Ceruloplasmin upregulation in retina of murine and human glaucomatous eyes.

PURPOSE: Ceruloplasmin (Cp) expression is increased locally as a response to many neurodegenerative conditions. The purposes of this study were to confirm findings of Cp upregulation in glaucoma, detect the time course of this upregulation in a glaucoma model, and better localize its expression in the retina. METHODS: mRNA and protein were extracted from the retina and brain of DBA/2 and C57BL/6 mice and were subjected to analysis by RT-PCR and immunoblotting. In addition, eyes from the same mouse strains were subjected to immunohistochemistry using antibodies specific for Cp. Eyes from human subjects with or without glaucoma were also subjected to immunohistochemical analysis for Cp. RESULTS: Cp mRNA and Cp protein were upregulated in the retinas of glaucomatous DBA/2 mice. Upregulation of Cp occurred at approximately the time of extensive retinal ganglion cell (RGC) death and increased with increasing age to 15 months in the retinas but not in the brains of these animals. No age-related Cp upregulation was detected in the reference normal mouse strain (C57BL/6), which can develop significant nonglaucomatous RGC loss toward the end of the same time frame. Cp upregulation was also detected in most eyes from the patients with glaucoma. Cp upregulation was localized to the Müller cells within the retinas and in the area of the inner limiting membrane. CONCLUSIONS: Cp is upregulated in the retina of a commonly used glaucoma model (the DBA/2 mouse) and in most human glaucomatous eyes. The timing of this upregulation suggests that it may represent a reactive change of the retina in response to a noxious stimulus or to RGC death. Such Cp upregulation may represent a protective mechanism within the retina.

Vascular changes in the posterior eye segment of secondary angle-closure glaucoma: cause or consequence?

BACKGROUND: To study the role of choroidal and retinal vessels in the pathology of secondary angle-closure glaucoma. METHODS: DBA/2NNia and non-glaucomatous C57BL/6J mice over the age range 2-20 months were investigated. Corrosion cast preparations of the vasculature were studied using scanning electron microscopy. Whole mounts of the retina and choroid were stained enzyme-histochemically for NADPH diaphorase as an indicator for nitric oxide synthase activity. Semi- and ultra-thin sections of the posterior eye segment were performed and evaluated. RESULTS: DBA/2NNia mice showed loss of choroidal pigmentation and a decrease in choriocapillary density already at 4 months of age. In animals 9 months and older, a decrease of choroidal NADPH-diaphorase positive nerve fibers was evident. The retinal vasculature showed only mild changes in NADPH-diaphorase staining, even in the oldest animals. The ultrastructural appearance of the retinal vessels was similar in both mouse strains and for all ages investigated. CONCLUSIONS: Choroidal changes in the DBA/2NNia mouse are similar to that seen in other glaucoma models. The lack of retinal vasculature changes in adult and senescent DBA/2NNia mice suggests a normal blood supply of the retina during the progress of secondary angle-closure glaucoma in these animals.

Complement component 1Q (C1Q) upregulation in retina of murine, primate, and human glaucomatous eyes.

PURPOSE: Complement has been implicated in the pathogenesis of neurodegenerative diseases. The purpose of this study was to investigate whether complement activation is part of the pathogenesis of retinal ganglion cell (RGC) loss in glaucoma. METHODS: mRNA and protein was extracted from the retina and brain of DBA/2 and C57/BL6 mice and subjected to RT-PCR and immunoblot analysis, respectively. In addition, eyes from the same mouse strains were subjected to immunohistochemistry with antibodies specific to complement component 1q (C1q). Eyes from monkeys with unilateral experimental glaucoma were also subjected to immunohistochemical analysis, as were eyes from human subjects with or without glaucoma. RESULTS: C1q mRNA and C1q protein were found to be upregulated in the retina of glaucomatous DBA/2 mice. Upregulation of C1q preceded the time of extensive RGC death and increased with increasing age to 15 months in the retina, but not in the brain. No age-related C1q upregulation was detected in the reference mouse strain (C57BL/6), which develops significant nonglaucomatous RGC loss toward the end of the same time frame. C1q upregulation was also detected in laser-induced glaucomatous monkey eyes and in some (but not all) eyes of patients with glaucoma. C1q upregulation was localized to the Müller cells within the retina and in the area of the inner limiting membrane. CONCLUSIONS: Complement expression is upregulated in the retina of two commonly used glaucoma models (in the DBA/2 mouse and the monkey) and in some human glaucomatous eyes. The timing of this upregulation suggests that complement activation plays a significant role in the pathogenesis of glaucoma.

Optic nerve degeneration in the DBA/2NNia mouse: is the lamina cribrosa important in the development of glaucomatous optic neuropathy?

To study optic nerve (ON) degeneration in the DBA/2NNia (DBA) mouse, a species lacking a lamina cribrosa and a model for secondary angle-closure glaucoma, serial semi- and ultra-thin sectioning of the myelinated ON and of the ON head was performed and sections evaluated qualitatively and quantitatively by light and electron microscopy. Immunohistochemistry was performed using antibodies against collagen type I, III, VI, laminin, and connexin43. The major finding on the myelinated ON was a significant decrease in cross section area during ON degeneration which was paralleled by a loss of axons and an increase in microglia. The number of astrocytes and blood vessels did not change. The major findings on the ON papilla were that ON heads with only mild degeneration showed a pronounced focal degeneration around the central retinal artery. In more severely degenerated ON, newly formed bundles of collagen type VI were located between astrocyte processes within the ON head. In a species that has no lamina cribrosa, DBA mice can develop typical signs of glaucomatous optic neuropathy. The entrance of the central retinal vessels into the ONH seems to be a preferentially vulnerable region for axon loss in this mouse model. In addition, astrocytes in the ON head form extracellular material similar to that found in human glaucomatous eyes.

Characterization of retinal damage in the episcleral vein cauterization rat glaucoma model.

Episcleral vein cauterization (EVC) is used in rats to generate a glaucoma model with high intraocular pressure (IOP). The long-term retinal damage in this glaucoma model, however, has not been accurately quantified. We report the location and amount of retinal ganglion cell (RGC) damage caused by (EVC) induced IOP elevation in two rat strains. IOP was raised in one eye of Wistar (N = 5) and Brown-Norway(B-N)(N = 7) rats by EVC and monitored monthly until IOP in contralateral eyes equalized at 5 months post-surgery. Animals were maintained for 3.5-4.5 additional months. B-N rats (N = 7) that had no EVC served as controls for this strain. Scotopic flash ERGs were recorded at baseline and just prior to euthanasia. Automated counts of all retrogradely labeled RGCs in retinal flat-mounts were determined and compared between contralateral eyes. RGC density maps were constructed and RGC size distribution was determined. Oscillatory potentials in the group of eyes which had elevated IOP were decreased at the time of euthanasia, when IOP had returned to normal. The group of normal B-N rats had similar RGC counts between contralateral eyes. In the experimental group the mean number of RGCs was not significantly different between control and experimental eyes, but 1 of 5 Wistar and 2 of 7 B-N experimental eyes had at least 30% fewer RGCs than contralateral control eyes. Total retinal area in B-N experimental eyes was higher compared to contralateral eyes. Cumulative IOP exposure of the experimental eyes was modestly correlated with RGC loss while oscillatory potentials appeared to be inversely related to RGC loss. In retinas with extensive (> 30% RGC loss) but not complete damage, smaller cells were preserved better than larger ones. The above results indicate that RGC loss in both Wistar and B-N strains is variable after a prolonged elevation of IOP via EVC. Such variability despite equivalent IOP levels and ERG abnormalities, suggests unknown factors that can protect IOP-stressed RGCs. Identification and enhancement of such factors could prove useful for glaucoma therapy.

Neuronal nitric oxide synthase (nNOS) positive retinal amacrine cells are altered in the DBA/2NNia mouse, a murine model for angle-closure glaucoma.

PURPOSE: To characterize retinal amacrine cell changes in eyes of DBA/2NNia (DBA) mice that develop an inherited angle-closure glaucoma. METHODS: DBA and non-glaucomatous C57BL/6J mice of different age groups (2 to 23 months of age) were studied and compared. Morphologic investigations included NADPH-diaphorase staining of retinal whole mounts and fluorescence immunohistochemistry of cryosections with antibodies against neuronal nitric oxide synthase (nNOS), tyrosin hydroxylase (TH), gamma aminobutyric acid (GABA), and vesicular acetylcholine transporter (VAChT). RESULTS: Immunohistochemistry of amacrine cell subpopulations in the retinae of DBA mice revealed no significant staining differences in the two mouse strains at all ages using antibodies against TH, GABA, and VAChT. However, staining with nNOS and NADPH diaphorase revealed significant differences between the DBA strain and the C57BL/6J mice. With the onset of elevated IOP and glaucoma beginning at around 6 months in the DBA mice, the total number of NOS positive amacrine cells continuously decreased from 1000 cells at 6 months of age down to 480 cells in animals older than 20 months of age, but did not decline in age-matched C57 mouse retinas. CONCLUSION: We previously described a parafoveal loss of nNOS positive amacrine cells in the monkey glaucoma model. The fact that there is also a significant decrease of nNOS amacrine cells in the glaucomatous mouse eye indicates a specific response of nNOS positive amacrine cells in glaucomatous retinopathy.

Glutamate-induced glutamine synthetase expression in retinal Muller cells after short-term ocular hypertension in the rat.

PURPOSE: To determine the effect of intraocular pressure (IOP) elevation on glutamate-induced expression of glutamine synthetase (GS) in retinal Müller cells of rat eyes. METHODS: Six groups of three rats each were studied. Group I was a normal control group. In Group II, one eye received an intravitreal glutamate injection (75 nmoles) while the contralateral eye served as control. In Groups III and IV, IOP was raised in one eye by episcleral vein cauterization. Moderately elevated IOP was maintained for 1 day in Group III or 1 week in Group IV (35 +/- 1.9-45 +/- 5.2 mm Hg). An additional two groups of rats received bilateral intravitreal glutamate injections (75 nmoles) immediately (Group V), or 6 days (Group VI), after induction of IOP elevation in one eye. One day after glutamate injection the rats in all groups were killed, and the eyes enucleated and fixed. Retinas from left and right eyes of each animal were embedded together in LR White resin (Ted Pella, Redding, CA). Sections were processed for GS immunolabeling with antibodies to GS by two-stage immunogold labeling with silver enhancement. Images of labeled retinas from the two eyes were captured under identical light microscopic conditions and the GS immunoreactivity in Müller cells was compared between the left and right retinas in the same section by image analysis. An additional five rats were included in Group II and the retinas were analyzed by Western blot analysis to confirm immunohistochemical findings. RESULTS: Similar to the finding in the control group (Group I), the GS immunoreactivity of the left and right eyes of Group III and IV remained unchanged even though the right eyes in the two groups had elevation of IOP lasting for 24 hours and 1 week, respectively. However, GS levels were significantly increased by 40 +/- 5.7% in normotensive eyes 24 hours after intravitreal injection of glutamate (Group II). The rise in GS immunoreactivity was abolished in eyes with acute IOP elevation (Group V). In contrast, when the eyes were exposed to high IOP for 1 week (Group VI), the glutamate-induced increase in GS immunoreactivity was restored. CONCLUSIONS: Elevated levels of vitreal glutamate can increase the expression of GS in retinal Müller cells. This increase was blocked if IOP was acutely elevated for 24 hours but was restored if IOP remained elevated for 1 week. This finding suggests that moderate elevation of IOP causes only short-term functional changes of glutamate metabolism (amidation) by retinal Müller cells. However, it is not known to what extent endogenous extracellular glutamate can regulate GS expression in normal eyes or in eyes with glaucoma.

Aqueous humor dynamics in monkeys after topical 8-iso PGE(2).

PURPOSE: To determine in normotensive cynomolgus monkeys, the effects of topical 8-iso prostaglandin (PG)E(2) on intraocular pressure (IOP), aqueous humor formation (AHF), uveoscleral outflow (Fu), and total and trabecular outflow facility. METHODS: IOP was measured by Goldmann applanation tonometry under ketamine anesthesia after single or twice-daily topical treatments with 8-iso PGE(2). With animals under pentobarbital anesthesia, AHF and flow to blood (equated to trabecular outflow) were determined by anterior chamber perfusion with radioactively labeled albumin solution. Fu and trabecular outflow facility were calculated from these measurements. Total outflow facility was measured by two-level, constant-pressure perfusion. RESULTS: IOP was not significantly changed after single or multiple 10- micro g doses of 8-iso PGE(2). The 25- micro g dose significantly decreased IOP by 2 to 3 mm Hg compared to the contralateral vehicle-treated control 4 to 6 hours after a single dose and by 3 to 5 mm Hg within 1.5 hours after twice-daily treatments for 4 to 5 days. Total outflow facility corrected for control eye washout was increased by an apparent 37% (P < 0.02, n = 7) from 2 to 3.5 hours after the ninth dose, largely due to outlier values obtained in one monkey. Isotope studies performed after twice-daily treatments totaling 9 to 29 doses showed no change in AHF, trabecular outflow facility, or total outflow facility. Relative to AHF, trabecular outflow was significantly decreased, and the calculated Fu was significantly increased when all data were analyzed. CONCLUSIONS: The present findings are consistent with lowering of IOP by 8-iso PGE(2), primarily by increasing Fu. A direct effect on the trabecular meshwork was not indicated by these in vivo studies.

Quantitative analysis of retinal ganglion cell (RGC) loss in aging DBA/2NNia glaucomatous mice: comparison with RGC loss in aging C57/BL6 mice.

PURPOSE: To quantify the extent and pattern of retinal ganglion cell (RGC) loss in the DBA2/NNia glaucomatous mouse strain as a function of age and compare it with ganglion cell loss in a nonglaucomatous strain. METHODS: All the ganglion cells in retinas of DBA/2NNia and C57/BL6 mice of various ages (five eyes per age group in 3-month intervals from 3 to 18 months of age) were counted. A novel counting method that does not rely on sampling and that uses retrograde labeling of RGCs with Fluorogold (Fluorochrome; Englewood, CO) was used. RGC loss in the glaucomatous DBA/2NNia mouse strain was contrasted to RGC loss in C57 mice at the same ages. The total number of Fluorogold-labeled cells per retina was compared within and among the two strains as a function of age. In addition, RGC density maps were constructed for each retina, and the range of densities for each age group was compared within and among the two strains. IOP in awake, nonsedated DBA/2NNia mice was measured with a rebound tonometer. RESULTS: RGC loss started between 12 and 15 months of age in C57 mice and led to an approximate 46% reduction by 18 months of age. The rate of loss was best approximated by a second-order polynomial curve. In comparison, DBA/2NNia mice also began showing RGC loss at approximately 12 months of age, but it proceeded at a much faster rate, with approximately 64% of their RGCs dying by the 15th month of age but little additional loss thereafter. RGC loss in the DBA animals had a focal pattern that appeared more patchy and showed greater variability than the age-related loss in C57 mice, which was more diffuse. IOP and total retinal area in DBA/2NNia mice began to increase at approximately 6 months of age. IOP normalized after the 12th month of age. CONCLUSIONS: Age-related RGC loss of up to 50% can occur in the C57 mouse by 18 months of age. The loss does not proceed linearly with age, as is often assumed, and differs both in extent and locational pattern from pathologic RGC loss secondary to glaucoma in DBA/2NNia mouse retinas.

Method for the noninvasive measurement of intraocular pressure in mice.

PURPOSE: To evaluate the applicability of rebound tonometry for measurement of IOP in the mouse eye. METHODS: An induction-impact (I/I) tonometer, which operates on the rebound principle, was scaled down and adapted to determine IOP of the mouse eye. IOP measurement using this concept is based on contacting the eye with a probe and detecting the motion as the probe collides with the eye and bounces back. The motion parameters of the probe vary according to eye pressure and are used to calculate IOP. A prototype instrument was constructed for measurement of IOP in mouse eyes, and its ability to accurately and reliably measure IOP was tested by comparing the measurements against the manometric (true) IOP determined in cannulated mouse eyes ex vivo. The I/I tonometer was also used to measure IOP in vivo in anesthetized adult C57BL/6 mice. RESULTS: A strong correlation between the true IOP and the I/I measurements (R(2) = 0.95) was found for IOPs in the range of 3.7 to 44.1 mm Hg in cannulated mouse eyes. Repeat determinations in individual eyes showed a low degree of variability in the relationship of the measured IOP with the true IOP. In anesthetized mice, mean IOP +/- SD as determined by rebound tonometry was 9.8 +/- 3.9 mm Hg when the animals were anesthetized with ketamine alone and 7.6 +/- 1.9 mm Hg when a mixture of ketamine, acepromazine, and xylazine was used. Contralateral eyes differed by 0.9 +/- 2.5 and 0.1 +/- 2.7 mm Hg, respectively, for the two anesthetic regimens. CONCLUSIONS: The I/I tonometer can be used for noninvasive, in vivo IOP measurement in mouse eyes. The availability of an easy-to-use, reliable tonometer for IOP measurements in mice will allow more extensive use of the mouse as a model for glaucoma.

Non-invasive determination of intraocular pressure in the rat eye. Comparison of an electronic tonometer (TonoPen), and a rebound (impact probe) tonometer.

BACKGROUND: The reproducibility and accuracy of the induction/impact (I/I) probe device (rebound tonometer) and the TonoPen XL electronic tonometer were compared through the measurement of intraocular pressure (IOP) differences between contralateral rat eyes. METHODS: IOP was measured in 18 female Wistar rats with variable, modest elevations of IOP in one eye. Mean IOP from five measurements on each of the 36 eyes was determined using the rebound tonometer, followed by the TonoPen XL. Following cannulation, true (manometrically determined) IOP was recorded with a calibrated pressure transducer. Differences between the operated and normal eye of each animal for each tonometric method of measurement were correlated with the true difference in IOP. RESULTS: IOP differences between the operated and contralateral normal eye ranged from +5.9 to -1.7 mmHg (mean +1.7 mmHg) when measured by cannulation and from +7.2 to -1.4 mmHg (mean +2.4 mmHg) and +9.8 to -3.2 mmHg (mean +3.6 mmHg) when measured with the rebound tonometer and TonoPen XL respectively. Differences between eyes recorded by rebound tonometer ( y) correlated with those determined by cannulation ( x) in a linear fashion ( y=0.8243 x+1.0721; R(2)=0.6603). Correlation for the TonoPen XL was much weaker ( y=0.8675 x+2.1672; R(2)=0.2077). IOP measurements using the rebound tonometer did not differ significantly from true IOP, whereas TonoPen XL increasingly underestimated IOP with increasing IOP (9-20 mmHg). CONCLUSION: The rebound tonometer displayed greater accuracy and less variability than TonoPen XL in measuring the IOP of the rat eye (range 9-20 mmHg).

Prospects for relevant glaucoma models with retinal ganglion cell damage in the rodent eye.

Retinal ganglion cell (RGC) death is the end result of practically all diseases of the optic nerve, including glaucomatous optic neuropathy. Understanding the factors determining susceptibility of the retina or the optic nerve to glaucomatous damage, and the means to prevent it, requires good animal models. Here we review the different, current models in rodents that have been used to study RGC damage, discuss their value, and their adequacy as models for human glaucoma.

Cytoarchitecture of the retinal ganglion cells in the rat.

PURPOSE: To determine the number and cytoarchitecture of retinal ganglion cells (RGCs) in the female Wistar rat, by using a newly devised procedure for rapid RGC counting in the entire retina that avoids assumptions about RGC spatial arrangement. METHODS: RGCs of normal female Wistar rats were retrogradely labeled with a fluorescent tracer. Automated counting was accomplished by applying standard imaging software to analysis of all labeled cells in retinal flatmounts. The method was validated by comparison of automated and manual counts of 70,000 RGCs in frames covering the density range in the normal rat retina of 600 to 3600 RGC/mm(2). RGC numbers were determined for each retina and compared with the contralateral retina of the same animal. RGC density maps were constructed for each retina. RGC size distribution was determined. RESULTS: Automated RGC counting showed a good linear correlation with manual counting (R(2) = 0.9416). Mean total RGC count in 10 rat eyes was 97,609 +/- 3,930 (SEM) per eye. Contralateral eyes differed by an average of 4.1% (3983 plus minus 5098 RGCs). Size analysis calculated from cell areas confirmed that the majority of rat RGCs are between 7 and 21.5 microm in equivalent diameter. The RGC counts for all frames at the same eccentricity in all 10 of the retinas showed that variability increased with eccentricity and increased further as the fractional area of the retina sampled at each eccentricity was reduced. There was also significant variability in the spatial density of the RGCs at the same eccentricity location between different eyes. Comparison of total RGC counts between left and right eyes estimated from RGC counts in sectors of the retina (hemiretinas or quadrants) showed increased variability compared with counting all the RGCs in a retina. CONCLUSIONS: RGCs in the Wistar rat display significant variability in their cytoarchitecture. Such variability can make quantification by sampling problematic for diffuse, and particularly, for focal RGC losses resulting from experimental interventions, unless virtually the entire RGC population is counted.