1α,25-Dihydroxyvitamin D(3) and its analog, 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D(3) (2MD), suppress intraocular pressure in non-human primates.

Ocular hypertension is the greatest known risk factor for glaucoma that affects an estimated 70 million people worldwide. Lowering intraocular pressure (IOP) remains the mainstay of therapy in the management of glaucoma. By means of microarray analysis, we have discovered that 1α,25-dihydroxyvitamin D(3) (1α,25-(OH)(2)D(3)) regulates genes that are known to be involved in the determination of intraocular pressure (IOP). Topical administration of 1α,25-(OH)(2)D(3) or its analog, 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D(3) (2MD), markedly reduces IOP in non-human primates. The reduction in IOP is not the result of reduced aqueous humor formation, while a 35% increase in aqueous humor drainage by 1α,25-(OH)(2)D(3) was found but this increase did not achieve significance. Nevertheless, our results suggest that 1α,25-(OH)(2)D(3), or an analog thereof, may present a new approach to the treatment of glaucoma.

Effect of nitric oxide compounds on monkey ciliary muscle in vitro.

The effects of various nitric oxide compounds and their inhibitors on monkey ciliary muscle contraction in vitro were investigated in both the longitudinal and circular vectors. The responses to nitric oxide compounds in carbachol precontracted ciliary muscle consisted of an initial relaxation often followed by recovery to near carbachol precontracted levels while the compound was still present. Sodium nitroprusside produced the greatest relaxation responses (nearly 100% relaxation in both vectors at 10(-3) M). The highest concentrations of isosorbide dinitrate (10(-4) M) and L-arginine (10(-3) M) produced relaxation responses of approximately 50% in both vectors. 8-Bromo cyclic GMP produced the smallest relaxation responses (25-35%). Nitric oxide synthase inhibition enhanced carbachol contraction up to 20% in the longitudinal but not the circular vector. Phosphodiesterase inhibition did not further enhance the relaxation response to L-arginine. Guanylate cyclase inhibition partially attenuated the relaxation response to sodium nitroprusside. Nitric oxide generating compounds were effective in relaxing precontracted monkey ciliary muscle in vitro. Endogenous production of nitric oxide is likely involved in the regulation of the contractile response in monkey ciliary muscle. Nitric oxide generating compounds may have potential value in therapeutic areas where modulation of ciliary muscle tension is desirable.

Heparin II domain of fibronectin mediates contractility through an alpha4beta1 co-signaling pathway.

In the trabecular meshwork (TM) of the eye, regulation of tissue contractility by the PPRARI sequence within the Heparin II (HepII) domain of fibronectin is believed to control the movement of aqueous humor and dictate the level of intraocular pressure. This study shows that the HepII domain utilizes activated alpha4beta1 integrin and collagen to mediate a co-signaling pathway that down-regulates contractility in TM cells. siRNA silencing of alpha4beta1 integrin blocked the actin disrupting effects of both PPRARI and the HepII domain. The down-regulation of the actin cytoskeleton and contractility did not involve syndecan-4 or other heparan sulfate proteoglycans (HSPGs) since siRNA silencing of syndecan-4 expression or heparitinase removal of cell surface HSPGs did not prevent the HepII-mediated disruption of the actin cytoskeleton. HepII-mediated disruption of the cytoskeleton depended upon the presence of collagen in the extracellular matrix, and cell binding studies indicated that HepII signaling involved cross-talk between alpha4beta1 and alpha1/alpha2beta1 integrins. This is the first time that the PPRARI sequence in the HepII domain has been shown to serve as a physiological alpha4beta1 ligand, suggesting that alpha4beta1 integrin may be a key regulator of tissue contractility.

Sodium orthovanadate effect on outflow facility and intraocular pressure in live monkeys.

Sodium orthovanadate (Na(3)VO(4)) is reported to reduce IOP by affecting aqueous formation, but whether it also affects outflow facility (OF) is unclear. We tested the effect of Na(3)VO(4) on OF and intraocular pressure (IOP) in live cynomolgus monkeys, and on actin and cell adhesion organization in cultured human trabecular meshwork (HTM) cells. Total OF (n = 12) was measured by 2-level constant pressure perfusion of the monkey anterior chamber (AC) before and after exchange with 1 mM Na(3)VO(4) or vehicle in opposite eyes. Topical 1% Na(3)VO(4) or vehicle only was given twice daily (each 2 × 20 µL drops) for 4 days to opposite eyes (n = 8), and Goldmann IOP was measured before and hourly after treatment for 6 h on Days 1 and 4. Filamentous actin and vinculin-containing cell adhesions were examined by epifluorescence microscopy after the cells had been incubated with 1 mM Na(3)VO(4) for 24 h. A) In monkeys, Na(3)VO(4) increased OF by 29.3 ± 8.8% (mean ± s.e.m.) over the perfusion interval when adjusted for baseline and contralateral eye washout (p = 0.01; n = 12). B) Day 1 baseline IOP was 16.2 ± 1.5 mmHg in treated eyes and 15.9 ± 1.3 mmHg in the contralateral control eyes. Following treatment on Day 1, IOP was no different (p > 0.05) between treated eyes and control eyes at any time-point or compared to baseline. Day 4 mean IOP averaged over hours 2-6 was 13.5 ± 0.8 mmHg in treated eyes and 16.1 ± 0.2 mmHg in control eyes. Treated eye IOP was lower than its Day 4 baseline (p < 0.005), lower than control eyes for the same Day 4 interval (p = 0.009), and lower than the Day 1 baseline (p = 0.0000). Control eye IOP on Day 4 was not significantly different from baseline on Day 1. C) Incubation of HTM cells with 1 mM Na(3)VO(4) for 24 h caused a loss of actin stress fibers and vinculin-containing adhesions. Cell retraction and separation was also observed in vanadate-treated cultures. Reformation of actin stress fibers, vinculin-containing adhesions and confluent monolayers occurred within 24 h after Na(3)VO(4)-containing culture medium was replaced with Na(3)VO(4)-free medium. Ocular administration of Na(3)VO(4) to live monkeys significantly increases OF and reduces IOP. Na(3)VO(4) reversibly disrupts actin and cell adhesion organization and causes retraction and separation of cultured HTM cells. Na(3)VO(4) increases pressure-dependent outflow in live monkeys. Altered actin architecture in the TM may play a part in this increased OF.

The role of the actomyosin system in regulating trabecular fluid outflow.

Abnormally high resistance to aqueous humor drainage via the trabecular meshwork and Schlemm's canal is highly correlated with the development of primary open-angle glaucoma. Contractility of the actomyosin system in the trabecular cells or inner wall endothelium of Schlemm's canal is an important factor in the regulation of outflow resistance. Cytoskeletal agents, affecting F-actin integrity or actomyosin contractility, or gene therapies, employing overexpression of caldesmon or Rho-A inhibition, can decrease outflow resistance in the drainage pathway. In this review, we discuss the mechanisms underlying these and similar effects on trabecular outflow resistance in living animals and/or in cultured ocular anterior segments from enucleated animal or human eyes.

Update on the mechanism of action of topical prostaglandins for intraocular pressure reduction.

A decade has passed since the first topical prostaglandin analog was prescribed to reduce intraocular pressure (IOP) for the treatment of glaucoma. Now four prostaglandin analogs are available for clinical use around the world and more are in development. The three most efficacious of these drugs are latanoprost, travoprost, and bimatoprost, and their effects on IOP and aqueous humor dynamics are similar. A consistent finding is a substantial increase in uveoscleral outflow and a less consistent finding is an increase in trabecular outflow facility. Aqueous flow appears to be slightly stimulated as well. Prostaglandin receptors and their associated mRNAs have been located in the trabecular meshwork, ciliary muscle, and sclera, providing evidence that endogenous prostaglandins have a functional role in aqueous humor drainage. Earlier evidence found that topical PG analogs release endogenous prostaglandins. One well-studied mechanism for the enhancement of outflow by prostaglandins is the regulation of matrix metalloproteinases and remodeling of extracellular matrix. Other proposed mechanisms include widening of the connective tissue-filled spaces and changes in the shape of cells. All of these mechanisms alter the permeability of tissues of the outflow pathways leading to changes in outflow resistance and/or outflow rates. This review summarizes recent (since 2000) animal and clinical studies of the effects of topical prostaglandin analogs on aqueous humor dynamics and recent cellular and molecular studies designed to clarify the outflow effects.

SB772077B, A New Rho Kinase Inhibitor Enhances Aqueous Humour Outflow Facility in Human Eyes.

We investigated the effect of a new Rho kinase inhibitor, SB772077B (SB77) on aqueous outflow facility (OF) in human eyes using human organ-cultured anterior segment (HOCAS). IOP was monitored for 24 h post-treatment with either SB77 (0.1/10/50 µM) or vehicle after a stable baseline pressure. The hydrodynamic pattern of aqueous outflow was analysed by labelling outflow pathway with red fluorescent microspheres. The effect of SB77 on cell morphology, actin stress fibers, focal adhesions, ECM, status of RhoA activation and myosin light chain phosphorylation (p-MLC) were evaluated and compared with Y27632, by immunostaining using primary human trabecular meshwork (HTM) cells. Following 24 h treatment, SB77 increased OF by 16% at 0.1 µM (N = 6), 29% at 10 µM (N = 8; p = 0.018) and 39% at 50 µM (N = 8; p = 0.004) in human eyes. There was an overall increase in tracer quantity and in area along inner wall of Schlemm's canal. Treatment with SB77 showed no evidence of cytotoxicity and caused a significant reduction in the expression of fibrotic markers compared to Y27632. The present findings indicate that SB77 treatment was effective in enhancing OF and reducing fibrotic markers in an ex vivo model. Thus SB77 may be a potential clinical candidate for the management of glaucoma.

Proteasome Inhibition Increases the Efficiency of Lentiviral Vector-Mediated Transduction of Trabecular Meshwork.

Purpose: To determine if proteasome inhibition using MG132 increased the efficiency of FIV vector-mediated transduction in human trabecular meshwork (TM)-1 cells and monkey organ-cultured anterior segments (MOCAS). Methods: TM-1 cells were pretreated for 1 hour with 0.5% dimethyl sulfoxide (DMSO; vehicle control) or 5 to 50 µM MG132 and transduced with FIV.GFP (green fluorescent protein)- or FIV.mCherry-expressing vector at a multiplicity of transduction (MOT) of 20. At 24 hours, cells were fixed and stained with antibodies for GFP, and positive cells were counted, manually or by fluorescence-activated cell sorting (FACS). Cells transduced with FIV.GFP particles alone were used as controls. The effect of 20 µM MG132 treatment on high- and low-dose (2 × 107 and 0.8 × 107 transducing units [TU], respectively) FIV.GFP transduction with or without MG132 was also evaluated in MOCAS using fluorescence microscopy. Vector genome equivalents in cells and tissues were quantified by quantitative (q)PCR on DNA. Results: In the MG132 treatment groups, there was a significant dose-dependent increase in the percentage of transduced cells at all concentrations tested. Vector genome equivalents were also increased in TM-1 cells treated with MG132. Increased FIV.GFP expression in the TM was also observed in MOCAS treated with 20 µM MG132 and the high dose of vector. Vector genome equivalents were also significantly increased in the MOCAS tissues. Increased transduction was not seen with the low dose of virus. Conclusions: Proteasome inhibition increased the transduction efficiency of FIV particles in TM-1 cells and MOCAS and may be a useful adjunct for delivery of therapeutic genes to the TM by lentiviral vectors.

Effects of Latanoprost and Bimatoprost on the Expression of Molecules Relevant to Ocular Inflow and Outflow Pathways.

BACKGROUND AND PURPOSE: The intraocular pressure (IOP)-lowering and side effects in response to different prostaglandin F2α analogues can be variable, but, the underlying basis for this difference remains unknown. This study investigated the differential changes of cellular proteins relevant to IOP-lowering effects of latanoprost and bimatoprost. METHODS: The human T lymphoblast (MOLT-3) cell line and immortalized human trabecular meshwork (iHTM) cells were studied by quantitative PCR and by immunofluorescence after treatment with either latanoprost or bimatoprost. New Zealand white rabbit eyes were treated topically with each agent and, following euthanasia, anterior segment tissues were studied with immunostaining. RESULTS: In cultured MOLT-3 cells, mRNA expression of both c-fos and matrix metalloproteinase 9 increased significantly in response to each agent. In addition, there was little change in tissue inhibitor of metalloproteinase (TIMP)-3 mRNA, but a significant decrease in TIMP-4. Fibronectin mRNA in MOLT-3 cells was down-regulated with bimatoprost, but was up-regulated with latanoprost. Immunofluorescence analysis of iHTM cells showed that intracellular fibronectin was significantly decreased by bimatoprost, but was increased by latanoprost. Both latanoprost and bimatoprost increased mRNA expression of NF-кB p65 and decreased that of IкBα. Aquaporin-1 mRNA expression was significantly down-regulated by bimatoprost. Immunostaining also revealed a significant decrease of aquaporin-1 in the ciliary epithelium of New Zealand white rabbits after bimatoprost treatment. CONCLUSIONS: Similarities in protein expression produced by latanoprost and bimatoprost in vitro may be relevant to the mechanism for their IOP-lowering effects in vivo. Differences in fibronectin expression and in aquaporin-1 expression in response to each agent may contribute to variability in the IOP-lowering efficacy in some studies.

Aqueous humor dynamics in monkeys after topical R-DOI.

PURPOSE: To determine the effects of R-DOI, a selective 5-HT2 agonist, on intraocular pressure (IOP) and aqueous humor dynamics in monkeys. METHODS: Normotensive cynomolgus monkeys (n = 8) were treated topically once daily with four 5-muL drops of 0.5% R-DOI in one eye, vehicle in the opposite eye. The 6-hour IOP response (Goldmann applanation tonometry) was determined before the drug application and on the third day of treatment. Aqueous humor formation, or flow (AHF, measured by fluorophotometry), was measured from hours 3 to 8 after the third dose. Beginning 3.5 hours after the fourth or fifth dose, AHF was measured by dilution of radio-iodinated monkey albumin perfused through the anterior chamber and flow to blood by accumulation of albumin in the general circulation. Uveoscleral outflow (Fu) was calculated. Flow to blood was determined at spontaneous and elevated pressures, allowing calculation of trabecular outflow facility. Total outflow facility was determined by two-level constant pressure perfusion from 3.5 to 5 hours and from 5.5 to 6.25 hours after R-DOI treatment. RESULTS: Reduction of IOP in treated eyes was compared to the opposite control eyes corrected for the 6-hour IOP baseline before the first dose. After the third dose of R-DOI, IOP was significantly (P < 0.01, n = 7) decreased by 1.4 to 4.7 mm Hg over the 6 hours. AHF (by fluorophotometry) increased by 13% (P < 0.05, n = 8) in treated compared with control eyes corrected for baseline. AHF (isotope dilution) increased by 30% (P < 0.01, n = 8), flow to blood decreased by 28% (n = 5), and Fu increased by 241% (P < 0.05, n = 5). Total and trabecular outflow facility were unchanged. CONCLUSIONS: R-DOI caused a small but significant increase in AHF and lowered IOP in normotensive monkeys primarily by increasing Fu.

The effect of C3 transgene expression on actin and cellular adhesions in cultured human trabecular meshwork cells and on outflow facility in organ cultured monkey eyes.

PURPOSE: To determine the effects of adenovirus-delivered exoenzyme C3 transferase (C3) gene expression on cultured human trabecular meshwork (HTM) cells and on outflow facility in organ cultured monkey anterior segments. METHODS: An adenoviral (Ad) vector expressing both C3 and green fluorescent protein (GFP) was used to transduce cultured HTM cells. Changes in cell morphology and the organization of actin, vinculin, and beta-catenin were assessed using immunofluorescence. Cultured monkey eye anterior segments were used to test the effects of AdC3GFP on outflow facility. RESULTS: Treatment of HTM cells with AdC3GFP resulted in dose-dependent morphological changes 3 or 4 days post-transduction. The AdC3GFP-transduced cells were either partially retracted, rounded, or very elongated compared to non-transduced cells. Compared to AdGFP-transduced cells, AdC3GFP-transduced cells demonstrated disrupted actin cytoskeleton, reduced vinculin-positive focal adhesions, and loss of beta-catenin staining. Cells transduced with AdGFP did not round up or retract. In organ culture studies, outflow facility was increased by 90+/-21% (n=15, p<0.001) in AdC3GFP-transduced eyes compared to baseline and corrected for AdGFP-transduced control eye washout on days 3-6 after transduction. CONCLUSIONS: C3 transduction is effective in disrupting actin filaments, cytoskeleton, and cellular adhesions in HTM cells and in increasing outflow facility in organ cultured monkey anterior segments, suggesting that expressing the C3 gene in the trabecular meshwork may be an effective approach for glaucoma therapy.

Vitreous glutamate concentration and axon loss in monkeys with experimental glaucoma.

OBJECTIVE: To evaluate vitreous glutamate concentration and axon loss in monkeys with experimental glaucoma. METHODS: We induced unilateral chronic glaucoma by means of laser trabecular destruction in 14 rhesus and 6 cynomolgus monkeys. Intraocular pressure (IOP) was monitored weekly. We assessed optic nerve damage clinically and photographically. Vitreous, sampled immediately before enucleation, was analyzed for glutamate concentration by means of high-performance liquid chromatography. We quantified percentage of axon loss after histopathologic sectioning of the optic nerve, compared median glutamate concentration ratios, and assessed correlation of glutamate concentration, axon count, IOP, cup-disc ratio, duration of IOP elevation, and age. RESULTS: Median vitreous glutamate concentration in glaucomatous eyes was 7.0 micromol/L (range, 3.0-88.6 micromol/L) vs 6.7 micromol/L (range, 2.8-87.4 micromol/L) in control eyes. The ratio (glaucomatous to control eyes) was 1.08. We found no significant correlation between vitreous glutamate concentration ratio and any of the other variables. The IOP, disc cupping, and axon loss were correlated. CONCLUSIONS: We found no difference between vitreous glutamate concentration in glaucomatous and contralateral control monkey eyes when the entire data set was examined and no evidence of correlation between vitreous glutamate concentration and axon loss. CLINICAL RELEVANCE: Vitreous concentration of the excitotoxic amino acid glutamate, thought to be associated with retinal ganglion cell death in glaucoma, was not altered in this study.

Viral Vector Effects on Exoenzyme C3 Transferase-Mediated Actin Disruption and on Outflow Facility.

PURPOSE: Purified Clostridium botulinum exoenzyme C3 transferase (C3) effects on the actin cytoskeleton in human trabecular meshwork cells (HTM) and on the outflow facility response in monkey organ-cultured anterior segments (MOCAS) were determined in the presence or absence of viral vectors. METHODS: Human adenovirus type 5 (AdV) and feline immunodeficiency virus (FIV) vectors were produced using kits. Cell soluble purified C3 (C3cs) was purchased commercially. Recombinant C3 (C3rec) cDNA was overexpressed in Escherichia coli and purified. The HTM cells were incubated with up to 10 µg/mL C3cs or with 5 µg of C3rec and/or viral vector (multiplicity of infection [MOI] = 25). Cells then were fixed and stained for actin. Outflow facility in MOCAS was measured at baseline, 4 hours, 24 hours, and 3 to 4 days following bolus injection of AdV (1.6 × 107 transducing units) and/or 2.5 µg C3rec. RESULTS: The HTM cells treated for 4 hours with C3cs (all doses) or for 24 hours with C3rec developed a rounded morphology and lost stress fibers. Cells transduced with vectors alone showed no changes at any time point. Cells exposed to C3rec and cotransduced with either viral vector showed significant disruption of the actin cytoskeleton within 4 hours after exposure, which persisted at 24 hours. In MOCAS, the AdV vector alone had no effect on outflow facility, but enhanced the response to C3rec at 4 hours. CONCLUSIONS: Coadministration of viral vectors enhances the ability of C3 transferase to disrupt actin stress fiber formation in HTM cells and increase outflow facility in MOCAS. Viral vectors potentially could be used to increase the bioavailability of proteins for cells that are difficult to transfect.

Rat, mouse, and primate models of chronic glaucoma show sustained elevation of extracellular ATP and altered purinergic signaling in the posterior eye.

PURPOSE: The cellular mechanisms linking elevated IOP with glaucomatous damage remain unresolved. Mechanical strains and short-term increases in IOP can trigger ATP release from retinal neurons and astrocytes, but the response to chronic IOP elevation is unknown. As excess extracellular ATP can increase inflammation and damage neurons, we asked if sustained IOP elevation was associated with a sustained increase in extracellular ATP in the posterior eye. METHODS: No ideal animal model of chronic glaucoma exists, so three different models were used. Tg-Myoc(Y437H) mice were examined at 40 weeks, while IOP was elevated in rats following injection of hypertonic saline into episcleral veins and in cynomolgus monkeys by laser photocoagulation of the trabecular meshwork. The ATP levels were measured using the luciferin-luciferase assay while levels of NTPDase1 were assessed using qPCR, immunoblots, and immunohistochemistry. RESULTS: The ATP levels were elevated in the vitreal humor of rats, mice, and primates after a sustained period of IOP elevation. The ecto-ATPase NTPDase1 was elevated in optic nerve head astrocytes exposed to extracellular ATP for an extended period. NTPDase1 was also elevated in the retinal tissue of rats, mice, and primates, and in the optic nerve of rats, with chronic elevation in IOP. CONCLUSIONS: A sustained elevation in extracellular ATP, and upregulation of NTPDase1, occurs in the posterior eye of rat, mouse, and primate models of chronic glaucoma. This suggests the elevation in extracellular ATP may be sustained in chronic glaucoma, and implies a role for altered purinergic signaling in the disease.

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.

Neurochemical correlates of cortical plasticity after unilateral elevated intraocular pressure in a primate model of glaucoma.

PURPOSE: To examine the time course of changes in the expression patterns of several synaptic plasticity markers in the primary visual cortex after unilateral elevated intraocular pressure (IOP) in a primate model of glaucoma. METHODS: A monkey model of experimental glaucoma was combined with immunohistochemical and histochemical methods to assess changes in expression patterns and metabolic activity of cortical neurons in V1. RESULTS: Experimental unilateral glaucoma altered the spatial and temporal distribution of several neurochemicals associated with cortical plasticity in V1 of the primate. Within-animal comparisons of immunohistochemical studies revealed that GABAa receptor protein and GAP-43 were significantly lower in glaucomatous versus normal eye bands after 2, 4, and 7 months of elevated IOP. SYN immunoreactivity was also lower in the glaucomatous versus the normal eye bands but only at 4 months of elevated IOP. CAMKIIalpha immunoreactivity levels were higher in the glaucomatous versus the normal eye bands. Between-animal comparisons revealed that the levels of GAP-43 and SYN were upregulated, whereas levels of GABAa receptor protein were downregulated, in glaucomatous eyes when compared with levels in the visual cortex of normal animals. CONCLUSIONS: Unilateral elevation of IOP affects both the metabolic activity of cortical neurons and the expressed levels of GAP-43, SYN, GABAa receptor protein, and CAMKIIalpha, as measured immunohistochemically in the primary visual cortex of adult monkeys. Because these neurochemicals are thought to be necessary for synaptic plasticity, their redistribution may support functional recovery of cortical neurons after damage to retinal ganglion cells induced by elevated IOP.

H-7 effect on outflow facility after trabecular obstruction following long-term echothiophate treatment in monkeys.

PURPOSE: To determine whether H-7 can enhance outflow facility after trabecular meshwork obstruction by extracellular material that accumulates after long-term treatment of monkeys with the cholinesterase inhibitor echothiophate iodide (ECHO). METHODS: Cynomolgus monkeys were treated topically with 150 microg ECHO in one (n = 4 eyes) or both (n = 8 eyes) eyes for up to 48 weeks. Accommodation response to topical pilocarpine was monitored periodically. Outflow facility response to H-7 was measured by two-level constant pressure perfusion on three or four different occasions after intraocular pressure was elevated for 12 to 18 weeks. RESULTS: Long-term treatment with ECHO decreased the accommodative response to pilocarpine and increased intraocular pressure, as has been reported. Baseline outflow facility was decreased by 46% +/- 7% (n = 12, P < 0.001). H-7 partially restored baseline outflow facility measured during subsequent perfusions while ECHO treatment was continued. Concurrent H-7 enhanced outflow facility by 73% +/- 18% (n = 12, P < 0.005) beyond the same-day baseline in ECHO-treated eyes. Cessation of ECHO treatment further restored baseline outflow facility, and the outflow facility response to H-7. CONCLUSIONS: H-7 can enhance OF in the presence of trabecular obstruction produced by long-term ECHO treatment. This suggests that H-7 may be useful in treating glaucoma, even in the presence of accumulated plaque material that has been described previously.

Viscocanalostomy in rhesus monkeys.

OBJECTIVE: To examine structural changes and aqueous humor outflow after viscocanalostomy in live normal monkey eyes. METHODS: Viscocanalostomy surgery was performed in 1 eye of each of 4 rhesus monkeys. Outflow facility was determined before and after surgery. All eyes were fixed and examined by light and/or electron microscopy 36 or 63 days postoperatively. RESULTS: Schlemm canal was replaced by scar tissue at the surgical site. The juxtacanalicular zone contained homogeneous material, probably high-molecular-weight 1.4% sodium hyaluronate. The sclera external to Schlemm canal was overhydrated, and remains of a scleral lake were present in 1 animal. Multiple defects were present in the endothelial lining of Schlemm canal inner and outer wall. Fine fibrillar material and sheath-derived plaque material partly bridged the defects. Along the inner wall, aggregations of thrombocytes covered some defects in the endothelial lining of the canal. At 90 degrees to 180 degrees from the surgical site, small and fewer breaks in the inner wall were seen. Postsurgery outflow facility (n = 2) was approximately 30% higher in the treated eye than in the contralateral control, corrected bilaterally for presurgery baseline. CONCLUSIONS: The most likely explanations for the increase in outflow facility in monkeys after viscocanalostomy are focal disruptions of the inner wall endothelium of Schlemm canal and disorganization of the juxtacanalicular zone, resulting in direct communication of juxtacanalicular zone extracellular spaces with the lumen of Schlemm canal. The continuous presence of sodium hyaluronate might prevent repair of these defects by interfering with thrombocyte function. CLINICAL RELEVANCE: In nonhuman primates, viscocanalostomy appears to decrease outflow resistance through persisting focal disruption of the inner wall endothelium and opening of the juxtacanalicular or cribriform region of the trabecular meshwork, the tissue most affected by pathologic changes in primary open-angle glaucoma in humans.

Effects of prostaglandins on the aqueous humor outflow pathways.

Topical treatments with certain prostaglandins (PGs), including FP receptor agonists, lower intraocular pressure by increasing uveoscleral outflow. Although the precise mechanism for the increased uveoscleral outflow is not known, there appears to be activation of a molecular transduction cascade and an increase in the biosynthesis of certain metalloproteinases. This leads to reduction of extracellular matrix components within the ciliary muscle, iris root, and sclera. It is possible that this reduction of extracellular matrix present within portions of the uveoscleral pathway may contribute to the mechanism of increased uveoscleral outflow. Additional mechanisms that may contribute to the PG-mediated increase of uveoscleral outflow include relaxation of the ciliary muscle, cell shape changes, cytoskeletal alteration, or compaction of the extracellular matrix within the tissues of the uveoscleral outflow pathway. Future studies should clarify the importance of these various responses that may contribute to increased uveoscleral outflow. At present, there is no compelling evidence for a substantial facility-increasing effect on the trabecular meshwork outflow for any of these compounds.

Differential expression of matrix metalloproteinases in monkey eyes with experimental glaucoma or optic nerve transection.

Extracellular matrix (ECM) remodeling after neuronal injury and reactive gliosis is carried out by activation of matrix metalloproteinases (MMPs) regulated by their tissue inhibitors (TIMPs). In glaucoma, there is a loss of retinal ganglion cells and extensive ECM remodeling (cupping) at the level of the optic nerve head, frequently associated with elevated intraocular pressure. To determine whether ECM remodeling in the glaucomatous optic nerve head occurs in response to loss of axons or to elevated intraocular pressure we compared the patterns of MMP and TIMP expression in the eyes of monkeys with laser-induced glaucoma or with optic nerve transection. MT1-MMP and MMP1 expression was markedly increased in reactive astrocytes in optic nerve heads with experimental glaucoma but not in the optic nerve head of transected eyes. In normal control eyes retinal ganglion cells expressed MMP2, TIMP1 and TIMP2 constitutively, and the proteins were detected in their axons. At the site of transection, MT1-MMP, MMP1, MMP2, TIMP1 and TIMP2 were expressed by reactive astrocytes. Inflammatory cells, fibroblasts and reactive astrocytes at the transected site expressed MMP3 and MMP9, which were undetectable in the retina and optic nerve head in any condition. Constitutive expression of MMP2, TIMP1 and TIMP2 in retinal ganglion cells suggests a role in maintenance of synaptic integrity and plasticity and maintenance of the periaxonal space. Increased MMP1 and MT1-MMP1 expression in the glaucomatous optic nerve head is specific to tissue remodeling due to elevated intraocular pressure and not secondary to loss of axons.