Rong-Fang Wang

Differential vulnerability of neurochemically identified subpopulations of retinal neurons in a monkey model of glaucoma

The vulnerability of subpopulations of retinal neurons delineated by their content of cytoskeletal or calcium-binding proteins was evaluated in the retinas of cynomolgus monkeys in which glaucoma was produced with an argon laser. We quantitatively compared the number of neurons containing either neurofilament (NF) protein, parvalbumin, calbindin or calretinin immunoreactivity in central and peripheral portions of the nasal and temporal quadrants of the retina from glaucomatous and fellow non-glaucomatous eyes. There was no significant difference between the proportion of amacrine, horizontal and bipolar cells labeled with antibodies to the calcium-binding proteins comparing the two eyes. NF triplet immunoreactivity was present in a subpopulation of retinal ganglion cells, many of which, but not all, likely correspond to large ganglion cells that subserve the magnocellular visual pathway. Loss of NF protein-containing retinal ganglion cells was widespread throughout the central (59-77% loss) and peripheral (96-97%) nasal and temporal quadrants and was associated with the loss of NF-immunoreactive optic nerve fibers in the glaucomatous eyes. Comparison of counts of NF-immunoreactive neurons with total cell loss evaluated by Nissl staining indicated that NF protein-immunoreactive cells represent a large proportion of the cells that degenerate in the glaucomatous eyes, particularly in the peripheral regions of the retina. Such data may be useful in determining the cellular basis for sensitivity to this pathologic process and may also be helpful in the design of diagnostic tests that may be sensitive to the loss of the subset of NF-immunoreactive ganglion cells.

A comparative study of latanoprost (Xalatan) and isopropyl unoprostone (Rescula) in normal and glaucomatous monkey eyes

Latanoprost (PhXA41, Xalatan) and isopropyl unoprostone (UF-021, unoprostone, Rescula) two new prostanoid derivatives, have been shown to reduce intraocular pressure (IOP) significantly in patients with glaucoma or ocular hypertension. This study was designed to compare the ocular hypotensive effects of latanoprost and unoprostone in cynomologus monkeys with glaucoma and characterizes the prostanoids mechanisms of action in normal cynomolgus monkey eyes. Intraocular pressure was measured daily at 0, 0.5, and 1 hour and hourly for 5 additional hours during 1 baseline day, 1 vehicle-treated day, and 5 days of therapy with either 0.005% latanoprost or 0.12% unoprostone applied twice daily, at 9:30 AM and 3:30 PM, to the glaucomatous eye of eight monkeys with unilateral laser-induced glaucoma. Outflow facility was measured in six normal monkeys 3 hours prior to dosing and 1 hour after unilateral dosing with either drug. Aqueous humor flow rates were measured in six normal monkeys hourly for 4 hours on 1 baseline day and on 1 treatment day beginning 1 hour after administration of either drug to one eye. Intraocular pressure was significantly (P < 0.005) reduced after the first application for 4 hours with latanoprost and for 2 hours with unoprostone, up to 5.4 +/- 0.8 mm Hg (mean +/- SEM) (latanoprost) and 3.8 +/- 0.5 mm Hg (unoprostone). Intraocular pressure was significantly (P < 0.005) reduced for at least 18 hours following each PM dose of latanoprost. Intraocular pressure was not reduced (P > .05) 18 hours after each PM dose of unoprostone. An enhancement of the ocular hypotensive effect was observed from day 1 to day 5 with repeated dosing of either drug. Latanoprost produced a greater magnitude of IOP reduction for a longer duration of time than unoprostone after each application. Neither drug altered outflow facility or aqueous humor flow rates. Latanoprost and unoprostone appear to reduce IOP in monkeys by enhancing uveoscleral outflow. Latanoprost appears to be more efficacious and potent than unoprostone in reducing IOP in glaucomatous monkey eyes.

Effect of 5-mca-nat, a Putative Melatonin Mt3 Receptor Agonist, on Intraocular Pressure in Glaucomatous Monkey Eyes

Purpose5-MCA-NAT, a putative melatonin MT3 receptor agonist, reduced intraocular pressure (IOP) in ocular normotensive rabbit eyes. This study evaluates the effect of topical application of 5-MCA-NAT on IOP in monkey eyes with laser-induced unilateral glaucoma. MethodsA multiple-dose study was performed in 8 glaucomatous monkey eyes. One 25-μL drop of 5-MCA-NAT (2%) was applied topically to the glaucomatous eye at 9:30 am and 3:30 pm for 5 consecutive days. IOP was measured hourly for 6 hours beginning at 9:30 am for one baseline day, one vehicle-treated day, and treatment days 1, 3, and 5 with 5-MCA-NAT. ResultsCompared with vehicle treatment, twice daily administration of 5-MCA-NAT for 5 days reduced (P < 0.05) IOP from 1 hour to 5 hours after the first dose, and the IOP-lowering effects were shown to last at least 18 hours following administration, based on IOP measurements made after the fourth and eighth doses. The ocular hypotensive effect of 5-MCA-NAT was enhanced with repeated dosing. The maximum reduction (P < 0.001) of IOP occurred at 3 hours after each morning dose, and was 4.0 ± 0.5 (mean ± SEM) mm Hg (10%) on day 1, 5.6 ± 0.8 mm Hg (15%) on day 3, and 7.0 ± 1.1 mm Hg (19%) on day 5. Adverse ocular or systemic side effects were not observed during the 5 days of treatment. Conclusions5-MCA-NAT, a putative melatonin MT3 receptor agonist, reduces IOP in glaucomatous monkey eyes. Melatonin agonists with activity on the putative MT3 receptor may have clinical potential for treating elevated IOP.

Glutamate Receptor Subunit GluR2 and NMDAR1 Immunoreactivity in the Retina of Macaque Monkeys with Experimental Glaucoma Does Not Identify Vulnerable Neurons

Excitatory amino acid neurotoxicity has been proposed as a mechanism underlying selective neuronal death in glaucoma. The relationships between the cellular distribution of glutamate receptor subunit proteins GluR2 and NMDAR1 and the vulnerability of restricted retinal neuron subpopulations was explored in experimental glaucoma in macaque monkeys, produced by treating the trabecular meshwork in one eye with argon or diode laser burns. Immunostaining of retinal segments was performed using specific monoclonal antibodies to the GluR2 and NMDAR1 subunit proteins as well as neurofilament protein. The distribution of immunoreactivity was qualitatively assessed in the retina, and ganglion cells were counted in the paracentral and peripheral regions of each retinal segment. Immunoreactivity for both of these glutamate receptor subunit proteins was widely distributed in most retinal neuron types in control eyes and was colocalized with neurofilament protein in ganglion cells. In the glaucomatous eyes, densities of GluR2- and NMDAR1-immunoreactive ganglion cells were dramatically reduced compared to unaffected fellow eyes, but GluR2- and NMDAR1-immunoreactive populations of horizontal, bipolar, and amacrine cells were not affected. These data parallel previous observations on the selective vulnerability of ganglion cells in this experimental model of glaucoma. However, GluR2 and NMDAR1 subunits do not constitute cell type-specific markers of vulnerability in glaucoma as they are present in neurons prone to degeneration as well as in resistant ones. While retinal pathology in glaucoma involves excitotoxic mechanisms that may be related to glutamate receptor subunits regulating calcium fluxes, the specific pattern of neuronal vulnerability clearly depends on other cellular characteristics such as morphology, connectivity, and other aspects of the neurochemical phenotype.

Effect of 0.04% AR-13324, a ROCK, and norepinephrine transporter inhibitor, on aqueous humor dynamics in normotensive monkey eyes.

Purpose:To determine the mechanism by which topically applied AR-13324, a rho kinase inhibitor, and an inhibitor of the norepinephrine transporter, reduces intraocular pressure (IOP) in normotensive monkey eyes. Methods:Seven normotensive monkeys were used. Tonographic outflow facility (C) was measured before drug administration and repeated 6 hours after administration of 50 µL (25 µL×2) of 0.04% AR-13324 to 1 eye and an equal volume of vehicle to the contralateral control eye. Baseline aqueous humor flow rates (F) were measured hourly for 6 hours beginning at 10:00 AM on day 1. On day 2, 50 µL (25 µL×2) of 0.04% AR-13324 was applied to 1 eye of each animal and vehicle to the fellow eye at 8:00 AM. Aqueous humor flow rates were measured at the same times as on the baseline day beginning 2 hours after dosing. Results:Six hours after a single dose of 0.04% AR-13324 to 7 normal monkey eyes, C was increased (P<0.05) by 53% in drug-treated eyes compared with either contralateral vehicle-treated control eyes or baseline measurements. The IOP measured by pneumatonometer in treated eyes was reduced (P<0.005) by 25% when compared with baseline measurements and by 24% when compared with contralateral vehicle-treated eyes. For 6 hours after a single dose of 0.04% AR-13324, F was reduced (P<0.05) by 20% and 23% when compared with contralateral vehicle-treated eyes and baseline values, respectively. Conclusions:AR-13324 reduces IOP in normotensive monkey eyes. A dual mechanism of action, increase in tonographic outflow facility, and decrease of aqueous humor flow rates, accounts for the IOP reduction in normotensive monkey eyes.

A Comparison of Argon Laser and Diode Laser Photocoagulation of the Trabecular Meshwork to Produce the Glaucoma Monkey Model

Purpose: To create an experimental glaucoma monkey model using high-power diode laser photocoagulation of the trabecular meshwork, and to compare this with the experimental glaucoma monkey model induced by argon laser photocoagulation of the trabecular meshwork.Methods: One eye each of eight adult cynomolgus monkeys underwent repeated application of diode laser photocoagulation of the trabecular meshwork until sustained intraocular pressure (IOP) elevation was achieved. 50 to 120 spots were applied to midtrabecular meshwork for 360°; spot size, 75 μm; power, 1.2 W; duration, 0.5 seconds. Intraocular pressure, tonographic outflow facility, and ophthalmoscopically and photographically documented optic nerve head evaluations were carried out before and after treatment. Data were compared retrospectively with similar data from an experimental glaucoma monkey model after argon laser photocoagulation of the trabecular meshwork (n = 10).Results: The average number of laser treatments to achieve stable IOP elevation was 3.0 with both diode and argon laser trabecular treatments (p > 0.99). On week 4 after initial pressure elevation, peak IOP was greater—(p < 0.05) 43.0 mmHg ± 2.4 mmHg (mean ± SEM) and 37.4 mmHg ±1.3 mmHg—in the diode laser-induced than in the argon laser-induced glaucomatous eyes, respectively. Outflow facility (μl/min/ mmHg) was reduced (p < 0.001) in both diode (0.09 ± 0.01 μl/min/mmHg) and argon (0.10 ± 0.01 μl/min/mmHg) laser-induced glaucomatous eyes compared with untreated fellow eyes. Both the diode and argon laser techniques produced the earliest signs of optic nerve head excavation within about one month of IOP elevation.Conclusions: Repeat diode laser photocoagulation of the trabecular meshwork produced higher (p < 0.05) IOP elevation than argon laser photocoagulation of the trabecular meshwork in this study. No significant differences in outflow facility and optic nerve head change were observed between these two laser techniques. The experimental glaucoma monkey model can be created with either the diode or argon laser photocoagulation of the trabecular meshwork.

Comparison of the ocular hypotensive effect of brimonidine, dorzolamide, latanoprost, or artificial tears added to timolol in glaucomatous monkey eyes.

Purpose: To investigate the additive ocular hypotensive effect of brimonidine, dorzolamide, latanoprost, or artificial tears to timolol in monkey eyes with laserinduced unilateral glaucoma. Methods: Eight monkeys were used and each animal received all four combinations of drugs in a randomized fashion during the study. The washout period between each combination was at least 2 weeks. Intraocular pressure (IOP) was measured at 8:30 AM, 11:00 AM, 1:00 PM, and 3:30 PM on day 1 (untreated baseline), day 2 (timolol treatment alone), and days 3 through 5 (combination therapy with two drugs). One drop of 0.5% timolol was topically applied at 3:45 PM on day 1 and at 8:45 AM and 3:45 PM on days 2 through 5. One drop of 0.2% brimonidine or 2% dorzolamide or artificial tears was added on day 2 at 4:00 PM and at 9:00 AM and 4:00 PM on days 3 through 5, or latanoprost was added at 9:00 AM on days 3 through 5. Results: Compared with timolol alone, the maximal additive reduction in IOP which occurred on day 5 was 4.8 ± 0.8 mm Hg (mean ± standard error of the mean) with timolol plus brimonidine, 5.6 ± 1.0 mm Hg with timolol plus dorzolamide, 4.3 ± 1.0 mm Hg with timolol plus latanoprost, and 2.0 ± 0.5 mm Hg with timolol plus artificial tears (P < 0.01). At all measurements, timolol plus brimonidine, timolol plus dorzolamide, and timolol plus latanoprost caused greater (P < 0.05) IOP reductions than did timolol plus artificial tears. The additive IOP‐lowering effect was similar (P > 0.60) when comparing timolol plus brimonidine and timolol plus dorzolamide, timolol plus brimonidine and timolol plus latanoprost, timolol plus dorzolamide and timolol plus latanoprost at all measurements, but timolol plus dorzolamide caused a greater (P < 0.05) reduction of IOP than did timolol plus latanoprost at 0 hours on day 5. Conclusions: The addition of brimonidine, dorzolamide, or latanoprost to timolol caused similar additional reductions of IOP in glaucomatous monkey eyes.

Effect of flunarizine, a calcium channel blocker, on intraocular pressure and aqueous humor dynamics in monkeys.

PurposeTo evaluate the effects of flunarizine, a nonselective calcium channel blocker, on intraocular pressure (IOP) in monkeys with laser-induced unilateral glaucoma and on aqueous humor dynamics in normal monkeys. MethodsThe IOP was measured before and hourly for 6 hours after single-dose administration of 0.5%, 1%, or 2% flunarizine to the glaucomatous eye of 8 monkeys with unilateral laser-induced glaucoma. In a separate multiple-dose study, 0.5% flunarizine was applied twice daily for 5 consecutive days to the glaucomatous eye of the same 8 monkeys. IOP was measured at untreated baseline, after treatment with vehicle only, and on treatment days 1, 3, and 5. Tonographic outflow facility and fluorophotometric flow rates of aqueous humor were measured in 7 normal monkeys before and after the fifth dose of twice-daily treatment with 0.5% flunarizine. ResultsUnilateral application of 50 μL of 0.5%, 1%, or 2% flunarizine reduced IOP bilaterally. In the treated glaucomatous eyes, flunarizine reduced the IOP for 2, 3, or 5 hours, with a maximum reduction of 2.5±0.5 (mean±SEM) mm Hg (9%), 3.0±0.4 mm Hg (10%), and 5.0±0.8 mm Hg (18%) following the 0.5%, 1%, and 2% concentrations, respectively (P<0.01). The maximum reductions in IOP in the contralateral untreated eyes were 1.3±0.5 mm Hg, 1.5±0.3 mm Hg, and 2.9±0.7 mm Hg following the 0.5%, 1%, and 2% concentrations, respectively (P<0.05). Both the magnitude and duration of the ocular hypotensive effect of 0.5% flunarizine were enhanced with twice-daily administration for 5 days. Outflow facility in normal monkey eyes was increased (P<0.05) by 39% in the treated eyes compared with vehicle-treated contralateral eyes and by 41% compared with baseline values, and aqueous humor flow rates were unchanged (P>0.30). ConclusionsFlunarizine reduces IOP in a dose-dependent manner when administered to glaucomatous monkey eyes, but also has an ocular hypotensive effect on the contralateral untreated eyes. An increase in tonographic outflow facility seems to account for the IOP reduction in normal monkey eyes.

Effect of pilocarpine 4% in combination with latanoprost 0.005% or 8-iso prostaglandin E2 0.1% on intraocular pressure in laser-induced glaucomatous monkey eyes.

PurposeTo compare the effect of pilocarpine, an agent that reduces uveoscleral outflow, on the ocular hypotensive efficacy of latanoprost and 8-iso prostaglandin E2 (PGE2). MethodsEach of the two treatment groups was composed of the same eight monkeys with unilateral laser-induced glaucoma. Intraocular pressure (IOP) was measured hourly for 6 hours beginning at 9:00 am on the baseline day (Thursday before treatment week) and on treatment days 1, 3, and 5 (Monday, Wednesday, and Friday). On all five treatment days, one drop of pilocarpine 4% was administered at 9:00 am and 3:00 pm, and one drop of latanoprost 0.005% or 25 &mgr;L of 8-iso PGE2 0.1% was administered at 10:00 am and 4:00 pm. ResultsOne hour after pilocarpine instillation on day 1, the reduction of IOP was similar (P > 0.90) in both treatment groups, 7.6 ± 1.1 mm Hg (mean ± standard error of the mean ) in the latanoprost group and 7.4 ± 0.8 mm Hg in the 8-iso PGE2 group. However, the IOP effects of the two treatment groups became significantly different (P < 0.05) beginning 2 hours after dosing with latanoprost or 8-iso PGE2 on day 1. A difference (P < 0.05) between the two groups persisted at all subsequent measurements. The reduction of IOP lessened with repeated dosing in the latanoprost and 8-iso PGE2 groups. Three hours after dosing with pilocarpine and two hours after dosing with the prostanoids, the IOP reduction was 8.3 ± 0.9 mm Hg in the latanoprost group and 9.9 ± 0.6 mm Hg in the 8-iso PGE2 group on day 1, and 2.1 ± 1.0 mm Hg in the latanoprost group and 7.3 ± 0.9 mm Hg in the 8-iso PGE2 group on day 5. ConclusionsThe smaller reductions in IOP with pilocarpine and latanoprost than with pilocarpine and 8-iso PGE2 show that pilocarpine blocks much more of the ocular hypotensive effect of latanoprost than of 8-iso PGE2. The results also indicate that pilocarpine and latanoprost are mutually antagonistic. Enhancement of uveoscleral outflow appears to account for most of the ocular hypotensive effect of latanoprost and for much less of the ocular hypotensive effect of 8-iso prostaglandin E2.

The effect of MK-927, a topical carbonic anhydrase inhibitor, on IOP in glaucomatous monkeys

MK-927 is a water soluble, potent inhibitor of human carbonic anhydrase (CA) II in vitro. Topical administration of MK-927 reduces intraocular pressure (IOP) in rabbits. Elevated IOP was produced in cynomolgus monkey eyes by argon laser photocoagulation of the trabecular meshwork. IOP was measured at 0 hr, 0.5 hr and hourly for 8 hrs in 8 eyes for two baseline days, one day on vehicle and five days of therapy with 2% MK-927 b.i.d., after initial single-dose trials of various concentrations. IOP was not significantly different comparing baseline and vehicle treated days. Significant (p less than 0.05) reductions of IOP occurred for five days lasting at least 8 hrs after each dosing. At 3 hrs after treatment with vehicle the IOP was 31.6 +/- 3.4 (SE) mm Hg. Maximum reduction of IOP occurred at 3 hrs after application of MK-927, the IOP decreasing from day 1 (19.9 +/- 1.0 mm Hg) to day 5 (16.5 +/- 1.6 mm Hg). MK-927 appears to have great clinical potential.