Effects of experimental glaucoma in Lama1nmf223 mutant mice.

To identify changes in response to experimental intraocular pressure (IOP) elevation associated with the laminin α1 nmf223 mutation in mice. Laminin mutant (LM) mice (Lama1nmf223) and C57BL/6J (B6) mice in two age groups each (4-5 months and >1 year) underwent intracameral microbead injections to produce unilaterally elevated IOP. We assessed axonal transport block of immunofluorescently labeled amyloid precursor protein (APP) after 3 days and retinal ganglion cell (RGC) axon loss after 6 weeks. Light, electron and fluorescent microscopy was used to study baseline anatomic differences and effects of 3-day IOP elevation in younger LM mice. In younger mice of both LM and B6 strains, elevated IOP led to increased APP block in the retina, prelaminar optic nerve head (preONH), unmyelinated optic nerve (UON), and myelinated optic nerve (MON). APP blockade not significantly different between younger B6 and LM mouse strains. Older LM mice had greater APP accumulation in both control and glaucoma eyes compared to older B6, however, accumulation was not significantly greater in LM glaucoma eyes compared to LM controls. Axon loss at 6 weeks was 12.2% in younger LM and 18.7% in younger B6 mice (difference between strains, p = 0.22, Mann Whitney test). Untreated LM optic nerve area was lower compared to B6 (nerve area, p < 0.0001, t-test). Aberrant axon bundles, as well as defects, thickening and reduplication of pia mater, were seen in the optic nerves of younger LM mice. Axonal transport blockade significantly differed between old B6 and old LM mice in control and glaucoma eyes, and younger LM mice had abnormal axon paths and lower optic nerve area.

Quantitative Microstructural Analysis of Cellular and Tissue Remodeling in Human Glaucoma Optic Nerve Head.

Purpose: To measure quantitatively changes in lamina cribrosa (LC) cell and connective tissue structure in human glaucoma eyes. Methods: We studied 27 glaucoma and 19 age-matched non-glaucoma postmortem eyes. In 25 eyes, LC cross-sections were examined by confocal and multiphoton microscopy to quantify structures identified by anti-glial fibrillary acidic protein (GFAP), phalloidin-labeled F-actin, nuclear 4',6-diamidino-2-phenylindole (DAPI), and by second harmonic generation imaging of LC beams. Additional light and transmission electron microscopy were performed in 21 eyes to confirm features of LC remodeling, including immunolabeling by anti-SOX9 and anti-collagen IV. All glaucoma eyes had detailed clinical histories of open-angle glaucoma status, and degree of axon loss was quantified in retrolaminar optic nerve cross-sections. Results: Within LC pores, the proportionate area of both GFAP and F-actin processes was significantly lower in glaucoma eyes than in controls (P = 0.01). Nuclei were rounder (lower median aspect ratio) in glaucoma specimens (P = 0.02). In models assessing degree of glaucoma damage, F-actin process width was significantly wider in glaucoma eyes with more damage (P = 0.024), average LC beam width decreased with worse glaucoma damage (P = 0.042), and nuclear count per square millimeter rose with worse damage (P = 0.019). The greater cell count in LC pores represented 92.3% astrocytes by SOX9 labeling. The results are consistent with replacement of axons in LC pores by basement membrane labeled by anti-collagen IV and in-migrating astrocytes. Conclusions: Alteration in LC structure in glaucoma involves migration of astrocytes into axonal bundles, change in astrocyte orientation and processes, production of basement membrane material, and thinning of connective tissue beams.

The role of aquaporin-4 in optic nerve head astrocytes in experimental glaucoma.

PURPOSE: To study aquaporin channel expression in astrocytes of the mouse optic nerve (ON) and the response to IOP elevation in mice lacking aquaporin 4 (AQP4 null). METHODS: C57BL/6 (B6) and AQP4 null mice were exposed to bead-induced IOP elevation for 3 days (3D-IOP), 1 and 6 weeks. Mouse ocular tissue sections were immunolabeled against aquaporins 1(AQP1), 4(AQP4), and 9(AQP9). Ocular tissue was imaged to identify normal AQP distribution, ON changes, and axon loss after IOP elevation. Ultrastructure examination, cell proliferation, gene expression, and transport block were also analyzed. RESULTS: B6 mice had abundant AQP4 expression in Müller cells, astrocytes of retina and myelinated ON (MON), but minimal AQP4in prelaminar and unmyelinated ON (UON). MON of AQP4 nulls had smaller ON area, smaller axon diameter, higher axon density, and larger proportionate axon area than B6 (all p≤0.05). Bead-injection led to comparable 3D-IOP elevation (p = 0.42) and axonal transport blockade in both strains. In B6, AQP4 distribution was unchanged after 3D-IOP. At baseline, AQP1 and AQP9 were present in retina, but not in UON and this was unaffected after IOP elevation in both strains. In 3D-IOP mice, ON astrocytes and microglia proliferated, more in B6 than AQP4 null. After 6 week IOP elevation, axon loss occurred equally in the two mouse types (24.6%, AQP4 null vs. 23.3%, B6). CONCLUSION: Lack of AQP4 was neither protective nor detrimental to the effects of IOP elevation. The minimal presence of AQP4 in UON may be a vital aspect of the regionally specific phenotype of astrocytes in the mouse optic nerve head.

A method to quantify regional axonal transport blockade at the optic nerve head after short term intraocular pressure elevation in mice.

Axonal transport blockade is an initial step in retinal ganglion cell (RGC) degeneration in glaucoma and targeting maintenance of normal axonal transport could confer neuroprotection. We present an objective, quantitative method for assessing axonal transport blockade in mouse glaucoma models. Intraocular pressure (IOP) was elevated unilaterally in CD1 mice for 3 days using intracameral microbead injection. Longitudinal sections of optic nerve head (ONH) were immunofluorescently labeled for myelin basic protein (MBP) and amyloid precursor protein (APP), which is transported predominantly orthograde by neurons. The beginning of the myelin transition zone, visualized with the MBP label, was more posterior with elevated IOP, 288.8 ± 40.9 µm, compared to normotensive control eyes, 228.7 ± 32.7 µm (p = 0.030, N = 6 pairs). Glaucomatous regional APP accumulations in retina, prelaminar ONH, unmyelinated ONH, and myelinated optic nerve were identified by objective qualification of pixels with fluorescent intensity greater than the 97.5th percentile value of control eyes (suprathreshold pixels). This method segregated images with APP blockade from those with normal transport of APP. The fraction of suprathreshold pixels was significantly higher following IOP elevation than in normotensive controls in the unmyelinated ONH and myelinated nerve regions (paired analyses, p = 0.02 and 0.003, respectively, N = 12), but not in retina or prelaminar ONH (p = 0.91 and 0.08, respectively). The mean intensity of suprathreshold pixels was also significantly greater in glaucoma than in normotensive controls in prelaminar ONH, unmyelinated ONH and myelinated optic nerve (p = 0.01, 0.01, 0.002, respectively). Using this method, subconjunctival glyceraldehyde, which is known to worsen long-term RGC loss with IOP elevation, also produced greater APP blockade, but not statistically significant compared to glaucoma alone. Systemic losartan, which aids RGC axonal survival in glaucoma, reduced APP blockade, but not statistically significant compared to glaucoma alone. The method provides a short-term assessment of axonal injury for use in initial tests of neuroprotective therapies that may beneficially affect RGC transport in animal models of glaucoma.

Rho-Kinase Inhibition Reduces Myofibroblast Differentiation and Proliferation of Scleral Fibroblasts Induced by Transforming Growth Factor ß and Experimental Glaucoma.

PURPOSE: We evaluated prevention of transforming growth factor ß (TGFß)-induced transdifferentiation of cultured scleral fibroblasts to myofibroblasts by rho-associated protein kinase (ROCK) inhibitors. Additionally, we tested whether local delivery of ROCK inhibitors reduced scleral fibroblast proliferation in response to chronic intraocular pressure (IOP) elevation. METHODS: Primary human peripapillary sclera (PPS) fibroblasts were cultured and treated with TGFß to induce myofibroblast transdifferentiation, as determined by immunoblot assessment of α smooth muscle actin (SMA) levels and collagen gel contraction. Cells were treated with the ROCK inhibitors Y27632, fasudil, and H1152 before TGFß treatment. ROCK activity in TGFß-treated fibroblasts and sclera from ocular hypertensive mice was assessed by measuring phosphorylation of the ROCK substrate MYPT1 at Thr696. Fibroblast proliferation following IOP elevation and ROCK inhibitor treatment was assessed by an enzyme-linked immunosorbent (ELISA) assay. RESULTS: ROCK inhibitors H1152 (10µM), Y27632 (10 µM), and fasudil (5µM) reduced SMA expression 72%, 85%, and 68%, respectively. Collagen gel contraction was reduced by 36% (P < 0.001), 27% (P = 0.0003), and 33% (P = 0.0019) following treatment with fasudil (25 µM), Y27632 (10 µM), and H1152 (10µM). ROCK activity induced by TGFß rose 4.74 ± 1.9 times over control at 4 hours (P = 0.0004) and 2.4 ± 0.47-fold (P = 0.0016) in sclera after IOP elevation. Proliferation of scleral fibroblasts after chronic IOP elevation was reduced 77% by Y27632 (P = 0.001) and 84% by fasudil (P = 0.0049). CONCLUSIONS: ROCK inhibitors reduce TGFß-induced myofibroblast transdifferentiation and glaucoma-induced scleral cell proliferation. TRANSLATIONAL RELEVANCE: These findings suggest altered fibroblast activity promoted by ROCK inhibitors could modify scleral biomechanics and be relevant to glaucoma treatment.

Sustained Dorzolamide Release Prevents Axonal and Retinal Ganglion Cell Loss in a Rat Model of IOP-Glaucoma.

PURPOSE: To determine if one injection of a sustained release formulation of dorzolamide in biodegradable microparticles (DPP) reduces retinal ganglion cell (RGC) loss in a rat model of glaucoma. METHODS: We injected either DPP or control microparticles intravitreally in rats. Two days later, unilateral ocular hypertension was induced by translimbal, diode laser treatment by a surgeon masked to treatment group. IOP and clinical exams were performed until sacrifice 6 weeks after laser treatment. RGC loss was measured by masked observers in both optic nerve cross-sections and RGC layer counts from retinal whole mounts. RESULTS: Cumulative IOP exposure was significantly reduced by DPP injection (49 ± 48 mm Hg × days in treated versus 227 ± 191 mm Hg × days in control microparticle eyes; P = 0.012, t-test). While control-injected eyes increased in axial length by 2.4 ± 1.7%, DPP eyes did not significantly enlarge (0.3 ± 2.2%, difference from control, P = 0.03, t-test). RGC loss was significantly less in DPP eyes compared with control microparticle injection alone (RGC axon count reduction: 21% vs. 52%; RGC body reduction: 25% vs. 50% [beta tubulin labeling]; P = 0.02, t-test). CONCLUSIONS: A single injection of sustained release DPP protected against RGC loss and axial elongation in a rat model of IOP glaucoma. TRANSLATIONAL RELEVANCE: Sustained release IOP-lowering medications have the potential to stop glaucoma progression.

Losartan Treatment Protects Retinal Ganglion Cells and Alters Scleral Remodeling in Experimental Glaucoma.

PURPOSE: To determine if oral losartan treatment decreases the retinal ganglion cell (RGC) death caused by experimental intraocular pressure (IOP) elevation in mice. METHODS: We produced IOP increase in CD1 mice and performed unilateral optic nerve crush. Mice received oral losartan, spironolactone, enalapril, or no drug to test effects of inhibiting angiotensin receptors. IOP was monitored by Tonolab, and blood pressure was monitored by tail cuff device. RGC loss was measured in masked axon counts and RGC bodies by ß-tubulin labeling. Scleral changes that could modulate RGC injury were measured including axial length, scleral thickness, and retinal layer thicknesses, pressure-strain behavior in inflation testing, and study of angiotensin receptors and pathways by reverse transcription polymerase chain reaction, Western blot, and immunohistochemistry. RESULTS: Losartan treatment prevented significant RGC loss (median loss = 2.5%, p = 0.13), while median loss with water, spironolactone, and enalapril treatments were 26%, 28% and 43%; p < 0.0001). The lower RGC loss with losartan was significantly less than the loss with spironolactone or enalapril (regression model p = 0.001; drug treatment group term p = 0.01). Both losartan and enalapril significantly lowered blood pressure (p< 0.001), but losartan was protective, while enalapril led to worse than water-treated RGC loss. RGC loss after crush injury was unaffected by losartan treatment (difference from control p = 0.9). Survival of RGC in cell culture was not prolonged by sartan treatment. Axonal transport blockade after 3 day IOP elevations was less in losartan-treated than in control glaucoma eyes (p = 0.007). Losartan inhibited effects of glaucoma, including reduction in extracellular signal-related kinase activity and modification of glaucoma-related changes in scleral thickness and creep under controlled IOP. CONCLUSIONS: The neuroprotective effect of losartan in mouse glaucoma is associated with adaptive changes in the sclera expressed at the optic nerve head.

Effect of CNTF on retinal ganglion cell survival in experimental glaucoma.

PURPOSE: To assess the neuroprotective effect of virally mediated overexpression of ciliary-derived neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in experimental rat glaucoma. METHODS: Laser-induced glaucoma was produced in one eye of 224 Wistar rats after injection of adenoassociated viral vectors (type 2) containing either CNTF, BDNF, or both, with saline-injected eyes and noninjected glaucomatous eyes serving as the control. IOP was measured with a hand-held tonometer, and semiautomated optic nerve axon counts were performed by masked observers. IOP exposure over time was adjusted in multivariate regression analysis to calculate the effect of CNTF and BDNF. RESULTS: By multivariate regression, CNTF had a significant protective effect, with 15% less RGC axon death (P < 0.01). Both combined CNTF-BDNF and BDNF overexpression alone had no statistically significant improvement in RGC axon survival. By Western blot, there was a quantitative increase in CNTF and BDNF expression in retinas exposed to single viral vectors carrying each gene, but no increase with sequential injection of both vectors. CONCLUSIONS: These data confirm that CNTF can exert a protective effect in experimental glaucoma. The reason for the lack of observed effect in the BDNF overexpression groups is unclear, but it may be a function of the level of neurotrophin expression achieved.

Prolonged blockade of VEGF family members does not cause identifiable damage to retinal neurons or vessels.

Several ocular diseases complicated by neovascularization are being treated by repeated intraocular injections of vascular endothelial growth factor (VEGF) antagonists. While substantial benefits have been documented, there is concern that unrecognized damage may be occurring, because blockade of VEGF may damage the fenestrated vessels of the choroicapillaris and deprive retinal neurons of input from a survival factor. One report has suggested that even temporary blockade of all isoforms of VEGF-A results in significant loss of retinal ganglion cells. In this study, we utilized double transgenic mice with doxycycline-inducible expression of soluble VEGF receptor 1 coupled to an Fc fragment (sVEGFR1Fc), a potent antagonist of several VEGF family members, including VEGF-A, to test the effects of VEGF blockade in the retina. Expression of sVEGFR1Fc completely blocked VEGF-induced retinal vascular permeability and significantly suppressed the development of choroidal neovascularization at rupture sites in Bruch's membrane, but did not cause regression of established choroidal neovascularization. Mice with constant expression of sVEGFR1Fc in the retina for 7 months had normal electroretinograms and normal retinal and choroidal ultrastructure including normal fenestrations in the choroicapillaris. They also showed no significant difference from control mice in the number of ganglion cell axons in optic nerve cross sections and the retinal level of mRNA for 3 ganglion cell-specific genes. These data indicate that constant blockade of VEGF for up to 7 months has no identifiable deleterious effects on the retina or choroid and support the use of VEGF antagonists in the treatment of retinal diseases.

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 effect of experimental glaucoma and optic nerve transection on amacrine cells in the rat retina.

PURPOSE: To detect alterations in amacrine cells associated with retinal ganglion cell (RGC) depletion caused by experimental optic nerve transection and glaucoma. METHODS: Intraocular pressure (IOP) was elevated unilaterally in 18 rats by translimbal trabecular laser treatment, and eyes were studied at 1 (n = 6), 2 (n = 5), and 3 (n = 7) months. Complete optic nerve transection was performed unilaterally in nine rats with survival for 1 (n = 4) and 3 (n = 5) months. Serial cryosections (five per eye) were immunohistochemically labeled with rabbit anti-gamma-aminobutyric acid (GABA) and anti-glycine antibodies. Cells in the ganglion cell and inner nuclear layers that labeled for GABA or glycine were counted in a masked fashion under bright-field microscopy. Additional labeling with other RGC and amacrine antigens was also performed. RGC loss was quantified by axon counts. RESULTS: Amacrine cells identified by GABA and glycine labeling were not significantly affected by experimental glaucoma, with a mean decrease of 15% compared with bilaterally untreated control cells (557 +/- 186 neurons/mm [glaucoma] versus 653.9 +/- 114.4 neurons/mm [control] of retina; P = 0.15, t-test). There was no significant trend for amacrine cell counts to be lower in eyes with fewer RGCs (r = -0.39, P = 0.11). By contrast, there was highly significant loss of GABA and glycine staining 3 months after nerve transection, both in the treated and the fellow eyes (P < 0.0001, t-test). However, there was a substantial number of remaining amacrine cells in transected retinas, as indicated by labeling for calretinin and calbindin. CONCLUSIONS: Experimental glaucoma causes minimal change in amacrine cells and their expression of neurotransmitters. After nerve transection, neurotransmitter presence declines, but many amacrine cell bodies remain. Differences among optic nerve injury models, as well as effects on "untreated" fellow eyes, should be recognized.

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.

Intraocular injection of dibutyryl cyclic AMP promotes axon regeneration in rat optic nerve.

The optic nerve is a CNS pathway containing molecules capable of inhibiting axon elongation. The growth program in embryonic retinal ganglion cell (RGC) neurons enables axons to regenerate in the optic nerve through at least two mechanisms. Namely, high cyclic AMP (cAMP) levels abrogate the ability of CNS molecules to inhibit elongation, and the pattern of gene expression enables axons to undergo rapid, sustained, and lengthy elongation. In adult mammals, recovery of visual function after optic nerve injury is limited by both the death of most RGC neurons and the inability of surviving axons to regenerate. We now report that a single intraocular injection of the membrane-permeable cAMP analogue dibutyryl cAMP (db cAMP) promotes the regeneration of RGC axons in the optic nerves of adult rats, but does not prevent the death of RGC neurons. This regeneration in optic nerves crushed within the orbit (2 mm from the eye) was equally effective either 1 day before or 1 day after db cAMP injection. The number of regenerating axons, which was maximal 14 days after crush, declined with increasing time after injury (i.e., 28, 56, and 112 days) and distance beyond the crush site (i.e., 0.25, 0.5, and 1.0 mm). Thus, db cAMP promotes optic nerve regeneration without increasing the survival of axotomized RGC neurons. Furthermore, since db cAMP does not enable axons to undergo rapid, sustained, and lengthy elongation, strategies that increase survival and promote these changes in elongation may critically complement the ability of db cAMP to promote regeneration.

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.

Caspase activation and amyloid precursor protein cleavage in rat ocular hypertension.

PURPOSE: Retinal ganglion cell (RGC) death in glaucoma involves apoptosis. Activation of caspases and abnormal processing of amyloid precursor protein (APP) are important events in other chronic neurodegenerations, such as Alzheimer's disease (AD). The retinal expression and activation of caspases and the patterns of caspase-3-mediated APP processing in ocular hypertensive models of rat glaucoma were investigated. METHODS: RGC death was produced in one eye by chronic exposure to increased intraocular pressure (IOP) or by optic nerve transection. Elevated IOP was produced by obstruction of aqueous humor outflow with laser coagulation or limbal hypertonic saline injection. Caspase activity and APP processing in the retina were examined by RNase protection assay (RPA), immunocytochemistry, immunoblot assay, and colorimetric assay. RESULTS: RPA revealed elevations of caspase-3 mRNA, as well as other apoptosis-related mRNAs. Immunocytochemistry showed caspase-3 activation in RGCs damaged by ocular hypertension. The generation of the caspase-3-mediated APP cleavage product (DeltaC-APP) was also increased in ocular hypertensive RGCs. Western immunoblot assay and colorimetry revealed significantly more activated caspase-3 in ocular hypertensive retinas than in control retinas. The activated form of caspase-8, an initiator caspase, and amyloid-beta, a product of APP proteolysis and a component of senile plaques in AD, were detected in RGCs by immunohistochemistry significantly more often in ocular hypertensive than in control retinas. The amounts of full-length APP were reduced and amyloid-beta-containing fragments were increased in ocular hypertensive retinas by Western immunoblot assay. CONCLUSIONS: Rat RGCs subjected to chronic ocular hypertension demonstrate caspase activation and abnormal processing of APP, which may contribute to the pathophysiology of 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.