonstrate that mouse iPSCs can be induced to differentiate into TM-like cells suitable for autologous transplantation. Methods Directed induction of stem cell differentiation was achieved through coculture of mouse iPSCs with human TM cells for up to 21 days. The resultant TM-like cells (iPSC-TM) were characterized morphologically, immunohistochemically, and functionally. Results The iPSC-TM cells closely resembled cultured human TM cells morphologically and began to express many markers of TM cells while ceasing to express pluripotency markers such as Nanog, Oct4, and Sox2. Functionally, these cells developed the ability to phagocytose particles. Finally, exposure to dexamethasone or phorbol 12-myristate acetate caused a distinct increase in the production and secretion of myocilin and matrix metalloproteinase-3, respectively, behavior characteristic of TM cells. Conclusions Our data demonstrate that iPSCs can be induced to assume a phenotype that resembles native TM cells in many important aspects. Not only do these cells represent a valuable research tool, but transplantation into glaucomatous eyes with elevated IOP may also restore function to the TM, resulting in re-establishment of IOP.

Glaucoma and age-related macular degeneration (AMD) are the two leading causes of visual loss in the United States. We utilized a novel study design to perform a genome-wide association for both primary open angle glaucoma (POAG) and AMD. This study design utilized a two-stage process for hypothesis generation and validation, in which each disease cohort was utilized as a control for the other. A total of 400 POAG patients and 400 AMD patients were ascertained and genotyped at 500,000 loci. This study identified a novel association of complement component 7 (C7) to POAG. Additionally, an association of central corneal thickness, a known risk factor for POAG, was found to be associated with ribophorin II (RPN2). Linked monogenic loci for POAG and AMD were also evaluated for evidence of association, none of which were found to be significantly associated. However, several yielded putative associations requiring validation. Our data suggest that POAG is more genetically complex than AMD, with no common risk alleles of large effect.

PURPOSE. The degeneration of retinal ganglion cells (RGC) in the glaucomatous retina is accompanied by activation of the classical complement cascade. The purpose of this study was to evaluate whether complement component C1q binding and activation of the complement cascade in the glaucomatous retina requires the presence of immunoglobulins. METHODS. Experimental glaucoma was induced in normal mice and those carrying a targeted deletion of the RAG1 gene. Binding of C1q to RGC and accumulation of C3 and C5b-9 was investigated using immunohistochemical and proteomic approaches. Damage to the optic nerve and RGC was determined and compared between the two strains. Complement activation and accumulation were also evaluated in vitro using dissociated retinal cell cultures. RESULTS. C1q was detected in the RGC layer in both RAG1 � /� and control mice with elevated IOP, but not in mice with normal IOP. Proteomic analysis of retinal membrane fractions indicated that C1q and C3 are membrane bound to a similar degree in RAG1 � /� and control mice with elevated IOP. The absence of Ig does not affect the rate of axonal damage or RGC loss. Furthermore, cultured RGC maintained in serum-free media are also C1q and C3 immunoreactive, demonstrating that Ig is not required for C1q binding to damaged RGC. CONCLUSIONS. Our data demonstrate that lack of immunoglobulins and mature T/B cells does not influence the progression of glaucoma. Furthermore, immunoglobulins do not appear to be required for C1q binding and complement cascade activation on damaged RGC. These findings suggest that C1q recognizes an alternative binding partner expressed by stressed RGC. (Invest Ophthalmol Vis Sci. 2012;53:6370‐6377) DOI: 10.1167/iovs.12-10442

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.

Purpose The pathophysiological events that occur in advanced glaucoma are not well characterized. The principal purpose of this study is to characterize the gene expression changes that occur in advanced glaucoma. Methods Retinal RNA was obtained from canine eyes with advanced glaucoma as well as from healthy eyes. Global gene expression patterns were determined using oligonucleotide microarrays and confirmed by real-time PCR. The presence of tumor necrosis factor (TNF) and its receptors was evaluated by immunolabeling. Finally, we evaluated the presence of serum autoantibodies directed against retinal epitopes using western blot analyses. Results We identified over 500 genes with statistically significant changes in expression level in the glaucomatous retina. Decreased expression levels were detected for large number of functional groups, including synapse and synaptic transmission, cell adhesion, and calcium metabolism. Many of the molecules with decreased expression levels have been previously shown to be components of retinal ganglion cells. Genes with elevated expression in glaucoma are largely associated with inflammation, such as antigen presentation, protein degradation, and innate immunity. In contrast, expression of many other pro-inflammatory genes, such as interferons or interleukins, was not detected at abnormal levels. Conclusions This study characterizes the molecular events that occur in the canine retina with advanced glaucoma. Our data suggest that in the dog this stage of the disease is accompanied by pronounced retinal neuroinflammation.

Glaucoma is characterized as a progressive optic neuropathy with characteristic optic disc changes and progressive visual field deficits.1 Elevated intraocular pressure (IOP) is considered a primary risk factor for the initiation and progression of glaucomatous neuropathy.1,2 However, in many patients, despite adequate control of the IOP, the loss of vision continues to progress, which necessitates further identification of molecular mechanisms responsible for the glaucomatous neurodegeneration and development of novel therapeutic modalities that directly target disease-affected retinal ganglion cells (RGCs).2–4 Despite new discoveries, the underlying genetic abnormalities and environmental factors that may contribute to the development and progression of glaucoma have not been clearly defined.5,6 Glaucoma in humans is typically a chronic and slowly progressive disease that may cause significant damage to the optic nerve before any symptoms of the disease are recognized. The gradual and often silent progression of the disease creates difficulties in early diagnosis and understanding of early changes in glaucomatous eyes. To better understand early glaucomatous changes and to develop effective diagnostic and therapeutic modalities, it is essential to create animal models that recapitulate the silent and slow development of the disease characterized by a progressive loss of RGC function. Numerous inducible animal models of glaucoma have been used successfully to test different therapeutic strategies and to evaluate molecular mechanisms of RGC damage resulting from chronic elevation of IOP.2,7,8 However, the major obstacles in many of these models is a very high elevation of IOP immediately after glaucoma induction surgery,9–11 the inability to sustain elevated IOP longer than a period of a several months,12–17 and the size of the eye, which is dramatically smaller than the human eye, posing a problem for effective translation of therapeutic options to the human patients.18,19 Spontaneously occurring animal models of glaucoma in mice and dogs have also been described and have provided important information about the structural and molecular events occurring during the development of glaucomatous optic neuropathy.20–26 Spontaneously occurring large animal models of glaucoma offer a unique opportunity to collect data by using instrumentation identical to that used in human patients with glaucoma. They are an excellent tool for the rapid development and translation of novel medical and surgical therapies from animals to human patients, and they provide interesting data correlating morphologic or biochemical findings to functional parameters. The principal purpose of this manuscript was to describe the morphologic and functional changes in a canine model of hereditary primary angle-closure glaucoma (PACG). We hope this model will further advance our understanding of early functional, structural, and molecular changes in glaucomatous eyes.

Glaucoma is one of the leading causes of blindness worldwide. This review discusses the clinical features, genetics, molecular biology, and cell biology of glaucoma. The authors present a typical case of glaucoma, together with the ocular findings.

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

Mutations in myocilin (MYOC) are the most common genetic cause of primary open angle glaucoma (POAG), but the mechanisms underlying MYOC-associated glaucoma are not fully understood. Here, we report the development of a transgenic mouse model of POAG caused by the Y437H MYOC mutation; the mice are referred to herein as Tg-MYOC(Y437H) mice. Analysis of adult Tg-MYOC(Y437H) mice, which we showed express human MYOC containing the Y437H mutation within relevant eye tissues, revealed that they display glaucoma phenotypes (i.e., elevated intraocular pressure [IOP], retinal ganglion cell death, and axonal degeneration) closely resembling those seen in patients with POAG caused by the Y437H MYOC mutation. Mutant myocilin was not secreted into the aqueous humor but accumulated in the ER of the trabecular meshwork (TM), thereby inducing ER stress in the TM of Tg-MYOC(Y437H) mice. Furthermore, chronic and persistent ER stress was found to be associated with TM cell death and elevation of IOP in Tg-MYOC(Y437H) mice. Reduction of ER stress with a chemical chaperone, phenylbutyric acid (PBA), prevented glaucoma phenotypes in Tg-MYOC(Y437H) mice by promoting the secretion of mutant myocilin in the aqueous humor and by decreasing intracellular accumulation of myocilin in the ER, thus preventing TM cell death. These results demonstrate that ER stress is linked to the pathogenesis of POAG and may be a target for treatment in human patients.

Inappropriate activity of the complement cascade contributes to the pathophysiology of several neurodegenerative conditions. This study sought to determine if components of the complement cascade are synthesized in the retina following the development of ocular hypertension (OHT) and if complement accumulates in association with retinal ganglion cells. Toward this goal the gene expression levels of complement components 1qb (C1qb) and 3 (C3) were determined in the retina by quantitative polymerase chain reaction in human eyes with elevated intraocular pressure (IOP) and healthy retinal tissue as well as in a rat model of OHT induced by laser cauterization of the trabecular meshwork and episcleral veins. Immunohistochemical methods were employed to determine the sites of complement deposition in the retina and optic nerve head. Our data demonstrate that transcript levels for C1q and C3 are significantly elevated in retinae subjected to OHT, both in the animal model as well as in human eyes. Immunohistochemical analyses indicate that C1q and C3 accumulate specifically in the retinal ganglion cell layer and the nerve fiber layer. In addition, we demonstrate that the terminal complement complex, or membrane attack complex, is formed both in the human and rat model as a consequence of OHT. Complement activation, particularly formation of membrane attack complexes, has the potential to exacerbate ganglion cell death through bystander lysis or glial cell activation. The results show that complement activation occurs in the retina that has been subjected to elevated IOP, and may have implications in pathophysiology of glaucoma. 2006 Elsevier Ltd. All rights reserved.

Dysregulation of blood flow with subsequent tissue hypoxia, secondary to or independent from elevated intraocular pressure (IOP) in glaucoma, has been implicated as a component of the pathogenic mechanisms of optic nerve degeneration and retinal ganglion cell (RGC) loss. Many patients with glaucoma exhibit vascular abnormalities such as vasospasm, systemic hypotension, angiographic vascular perfusion defects, and alterations in blood flow parameters that may result in reduced vascular perfusion in the optic nerve head and retina.1–5 Besides these clinical findings, hypoxic tissue stress is evident in the glaucomatous optic nerve head and retina by increased expression of a hypoxia-induced transcription factor, hypoxia-inducible factor (HIF)-1α.6 HIF-1α is known to activate transcription of a wide variety of genes with products that increase oxygen delivery and represent an adaptive response to hypoxia.7 Of interest, the retinal regions exhibiting increased HIF-1α immunolabeling in some of the donor eyes with glaucoma have been found to exhibit a close concordance with the location of visual field defects recorded in these eyes.6 It has long been known that the vertebrate retina consumes large amounts of oxygen and that the main oxygen consumption in the inner retina takes place in RGCs.8 However, although the maintenance of an adequate oxygen supply is critical for neuronal viability and function, current understanding of RGC oxygenation is incomplete. The oxygen demand of RGCs is supplied mainly by retinal arteries in the retina and short posterior ciliary arteries in the optic nerve head. Despite a heterogeneity in vascular response to increased IOP, oxygen tension in the inner retina is relatively unaffected by IOP changes, owing to effective autoregulation of retinal circulation.8–10 Similarly, optic nerve head perfusion pressure is adequately compensated by vascular autoregulation under normal conditions.11 However, in glaucomatous eyes, several risk factors compromising the autoregulatory control have been proposed to reduce blood flow, particularly in intermittent episodes affected by circadian variations in IOP, systemic blood pressure, and ocular perfusion pressure.1–5 In addition to these vascular factors, another important aspect of tissue oxygen delivery that is not well understood is extravascular oxygen transport. Although tissue perfusion is known to involve gas diffusion depending on the oxygen tension gradient and diffusion distance, it is unclear whether there is a facilitated mechanism for oxygen transport to RGCs from retinal capillaries, which are known to be surrounded by glial cells. This mechanism may be particularly important in glaucomatous conditions in which glial cells are in an activated state and may require increased oxygen consumption.12 Regarding the optic nerve head, the load-bearing connective tissue encasing the lamina cribrosa vasculature possibly makes RGC axons even more dependable for a facilitated oxygen transport. Besides a limited understanding of tissue oxygen transport within the retina and optic nerve head tissues, mechanisms controlling intracellular oxygen transport to mitochondria for oxidative phosphorylation also remain unclear. Hemoglobin (Hb) is a more than 600 million-year-old respiratory protein that reversibly binds oxygen and mediates oxygen transport function in blood erythrocytes.13 Repeated detection of Hb in our proteomic studies and recent discovery of Hb expression in the retinal pigment epithelium14 stimulated us to determine whether this hemeprotein may also be present in the inner retina and optic nerve head and whether the oxygen transport function of Hb may be involved in RGC oxygenation in normal or glaucomatous eyes. To answer these questions, we performed a series of experiments determining expression and cellular localization of Hb in animal tissues as well as in glaucomatous and nonglaucomatous human donor eyes, with particular attention to RGCs and macroglia. To determine whether this protein is regulated by ambient oxygen concentrations, we also performed in vitro experiments with primary cultures of rat RGCs and macroglia incubated in the absence and presence of hypoxia. Herein, we present new evidence that Hb is expressed by these cell types as repeatedly detected in the retinal proteome and exhibits an upregulation in ocular hypertensive rat eyes and glaucomatous human donor eyes. Our in vitro findings revealed that hypoxia boosts glial Hb expression through glial erythropoietin (EPO), a well-known target of hypoxic HIF-1α signaling. In addition to this autocrine loop, hypoxia also upregulates Hb expression in RGCs in a paracrine manner. These findings support that in addition to tissue gas diffusion, Hb is likely to be involved in tissue oxygen transport in the inner retina and optic nerve head and that, besides vascular autoregulation, this oxygen-binding protein may provide an intrinsic mechanism for regulation of cellular oxygenation. Based on other predicted functions of Hb in free radical scavenging and nitric oxide detoxification, such an intrinsic protective mechanism against hypoxic/oxidative injury may have important implications in glaucomatous neurodegeneration.