Changes in gene expression in experimental glaucoma and optic nerve transection: the equilibrium between protective and detrimental mechanisms.

PURPOSE: The authors studied retinal gene expression changes in rats after experimental intraocular pressure elevation and optic nerve transection to elucidate molecular mechanisms of retinal ganglion cell (RGC) death. METHODS: Translimbal laser photocoagulation was used to induce unilateral IOP elevation in 41 albino Wistar rats. In 38 additional animals, unilateral transection of the optic nerve was performed. Retinas were harvested 1 day, 3 days, 1 week, 2 weeks, 4 weeks, and 8 weeks after each treatment, and total RNA was isolated. Pooled RNA from each time point was analyzed with rat genome arrays. Array results were confirmed by real-time PCR, and localization studies were performed using in situ hybridization for select genes. RESULTS: Genes that were upregulated in glaucoma, but not after transection, included Cyclin D2, Stat1, Stat3, c-Fos, Junb, Anxa1, Anxa 3, and CCAAT/enhancer binding protein (Cebp-delta). In glaucoma and transection models, the upregulation of c-Jun, Activating transcription factor 3, Heat shock protein 27, and Timp1 were observed. Comparisons among microarray databases were performed between our data and reports of retinal and optic nerve injury models in mice, rats, and monkeys. CONCLUSIONS: Gene expression changes specific to experimental glaucoma injury were identified. The present analysis supports the importance of neuroinflammation and the participation of the tumor necrosis factor alpha signaling pathway in glaucoma injury. The alterations observed include processes that are both protective of and detrimental to the survival of RGCs.

Characteristics of progenitor cells derived from adult ciliary body in mouse, rat, and human eyes.

PURPOSE: To isolate and characterize progenitor cells derived from adult mammalian ciliary body. METHODS: The authors isolated progenitor cells from the ciliary body of adult mice, rats, and human cadaver eyes and determined quantitative growth characteristics of groups of progenitor cells called neurosphere (NS) cells, including individual cell diameter, NS diameter, percentage of NS-forming cells, and cell number per eye in mouse, rat, and human eyes. The immunolabeling and ultrastructure of NS cells were investigated by confocal and transmission electron microscopy. RESULTS: Average diameters of individual cells and neurospheres after 1 week in culture were similar in mice, rats, and humans (cell diameters: 22 +/- 1.1, 21 +/- 0.3, 25 +/- 0.4 mum; NS diameters: 139 +/- 22, 137 +/- 9, 141 +/- 11 mum, respectively). Mean numbers of cells per NS were estimated to be 1183 in mice, 5360 in rats, and 685 in humans. Molecules that were identified by immunolabeling in NS cells included nestin, Chx-10, vimentin, GFAP, and Pax-6. Thy-1 was expressed in some NS cells. Ultrastructurally, NS cells displayed abundant rough endoplasmic reticulum and many cellular processes but no characteristics of mature retinal neurons or glia. CONCLUSIONS: Progenitor cells from adult mammalian ciliary body have significant, but limited, proliferation potential and express markers characteristic of other progenitor cells and seen during early retinal development. The ciliary body could be a source of cells for transplantation in experimental rodent eyes and for autotransplantation in human eyes.

Optic nerve dynein motor protein distribution changes with intraocular pressure elevation in a rat model of glaucoma.

Acute intraocular pressure (IOP) elevation causes accumulation of retrogradely-transported brain derived neurotrophic factor and its receptor at the optic nerve head (ONH) in rats and monkeys. Obstruction of axonal transport may therefore be involved in glaucoma pathogenesis, but it is unknown if obstruction is specific to certain transported factors or represents a generalized failure of retrograde axonal transport. The dynein motor complex mediates retrograde axonal transport in retinal ganglion cells (RGC). Our hypothesis was that elevated IOP interferes with dynein-mediated axonal transport. We studied the distribution of dynein subunits in the retina and optic nerve after acute and chronic experimental IOP elevation in the rat. IOP was elevated unilaterally in 54 rats. Dynein subunit distribution was compared in treated and control eyes by immunohistochemistry and Western blotting at 1 day (n=12), 3 days (n=4), 1 week (n=15), 2 weeks (n=12) and 4 weeks (n=11). For immunohistochemistry, sections through the ONH were probed with an anti-dynein heavy chain (HC) antibody and graded semi-quantitatively by masked observers. Other freshly enucleated eyes were microdissected for separate Western blot quantification of dynein intermediate complex (IC) in myelinated and unmyelinated optic nerve, ONH and retina. Immunohistochemistry showed accumulation of dynein HC at the ONH in IOP elevation eyes compared to controls (P<0.001, Wilcoxon paired sign-rank test, n=29). ONH dynein IC was elevated by 46.5% in chronic IOP elevation eyes compared to controls by Western blotting (P<0.001, 95% CI=25.9% to 67.8%, n=17). The maximum increase in ONH dynein IC was 78.7% after 1 week (P<0.05, n=5), but significant increases were also detected after 4 h and 4 weeks of IOP elevation (P<0.05, n=4 rats per group). Total retinal dynein IC was increased by 8.7% in chronic IOP elevation eyes compared to controls (P<0.03, 95% CI 1.4% to 16.1%, n=24). In the retina, IOP elevation particularly affected the 72 kD subunit of dynein IC, which was 100.7% higher in chronic IOP elevation eyes compared to controls (P<0.00001, 95% CI 71.0% to 130.4%, n=21). Dynein IC changes in myelinated and unmyelinated optic nerve were not significant (P>0.05). We conclude that dynein accumulates at the ONH with experimental IOP elevation in the rat, supporting the hypothesis that disrupted axonal transport in RGC may be involved in the pathogenesis of glaucoma. The effect of IOP elevation on other motor proteins deserves further investigation in the future.

The transcription factor c-jun is activated in retinal ganglion cells in experimental rat glaucoma.

This study investigates the role of the MAP kinase pathway including c-jun, ATF-2 and JNK in glaucomatous eyes of rats and in optic nerve transection. Glaucoma was induced in one eye of 51 adult Wistar rats by laser treatment to the trabecular meshwork. Eighteen further rats underwent unilateral optic nerve transection. We studied the transcription factor c-jun, its activated form, phospho-c-jun, the transcription factor p-ATF-2, and the enzyme JNK by immunohistochemistry. The activation of p-c-jun was also investigated using western blot analysis. Treated and control eyes were compared in a masked way at multiple time points after injury. We found a statistically significant increase in immunolabelling for c-jun and phospho-c-jun in retinal ganglion cells (RGCs) from 1 day to 4 weeks after intraocular pressure (IOP) elevation. At 1 and 2 days after the laser treatment, a mean of 2.9+/-3.3 RGCsmm(-1) were positive for c-jun (n=12, p=0.005, t-test), increasing to a mean of 13.4+/-7.5 cells mm(-1) at 1 week (n=18, p=0.00005), and decreasing to 2.3+/-2.0 cells mm(-1) at 2 weeks (n=5, p=0.04) and 0.1+/-0.1 cells mm(-1) at 2 months. Few of the 47 control eyes had any labelling for c-jun or phospho-c-jun, while between 80 and 100% of elevated IOP eyes showed positivity during the first 2 weeks of experimental glaucoma. After optic nerve transection, c-jun and phospho-c-jun were also significantly activated at 1, 2 and 9 days (p<0.03, t-test). Western blot analysis demonstrated significantly increased phospho-c-jun amounts in both transected and glaucomatous eyes compared to control fellow eyes 1 week following treatment. JNK was not significantly activated in glaucoma or optic nerve transection. P-ATF-2 was not significantly activated in glaucoma, but was significantly increased 2 days after optic nerve transection. We conclude that the process leading to RGC death in experimental glaucoma and after optic nerve transection involves the activation of c-jun at the RGC layer. C-jun is activated more gradually in glaucoma then after optic nerve transection.

Effect of glatiramer acetate on primary and secondary degeneration of retinal ganglion cells in the rat.

PURPOSE: After crush injury to the optic nerve, elevated intraocular pressure, and glutamate toxicity, the immune modulator glatiramer acetate (GA, Cop-1; Copaxone; Teva Pharmaceutical Industries, Pitach Tikva, Israel) has been shown to reduce the delayed cell death of retinal ganglion cells (RGCs). This study was undertaken to confirm the protective effect of GA on secondary degeneration of RGCs in the rat, by using a spatial, rather than temporal, model. METHODS: A total of 131 Wistar rats divided into 10 groups underwent bilateral stereotactic injection of fluorescent tracer (Fluorogold; Fluorochrome, Denver, CO) into the superior colliculus to label RGCs. They received a concurrent subcutaneously injection of (1) GA mixed with complete Freund's adjuvant (CFA), (2) CFA alone, or (3) saline. One week later, the superior one third of the left optic nerve was transected in animals in the six partial transection groups. Optic nerves in four additional groups underwent full transection. Rats were killed and retinas harvested from both eyes 1 or 4 weeks after partial transection and 1 or 2 weeks after full transection. RGC densities were calculated from retinal wholemounts, and differences between right (control) and left (transected) eyes were compared across treatment groups. RESULTS: Among the partial transection groups, differences in the mean percentage of RGC loss in the inferior retinas were not significant at 1 or 4 weeks (ANOVA; P = 0.20, P = 0.12, respectively). After full transection, there was significantly more RGC loss in the GA group than in the CFA group when comparing whole retinas at 1 week, but not at 2 weeks (two-tailed t-test; P = 0.04, P = 0.36, respectively). CONCLUSIONS: There is no evidence that GA has a neuroprotective effect after optic nerve transection, either for primarily injured or secondarily involved RGC.

Increased expression of iron-regulating genes in monkey and human glaucoma.

PURPOSE: To understand the mechanisms mediating retinal ganglion cell loss in glaucoma, the gene expression patterns were compared for transferrin, ceruloplasmin, and ferritin between normal and glaucomatous retina in monkey and human eyes. METHODS: Laser photocoagulation was used to produce unilateral experimental glaucoma in monkeys. Gene expression was assessed by in situ hybridization and quantitative reverse transcription polymerase chain reaction (PCR). Immunohistochemistry was used to examine the retinal expression of iron-related proteins in the retina in experimental monkey glaucoma and human glaucoma. RESULTS: Comparison of glaucomatous with control monkey retinas demonstrated increased mRNA expression of transferrin, ceruloplasmin, and ferritin heavy and light chains. In situ hybridization localized retinal gene expression of transferrin mainly to the inner nuclear layer and ferritin to both the inner and outer nuclear layers. Immunohistochemical examination of monkey and human glaucoma for these iron-related proteins demonstrated increases at the protein level. CONCLUSIONS: Increased mRNA and protein levels of the iron-regulating proteins transferrin, ceruloplasmin, and ferritin are present in glaucoma. Together, these results suggest the involvement of iron and copper metabolism and associated antioxidant systems in the pathogenesis of glaucoma.

Gene therapy with brain-derived neurotrophic factor as a protection: retinal ganglion cells in a rat glaucoma model.

PURPOSE: To develop a modified adenoassociated viral (AAV) vector capable of efficient transfection of retinal ganglion cells (RGCs) and to test the hypothesis that use of this vector to express brain-derived neurotrophic factor (BDNF) could be protective in experimental glaucoma. METHODS: Ninety-three rats received one unilateral, intravitreal injection of either normal saline (n = 30), AAV-BDNF-woodchuck hepatitis posttranscriptional regulatory element (WPRE; n = 30), or AAV-green fluorescent protein (GFP)-WPRE (n = 33). Two weeks later, experimental glaucoma was induced in the injected eye by laser application to the trabecular meshwork. Survival of RGCs was estimated by counting axons in optic nerve cross sections after 4 weeks of glaucoma. Transgene expression was assessed by immunohistochemistry, Western blot analysis, and direct visualization of GFP. RESULTS: The density of GFP-positive cells in retinal wholemounts was 1,828 +/- 299 cells/mm(2) (72,273 +/- 11,814 cells/retina). Exposure to elevated intraocular pressure was similar in all groups. Four weeks after initial laser treatment, axon loss was 52.3% +/- 27.1% in the saline-treated group (n = 25) and 52.3% +/- 24.2% in the AAV-GFP-WPRE group (n = 30), but only 32.3% +/- 23.0% in the AAV-BDNF-WPRE group (n = 27). Survival in AAV-BDNF-WPRE animals increased markedly and the difference was significant compared with those receiving either AAV-GFP-WPRE (P = 0.002, t-test) or saline (P = 0.006, t-test). CONCLUSIONS: Overexpression of the BDNF gene protects RGC as estimated by axon counts in a rat glaucoma model, further supporting the potential feasibility of neurotrophic therapy as a complement to the lowering of IOP in the treatment of glaucoma.

A model to study differences between primary and secondary degeneration of retinal ganglion cells in rats by partial optic nerve transection.

PURPOSE: To use a rat model of optic nerve injury to differentiate primary and secondary retinal ganglion cell (RGC) injury. METHODS: Under general anesthesia, a modified diamond knife was used to transect the superior one third of the orbital optic nerve in albino Wistar rats. The number of surviving RGC was quantified by counting both the number of cells retrogradely filled with fluorescent gold dye injected into the superior colliculus 1 week before nerve injury and the number of axons in optic nerve cross sections. RGCs were counted in 56 rats, with 24 regions examined in each retinal wholemount. Rats were studied at 4 days, 8 days, 4 weeks, and 9 weeks after transection. The interocular difference in RGCs was also compared in five control rats that underwent no surgery and in five rats who underwent a unilateral sham operation. It was confirmed histologically that only the upper optic nerve had been directly injured. RESULTS: At 4 and 8 days after injury, superior RGCs showed a mean difference from their fellow eyes of -30.3% and -62.8%, respectively (P = 0.02 and 0.001, t-test, n = 8 rats/group), whereas sham-operation eyes had no significant loss (mean difference between eyes = 1.7%, P = 0.74, t-test). At 8 days, inferior RGCs were unchanged from control, fellow eyes (mean interocular difference = -4.8%, P = 0.16, t-test). Nine weeks after transection, inferior RGC had 34.5% fewer RGCs than their fellow eyes, compared with 41.2% fewer RGCs in the superior zones of the injured eyes compared with fellow eyes. Detailed, serial section studies of the topography of RGC axons in the optic nerve showed an orderly arrangement of fibers that were segregated in relation to the position of their cell bodies in the retina. CONCLUSIONS: A model of partial optic nerve transection in rats showed rapid loss of directly injured RGCs in the superior retina and delayed, but significant secondary loss of RGCs in the inferior retina, whose axons were not severed. The findings confirm similar results in monkey eyes and provide a rodent model in which pharmacologic interventions against secondary degeneration can be tested.

Measurement of amino acid levels in the vitreous humor of rats after chronic intraocular pressure elevation or optic nerve transection.

PURPOSE: To investigate whether the levels of free amino acids and protein in the vitreous of rat eyes are altered with chronic intraocular pressure (IOP) elevation or after optic nerve transection. MATERIALS AND METHODS: The concentrations of 20 amino acids in the vitreous humor were measured by high-performance liquid chromatography in both eyes of 41 rats with unilateral IOP elevation induced by translimbal photocoagulation. Eyes were studied 1 day and 1, 2, 4, and 9 weeks after initial IOP elevation. The same amino acids were measured in 41 rats 1 day and 2, 4, and 9 weeks after unilateral transection of the orbital optic nerve. The intravitreal protein level was assayed in additional 22 rats with IOP elevation and 12 rats after nerve transection. Two masked observers evaluated the amount of optic nerve damage with a semiquantitative, light-microscopic technique. RESULTS: In rats with experimental glaucoma, amino acid concentrations were unchanged 1 day after treatment. At 1 week, 4 of 20 amino acids (aspartate, proline, alanine, and lysine) were higher than in control eyes ( < or = 0.01), but this difference was nonsignificant after Bonferroni correction for multiple simultaneous amino acid comparisons (none achieved < 0.0025). No amino acid was significantly different from control in the nerve transection groups (all > 0.05). Vitreous protein level was significantly higher in glaucomatous eyes than their paired controls at 1 day ( < 0.0001) and 1 week ( < 0.002). One day and 1 week after optic nerve transection, vitreal proteins were significantly elevated compared with control eyes from untreated animals ( < 0.0020 and < 0.0022, respectively), though not compared with their fellow eyes ( = 0.25 and 0.10). CONCLUSION: Chronic experimental glaucoma and transection of the optic nerve increase the amount of protein in the rat vitreous above control levels. In the vitreous of rats with experimental glaucoma, a number of free amino acids were transiently elevated to a modest degree, but no significant difference in vitreous glutamate concentration was detected ( > 0.01).

Retinal glutamate transporter changes in experimental glaucoma and after optic nerve transection in the rat.

PURPOSE: High levels of glutamate can be toxic to retinal ganglion cells. Effective buffering of extracellular glutamate by retinal glutamate transporters is therefore important. This study was conducted to investigate whether glutamate transporter changes occur with two models of optic nerve injury in the rat. METHODS: Glaucoma was induced in one eye of 35 adult Wistar rats by translimbal diode laser treatment to the trabecular meshwork. Twenty-five more rats underwent unilateral optic nerve transection. Two glutamate transporters, GLAST (EAAT-1) and GLT-1 (EAAT-2), were studied by immunohistochemistry and quantitative Western blot analysis. Treated and control eyes were compared 3 days and 1, 4, and 6 weeks after injury. Optic nerve damage was assessed semiquantitatively in epoxy-embedded optic nerve cross sections. RESULTS: Trabecular laser treatment resulted in moderate intraocular pressure (IOP) elevation in all animals. After 1 to 6 weeks of experimental glaucoma, all treated eyes had significant optic nerve damage. Glutamate transporter changes were not detected by immunohistochemistry. Western blot analysis demonstrated significantly reduced GLT-1 in glaucomatous eyes compared with control eyes at 3 days (29.3% +/- 6.7%, P = 0.01), 1 week (55.5% +/- 13.6%, P = 0.02), 4 weeks (27.2% +/- 10.1%, P = 0.05), and 6 weeks (38.1% +/- 7.9%, P = 0.01; mean reduction +/- SEM, paired t-tests, n = 5 animals per group, four duplicate Western blot analyses per eye). The magnitude of the reduction in GLT-1 correlated significantly with mean IOP in the glaucomatous eye (r(2) = 0.31, P = 0.01, linear regression). GLAST was significantly reduced (33.8% +/- 8.1%, mean +/- SEM) after 4 weeks of elevated IOP (P = 0.01, paired t-test, n = 5 animals per group). In contrast to glaucoma, optic nerve transection resulted in an increase in GLT-1 compared with the control eye (P = 0.01, paired t-test, n = 15 animals). There was no significant change in GLAST after transection. CONCLUSIONS: GLT-1 and GLAST were significantly reduced in an experimental rat glaucoma model, a response that was not found after optic nerve transection. Reductions in GLT-1 and GLAST may increase the potential for glutamate-induced injury to RGC in glaucoma.

Translimbal laser photocoagulation to the trabecular meshwork as a model of glaucoma in rats.

PURPOSE: To develop and characterize a model of pressure-induced optic neuropathy in rats. METHODS: Experimental glaucoma was induced unilaterally in 174 Wistar rats, using a diode laser with wavelength of 532 nm aimed at the trabecular meshwork and episcleral veins (combination treatment group) or only at the trabecular meshwork (trabecular group) through the external limbus. Intraocular pressure (IOP) was measured by a tonometer in rats under ketamine-xylazine anesthesia. Possible retinal vascular compromise was evaluated by repeated fundus examinations and by histology. The degree of retinal ganglion cell (RGC) loss was assessed by a masked, semiautomated counting of optic nerve axons. Effects of laser treatment on anterior ocular structures and retina were judged by light microscopy. RESULTS: After the laser treatment, IOP was increased in all eyes to higher than the normal mean IOP of 19.4 +/- 2.1 mm Hg (270 eyes). Peak IOP was 49.0 +/- 6.1 mm Hg (n = 108) in the combination group that was treated by a laser setting of 0.7 seconds and 0.4 W and 34.0 +/- 5.7 mm Hg (n = 46) in the trabecular group. Mean IOP after 6 weeks was 25.5 +/- 2.9 mm Hg in glaucomatous eyes in the combination group compared with 22.0 +/- 1.8 mm Hg in the trabecular group. IOP in the glaucomatous eyes was typically higher than in the control eyes for at least 3 weeks. In the combination group, RGC loss was 16.1% +/- 14.4% at 1 week (n = 8, P = 0.01), 59.7% +/- 25.7% at 6 weeks (n = 88, P < 0.001), and 70.9% +/- 23.6% at 9 weeks (n = 12, P < 0.001). The trabecular group had mean axonal loss of 19.1% +/- 14.0% at 3 weeks (n = 9, P = 0.004) and 24.3% +/- 20.2% at 6 weeks (n = 25, P < 0.001), increasing to 48.4% +/- 32.8% at 9 weeks (n = 12, P < 0.001). Laser treatment led to closure of intertrabecular spaces and the major outflow channel. The retina and choroid were normal by ophthalmoscopy at all times after treatment. Light microscopic examination showed only loss of RGCs and their nerve fibers. CONCLUSIONS: Increased IOP caused by a laser injury to the trabecular meshwork represents a useful and efficient model of experimental glaucoma in rats.