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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

To determine alterations which occur in the size and shape of lamina cribrosa (LC) pores in glaucomatous eyes over a period of time.Baseline and follow up optic disc photographs were retrospectively studied in 39 eyes of 39 patients with glaucoma. Only eyes with a vertical cup to disc ratio equal to or greater than 0.6 were included in the study. In addition, all selected eyes had to have serial optic disc photographs obtained at least 3 years apart allowing clear visualisation of LC surface. The association of the alterations in LC surface morphology with patient specific and eye specific characteristics was statistically analysed.During a mean study period of 3.90 (SD 0.7) years, individual pore size (mean pore area to disc area ratio) exhibited a significant decrease between baseline and follow up measurements of each eye (p0.0001). However, during the study period, total pore area to disc area ratio did not change (p0.05), and the change in pore shape in some eyes (from circular to more oval and elongated) was statistically insignificant (p = 0.12). Although a relation was detectable between the optic disc and lamina cribrosa parameters at a given time, which reflects cumulative effects, during the study period, there was no significant association between the changes of the LC parameters and neural tissue damage. The rate and the magnitude of the changes in individual pore size during the study period were not significantly different among the eyes exhibiting progressive neural rim damage and those staying stable (p0.05).These findings demonstrate that the LC surface morphology exhibits changes along with the glaucomatous optic disc damage. However, the clinical appearance of LC surface in glaucomatous eyes may continue to change, even when the neural rim damage is clinically stable. These findings are probably associated with the chronic cellular events of tissue remodelling that occur in the glaucomatous optic nerve head.

To study serum titers of antibodies against heat shock proteins in glaucoma patients from two different ethnic populations and to examine the relationship between serum antibody titers and glaucomatous damage.Comparative, cross-sectional study.Twenty-seven age-matched patients with primary open-angle glaucoma, 28 patients with normal pressure glaucoma, and a control group of 26 healthy subjects from Japan; and 21 age-matched patients with primary open-angle glaucoma, 40 patients with normal pressure glaucoma, and a control group of 20 healthy subjects from the United States.Measurement of serum antibody titers and examination of optic disc and visual field parameters.Serum immunoreactivity against heat shock proteins, including hsp27, alphaB-crystallin, and human and bacterial hsp60, was studied by enzyme-linked immunosorbent assay (ELISA). The relationship of serum antibody titers to glaucoma parameters, including mean deviation, neural rim area-to-disc area ratio, and peripapillary atrophy area-to-disc area ratio was examined.The serum ELISA titers of antibodies, including hsp27, alphaB-crystallin, human hsp60, and bacterial hsp60 antibodies, were higher in glaucoma patients compared with control subjects in both the Japanese and American groups (P:0.05). There was no association between the serum antibody titers and optic disc parameters in either group from Japan or the United States (P:0.05).These findings suggest that increased titers of circulating antibodies against heat shock proteins occur in both Japanese and American patients with glaucoma. The lack of a relationship between the level of serum antibody titers and the degree of glaucomatous damage in either ethnic group suggests that increased antibodies to heat shock proteins do not occur as an epiphenomenon of the glaucomatous neurodegeneration process.

Purpose To examine immunostaining of 60-kd and 27-kd heat shock proteins (HSP 60 and HSP 27), which are known to increase cell survival in response to stress, in glaucomatous retina and optic nerve head. Methods Six postmortem eyes from patients with primary open-angle glaucoma, 6 eyes from patients with normal-pressure glaucoma, and 6 eyes from age-matched normal subjects were studied by immunohistochemistry. The sections of the retina and optic nerve head were examined after immunostaining with antibodies to HSP 60 and HSP 27. Results The intensity of the immunostaining and the number of labeled cells for heat shock proteins (HSPs) were greater in retina sections from glaucomatous eyes than in sections from normal eyes from age-matched donors. Retinal immunostaining of HSP 60 was prominent in the retinal ganglion cells and photoreceptors, whereas immunostaining of HSP 27 was prominent in the nerve fiber layer and ganglion cells as well as in the retinal vessels. In addition, retinal immunostaining of these HSPs exhibited regional and cellular differences. Optic nerve heads of glaucomatous eyes exhibited increased immunostaining of HSP 27, but not HSP 60, which was mostly associated with astroglial cells in the lamina cribrosa. Conclusion The increased immunostaining of HSP 60 and HSP 27 in the glaucomatous eyes may reflect a role of these proteins as a cellular defense mechanism in response to stress or injury in glaucoma. Clinical Relevance These findings suggest that immunoregulation is an important component of glaucomatous optic neuropathy.

BACKGROUND/AIMS Mechanical forces are thought to induce cellular responses through activation of signalling pathways. Cells within the intraocular environment are exposed to constant changes in the levels of intraocular pressure. In this study, an attempt was made to determine the acute effects of elevated hydrostatic pressure on different intraocular cells grown in culture. METHODS Different cell lines derived from ocular tissues including non-pigmented and pigmented ciliary epithelium, trabecular meshwork, retina, and lamina cribrosa were incubated in a pressurised chamber at 50 mm Hg in a culture incubator at 37°C for up to 6 hours. Control cells were incubated at atmospheric pressure. The viability of the cells was examined using their intracellular esterase activity. The morphology and cytoskeleton of the cells were investigated using microscopy and phalloidin staining. Adenylyl cyclase activity was assessed by measuring the conversion of [3H]-cAMP from [3H]-ATP in response to elevated hydrostatic pressure for 1–6 hours. In addition, at the end of incubation period under elevated hydrostatic pressure the recovery of adenylyl cyclase activity to control levels was examined. RESULTS Cell viability did not change following exposure to elevated hydrostatic pressure for 6 hours. Cells subjected to elevated hydrostatic pressure demonstrated morphological differences characterised by a more rounded shape and a redistribution of actin stress fibres that was most prominent in lamina cribrosa astrocytes. A time dependent increase in basal adenylyl cyclase activity, and a decrease in maximum forskolin stimulated activity were observed in all cell lines following exposure to elevated hydrostatic pressure. CONCLUSION These observations demonstrate that cell lines from different ocular tissues are sensitive to changes in external pressure in vitro. They exhibit morphological and cytoskeletal changes as well as significant alterations of intracellular adenylyl cyclase activity following exposure to acute and sustained levels of elevated hydrostatic pressure of up to 6 hours9 duration.

Background Serum autoantibodies that cross-react with glycosaminoglycans have been proposed to play a significant role in specific tissue injury in patients with systemic autoimmune diseases. Objective To investigate whether serum immunoreactivity to glycosaminoglycans is present in patients with glaucoma who have aberrant serum autoantibodies to DNA, RNA, nuclear proteins, or retinal proteins, as proteoglycans and their glycosaminoglycan side chains are important components of the optic nerve head and its vasculature. Methods We performed Western blotting using patient serum samples and human optic nerve head homogenates that were treated with or without specific glycosaminoglycan degrading enzymes. Monoclonal antibodies that recognize different determinants of glycosaminoglycans were used to identify specific substrate antigenicity. We compared the serum immunoreactivity to glycosaminoglycans in 60 age-matched patients with normal-pressure glaucoma, 36 patients with primary open-angle glaucoma, and 20 control subjects by enzyme-linked immunosorbent assay. In addition, immunohistochemistry was performed to compare the distribution patterns of glycosaminoglycans in the optic nerve head of postmortem eyes of age-matched patients with normal-pressure glaucoma, primary open-angle glaucoma, and control subjects. Results Western blotting demonstrated that serum samples from patients with glaucoma who have circulating autoantibodies can recognize optic nerve head proteoglycans, including chondroitin sulfate and heparan sulfate. The level of serum autoantibodies binding purified chondroitin sulfate and heparan sulfate glycosaminoglycans in an enzyme-linked immunosorbent assay was approximately 100% higher in patients with normal-pressure glaucoma than that in control subjects and approximately 50% higher than that in patients with primary open-angle glaucoma. We also observed increased immunostaining of glycosaminoglycans in the optic nerve head of eyes with glaucoma, particularly those with normal intraocular pressure, compared with control eyes. Conclusion There are increased levels of autoantibodies recognizing glycosaminoglycans of the optic nerve head in the serum samples of some patients with glaucoma. Clinical Relevance These autoantibodies may increase the susceptibility of the optic nerve head to damage in these patients by changing the functional properties of the lamina cribrosa, its vasculature, or both.

PURPOSE: Increased serum immunoreactivity to retinal proteins may have a role in the disease process of some glaucoma patients. We describe a patient with normal- pressure glaucoma whose serum immunoreactivity to retinal proteins regressed after methotrexate treatment for rheumatoid disease. METHOD: Case report. RESULTS: In a 66-year-old white female with normal-pressure glaucoma and rheumatoid disease, sequential Western blots using patient sera against retinal proteins demonstrated a decrease in the immunoreactive bands after treatment. During the treatment period of 3 years, her visual fields appeared to have improved. Optic disk examination during the short periods without treatment, however, disclosed new, bilateral splinter hemorrhages on the optic disks. CONCLUSION: These observations suggest a potential role for immune-based intervention in similar patients.

Objective: To determine whether parapapillary chorioretinal atrophy is a risk factor for the development of glaucomatous optic disc or visual field damage. Methods: The initial morphometric parameters of the optic disc and parapapillary atrophy were retrospectively investigated in 350 eyes of 175 patients with ocular hypertension. The prognostic value of parapapillary atrophy at the baseline examination and its relationship with known risk factors for the development of glaucomatous damage were analyzed by multivariate analysis. Results: Visual field loss, optic disc damage, or both were detected in 98 eyes of 53 patients during the follow-up period of at least 10 years. By univariate analysis, the presence of parapapillary atrophy, as well as higher parapapillary atrophy area—disc area, zone β area—disc area, and parapapillary atrophy length—disc circumference ratios, at the baseline examination was associated with the conversion to glaucoma. In addition, higher intraocular pressure, larger vertical cupdisc ratio, and smaller neural rim area—disc area ratio at the baseline examination were associated with subsequent glaucomatous optic nerve damage. In a multivariate regression model adjusted for other factors, intraocular pressure (relative risk, 1.19), neural rim area—disc area ratio (relative risk, 0.72), and zone β area—disc area ratio (relative risk, 1.32) were found to be associated with the development of optic disc damage, visual field damage, or both. Conclusion: The presence and the size of parapapillary atrophy are related to the development of subsequent optic disc or visual field damage in patients with ocular hypertension.

To determine whether parapapillary chorioretinal atrophy in patients with ocular hypertension remained stationary or progressed along with glaucomatous optic nerve damage.The morphometric parameters and progression of parapapillary atrophy were retrospectively investigated, using serial photographs, in 350 eyes of 175 patients with ocular hypertension. The association of parapapillary atrophy progression with subsequent glaucomatous conversion and with other baseline patient- and eye-specific characteristics was analyzed.Progression in the area and extension of parapapillary atrophy before noticeable optic disc or visual field changes was observed in 48 (49.0%) of 98 eyes that converted to glaucoma, while parapapillary atrophy progression was noted in 25 (9.9%) of 252 ocular hypertensive eyes that did not develop glaucomatous damage (P.001). The predictive sensitivity and specificity of this observation were 49% and 90%, respectively. In a logistic multiple regression model, the progression of parapapillary atrophy was associated with a family history of glaucoma (odds ratio, 2.7) and the initial size of zone beta (odds ratio, 1.64, for an increase of 0.10 of the zone beta area-disc area ratio).The progression of parapapillary chorioretinal atrophy may be an early glaucomatous finding in some patients with ocular hypertension.

We studied morphometrically the color transparencies and fiuorescein angiograms of 5 patients with six exudative retinal arterial macroaneurysms. Our aim was to express the dependence of the exudate morphology upon the location of the macroaneurysm with an algebraic polynomial analysis. We derived the second-degree conical equations of the exudate curves and computed the location of their cardinal descriptive parameters. The relationship between the site of the macroaneurysms and the computed hypothetical parameters of their exudate curves revealed that the structural features of the exudates are dependent upon the distance of the macroaneurysm to the foveola, the gravitational force and the clearing capacity of the venous net. The point where the macroaneurysm develops approximately 3 mm from the center of the macula seems to be a ‘crucial point’. Macroaneurysms located closer than this point may cause the exudates that occupy the fovea and create severe visual loss.

Twenty-one macroaneurysms and related vessels of 19 patients were evaluated morphometrically. Macroaneurysms were classified into two groups as hemorrhagic and exudative in terms of their major clinical sign. Average diameters of the macroaneurysms were arranged in a Gaussian distribution curve (mean and standard deviation: 281.60 +/- 57.28 micrometers). Regarding the distribution curve based on this data macroaneurysm can be defined as being greater than 109.76 micrometers. Macroaneurysms were most frequent on the superotemporal vessels (52.38%), followed by the inferotemporal (38.10%), inferonasal (4.76%) and superonasal vessels. Average diameter (r = +0.68, p = 0.0006) and area (r = +0.71, p = 0.0003) of the macroaneurysms were significantly correlated to the diameter of the relevant arterial segments. The distribution of the macroaneurysms in respect to arterial bifurcation (chi 2 = 18.762, p = 0.0003) and arteriovenous crossings (chi 2 = 8.286, p = 0.0405) were nonrandom with macroaneurysms clustering near this points. Hemorrhagic macroaneurysms were significantly closer to the optic disc (p < 0.01) and were located on relatively larger arterioles (p < 0.01). They were also more circular (p < 0.01) in shape and greater in area (p < 0.01) and diameter (p < 0.01) than the exudative ones. These findings suggest that the location of the macroaneurysm is closely related to its clinical appearance.

To compare the initial and follow-up discernible glaucomatous neuroretinal rim (NRR) and parapapillary chorioretinal changes in patients with primary open-angle glaucoma (POAG) and exfoliative glaucoma (EG), we analyzed 40 optic discs from 40 patients with POAG, 40 optic discs from 40 patients with EG and 20 optic discs from 20 normal subjects. The mean intraocular pressure was higher in the EG group when compared with POAG (p < 0.001). Although the mean disc areas in both groups were not significantly different, the mean intial NRR area to disc area ratio was significantly smaller in patients with EG (p < 0.001). At the initial diagnosis the most prominent NRR defects of the patients with POAG were at the inferotemporal and superotemporal sectors, followed by the temporal and nasal sectors. However, the NRR in the EG group was noticed to decrease diffusely without such a sectorial preference. There was no significant difference between the mean loss of the NRR to disc area ratio in both groups at the end of the follow-up period of 1 year (p < 0.05). The values of parapapillary chorioretinal atrophy areas in POAG and EG patients were not significantly different from each other, neither at the initial examination nor during the follow-up period (p < 0.05). Those results suggest that high intraocular pressure in eyes with EG may constitute a major risk for rapid and progressive glaucomatous optic disc damage.

Objective To examine tissue hypoxia in the retina and optic nerve head of glaucomatouseyes by the assessment of a transcription factor, hypoxia-inducible factor1α (HIF-1α), which is tightly regulated by the cellular oxygenconcentration. Methods Using immunohistochemical analysis, the cellular localization of HIF-1αwas studied in the retina and optic nerve head of 28 human donor eyes withglaucoma compared with 20 control eyes from healthy donors matched for severalcharacteristics. The relationship between the retinal regions that exhibitedimmunostaining for HIF-1α and functional damage was examined using visualfield data. Results There was an increase in the immunostaining for HIF-1α in theretina and optic nerve head of glaucomatous donor eyes compared with the controleyes. In addition, the retinal location of the increased immunostaining forHIF-1α in some of the glaucomatous eyes was closely concordant withthe location of visual field defects recorded in these eyes. Conclusions Because the regions of HIF-1α induction represent the areas ofdecreased oxygen delivery and hypoxic stress, information obtained from thisstudy provides direct evidence that tissue hypoxia is present in the retinaand optic nerve head of glaucomatous eyes, and hypoxic signaling is a likelycomponent of the pathogenic mechanisms of glaucomatous neurodegeneration. Clinical Relevance These findings support the presence of tissue hypoxia in the retinaand optic nerve head of glaucomatous patients.

Reducing intraocular pressure (IOP) in glaucomatous eyes does not always prevent disease progression.To determine the clinical factors associated with progressive optic disc damage in glaucomatous eyes receiving treatment to reduce IOP.Baseline and follow-up optic disc photographs as well as demographic and clinical data were retrospectively studied in 186 eyes of 93 patients with primary open-angle glaucoma, and in 138 eyes of 69 patients with normal-pressure glaucoma. The patients with primary open-angle glaucoma were included in the study only if their treated IOPs during a follow-up period of 5 years were less than 21 mm Hg. The patients with normal-pressure glaucoma were included only if their IOPs were reduced by at least 20% during the follow-up period. The association of progressive optic disc damage with patient- and eye-specific characteristics was examined using multivariate analysis.During the 5-year study period, 141 (43.5%) of the 324 eyes exhibited progressive optic disc damage defined by at least a 5% decrease in the neural rim area-to-disc area ratio. Using multivariate analysis, the following were found to be strongly associated with progressive neural rim damage: a baseline smaller neural rim area-disc area ratio (P.001); a baseline larger zone beta area-disc area ratio (P =.04); a baseline larger parapapillary atrophy length-disc circumference ratio (P =.05); a diagnosis of normal-pressure glaucoma (P =.01); and combined medical and surgical treatment prior to the study period (P =.01).Clinical factors other than IOP may be important indicators of subsequent progression of glaucomatous optic disc damage. Our findings suggest that eyes with advanced glaucomatous optic disc damage and normal-pressure glaucoma are more likely to progress despite receiving treatment to reduce IOP.

Objective To study expression and location of matrix metalloproteinases (MMPs) and tumor necrosis factor α (TNF-α) in glaucomatous optic nerve heads, which are known to be secreted in response to a variety of neuronal injury. Methods Four postmortem eyes from patients with primary open-angle glaucoma, 7 eyes from patients with normal-pressure glaucoma, and 4 eyes from age-matched normal donors were studied by immunohistochemistry. The sections of the optic nerve heads were examined after immunostaining with antibodies to MMPs (MMP-1, MMP-2, and MMP-3), TNF-α, or TNF-α receptor 1. Results The intensity of the immunostaining and the number of stained cells for MMPs, TNF-α, or TNF-α receptor 1 were greater in the glaucomatous optic nerve heads, particularly in eyes with normal-pressure glaucoma compared with age-matched controls. Positive immunostaining was observed in all regions of the glaucomatous optic nerve heads, but most prominently in the postlaminar region. Immunostaining was observed mainly in glial cells and their processes around the axons and blood vessels and in pial septae. Conclusion There is increased immunostaining for MMPs, TNF-α and TNF-α receptor 1 in the glaucomatous optic nerve head, which suggests increased expression of these proteins in glaucoma and thereby implies a role in the tissue remodeling and degenerative changes seen in glaucomatous optic nerve heads. Clinical Relevance The MMPs and TNF-α may be components of astroglial activation that occurs in glaucomatous optic nerve heads. The biological alterations in the expression of these proteins may play a role in the progression of glaucomatous optic neuropathy.

Objective To study the histopathological changes of eyes from a patient with normal-pressure glaucoma whose clinical and laboratory findings were well documented. Methods Postmortem histopathological findings in a patient with normal-pressure glaucoma who had monoclonal gammopathy and serum immunoreactivity to retinal proteins were examined in comparison with those of an age-matched control subject. Clinicopathological correlations to laboratory findings were examined. Results Clinical and histopathological findings of the patient, including cavernous degeneration of optic nerve and characteristic optic nerve cupping, were similar to those in patients with glaucoma who had high intraocular pressure. In addition, we found immunoglobulin G and immonuglobulin A deposition in the ganglion cells, inner and outer nuclear layers of the retina, and evidence of apoptotic retinal cell death using terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling technique. Conclusions Serum antibodies to retinal proteins and retinal immunoglobulin deposition constitute novel findings in a patient with normal-pressure glaucoma and may contribute to better understanding of the mechanisms underlying glaucomatous optic neuropathy in this disorder.

PURPOSE Releasable scleral flap sutures have been effectively used in trabeculectomy. The suggested time for suture removal is within the first two postoperative weeks. The authors wished to determine whether the use of intraoperative antifibrotics alters the time of suture removal and the results of surgery. METHODS The charts of 324 patients (388 eyes) undergoing trabeculectomy and 141 patients (174 eyes) undergoing combined trabeculectomy with cataract extraction with a minimum follow-up of 12 months were retrospectively reviewed. All eyes received intraoperative antifibrotics; mitomycin C in 534 eyes and 5-fluorouracil in 29 eyes. Two releasable scleral flap sutures were used in all of the eyes. RESULTS The removal time of at least one of the two releasable sutures was later than 21 days postoperatively in 89 eyes of 83 patients with trabeculectomy (22.9%) and in 58 eyes of 55 patients with combined surgery (33.3%). The suture removal was deferred in 39 eyes because of an early complication such as hypotony (intraocular pressure (IOP) < or = 5 mm Hg), bleb leak, or shallow or flat anterior chamber. There was no complication in the other 108 eyes, but suture removal was delayed until the IOP was more than 10 mm Hg. Immediate IOP reduction was (mean +/- standard deviation) 6.3 +/- 2.8 mm Hg when suture removal was performed beyond 3 weeks. There was a decreased response as the postoperative time until suture removal increases (r = -0.57). No serious complication associated with late suture removal was noted during the follow-up period (mean +/- standard deviation, 19.8 +/- 10.1 months). CONCLUSION Intraoperative pharmacologic modulation of wound healing in trabeculectomy and combined trabeculectomy with cataract extraction extends the period that releasable suture removal is clinically effective. However the response decreases with a longer interval to releasable suture removal.

Purpose Endothelins (ET) have some effects on the regulation of aqueous humor dynamics. To investigate their possible role in glaucoma, we measured plasma and aqueous humor ET levels in patients with and without primary open-angle glaucoma. Methods Plasma and aqueous humor samples were obtained from 31 patients with primary open-angle glaucoma and 24 patients without glaucoma. Measurements were made by radioimmunoassay (RIA) for ETs with the following cross-reactivities: ET-1, ET-2, and big ET-1, 100%; and ET-3, 70%. Results The ages (mean +/- SD) of the patients with primary open-angle glaucoma (72.3 +/- 10 years) and normal subjects (72.8 +/- 8 years) were similar (p = 0.92). There was no significant difference between plasma ET levels of the two groups (p = 0.07). However, aqueous humor ET levels (mean +/- SD) were higher in the primary open-angle glaucoma group (44.26 +/- 2.6 pg ml-1) than in normal subjects (42.17 +/- 1.6 pg ml-1) (p = 0.001). The ratios of corresponding aqueous humor to plasma levels of ETs were approximately 10% higher in the primary open-angle glaucoma group (3.76) than in normal subjects (3.41) (p = 0.0002). Conclusions The small increase in aqueous humor endothelin levels in patients with primary open-angle glaucoma versus controls of similar age may be relevant to the understanding of the various roles of ETs in aqueous humor dynamics in these patients.