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.

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.

Gene delivery to the eye using adeno-associated viral vectors.

Adeno-associated virus (AAV) vectors provide a useful way to deliver genes to the eye. They have a number of important properties which make them suitable for this purpose, not least their lack of significant pathogenicity and the potential for long-term transfection of retinal cells. The optimal methods for AAV-mediated gene delivery are determined by the location and characteristics of the target cell type. Efficient gene delivery to photoreceptors and pigment epithelial cells following subretinal injection of AAV has been achieved in various animal models. AAV-mediated gene therapy has been shown to slow photoreceptor loss in rodent models of primary photoreceptor diseases and in dogs with a naturally occurring disease similar to human Leber's congenital amaurosis (LCA). Efficient gene delivery to other cell types such as retinal ganglion cells (RGCs), however, has been more problematic. In this article, we review the potential uses of AAV-mediated gene delivery to the eye. We describe the selection of an appropriate AAV vector for ocular gene transfer studies and discuss the techniques used to deliver the virus to the eye and to assess ocular transfection. We emphasize our techniques for successful gene transfer to retinal ganglion cells, which have often proven challenging to transfect with high efficiency. Using a modified AAV incorporating a chicken beta-actin (CBA) promoter and the woodchuck hepatitis posttranscriptional regulatory element, we describe how our techniques allow approximately 85% of rat retinal ganglion cells to be transfected within 2 weeks of a single intravitreal virus injection. Our techniques facilitate the study of the pathogenesis of RGC diseases such as glaucoma and the development of novel new treatments based on gene therapy.

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.