Search

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.

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.

Purpose Traumatic brain injury (TBI) is a risk factor for developing chronic neurodegenerative conditions including Alzheimer's disease (AD). The purpose of this study was to examine chronic effects of blast TBI on retinal ganglion cells (RGC), optic nerve, and brain amyloid load in a mouse model of AD amyloidosis. Methods Transgenic (TG) double-mutant APPswePSENd19e (APP/PS1) mice and nontransgenic (Non-TG) littermates were exposed to a single blast TBI (20 psi) at age 2 to 3 months. RGC cell structure and function was evaluated 2 months later (average age at endpoint = 4.5 months) using pattern electroretinogram (PERG), optical coherence tomography (OCT), and the chromatic pupil light reflex (cPLR), followed by histologic analysis of retina, optic nerve, and brain amyloid pathology. Results APP/PS1 mice exposed to blast TBI (TG-Blast) had significantly lower PERG and cPLR responses 2 months after injury compared to preblast values and compared to sham groups of APP/PS1 (TG-Sham) and nontransgenic (Non-TG-Sham) mice as well as nontransgenic blast-exposed mice (Non-TG-Blast). The TG-Blast group also had significantly thinner RGC complex and more optic nerve damage compared to all groups. No amyloid-β (Aβ) deposits were detected in retinas of APP/PS1 mice; however, increased amyloid precursor protein (APP)/Aβ-immunoreactivity was seen in TG-Blast compared to TG-Sham mice, particularly near blood vessels. TG-Blast and TG-Sham groups exhibited high variability in pathology severity, with a strong, but not statistically significant, trend for greater cerebral cortical Aβ plaque load in the TG-Blast compared to TG-Sham group. Conclusions When combined with a genetic susceptibility for developing amyloidosis of AD, blast TBI exposure leads to earlier RGC and optic nerve damage associated with modest but detectable increase in cerebral cortical Aβ pathology. These findings suggest that genetic risk factors for AD may increase the sensitivity of the retina to blast-mediated damage.

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.

Objective To perform detailed analysis of retinal changes in dogs with SARDS using optical coherence tomography (OCT), funduscopy, and molecular analysis. Animals Subjects were 29 dogs from 12 US states and Canada diagnosed with SARDS by 8 ophthalmologists. An additional 7 eyes from 5 deceased SARDS dogs were used for molecular and histological analysis. Procedures Dogs were evaluated using chromatic pupil light reflex testing (cPLR), and electroretinography (ERG); subjects underwent complete ophthalmic examination, including funduscopy, retinal photography, and OCT, in addition to complete laboratory analysis, blood pressure evaluation, abdominal and thoracic radiographs, and computerized tomography (CT) imaging to assess possible systemic abnormalities. Histology and immunohistochemistry analysis was performed in 2 SARDS eyes. Microarray analysis was performed in 5 SARDS retinas. Results Thirty‐eight percent of patients had

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.

The trabecular meshwork’s (TM) physiological role is to maintain normal intraocular pressure by regulating aqueous humor outflow. With age, and particularly in eyes with primary open angle glaucoma, the number of cells residing within the TM is markedly decreased and the function of the tissue is compromised. Here we evaluate if transplantation of induced pluripotent stem cell derived TM like cells (iPSC-TM) restores TM cellularity and function in human eyes obtained from older human donors. Human iPSC were differentiated into iPSC-TM and compared to primary TM cells by RNAseq. iPSC-TM were then injected into the anterior segments of human eyes maintained in perfusion culture. Seven and 14 days eyes after injection eyes that received iPSC-TM contained significantly more cells in the TM. Fewer than 1% of all cells appeared to be iPSC-TM, but significantly more cells in these eyes were immunopositive for Ki 67 and incorporated BrdU. Our study demonstrates that transplantation iPSC-TM stimulates proliferation of endogenous TM cells in perfusion cultured human eyes from aged donors. These data, in concert with our previous findings in animal models, suggest that functional regeneration of the TM may be possible in human eyes with primary open angle 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.

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.

Purpose The defining feature of glaucoma is excavation of the optic nerve head; however, the mechanism of this loss of tissue is not well understood. We recently discovered a copy number variation upstream of matrix metalloproteinase 19 (MMP19) in a large, autosomal dominant pedigree with a congenital malformation of the optic disc called cavitary optic disc anomaly (CODA). Patients with CODA have abnormal optic discs that exhibit an excavated shape similar to cupping seen in glaucoma. The goal of this study is to characterize the localization of MMP19 within the human optic nerve. Methods The MMP19 protein in the optic nerve was evaluated with western blot analysis and with immunohistochemistry in sagittal and en face/cross sections of optic nerves obtained from healthy human donor eyes. Results The MMP19 protein was detected in the human optic nerve, retina, and RPE/choroid with western blot analysis, with highest expression in the retina and the optic nerve. Using immunohistochemistry, MMP19 was localized within the optic nerve to the extracellular space within the septa that separate bundles of optic nerve axons into fascicles. The presence of MMP19 within the optic nerve septa was further confirmed by the colocalization of MMP19 to this structure with type IV collagen. Strong labeling of MMP19 was also detected in the arachnoid layer of the optic nerve sheath. Finally, immunohistochemistry of the optic nerve cross sections demonstrated that MMP19 shows a peripheral to central gradient, with more abundant labeling along the edges of the optic nerve and in the arachnoid layer than in the center of the nerve. Conclusions Abundant MMP19 was detected in the optic nerve head, the primary site of pathology in patients with CODA. The localization of MMP19 to the optic nerve septa is consistent with its predicted secretion and accumulation within the extracellular spaces of this tissue. Moreover, the lateral localization of MMP19 observed in the optic nerve cross sections suggests that it might have a role in regulating adhesion to the optic nerve to the scleral canal and remodeling the extracellular matrix that provides the structural integrity of the optic disc. Dysregulation of MMP19 production might, therefore, undermine the connections between the optic nerve and the scleral canal and cause a collapse of the optic disc and the development of CODA. Similar processes might also be at work in the formation of optic disc cupping in glaucoma.

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.

Recent reports have indicated that components of the complement cascade are synthesized during the degeneration of retinal ganglion cells (RGC) in glaucoma. While complement deposition in the retina may simply serve to aid phagocytosis of damaged RGC, activation of the complement cascade can also contribute to neuronal loss in neurodegenerative diseases. This study was designed to determine if disruption of the complement cascade affects RGC survival in a murine model of retinal ischemia-reperfusion (I/R) injury. We induced retinal ischemia in the eyes of normal mice and mice with a targeted disruption of the complement component 3 (C3) gene. Tissue was harvested 7 and 21 days after induction of I/R and retinal complement synthesis was determined by quantitative PCR and immunohistochemical methods. RGC death and associated axon loss was evaluated through histological examination of the optic nerve and retina. Our data show that retinal I/R induces the expression and deposition of complement components. C3 deficient mice clearly exhibited reduced optic nerve damage and substantial preservation of RGC 1 week after I/R when compared to normal animals (p=0.005). Three weeks after the ischemic event C3 deficient mice retained more RGC cell bodies although the degree of optic nerve damage was similar between both groups. These findings demonstrate that inhibition of the complement cascade delays optic nerve axonal and RGC degeneration in retinal I/R. It appears that injured RGC are targeted and actively destroyed through complement mediated processes. These results may have implications for the pathophysiology and clinical management of ischemic retinal conditions.

This study provides a detailed description of immunolocalization of two oxygen-binding proteins, neuroglobin (Ngb) and cytoglobin (Cygb), in the anterior segment of healthy human and canine eyes. Specific antibodies against Ngb and Cygb were used to examine their distribution patterns in anterior segment structures including the cornea, iris, trabecular meshwork, canal of Schlemm, ciliary body, and lens. Patterns of immunoreactivity (IR) were imaged with confocal scanning laser and conventional microscopy. Analysis of sectioned human and canine eyes showed Ngb and Cygb IR in the corneal epithelium and endothelium. In the iris, Ngb and Cygb IR was localized to the anterior border and the stroma, iridal sphincter, and dilator muscle. In the iridocorneal angle, Ngb and Cygb were detected in endothelial cells of the trabecular meshwork and canal of Schlemm in human. In the ciliary body, Ngb and Cygb IR was localized to the non-pigmented ciliary epithelium of the pars plana and pars plicata and in ciliary body musculature. Ngb and Cygb distribution was similar and colocalized within the same structures of healthy human and canine anterior eye segments. Based on their immunolocalization and previously reported biochemical features, we hypothesize that Ngb and Cygb may function as scavengers of reactive oxygen species. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 56:863–872, 2008)

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.

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.

Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss in the developed world. The study of a rare mendelian form of macular degeneration implicated fibulin genes in the pathogenesis of more common forms of this disease. We evaluated five fibulin genes in a large series of patients with AMD.We studied 402 patients with AMD and 429 control subjects from the same clinic population. Patients were examined by means of indirect ophthalmoscopy, slit-lamp microscopy, and fundus photography to establish the presence and phenotypic pattern of AMD. DNA samples were screened for sequence variations in five members of the fibulin gene family.Amino acid-altering sequence variations were found in all five fibulin genes, many of which were observed only in patients with AMD. Several of the altered residues have been conserved during evolution. Seven of the 402 patients with AMD had amino acid-altering sequence variations in the fibulin 5 gene, whereas none were observed among 429 control subjects (P0.01). In addition, these seven patients all had small, circular drusen, which are commonly referred to as basal laminar or cuticular drusen.Missense mutations in the fibulin 5 gene were found in 1.7 percent of patients with AMD. Many variations in other fibulin genes were also found in these patients, and the evolutionary conservation of the affected residues suggests that several of these variations may also be involved in AMD.

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

Purpose Autosomal recessive retinitis pigmentosa (ARRP) is a genetically heterogeneous condition characterized by progressive loss of retinal photoreceptor cells. In order to gain new insights into the pathogenesis of ARRP, we evaluated the morphological, biochemical, and gene expression changes in eyes from a human donor with ARRP due to mutations in the ABCA4 gene. Methods Eyes were obtained postmortem from a donor with end-stage retinitis pigmentosa. The coding sequences of the RDS, RHO, and ABCA4 genes were screened for disease-causing mutations. Morphological changes in different regions of the retina were examined histologically, and levels of lipofuscin-associated bisretinoids were measured. Gene expression was examined in retinal/choroidal tissue using microarray analysis, and all parameters were compared to those in unaffected control donors. Results Genetic analysis of the donor's DNA identified two mutations in the ABCA4 gene, IVS14+1G > C and Phe1440del1 cT, each on a separate allele. Morphological evaluation revealed complete loss of the outer nuclear layer, remodeling of the inner retina, loss of retinal vasculature, and regional neovascularization. The retinal pigment epithelium and choriocapillaris exhibited regional preservation. Microarray analysis revealed loss of photoreceptor cell-associated transcripts, with preservation of multiple genes expressed specifically in inner retinal neurons. Conclusions The persistence of transcripts expressed by inner retinal neurons suggests that despite significant plasticity that occurs during retinal degeneration, bipolar cells and ganglion cells remain at least partially differentiated. Findings from this study suggest that some forms of therapy currently under investigation may have benefit even in advanced retinal degeneration.

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.

Data from human genetics, histopathology, and animal models reveal a major role for the complement system in the development of age-related macular degeneration (AMD). Genetic variations in the complement factor H (CFH) gene are associated with an elevated risk of AMD. In this study we sought to determine whether eyes from donors with a high-risk genotype (homozygosity for the histidine allele at codon 402) exhibit altered levels of membrane attack complex (MAC) in the choroid, compared to eyes with a low risk genotype (homozygosity for tyrosine). Proteins were extracted from the RPE/choroid of 18 donors (10 low risk and 8 high risk) and levels of MAC were assessed using an ELISA assay. Eyes from donors homozygous for the histidine allele showed 69% higher levels of MAC than those homozygous for the tyrosine allele (p < 0.05), independent of whether the eyes showed signs of early AMD. Our results provide evidence that high-risk CFH genotypes may affect AMD risk by increased deposition of MAC around the aging choriocapillaris.

To evaluate the ability of mesenchymal stem cells (MSCs) engineered to produce and secrete brain-derived neurotrophic factor (BDNF) to protect retinal function and structure after intravitreal transplantation in a rat model of chronic ocular hypertension (COH).COH was induced by laser cauterization of trabecular meshwork and episcleral veins in rat eyes. COH eyes received an intravitreal transplant of MSCs engineered to express BDNF and green fluorescent protein (BDNF-MSCs) or just GFP (GFP-MSCs). Computerized pupillometry and electroretinography (ERG) were performed to assess optic nerve and retinal function. Quantification of optic nerve damage was performed by counting retinal ganglion cells (RGCs) and evaluating optic nerve cross-sections.After transplantation into COH eyes, BDNF-MSCs preserved significantly more retina and optic nerve function than GFP-MSC-treated eyes when pupil light reflex (PLR) and ERG function were evaluated. PLR analysis showed significantly better function (P = 0.03) in BDNF-MSC-treated eyes (operated/control ratio = 63.00% ± 11.39%) than GFP-MSC-treated eyes (operated/control ratio = 31.81% ± 9.63%) at 42 days after surgery. The BDNF-MSC-transplanted eyes also displayed a greater level of RGC preservation than eyes that received the GFP-MSCs only (RGC cell counts: BDNF-MSC-treated COH eyes, 112.2 ± 19.39 cells/section; GFP-MSC-treated COH eyes, 52.21 ± 11.54 cells/section; P = 0.01).The authors have demonstrated that lentiviral-transduced BDNF-producing MSCs can survive in eyes with chronic hypertension and can provide retina and optic nerve functional and structural protection. Transplantation of BDNF-producing stem cells may be a viable treatment strategy for glaucoma.

Purpose To determine the role of the recently discovered primary open angle glaucoma (POAG) risk factor mapped to chromosome 7q31 in glaucoma patients from Iowa and to determine the expression pattern of genes in the locus in human eyes. Methods A cohort of 545 POAG patients and 297 control subjects from Iowa were genotyped with a single nucleotide polymorphism (SNP; rs4236601) in the chromosome 7q31 locus using a quantitative polymerase chain reaction (PCR) assay. The expression of genes within the 7q31 locus, caveolin-1 (CAV1) and caveolin-2 (CAV2) in human eyes was investigated with immunohistochemistry. Results The minor allele frequency (MAF) of rs4236601 was 27% in control subjects and 29% in POAG patients. We detected no statistical difference when we compared the allele frequencies of rs4236601 between POAG patients and control subjects (p=0.5). Similarly, we detected no statistical difference in the frequency of the three possible rs4236601 genotypes between patients and controls (p=0.22). Immunohistochemistry showed caveolin expression in human retina, ciliary muscle, trabecular meshwork, and Schlemm’s canal. In our small cohort of donor eyes, the genotype of rs4236601 did not obviously influence labeling intensity or distribution of CAV1 and CAV2 in the retina. Conclusions A genome-wide association study of subjects from Iceland mapped the first common genetic risk factor for POAG to a small region of the genome on chromosome 7q31 that contains the caveolin genes CAV1 and CAV2. We were unable to detect this association in our patients from Iowa, suggesting that this risk factor may not have a strong effect in all populations.

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.

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.

To characterize the molecular and functional status of the rat retina and optic nerve after acute elevation of intraocular pressure (IOP). Retinal ischemia was induced in rats by increasing the IOP (110 mmHg/60 minutes). Microarray analysis, quantitative RT-PCR (qRT-PCR) and immunohistochemistry were used to characterize retinal tissue. PLGA microspheres containing neurotrophic factors (BDNF, GDNF, or CNTF) or empty microspheres were injected into the vitreous of operated animals 1 day after elevation of IOP. Pupil light reflex (PLR) parameters and electroretinograms (ERG) were monitored at multiple time points during the 60-day postoperative recovery period. Molecular analysis showed a significant intrinsic up-regulation of CNTF at 10 and 25 days after induction of the acute ocular hypertension (p = 0.0067). Molecular tissue analysis of GDNF and its receptors (GDNFR1, GDNFR2), and BDNF and its receptor (trkB) showed no change in expression. Animals that received CNTF microspheres had no significant functional recovery compared to animals which received blank microspheres (p > 0.05). Animals that received GDNF or BDNF microspheres showed significant PLR recovery (p

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.

PURPOSE. Best disease, or vitelliform macular degeneration, is an autosomal dominant form of macular degeneration that is caused by mutations in the gene encoding bestrophin. On clinical examination, Best disease is characterized by an elevated lesion beneath the neurosensory retina, resembling an egg yolk. The lesions in Best disease are primarily restricted to the macula, a small region of the retina responsible for central vision. The nature of the vitelliform material and the reason the development of such lesions is usually restricted to the macula are two unsolved questions in the pathogenesis of this disorder. METHODS. The expression of bestrophin protein and mRNA was evaluated by immunohistochemistry, Western blot, and quantitative PCR in a series of normal human eyes. The ultrastructure of the retinal pigment epithelium and the histopathology of two donors with clinically diagnosed Best disease were also examined. RESULTS. An eye from a Best disease donor with a T6R mutation was found to have deposits containing lipid and glycoconjugates within the central retinal scar. These deposits may be remnants of the vitelliform lesion. Immunohistochemical localization of bestrophin in a series of 22 unaffected eyes revealed a pattern in which macular labeling was less robust than labeling outside the macula in most (18/22) cases. This pattern was confirmed using quantitative PCR and Western blotting. CONCLUSIONS. Topographic differences in the levels of bestrophin protein may in part explain the propensity for the macula to develop lesions in Best disease.

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.

Purpose The goal of this study was to describe the detailed localization of the novel oxygen-binding molecules, neuroglobin (Ngb) and cytoglobin (Cygb), in mammalian retinas and to determine whether Ngb and Cygb are neuronal or glial proteins in the retina. Methods Antibodies directed against Ngb and Cygb were used to examine their patterns of distribution in normal canine retinas. Immunoblot analysis was performed to verify antibody specificity and the presence of Ngb and Cygb in canine tissues. Double-labeling immunohistochemistry was performed with the Ngb and Cygb antibodies along with antibodies against neuronal (MAP-2, class III beta-tubulin (TUJ1), PKCalpha, and calretinin) and glial antigens (vimentin and CRALBP). Tissue sections were analyzed with light and confocal microscopy. Results Ngb and Cygb proteins were observed in different retinal cells. Cygb (but not Ngb) was also present in canine kidney, liver, lung, and heart tissue. Immunohistochemical analysis of canine retinas demonstrated Ngb immunoreactivity (IR) in the ganglion cell layer (GCL), inner (INL) and outer (ONL) nuclear layers, inner (IPL) and outer plexiform (OPL) layers, photoreceptor inner segments (IS), and retinal pigment epithelium (RPE). Ngb IR was localized within retinal neurons, but not in glia. Cygb IR was found in neurons and their processes in the GCL, IPL, INL, and OPL and within the RPE, but not in glia. Conclusions Ngb and Cygb are widely distributed in retinal neurons and RPE, but not in glial cells of the canine retina. Their structure and distribution is suggestive of a possible role in oxygen transport in the mammalian retina.

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.

The iris plays an essential role in regulating the amount of light passing to the retina and is also important in many human diseases. Several diseases change iris pigmentation, including forms of oculocutaneous albinism, Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, Horner's syndrome, Waardenburg syndrome, and Fuchs' heterochromic iridocyclitis. In addition, other ocular diseases, such as pigment dispersion syndrome and exfoliation syndrome, involve disease-related morphologic changes to the pigmented tissues of the iris. Each of these diseases involves strong hereditary links, but much remains unknown concerning the underlying genetic pathways. In this study, we focused on three of these conditions: albinism, pigment dispersion syndrome, and exfoliation syndrome. In oculocutaneous albinism (OCA), there is reduced or absent pigmentation of the skin, hair, and eyes. Decreased melanin in the eyes can give rise to several ocular abnormalities, including foveal hypoplasia and decreased visual acuity; retinal ganglion cell axon misrouting; and strabismus, nystagmus, iris translucency, color vision impairment, and photophobia.1 The hereditary basis of OCA is complex. There are at least 4 genes that contribute to classic forms of OCA and at least another 12 associated with syndromic forms. The best understood form of OCA, and the most common in many populations, is OCA1.2,3 OCA1 is caused by mutations in the tyrosinase (TYR) gene, which encodes the rate-limiting enzyme necessary for melanin synthesis. Most people with OCA1 are believed to be compound heterozygotes, although in 15% of OCA1 cases, the second mutation remains unidentified.4 Interestingly, TYR appears to also influence many traits beyond pigmentation. For example, tyrosinase mutation is capable of rescuing a mouse model of pigment dispersion,5 but acts to worsen disease in mouse models of developmental glaucoma.6 Clearly, much remains unknown concerning TYR and its influences on ocular disease. In pigment dispersion syndrome, liberated pigment from the iris pigment epithelium becomes aberrantly deposited throughout the anterior chamber. As pigment accumulates in the iridocorneal angle, aqueous humor outflow resistance and intraocular pressure can become elevated.7,8 Although pigment dispersion syndrome has strong hereditary links,9,10 the genetic risk factors remain to be identified. DBA/2J mice develop a form of pigmentary glaucoma involving a pigment-dispersing iris disease, elevated intraocular pressure, and optic nerve damage.11,12 Mutations in two genes encoding melanosomal proteins, Tyrp1 and Gpnmb, are responsible for initiation of the DBA/2J disease process.13 To date, genetic studies of TYRP1 and GPNMB in human pigment dispersion patients have not detected mutations,13,14 suggesting that other genes in a pathway linked to TYRP1 and GPNMB may be the next most logical candidates worthy of consideration. In exfoliation syndrome, a primary diagnostic feature is the presence of fibrillar exfoliative material throughout the anterior chamber of the eye.15 The disease often also involves the dispersion of iris pigment and morphologic changes to the structure of the iris pigment epithelium.16,17 As with pigment dispersion syndrome, accumulations of material within the iridocorneal angle can obstruct aqueous humor outflow, resulting in elevated intraocular pressure and glaucoma. Recently, genetic variations in the LOXL1 gene have been linked with exfoliation syndrome.18 Because the same LOXL1 alleles associated with exfoliation syndrome also occur in the general population at a very high frequency, additional risk factors are presumed to exist. B6-Lystbg-J mice exhibit multiple ocular features resembling exfoliation syndrome, including the presence of an exfoliative-like material, pigment dispersion, and iris transillumination defects caused by an apparent loss of cell–cell adhesions within the iris pigment epithelium.17 Accordingly, LYST and other genes within the LYST genetic pathway are candidates that are likely to contribute to exfoliation syndrome in humans. We report global gene expression patterns of the iris in four strains of mice with identical genetic backgrounds: wild-type C57BL/6J mice with normal irides, albino mice with Tyr mutation, pigment dispersion–prone mice with Tyrp1 and Gpnmb mutations, and exfoliative-like mice with Lyst mutation. In each comparison between these strains, transcriptional changes are presented for select genes of functionally annotated clusters and are also presented according to the magnitude of the ratio of change.

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.

PURPOSE. Central corneal thickness (CCT) exhibits broad variability. For unknown reasons, CCT also associates with diseases not typically considered corneal, particularly glaucoma. The purpose of this study was to test the strain dependence of CCT variability among inbred mice and identify cellular and molecular factors associated with differing CCT. METHODS. Methodology for measuring murine CCT with ultrasound pachymetry was developed and used to measure CCT among 17 strains of mice. Corneas from three strains with nonoverlapping differences in CCT (C57BLKS/J, C57BL/6J, and SJL/J) were compared by histology, transmission electron microscopy, and expression profiling with gene microarrays. RESULTS. CCT in mice was highly strain dependent. CCT exhibited continuous variation from 89.2 m in C57BLKS/J to 123.8 m in SJL/J. Stromal thickness was the major determinant of the varying murine CCT, with epithelial thickness also contributing. Corneal expression levels of many genes differed between strains with differing CCT, but most of these changes did not correlate with the changes observed in previously studied corneal diseases nor did they correlate with genes encoding major structural proteins of the cornea. CONCLUSIONS. Murine CCT has been measured with a variety of different techniques, but only among a limited number of different strains. Here, pachymetry was established as an additional tool and used to conduct a broad survey of different strains of inbred mice. These results demonstrated that murine CCT was highly influenced by genetic background and established a baseline for future genetic approaches to further elucidate mechanisms regulating CCT and its disease associations. (Invest Ophthalmol Vis Sci. 2010;51:160‐171) DOI:10.1167/ iovs.09-3429