Faculty Bibliography

PURPOSE: To correlate aqueous humor soluble CD44 (sCD44) concentration, visual field loss, and glaucoma risk factors in primary open-angle glaucoma (POAG) patients. METHODS: Aqueous samples were obtained by paracentesis from normal and glaucoma patients who were undergoing elective surgery and analyzed for sCD44 concentration by enzyme-linked immunosorbent assay. RESULTS: In normal aqueous (n=124) the sCD44 concentration was 5.88+/-0.27 ng/mL, whereas in POAG aqueous (n=90) the sCD44 concentration was 12.76+/-0.66 ng/mL, a 2.2-fold increase (P<0.000001). In POAG patients with prior successful filtration surgery (n=13), the sCD44 concentration was decreased by 43% to 7.32+/-1.44 (P=0.001) in comparison with POAG patients without filtration surgery; however, the sCD44 concentration in the prior successful filtration subgroup with no medications and normal intraocular pressure was 12.62+/-3.81 (P=0.05) compared with normal. The sCD44 concentration of normal pressure glaucoma patients was 9.19+/-1.75 ng/mL, a 1.6-fold increase compared with normal (P=0.02). Race and intraocular pressure pulse amplitude were significant POAG risk factors in this cohort of patients. In both normal and POAG patients with mild and moderate visual field loss, sCD44 concentration was greater in African Americans than in whites (P=0.04). CONCLUSIONS: sCD44 concentration in the aqueous of POAG patients correlated with the severity of visual field loss in all stages in white patients and in mild to moderate stages in African American patients. sCD44 concentration in aqueous is a possible protein biomarker of visual field loss in POAG

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

PURPOSE: To report two cases of iris neovascularization associated with systemic cryoglobulinemia. DESIGN: Retrospective case report. METHODS: Patient chart review and review of literature. RESULTS: Two patients with iris neovascularization in the absence of retinal ischemia were subsequently found to have systemic cryoglobulinemia. Successful treatment of one patient's underlying lymphoma led to stabilization and resolution of neovascularization. CONCLUSIONS: Systemic cryoglobulinemia may be associated with anterior segment ischemia and neovascularization, and should be considered in the differential diagnosis of iris neovascularization in the absence of apparent retinal ischemia.

PURPOSE: Glaucoma is characterized by a progressive loss of retinal ganglion cells that results in a characteristic optic neuropathy associated with visual field loss. In previous studies, changes in the antibody profiles have been shown in the sera of patients with glaucoma, and these findings suggest a role for autoimmune involvement in the pathogenesis of glaucoma in some patients. The purpose of this study was to compare the antibody profiles against optic nerve antigens in patients with glaucoma in two different study populations from Germany and the United States. METHODS: One hundred twenty patients were included in the study, 60 from Germany and 60 from the United States: a control group (CTRL, n = 20), a group of patients with primary open-angle glaucoma (POAG, n = 20), and one group of patients with normal-pressure glaucoma (NPG, n = 20) from each country. Western blot analyses against bovine optic nerve antigens were used to detect the IgG antibody patterns present in the patients' sera. The complex antibody profiles were analyzed by multivariate statistical techniques. RESULTS: Complex IgG autoantibody repertoires were present in all patients with glaucoma as well as healthy subjects from both the German and the United States study population. A large similarity between all antibody profiles in both study populations was demonstrated in the number and frequency of both up- and downregulation of antibody reactivities in patients with glaucoma of both national cohorts. The multivariate analysis of discriminance found a significant difference between the glaucoma groups and healthy subjects against optic nerve antigens. As in previous studies, the NPG group revealed the highest variance from the control group (P < 0.01). Furthermore, a newly described antibody biomarker in both study populations was identified as alpha-fodrin. Western blot results revealed that there was an increased frequency and enhanced immunoreactivity to alpha-fodrin (120 kDa) in the sera of patients with NPG. The presence of alpha-fodrin autoantibodies were confirmed by ELISA, in which a highly elevated anti-alpha-fodrin titer in patients with NPG was found to be significantly greater than in the control subjects (P < 0.01) or age-matched patients with POAG (P < 0.04). CONCLUSIONS: Complex IgG antibody patterns against optic nerve antigens can be reproducibly identified in the serum of study populations from the United States and Germany. In both cohorts, patients with glaucoma have characteristic differences in serum autoantibody repertoires from those in control subjects. A newly described autoantibody to alpha-fodrin found in other neurodegenerative diseases such as Alzheimer's, further implicate a role for autoimmunity and the neurodegenerative processes in glaucoma. The high correspondence of the autoantibody patterns found in the study populations from different continents provides further evidence that serum autoantibody patterns may be useful biomarkers for glaucoma detection or for determining prognosis in future studies by means of pattern-matching algorithms.

Purpose: Corneal wound healing was compared with topical ocular applications of moxifloxacin ophthalmic solution 0.5%, gatifloxacin ophthalmic solution 0.3%, or BSS Sterile Irrigating Solution after a 6-mm penetrating corneal incision during a 1-week observation period or after creation of a corneal flap during a 3-week observation period. Methods: Fifteen pigmented rabbits were randomized into 3 test groups of 5 rabbits per group (group 1, moxifloxacin 0.5%; group 2, gatifloxacin 0.3%; group 3, BSS Sterile Irrigating Solution). All animals underwent surgery using a microkeratome to create a corneal flap approximately 8.5 mm in diameter and 160 mu m deep in the right eye (OD). The animals were dosed 4 times a day with the appropriate test article for 7 days after surgery. Biomicroscopic examinations and postmortem histopathology were conducted to compare the different treatments. In a second study, 9 pigmented rabbits were divided into 3 test groups of 3 rabbits per group. Each animal underwent bilateral surgery to create a 6-mm linear, penetrating incision into the central cornea, using a diamond keratome. The wound was subsequently sutured, and the animals were treated bilaterally (topical ocular) for 7 days with moxifloxacin (3 times a day), gatifloxacin (4 times a day), or BSS Sterile Irrigating Solution (4 times a day). On day 7, the left eye (OS) of each animal was processed and analyzed using standard hematoxylin-eosin histopathology, whereas the right eye of each animal underwent processing and analysis using scanning and transmission electron microscopy. Corneas were examined to evaluate wound healing progress, including the epithelial plug, cellular infiltrates, and stromal precipitates. Results: For both the corneal flap and linear incision studies, no appreciable differences were noted between groups with regard to slit-lamp or histopathologic examinations. In the linear incision study, electron microscopy revealed normal remodeling processes in all treatment groups, with no evidence of abnormal electron densities of the basal epithelial cells, basement membrane, anchoring filaments, collagen fibrils, extracellular matrix, or stromal keratocytes. Conclusions: Results showed that moxifloxacin and gatifloxacin were not significantly different from BSS in their effects on corneal wound healing and corneal haze after creation of both superficial and penetrating corneal wounds.

PURPOSE: The present study evaluated the applicability of a rebound tonometer in measuring intraocular pressure (IOP) in rats and mice. METHODS: The accuracy of the TonoLab rebound tonometer was determined in cannulated mouse and rat eyes. IOP was manipulated by changing reservoir height, and tonometer pressure readings were recorded by an independent observer. IOP values were recorded in conscious Wistar rats and in four different strains of mice. The effects of anesthesia on IOP were evaluated in two different strains of mice. RESULTS: The IOP readings generated by the rebound tonometer correlated very well with the actual pressure in the eye. In rats, this linear correlation had a slope of 0.96 +/- 0.05 (mean +/- SEM, n = 4) and a Y-intercept of -2.1 +/- 1.2. In mice, the slope was 0.99 +/- 0.05 (n = 3), and the Y-intercept was 0.8 +/- 1.4. Using this method, the resting IOP of conscious male Wistar rats was observed to be 18.4 +/- 0.1 mm Hg (n = 132). In mice, strain differences in IOP were detected. Baseline IOP values in Balb/c, C57-BL/6, CBA, and 11- to 12-month-old DBA/2J mice were 10.6 +/- 0.6, 13.3 +/- 0.3, 16.4 +/- 0.3, and 19.3 +/- 0.4 mm Hg (n = 12), respectively. In separated studies, anesthesia lowered IOP from 14.3 +/- 0.9 to 9.2 +/- 0.5 mm Hg (n = 8) in C57-BL/6 mice, and from 16.6 +/- 0.4 to 9.4 +/- 0.6 mm Hg (n = 10) in CBA mice. CONCLUSIONS: The rebound tonometer was easy to use and accurately measured IOP in rats and mice. This technique, together with advances in genetic and other biological studies in rodents, will be valuable in the further understanding of the etiology and pathology of glaucoma.

BACKGROUND: Over the past decade, several new medical therapies have become available for the treatment of primary open-angle glaucoma (POAG). A systematic evidence-based approach for identifying an optimal therapeutic agent is lacking. OBJECTIVES: The aims of this review were to critically evaluate published treatment recommendations for POAG and, based on a systematic review of the literature, to develop criteria that would define a 'gold standard' medical therapy that reflects new treatment advances and established therapeutic goals. METHODS: A MEDLINE search spanning the years 1966 to 2002 and using the search terms gold standard, drug of choice, agent of choice, benchmark, ophthalmology, eye, and glaucoma was conducted and the results reviewed by a panel of 15 experts in the field of glaucoma. Published treatment recommendations for POAG were discussed. Criteria, anchored to medical evidence, for distinguishing a standard of medical therapy for POAG were defined. RESULTS: The terms connoting a gold standard therapy were found in only 258 of approximately 368,000 ophthalmology-related citations and 53 of almost 23,000 glaucoma citations, validating the need to define therapeutic standards. The lack of recommendations for the use of new classes of ocular hypotensive agents was acknowledged. Criteria identified to evaluate intraocular pressure (IOP)-lowering agents as gold standards included the following: efficacy in reducing IOP consistently over a 24-hour period to a level that will preserve the visual field and protect the optic nerve without inducing tachyphylaxis and tolerance, paucity of local and systemic adverse effects, promotion of patient compliance, and applicability in diverse patient populations. CONCLUSIONS: These criteria should be employed as measures for evidence-based analyses to evaluate available and future IOP-lowering medical therapies for POAG. The conceptual framework presented may be applicable to other therapeutic areas

PURPOSE: To determine whether bimatoprost is hydrolyzed to its free acid after topical application in humans in vivo. DESIGN: Prospective, masked, and vehicle controlled. PARTICIPANTS: Thirty-one eyes of 31 patients with cataracts. METHODS: Beginning 7 days before scheduled cataract surgery, one eye of each patient was treated with bimatoprost 0.03% or vehicle once daily, with the last drop administered 2 to 12 hours before anterior chamber paracentesis before cataract surgery. In a masked fashion, aqueous humor specimens were assayed for bimatoprost and its free acid by high-pressure liquid chromatography and mass spectrometry. MAIN OUTCOME MEASURE: Detection of the free acid of bimatoprost in aqueous humor. RESULTS: Aqueous humor concentrations of the free acid of bimatoprost were 22.0+/-7.0 nmol/l (mean +/- standard error of the mean, n = 12) and 7.0+/-4.6 nmol/l (n = 8) at 2 and 12 hours, respectively, and below the limit of detection after vehicle (n = 10). Concentrations of bimatoprost (amide) were 5.7+/-1.4 and 1.1+/-0.4 nmol/l at 2 and 12 hours, respectively, and undetectable after vehicle. CONCLUSION: After topical application of bimatoprost in humans, a sufficient concentration of its free acid, a potent FPprostanoid receptor agonist, is found in the aqueous humor to account for its ability to reduce intraocular pressure.

OBJECTIVE: To examine tissue hypoxia in the retina and optic nerve head of glaucomatous eyes by the assessment of a transcription factor, hypoxia-inducible factor 1alpha (HIF-1alpha), which is tightly regulated by the cellular oxygen concentration. METHODS: Using immunohistochemical analysis, the cellular localization of HIF-1alpha was studied in the retina and optic nerve head of 28 human donor eyes with glaucoma compared with 20 control eyes from healthy donors matched for several characteristics. The relationship between the retinal regions that exhibited immunostaining for HIF-1alpha and functional damage was examined using visual field data. RESULTS: There was an increase in the immunostaining for HIF-1alpha in the retina and optic nerve head of glaucomatous donor eyes compared with the control eyes. In addition, the retinal location of the increased immunostaining for HIF-1alpha in some of the glaucomatous eyes was closely concordant with the location of visual field defects recorded in these eyes. CONCLUSIONS: Because the regions of HIF-1alpha induction represent the areas of decreased oxygen delivery and hypoxic stress, information obtained from this study provides direct evidence that tissue hypoxia is present in the retina and optic nerve head of glaucomatous eyes, and hypoxic signaling is a likely component of the pathogenic mechanisms of glaucomatous neurodegeneration. Clinical Relevance These findings support the presence of tissue hypoxia in the retina and optic nerve head of glaucomatous patients.

To evaluate the neuroprotective potential of drug candidates to treat human glaucoma, a short-term rodent model of retinal ganglion cell death was employed. Transient ischemia applied to the rodent retina, with subsequent reperfusion for 1-4 weeks, produces an experimental retinal ganglion cell death that is quantifiable. A widely used method to detect viable retinal ganglion cells involves surgical injection of labeling compounds into the superior colliculus of the rodent brain, the retrograde transport of the compounds along the axons to the retina, and subsequent microscopic evaluation of the retina. In order to circumvent the labor intensive and invasive surgery of this method, we sought an alternative means of assessing retinal ganglion cell survival that would be more suitable for high-throughput analysis. We therefore developed a method of immunolabeling whole retinas ex vivo with an antibody to Brn-3b, an antigen expressed in a subpopulation of retinal ganglion cells, that allows for detection of a representative retinal ganglion cell population. Fluorescently tagged Brn-3b immunolabeled retinas were flat-mounted, digitally imaged, and assessed using image analysis software. We determined that 60 min of ischemia caused a 49% and a 32% decrease in Brn-3b positive retinal ganglion cells in Lewis rats after 4 weeks reperfusion, and Sprague-Dawley rats after 2 weeks reperfusion, respectively. In Swiss Webster ND4 mouse retinas subjected to 45 min ischemia and 7 days reperfusion, we found a 70% decrease in Brn-3b positive cells. Thus, ex vivo immunolabeling of retinal ganglion cells using antibody to Brn-3b provides an alternative to other methods of quantifying retinal ganglion cells.