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Objectives: To evaluate the effects of chronic optic nerve ischemia in a nonhuman primate model and to evaluate the regional variability of axonal loss., Methods: Unilateral ischemic optic neuropathy was induced by administration of endothelin-1 to the retrobulbar space via osmotic pumps in 12 primates for 6 to 12 months. The transversely cut sections were stained and divided into 16 regions. Average axonal density in each region was quantified and compared with the untreated contralateral control eyes., Results: Mean axonal density was 208 310/mm(2) and 220 661/mm(2) in treated and control eyes, respectively (P = .03, 1-tailed paired t test), for the entire group. Two-way analysis of variance showed a significant effect of endothelin-1 on overall axonal density for the experimental group (P<.001). Among the nerves with significant axonal loss, the mean axonal loss was 11.6% (4%-21%). Regional mapping of the damage showed the axonal loss varied in the damaged nerves; the damaged regions often clustered within specific quadrants., Conclusion: Chronic ischemia induced by local administration of endothelin-1 causes significant loss of optic nerve axons with varying regional susceptibility. Clinical Relevance Localized damage occurs in other types of optic neuropathy, such as glaucoma, and may result from regional differences in anatomy, metabolism, or vasculature of the primate optic nerve.

While elevated intraocular pressure (IOP) undoubtedly plays a crucial role in many glaucoma patients, vascular dysregulation and chronic regional ischemia are also thought to contribute to the pathophysiology of glaucoma. In an effort to critically evaluate hypotheses involving vascular abnormalities in glaucoma, Cioffi, Van Buskirk and co-workers have developed a model of optic neuropathy based on chronic regional ischemia. The multifocal electroretinogram (MERG) has previously been used to assess function in non-human primates with experimental glaucoma induced by high-IOP. In this study, the MERG was used to monitor function in macaque monkeys with experimental glaucoma induced by chronic anterior optic nerve ischemia. Initial recordings from experimental eyes, which were later documented histologically to have moderate axon loss, revealed little difference from recordings of control eyes. This suggested that many of the signal components in the macaque MERG, which are known (from other studies) to be eliminated by intravitreal injections of NMDA/TTX or by high-IOP experimental glaucoma, may also be affected by the choice of anesthetic agents and MERG recording parameters. Subsequent experiments were performed to specifically evaluate the effects of bipolar versus monopolar signal derivation, anesthetic agents, MERG stimulus design and spatial scale. The results demonstrate that successful measurement of inner retinal and optic nerve head MERG components, especially those which have been shown by other investigators to originate with ganglion cell spiking activity, will depend critically upon the choice of anesthetic agents and recording parameters. One of the most important parameters seems to be use of a monopolar signal derivation, with the contralateral cornea serving as the reference position.

Purpose: Frequency Doubling Technology (FDT) perimetry is a novel perimetric test that provides rapid screening (45 to 60 seconds) and full-threshold (4 to 5 minutes) testing for detection of vision loss. The purpose of this study was to determine the specificity and sensitivity of FDT perimetry for the detection of ocular disease., Methods: A total of 130 participants (257 eyes of 42 men and 88 women) recruited from the community completed FDT perimetry, standard achromatic automated perimetry (SAP), anterior segment biomicroscopy, tonometry, and dilated ophthalmoscopy. FDT results were considered abnormal if 1 point was abnormal (depressed below the 5% level on the screening protocol C-20-5). SAP was considered abnormal if the glaucoma hemifield test or pattern standard deviation was outside normal limits (P < .05) or a hemifield cluster of 3 depressed points on the pattern deviation probability plot (P < .05) was present. An abnormal eye examination was defined as the presence of an abnormality in the anterior segment, lens, or posterior segment that was likely to cause a visual field defect or the presence of glaucomatous or other optic neuropathy., Results: The mean age (+/- SD) of participants was 55.5 years (+/- 10.3). Ethnic groups, as reported by participants, included 77 (59%) African Americans, 40 (31%) Caucasians, and 13 (10%) in other groups. On clinical examination, 116 eyes (45%) were normal, 9 eyes (3.5%) had a cataract with best corrected visual acuity worse than 20/30, 16 eyes (6%) had open-angle glaucoma, and 17 eyes (7%) had retinal findings or lesions that were likely to cause a visual field defect. For FDT perimetry, 22 (8.6%) of 257 tests were unreliable, and for SAP, 65 (25.3%) of 257 tests were unreliable. The sensitivity and specificity of FDT perimetry for detecting an abnormal clinical examination were 55% and 90% and for detecting an abnormal examination that included an abnormal SAP, 64% and 86%., Conclusions: FDP demonstrated reasonable discriminatory power for detecting eye disease.

Purpose: Brimonidine is a highly selective alpha2-adrenoreceptor agonist that lowers intraocular pressure. The aim of the present study was to analyze in vivo the vasomotor effects and the influence of brimonidine on blood flow within the optic nerve, by means of intraluminal microvascular corrosion casting technique and intravascular injection of colored microspheres., Methods: New Zealand white rabbits received either brimonidine tartrate 0.2% or placebo (vehicle) topical drops in one eye for 4 weeks. Intraocular pressures were measured at baseline and 4 weeks. The anterior optic nerve microvasculature of four rabbits was examined with corrosion castings for regions of focal vasoconstriction. Optic nerve blood flow was determined in 16 rabbits by means of nonradioactive colored microspheres., Results: The vasoconstriction values of the short posterior ciliary arterial branches in the brimonidine eyes were 16.7%+/-3.7%. In the fellow untreated eyes, the mean vasoconstriction was 16.6%+/-2.4%. In the placebo-treated eyes, the average constriction was 15.9%+/-3.2%; the fellow eyes showed a mean constriction value of 16.1%+/-5.3%. There was no statistical difference between any of the groups (P = .2). The optic nerve blood flow in the brimonidine-treated rabbits was 0.18+/-0.06 ml/mg/min and 0.17+/-0.04 ml/mg/min in the treated and the fellow eyes, respectively. The difference between the optic nerve blood flow in the brimonidine-treated eyes and the optic nerve blood flow in all of the untreated eyes (0.19+/-0.06 ml/mg/min) also was not statistically different (P = .82)., Conclusions: Long-term application of brimonidine 0.2% does not affect the blood flow or vasomotor activity of the anterior optic nerve.

Purpose: To assess whether smaller targets and a 24-2 stimulus presentation pattern would improve the ability of frequency doubling technology (FDT) perimetry to detect and characterize early glaucomatous visual field loss., Methods: One hundred normal subjects between the ages of 20 and 85 participated in this study. In addition, 53 patients who either had early glaucomatous visual field loss (n = 23) or were high-risk glaucoma suspects with normal conventional visual fields (n = 30) were evaluated with the commercial version of FDT perimetry (full threshold test) with 17 stimuli (four 10 degrees diameter square targets per quadrant and a central 5 degrees circular target) and a custom version of FDT perimetry using 54 stimuli (4 degrees targets with 6 degrees grid spacing) arranged in a 24-2 stimulus presentation pattern., Results: The custom FDT test using a 24-2 stimulus presentation pattern had a similar dynamic range, and demonstrated normal aging characteristics and test-retest reliability that were similar to the commercial version of FDT perimetry using 17 larger stimuli. Both FDT tests showed an age-related sensitivity reduction of approximately 0.6 dB per decade, and exhibited an average test-retest reliability of 1 to 1.5 dB. The custom 24-2 FDT perimetry test had a greater variation of sensitivity with eccentricity than the commercial version of FDT perimetry that was probably related to the difference in stimulus size. The custom 24-2 FDT perimetry test had a greater percentage of abnormal test locations than the commercial FDT test for both early glaucomas and high-risk glaucoma suspects., Conclusions: FDT perimetry can be performed with smaller targets using a presentation pattern that is similar to conventional automated perimetry. In comparison to the commercially available 17 target display, the 24-2 stimulus pattern appears to have modestly higher sensitivity for detection of early glaucomatous loss and provides better characterization of the pattern of visual field loss, but the test takes approximately twice as long.

Purpose: To determine the effect of the cardiac cycle on scanning laser Doppler flowmeter measurements of retinal capillary blood flow in rhesus monkeys and humans., Methods: Multiple scanning laser Doppler flowmetry images of rhesus monkey and human retinal capillary blood flow over a range of heart rates were obtained. Average flow values were determined for the 64 scan lines that compose the two-dimensional flow map. Cutaneous blood flow was measured simultaneously with a laser Doppler flowmeter. The temporal relationships between retinal capillary blood flow, peripheral arterial pulse, and cutaneous blood flow were determined. In addition, human retinal capillary blood flow in a 10 x 10-pixel area during different phases of the cardiac cycle was compared., Results: Regular oscillations in human and rhesus monkey retinal capillary blood flow are evident as alternating bright and dark horizontal bands in scanning laser Doppler flowmetry images. These fluctuations are temporally correlated with cutaneous blood flow. Linear regression of actual vs predicted heart rate based on peaks in retinal capillary flow yielded r = 0.999 in a rhesus monkey and 0.938 in a human. Retinal capillary blood flow in a 10 x 10-pixel area fluctuated as much as 50% depending on the phase of the cardiac cycle., Conclusions: The alternating bright and dark banding pattern observed in scanning laser Doppler flowmetry scans of retinal capillary blood flow is related to the cardiac pulse. The errors introduced by pulse-related fluctuations in retinal capillary blood flow are significant and must be minimized or corrected for accurate and reproducible measurements of ocular hemodynamics.

Malpractice suits related to glaucoma fall into four general categories: failure to make the diagnosis of glaucoma or its progression, complications of glaucoma therapy, iatrogenic (especially steroid) induction of the disease, and treatment intervention beyond the physician's level of skill. The ophthalmologist's knowledge of possible pitfalls and the ability to communicate about risks and reasonable expectations are the basis of a long-term, litigation-free relationship with the glaucoma patient.

Evidence that vascular factors contribute to the pathogenesis and development of glaucomatous optic neuropathy continues to accumulate. A higher than expected prevalence of systemic vascular disorders in individuals with glaucoma has been documented. New sophisticated in vivo analysis techniques, such as ultrasound color Doppler imaging, suggest that decreased blood flow velocity and increased vascular resistance are present in the vessels serving the optic nerve of human subjects with glaucoma, implying the presence of either organic or functional vascular disorders in these individuals. Recognizing that different analysis techniques have led to conflicting observations, experimental models have been developed to provide an additional tool with which to interpret the effects of compromised optic nerve perfusion.

Purpose: Proteoglycans may serve important roles in trabecular meshwork structure or function. Detailed molecular characterization and identification of specific trabecular proteoglycan core proteins has been limited., Methods: Radiolabeled proteoglycans were extracted from cultured human trabecular meshworks and subjected to ion exchange and molecular sieve chromatography. Peaks were subjected to glycosaminoglycan content analysis. Reverse transcription with polymerase chain reaction was used to identify trabecular mRNAs of several common proteoglycan core proteins. Western immunoblots of trabecular extracts were also utilized to identify these proteoglycan core proteins., Results: The proteoglycans elute from ion exchange columns at high salt and molecular sieve column profiles, and they exhibit broad peaks typical of the proteoglycan microheterogeneity seen in other tissues. The four common glycosaminoglycan side-chains were identified on these proteoglycans. Trabecular cells in organ or cell culture contain mRNAs coding for decorin, biglycan, versican, perlecan and a basement membrane glycoprotein, SPARC. Syndecan-1 transcripts were present at very low levels, while aggrecan transcripts were not detectable. Decorin, biglycan, versican and perlecan core proteins were also identified by immunoblots of trabecular cell extracts., Conclusions: Several common proteoglycans are expressed by trabecular cells in organ explant or cell culture. Their general characteristics are not unlike those found in other tissues. These proteoglycans may serve important functions in the trabecular outflow pathway.

Background: The influence of the contour line alignment software algorithm on the variability of the Heidelberg Retina Tomograph (HRT) parameters remains unclear., Methods: Nine discrete topographic images were acquired with the HRT from the right eye in six healthy, emmetropic subjects. The variability of topometric data obtained from the same topographic image, analyzed within different samples of images, was evaluated. A total of four mean topographic images was computed for each subject from: all nine discrete images (A), the first six of those images (B), the last six of those nine images (C), and the first three combined with the last three images (D). A contour line was computed on the mean topographic image generated from the nine discrete topographic images (A). This contour line was then applied to the three other mean topographic images (B, C, and D), using the contour line alignment in the HRT software. Subsequently, the contour line on the mean topographic images was applied to each of the discrete members of the particular images subsets used to compute the mean topographic image, and the topometric data for these discrete topographic images was computed successively for each subset. Prior to processing each subset, the contour line on the discrete topographic images was deleted. This strategy provided a total of three analyses on each discrete topographic image: as a member of the nine images (mean topographic image A), and as a member of two subsets of images (mean topographic image B, C, and/or D). The coefficient of variation (100 x SD/mean) of the topographic parameters within those three analyses was calculated for each discrete topographic image in each subject ("intraimage" coefficient of variation). In addition, a coefficient of variation between the nine discrete topographic images ("interimage" coefficient of variation) was calculated., Results: The "intraimage" and "interimage" variability for the various topographic parameters ranged between 0.03% and 3.10% and between 0.03% and 24.07% respectively. The "intraimage" coefficients of variation and "interimage" coefficients of variation correlated significant (r2 = 0.77; P < 0.0001)., Conclusion: A high "intraimage" variability, i.e. a high variability in contour line alignment between sequential images, might be an important source of test re-test variability between sequential images.

Purpose: To evaluate blood flow reduction and topographic optic nerve changes after the local administration of endothelin-1 in vivo, delivered to the perineural region of the anterior optic nerve in the rabbit., Methods: Endothelin-1 (five rabbits) in a dosage of 0.1 microgram/day or balanced salt solution (two rabbits) was delivered to the perineural region of the anterior optic nerve with osmotically driven minipumps. Optic nerve blood flow was determined by the colored microspheres technique after 14 days of local endothelin-1 or balanced salt solution administration to the microvasculature of the optic nerve. In addition, optic nerve blood flow was determined in two rabbits that had no minipump implants. The morphologic changes induced by reduction of blood flow were assessed in five additional rabbits implanted with osmotically driven minipumps containing endothelin-1 (0.1 microgram/day). These rabbits were observed for 8 weeks, and the morphologic optic nerve changes were monitored with a confocal scanning laser ophthalmoscope., Results: Independent of intraocular pressure, endothelin-1 induced a decrease in blood flow of approximately 38% in the experimental eye, compared to the decrease induced by balanced salt solution or to the decrease in rabbits without minipumps (analysis of covariance, P = 0.0092). Multivariate statistical analysis showed a significant change in topometric parameters (cup area, cup depth, rim volume) obtained with a confocal scanning laser ophthalmoscope, indicating an increase in optic nerve cupping and a decrease of the perineural rim volume in the experimental eyes (P = 0.017)., Conclusions: The current results suggest that morphologic optic nerve alterations can be induced experimentally in the rabbit model after ischemia produced by the local administration of endothelin-1 to the perineural region of the anterior optic nerve.

Purpose: The purpose of this study was to evaluate flow reduction to the optic nerve after chronic administration of endothelin-1 in primates., Materials and Methods: Endothelin-1 (three rhesus monkeys), in a dosage of 0.1 microgram/day, or balanced salt solution (three rhesus monkeys) was delivered to the perineural region of the anterior optic nerve via osmotically driven minipumps. Optic nerve blood flow was determined by means of a colored microspheres technique after 7 days of local endothelin-1 or balanced salt solution administration. The effect of endothelin-1 on optic nerve blood flow was analyzed by analysis of convariance (ANCOVA) in a 2 (between groups: endothelin-1, balanced salt solution) x 2 (within subject: blood flow minipump optic nerve, blood flow controls) design, with intraocular pressure as a changing covariate., Results: The decrease of optic nerve blood flow in the endothelin-1 eyes was significant compared to the balanced salt solution eyes (ANCOVA p = 0.015). Among the monkeys implanted with endothelin-1 minipumps, the decrease in optic nerve blood flow in the experimental eye compared to the contralateral eye (mean +/- SD 35.7 +/- 9.1%) was significant (p = 0.01), while that among the monkeys implanted with balanced salt solution minipumps (mean +/- SD 0.7 +/- 5.5%) was not (p = 0.73)., Conclusions: This new primate model of chronic optic nerve ischemia may represent a method to evaluate experimentally the implication of a local hemodynamic perturbation in various optic neuropathies.

Purpose: The mechanism by which laser trabeculoplasty reduces elevated intraocular pressure in primary open-angle glaucoma has been established. To test the hypothesis that trabecular extracellular matrix turnover is involved, stromelysin expression after laser treatment of anterior segment organ cultures was evaluated., Methods: Argon laser trabeculoplasty, using typical clinical treatment parameters, was applied to anterior segment organ cultures. Stromelysin levels and activity were then evaluated at various times by immunoblots of Western transfers and by zymography. Stromelysin mRNA levels were evaluated by dot blot and by reverse transcription, followed by polymerase chain reaction amplification. Stromelysin protein was localized by immunohistochemistry, and image analysis was used for quantitation. Stromelysin mRNA was localized by in situ hybridization., Results: Trabecular stromelysin protein, activity, and mRNA levels were detectably elevated by 8 hours and were several-fold higher by 24 hours after treatment. Stromelysin immunostaining was elevated dramatically in the juxtacanalicular and insert regions of the meshwork, but only modestly in other regions. Stromelysin mRNA increases also were localized primarily to these regions. The juxtacanalicular stromelysin immunostaining increase was sustained for at least 1 week, whereas the insert levels declined somewhat after day 2., Conclusions: A stromelysin increase, localized primarily to the juxtacanalicular region of the meshwork, the putative site of the aqueous humor outflow resistance, should degrade trabecular proteoglycans, the putative outflow resistance source, and allow their uptake and further degradation by the juxtacanalicular cells. If diminished juxtacanalicular extracellular matrix turnover is responsible for the glaucomatous reduction in aqueous humor outflow, an increase in stromelysin in this specific area of the meshwork should ameliorate the problem. Thus, the observations support the working hypothesis and may explain the efficacy of this treatment for glaucoma.

The objective of this study was to establish a nonradioactive technique to accurately assess blood flow in a small tissue such as the anterior optic nerve. Colored microspheres, 10.2 +/- 0.23 microns in diameter, were injected into the left atrium in each of 17 anesthetized rabbits. The rabbits were divided into four groups injected, respectively, with 5, 10, 50, or 100 million microspheres. Microsphere quantification in the tissue was performed after postmortem dissection and alkaline corrosion of the anterior optic nerve. Blood flow was evaluated by means of reference sample comparison method. While the relative interocular difference (range, 15.3-162.8%) was not significantly different between the rabbits injected with 5, 10, or 50 million microspheres (Kruskal-Wallis test, P = 0.11), injection with 100 million microspheres yielded a significantly lower relative interocular differences (range 2.3-12.8%) compared to the other three groups (Kruskal-Wallis test, P = 0.0048). In addition, reproducibility of microsphere counts was evaluated by ranking the difference between right and left optic nerve in percentage of the average number of microspheres per milligram of tissue in both optic nerves. The correlation between the relative interocular difference (range, 2.3-162.8%) and the average number of microspheres per milligram of tissue (1.2-78.7 microspheres/mg optic nerve tissue) was statistically significant (Spearman R, -0.90; P < 0.0001). The interocular variability in microsphere counts and interocular difference in intraocular pressure did not correlate (Spearman R, 0.33; P = 0.20). The rabbits injected with 100 million microspheres showed the highest average number of microspheres (range, 31.8-78.7 microspheres/mg optic nerve tissue). The optic nerve blood flow ranged between 0.14 and 0.24 microliters/mg/min among the rabbits injected with 100 million microspheres. The present experimental technique of optic nerve blood flow measurement is relatively inexpensive, highly reproducible, and obviates disposal of radioactive materials.

Objective: To determine in a two-part study whether misalignment between the patient and the laser scanner is a major source of variability with the Heidelberg Retina Tomograph (Heidelberg [Germany] Engineering)., Methods: Three topographic images of the right optic nerve were acquired with the Heidelberg Retina Tomograph in eight patients with glaucoma. The correlations between average cup volume, variability of cup volume estimates, and direction of imaging were evaluated. Furthermore, the correlations between average rim volume, variability of rim volume estimates, and direction of imaging were evaluated. Next, the optic nerve cup volume and rim volume estimates of a rabbit's left eye were compared between three series of five topographic images acquired from three slightly different directions., Results: Average cup volume, variability of cup volume estimates, and variability in the direction of imaging correlated significantly among the patients with glaucoma (multiple R2 = .95; P < .001). Average rim volume, variability of rim volume estimates, variability in the direction of imaging, and variability in the mean height of the contour line also correlated significantly (multiple R2 = .88; P = .03). In the rabbit eye, the cup volume and the rim volume differed significantly among the three image series (analysis of variance, P < .001 and P = .04, respectively)., Conclusion: Misalignment between the patient and the laser scanner may account for significant variability with the Heidelberg Retina Tomograph.

Aims: To examine, in vivo, the anterior optic nerve vasomotor effects of chronic apraclonidine hydrochloride in rabbits., Methods: After local treatment in one randomly chosen eye with apraclonidine hydrochloride 0.5% over 21 days, the microvasculature of the optic nerve was examined in five rabbits using an intraluminal microvascular corrosion casting technique. The investigators were masked as to which eye was treated. The vasoconstriction near the branching point of arterioles supplying the optic nerve was calculated as a percentage of the downstream vessel calibre. An average constriction was calculated and compared between the treated and the contralateral, untreated, eyes by means of a two tailed t test for paired variables. Constriction values of a total of 72 arterioles supplying the optic nerve were obtained for the five rabbits., Results: The average constriction in the treated and the control eyes was comparable (p = 0.96)., Conclusion: Chronic administration of apraclonidine hydrochloride 0.5% produces no observable optic nerve vasomotor effects in the rabbit eye.