Primary open-angle glaucoma.

Objective: To demonstrate optical coherence tomography for high-resolution, noninvasive imaging of the human retina. Optical coherence tomography is a new imaging technique analogous to ultrasound B scan that can provide cross-sectional images of the retina with micrometer-scale resolution. Design: Survey optical coherence tomographic examination of the retina, including the macula and optic nerve head in normal human subjects. Settings Research laboratory. Participants: Convenience sample of normal human subjects. Main Outcome Measures: Correlation of optical coherence retinal tomographs with known normal retinal anatomy. Results: Optical coherence tomographs can discriminate the cross-sectional morphologic features of the fovea and optic disc, the layered structure of the retina, and normal anatomic variations in retinal and retinal nerve fiber layer thicknesses with 10- μm depth resolution. Conclusion: Optical coherence tomography is a potentially useful technique for high depth resolution, cross-sectional examination of the fundus.

Optical Coherence Tomography of the Human Retina

The Gene Expression Omnibus (GEO) database is an international public repository that archives and freely distributes high-throughput gene expression and other functional genomics data sets. Created in 2000 as a worldwide resource for gene expression studies, GEO has evolved with rapidly changing technologies and now accepts high-throughput data for many other data applications, including those that examine genome methylation, chromatin structure, and genome-protein interactions. GEO supports community-derived reporting standards that specify provision of several critical study elements including raw data, processed data, and descriptive metadata. The database not only provides access to data for tens of thousands of studies, but also offers various Web-based tools and strategies that enable users to locate data relevant to their specific interests, as well as to visualize and analyze the data. This chapter includes detailed descriptions of methods to query and download GEO data and use the analysis and visualization tools. The GEO homepage is at http://www.ncbi.nlm.nih.gov/geo/.

The Gene Expression Omnibus Database.

Glaucoma is an optic neuropathy that is characterized by the progressive degeneration of the optic nerve, leading to visual impairment. Glaucoma is the main cause of irreversible blindness worldwide, but typically remains asymptomatic until very severe. Open-angle glaucoma comprises the majority of cases in the United States and western Europe, of which, primary open-angle glaucoma (POAG) is the most common type. By contrast, in China and other Asian countries, angle-closure glaucoma is highly prevalent. These two types of glaucoma are characterized based on the anatomic configuration of the aqueous humour outflow pathway. The pathophysiology of POAG is not well understood, but it is an optic neuropathy that is thought to be associated with intraocular pressure (IOP)-related damage to the optic nerve head and resultant loss of retinal ganglion cells (RGCs). POAG is generally diagnosed during routine eye examination, which includes fundoscopic evaluation and visual field assessment (using perimetry). An increase in IOP, measured by tonometry, is not essential for diagnosis. Management of POAG includes topical drug therapies and surgery to reduce IOP, although new therapies targeting neuroprotection of RGCs and axonal regeneration are under development.

The molecular basis of retinal ganglion cell death in glaucoma.

Understanding mechanisms of pressure-induced optic nerve damage.

Glaucoma is the second leading cause of blindness worldwide, affecting approximately 60 million people.1 Although many risk factors are associated with glaucoma, IOP is the most widely recognized, and lowering IOP is the goal of current glaucoma therapies. When IOP is experimentally elevated in nonhuman primates, the pattern of optic nerve head (ONH) cupping, optic nerve axon degeneration, and selective loss of retinal ganglion cells replicates the pathologic features of human glaucoma. Clinically, human glaucomatous optic neuropathy is characterized by optic disc cupping and a pattern of visual field loss.2 Cupping results from the loss of optic nerve axons and posterior bowing and remodeling of the support structures of the ONH.3,4 Often, these changes are most pronounced in the superior and inferior parts of the nerve head. The most characteristic visual field defect is the arcuate scotoma, which arches above or below central fixation and follows the pathways of the nerve fiber bundles as they converge on the superior and inferior poles of the ONH.2,5,6

Regional variation in laminar structure suggests less support and protection for axons, possibly explaining the apparent increased susceptibility of axons that pass through the superior and inferior ONH. In the face of increased or fluctuating IOP, movement of the lamina may result in preferential mechanical injury to neural tissues in these regions.7 In addition, because the vascular supply to the ONH tissue lies within the laminar beams, this pattern could result in regionally compromised blood flow.8 Current experimental evidence supporting either of these mechanisms is limited, and the cellular events that connect the regional pattern of glaucomatous optic nerve damage with the known structural anatomy of the ONH are still largely unknown. Regardless of mechanism, the variation in the structure of the ONH provides the only anatomic correlation with the characteristic pattern of glaucomatous optic nerve axon loss.

The apparent vulnerability of the ONH to pressure-induced axonal injury has led many investigators to examine changes in the composition of glaucomatous human and experimental monkey ONH tissues. These ONHs are characterized by axon loss, gliotic scaring, increased expression of matrix metalloproteinases, and abnormal deposition of extracellular matrix (ECM) materials, including collagens, tropoelastin, tenascin, and proteoglycans.9–20

In the rat, glaucoma can be modeled by sclerosing aqueous outflow pathways to produce a sustained elevation of IOP.21 In this model, pressure causes a selective loss of retinal ganglion cells and a characteristic pattern of axon degeneration that begins in the superior quadrant of the optic nerve.21,22 In addition, immunohistochemical studies of ONH reveal that pressure-induced injury is accompanied by deposition of collagens and other ECM components, similar to that in human glaucoma.23 This deposition of ECM is preceded by a loss of gap junctional connexin 43 immunolabeling and evidence of astrocytic proliferation.24 By immunohistochemical analysis, the initial evidence of ECM deposition coincided with a decrease in ONH labeling for neurotrophins and astrocytic GFAP.24

Although the ONH is recognized as the likely site of initial injury in human glaucoma and in experimental IOP elevation glaucoma models, very little is known about the changes in gene expression that accompany this injury. In this study, we used microarray analysis to identify the genes and functional gene classes most altered in expression in the rat ONH after exposure to experimentally elevated pressure that results in extensive and ongoing optic nerve degeneration. Then, we used real-time quantitative (q)PCR to verify selected changes in gene expression initially identified by microarray analysis and to examine several genes not included on the arrays. To expand this study, we included qPCR analyses of ONHs from eyes with focal regions of degeneration in optic nerve cross-sections. Focal injury occurs in eyes with more mild pressure elevations or those of shorter duration and probably reflects earlier responses to pressure-induced nerve injury. We compared injury in these ONHs to those in ONHs with more extensive injury due to elevated IOP and to those after optic nerve transection, to evaluate ONH responses to simple loss of axons. The expansion of the qPCR study to nerves with focal injuries allows us to explore the potential of discovering, by future microarray analyses, unique or more dramatic alterations in gene expression that occur early in the injury process. To our knowledge, this is the first genome-wide analysis of expression changes in the ONH in response to elevated IOP.

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Global changes in optic nerve head gene expression after exposure to elevated intraocular pressure in a rat glaucoma model.

PURPOSE. To characterize retinal functional consequences of elevated intraocular pressure (IOP) in a rat model of experimental glaucoma. METHODS. Unilateral elevation of IOP was produced by hypertonic saline injection into an episcleral vein in 20 adult male Brown-Norway rats. IOP was measured in both eyes of awake animals four to five times per week. After 5 weeks, animals were dark adapted overnight (12 hours) and full-field electroretinograms (ERGs) were obtained simultaneously from both eyes. Scotopic ERG stimuli were brief white flashes (‐ 6.64 ‐2.72 log cd-s/m 2 ). Photopic responses were also obtained (0.97‐2.72 log cd-s/m 2 ) after 15 minutes of light adaptation (150 cd/m 2 ). Eyes were processed the following day for histologic evaluation by light microscopy, including masked determination of optic nerve injury grade (ONIG; 1, normal; 5, severe, diffuse damage). RESULTS. Among experimental eyes, the group average IOP (SD) was 34.5 4.1 mm Hg, whereas the average for control eyes was 28.1 0.5 mm Hg (t 7.1, P 0.0001). The average ONIG for experimental and control eye groups, respectively, was 3.4 1.7 and 1.0 0.02 (t 6.3, P 0.0001). The ONIG increased with mean IOP in experimental eyes (r 2 0.78, P 0.0001) and was unrelated to mean IOP in control eyes (r 2 0.09, P 0.18). In experimental eyes with relatively mild IOP elevation (mean IOP 31 mm Hg) and no structural (histologic) damage to the optic nerve evident by light microscopy (ONIG 1.1 0.2, n 5), there was a selective reduction of the positive scotopic threshold response (pSTR; P 0.001), whereas other ERG components remained unaltered. In four of the five eyes, pSTR amplitude was reduced by more than 50%, whereas all five had normal scotopic a-wave, b-wave, and OP amplitudes. Eyes with mean IOP of more than 35 mm Hg had reduced a-wave, b-wave, and oscillatory potential (OP) amplitudes.

Selective Ganglion Cell Functional Loss in Rats with Experimental Glaucoma

PURPOSE To determine the effect of several common general anesthetics on intraocular pressure (IOP) after experimental aqueous outflow obstruction in the rat METHODS A single episcleral vein injection of hypertonic saline was used to sclerose aqueous humor outflow pathways and produce elevated IOP in Brown Norway rats Animals were housed in either standard lighting or a constant low-level light environment Awake IOPs were determined using a TonoPen (Mentor, Norwell, MA) immediately before induction of anesthesia by either isoflurane, ketamine, or a mixture of injectable anesthetics (xylazine, ketamine, and acepromazine) For each anesthetic, IOPs were measured immediately after adequate sedation (time 0) and at 5-minute intervals, up to 20 minutes RESULTS; Awake IOPs ranged from 18 to 52 mm Hg All anesthetics resulted in a statistically significant (P: < 001) reduction in measured IOP at every duration of anesthesia when compared with the corresponding awake IOP With increasing duration of anesthesia, measured IOP decreased approximately linearly for both the anesthetic mixture and isoflurane However, with ketamine, IOP declined to 48% +/- 11% (standard lighting) and 60% +/- 7% (constant light) of awake levels at 5 minutes of anesthesia, where it remained stable In fellow eyes, the SD of the mean IOP in animals under anesthesia was always greater than the corresponding SD of the awake mean Anesthesia's effects in normal eyes and eyes with elevated IOP were indistinguishable CONCLUSIONS All anesthetics resulted in rapid and substantial decreases in IOP in all eyes and increased the interanimal variability in IOPs Measurement of IOP in awake animals provides the most accurate documentation of pressure histories for rat glaucoma model studies

Effect of General Anesthetics on IOP in Rats with Experimental Aqueous Outflow Obstruction

Purpose To determine the diural intraocular pressure (IOP) response of Brown Norway rat eyes after sclerosis of the aqueous humor outflow pathways and its relationship to optic nerve damage. Methods Hypertonic saline was injected into a single episcleral vein in 17 animals and awake IOP measured in both the light and dark phases of the circadian cycle for 34 days. Mean IOP for light and dark phases during the experimental period were compared with the respective pressures of the uninjected fellow eyes. Optic nerve cross sections from each nerve were graded for injury by five independent masked observers. Results For fellow eyes, mean light- and dark-phase IOP was 21 +/- 1 and 31 +/- 1 mm Hg, respectively. For four experimental eyes, mean IOPs for both phases were not altered. Six eyes demonstrated significant mean IOP elevations only during the dark phase. Of these, five showed persistent, large circadian oscillations, and four had partial optic nerve lesions. The remaining seven eyes experienced significant IOP elevations during both phases, and all had extensive optic nerve damage. Conclusions Episcleral vein injection of hypertonic saline is more likely to increase IOP during the dark phase than the light. This is consistent with aqueous outflow obstruction superimposed on a circadian rhythm of aqueous humor production. Because these periodic IOP elevations produced optic nerve lesions, both light- and dark-phase IOP determinations are necessary for accurate correlation of IOP history to optic nerve damage in animals housed in a light- dark environment.

William O. Cepurna

Papers

Understanding mechanisms of pressure-induced optic nerve damage.

Global changes in optic nerve head gene expression after exposure to elevated intraocular pressure in a rat glaucoma model.

Selective Ganglion Cell Functional Loss in Rats with Experimental Glaucoma

Effect of General Anesthetics on IOP in Rats with Experimental Aqueous Outflow Obstruction

Patterns of intraocular pressure elevation after aqueous humor outflow obstruction in rats.