Cynomolgus Monkey Eyes Research Articles

The Effect of Rho-Associated Protein Kinase Inhibitor on Monkey Schlemm's Canal Endothelial Cells

To investigate the effect of a specific inhibitor of Rho-associated protein kinase, Y-27632, on monkey Schlemm's canal endothelial (SCE) cells. SCE cells were isolated from cynomolgus monkey eyes. The effects of Y-27632 on aqueous outflow facility were evaluated using enucleated monkey eyes and a constant-pressure perfusion system. The effect of Y-27632 on the barrier function of the confluent SCE-cell monolayer was evaluated by measuring transendothelial electrical resistance (TEER) and fluorescein permeability. Y-27632-induced changes in the intracellular localization of ZO-1, claudin-5, β-catenin, pan-cadherin, and filamentous actin (F-actin) were examined by immunofluorescence. Gene-expression changes induced by Y-27632 were analyzed with microarray, and the functional categories of changed genes were identified by gene ontology analysis. The concentrations of intracellular calcium ions were estimated using Fluo-4/AM and a fluorescence microscope system. Y-27632 significantly increased the outflow facility and the number of associated giant vacuoles, decreased TEER of the SCE-cell monolayer, and increased the transendothelial flux of fluorescein. Y-27632 disrupted ZO-1 and claudin-5 expression in a confluent SCE-cell monolayer. Among 12,544 genes, Y-27632 treatment increased the expression of 57 genes and decreased the expression of 15 genes. Gene ontology analysis revealed that changed genes were related to various cellular functions, including regulation of calcium ion transport into the cytosol. Y-27632 partially diminished the A23187-induced increase in intracellular calcium ions. Y-27632 increased the permeability of the SCE-cell monolayer in association with disruption of the tight junction, F-actin depolymerization, and changes in various cell functions, including calcium transfer.

Pharmacokinetics of latanoprost in the cynomolgus monkey. 3rd communication: tissue distribution after topical administration on the eye studied by whole body autoradiography. Glaucoma Research Laboratories.

Latanoprost (13,14-dihydro-15(R)-17-phenyl-18,19,20-trinor-PGF2 alpha-isopropyl ester, CAS 130209-82-4, PhXA41, Xalatan), an antiglaucoma drug, labelled with tritium at carbon 13 and 14 (4.8 micrograms, 20.8 MBq/eye) was administered topically on the eyes of cynomolgus monkeys. After 0.5 h the concentration of radioactivity in the cornea was estimated to be about 0.6 ng eq/mg tissue. The elimination half-life of total radioactivity in the cornea was about 4 h. The corneal epithelium contained a higher concentration of radioactivity than the stroma. Cornea seemed to act as a slow release depot to the anterior part of the eye. In the iris, anterior chamber and ciliary body the maximal concentrations were 217.0 +/- 12.9 pg eq/mg, 99.8 +/- 7.4 pg eq/mg and 54.0 +/- 4.9 pg eq/mg, respectively, 1 h after administration. The elimination half-life of total radioactivity from these tissues was 3-4 h. Trace amounts (0.4-9 pg eq/mg) remained in these tissues 24 h after administration. Initially the radioactivity was present in the conjunctiva, sclera and choroid as well as in the general circulation. Radioactivity passed through the lachrymal ducts and high concentrations were observed in the oesophagus, stomach content, small intestine, bile and urine of a monkey administered latanoprost topical on the eye 0.5 and 1 h before sacrifice. In this animal concentrations of radioactivity were found in the kidney, liver, wall of the small intestine and blood. All other tissues in this animal contained lower concentrations of radioiactivity than the blood. In an animal sacrificed 2 h after administration of tritium labelled latanoprost on one eye and 6 h after administration on the other eye the highest concentrations of radioactivity were found in urine, bile and in the stomach content. Low concentration of radioactivity remained in the kidney and the liver. In a monkey administered latanoprost 12 and 24 h before death low concentrations remained in the colon, bile and urine. The anterior parts of the eyes from the monkey sacrificed 0.5 and 1 h after administration of latanoprost were cut out from the tissue sections for HPLC analysis. The predominating peak present corresponded to acid of latanoprost (PhXA85). In the stomach the radioactivity chromatographed as latanoprost and the acid of latanoprost. In the small intestine and in bile the main radioactive peak corresponded to 1,2-dinor-acid of latanoprost and in addition several more polar metabolites were present. In conclusion, latanoprost penetrated the cornea, was hydrolysed and slowly released into the anterior parts of the eye the site of action. The maximum concentration in the eye was reached after 1 h with an elimination half-life of 3-4 h. In the body the distribution was limited mainly to the gastro-intestinal tract, the kidney, the gall- and urinary bladder.

Potential for serotonergic agents to treat elevated intraocular pressure and glaucoma: focus on 5-HT2 receptor agonists

Although several classes of drugs, including FP-receptor prostaglandin agonist analogs, are available to treat elevated intraocular pressure (IOP) associated with glaucoma, novel pharmaceuticals to treat this potentially blinding disease are still being sought. With recent demonstration of potent IOP-lowering activity of serotonin-2 receptor agonists in conscious ocular hypertensive Cynomolgus monkey eyes, the prospects of this unexampled class of compounds yielding new drugs for the treatment of elevated IOP look bright. This article reviews the ocular serotonergic literature pertaining to elevated IOP in patients with ocular hypertension and glaucoma, the data on ocular hypotensive serotonin-2 agonists, and discusses their potential mechanism(s) of action at the cellular and whole-animal level.

Serotonin-2 Receptor Agonists as Novel Ocular Hypotensive Agents and their Cellular and Molecular Mechanisms of Action: Novel Drug Targets for Glaucoma Treatment

The eye is innervated by numerous serotonergic nerves and serotonin (5-hydroxytryptamine; 5HT) is present in the aqueous humor of animal and human eyes. In an effort to delineate the role of the serotonergic system in modulating intraocular pressure (IOP) within the anterior segment of the eye, extensive topical ocular dosing studies were conducted with a variety of 5HT ligands and in various animal species. Even though certain 5HT(1A) agonists decreased IOP in rabbits, these compounds failed to affect IOP in normotensive or ocular hypertensive monkey eyes. In contrast, while 5HT(2) agonists induced significant IOP reductions in normotensive rat eyes and in eyes of ocular hypertensive Cynomolgus monkeys, these agents were inactive in ocular normotensive cats and rabbits. Additional studies indicated a strong involvement of 5HT(2A) receptors in mediating IOP-lowering in conscious ocular hypertensive Cynomolgus monkeys. As a result of further structure-activity investigations, AL-34662, a selective 5HT(2) agonist (relative to other 5HT receptor types and sub-types) with high affinity, potency and efficacy at 5HT(2A), 5HT(2B) and 5HT(2C) receptors was discovered that efficaciously lowered IOP in the monkey model of ocular hypertension (33 +/- 3 % reduction out to 6 hrs post with a 300 microg topical ocular dose). Due to unavailability of monkey ocular cells, extensive in vitro studies were conducted using relevant human ocular cells in order to correlate with and support the in vivo observations in the monkeys. RT-PCR and in situ hybridization studies revealed the presence of mRNAs for 5HT(2A-C) receptor subtypes in human ocular tissues involved in IOP modulation. The relative distribution and density of these mRNAs were as follows: ciliary body (CB) (5HT(2A) > 5HT(2B) > 5HT(2C)), ciliary epithelium (CE) (5HT(2A) > 5HT(2B) = 5HT(2C)) and trabecular meshwork (TM) (5HT(2A)= 5HT(2B) >> 5HT(2C)). Furthermore, quantitative autoradiography revealed a relatively high specific binding of [(3)H]-5HT and [(3)H]-ketanserin to 5HT(2) receptors in human CE and longitudinal ciliary muscle (CM). Second messenger studies revealed the presence of phospholipase C-coupled 5HT(2A) receptors in h-CM and h-TM cells where they stimulated phosphoinositide (PI) hydrolysis and mobilized intracellular Ca(2+) when challenged with a variety of 5HT(2A-C) receptor agonists (e.g. alpha-methyl-5HT, (R)-DOI, alpha-methyl-5HT, BW-723C86, MK-212, mCPP, cabergoline, AL-34662). These functional responses were blocked by selective 5HT(2) receptor antagonists with the 5HT(2A) antagonist, M-100970, exhibiting the highest potency. Thus, functional 5HT(2A) receptors are present in human ocular cells involved in IOP reduction and this correlates with the ability of 5HT(2A) agonists to lower IOP in Cynomolgus monkeys, a surrogate for human subjects.

Morphological and hydrodynamic correlates in monkey eyes with laser induced glaucoma

This study investigated the relationship between decreased outflow facility (C) and changes in hydrodynamic aqueous humor outflow patterns and morphology in cynomolgus monkey eyes with unilateral chronically elevated intraocular pressure (IOP). Argon laser photocoagulation burns to the trabecular meshwork (TM) were made in one eye of each monkey (N = 3), leaving the contralateral eye as a normotensive control. IOPs were followed by pneumatonometry for 16–70 months. C was measured by fluorophotometry before sacrifice. To label the hydrodynamic patterns of outflow, the eyes were enucleated and perfused with fluorescent microspheres (0.5 μm; 0.002%) at the last pressure measured before death minus 7 mmHg. The eyes were perfusion-fixed at the same pressure. Confocal images were taken along the inner wall (IW) of the Schlemm's canal (SC). The total length (TL) and the filtration length (FL) of the IW decorated by tracers were measured in frontal sections. The average percent effective filtration length (PEFL = FL/TL) was calculated for each eye. Sections exhibiting SC were processed and examined under light and electron microscopy. The average IOP was significantly higher in laser-treated eyes (mean ± SD = 61.33 ± 4.16 mmHg) than controls (22.67 ± 4.16 mmHg, P = 0.002). The average C was 13-fold lower in laser-treated eyes (0.03 ± 0.02 μl/min/mmHg) than controls (0.39 ± 0.17 μl/min/mmHg, P = 0.057). By confocal microscopy, in control eyes, SC was open and a segmental distribution of microspheres was found in the TM with a greater concentration near the collector channel (CC) ostia. Much less tracer labeling was seen along SC in laser-treated eyes than control eyes. The average PEFL in controls (47.47 ± 10.79%) was 6-fold larger than in laser-treated eyes (8.40 ± 4.81%, P = 0.048). The average distance between the inner and outer wall of SC was 5-fold greater in control eyes (18.99 ± 6.03 μm) than in laser-treated eyes (3.47 ± 0.33 μm, P = 0.048). By light microscopy, there was extensive pigmentation throughout the TM, denser extracellular matrix in the JCT region, and most of SC collapsed with focal herniations of the IW and JCT protruding into the CC ostia in laser-treated eyes. By electron microscopy, few or no microspheres were observed in laser-treated areas and the areas with SC collapse. More microspheres were observed near the CC ostia area in non-lasered areas. In conclusion, in the laser-induced glaucoma model, laser damage results in a reduction in the available area for outflow across the IW of SC which contributes to the decrease in C and thus elevation of the IOP. Constriction of SC, caused by the chronic elevation of IOP, further decreases the available area for outflow across the IW which decreases C even more in a vicious cycle. This study suggests that the available area for aqueous humor outflow across the IW of SC may play a role in regulating outflow resistance and maintaining IOP.

Effect of Anterior Zonule Transection on the Change in Lens Diameter and Power in Cynomolgus Monkeys during Simulated Accommodation

To quantify the role of anterior zonular tension on the optomechanical lens response during simulation of accommodation in primates. Postmortem cynomolgus monkey eyes (n = 14; age range, 3.0-11.5 years) were dissected leaving intact the lens, zonules, ciliary body, hyaloid membrane, anterior vitreous, and a scleral rim. The lens was mounted in a lens-stretching system and stretched radially in step-wise fashion. The load, and the lens diameter and power were measured at each step and the diameter- and power-load relationships were quantified. The anterior zonular fibers were then transected, and the experiment was repeated. The equatorial lens diameter and lens optical power before and after zonular transection were compared. Stretching increased the lens diameter by 0.25 +/- 0.09 mm (median +/- interquartile range) before and 0.25 +/- 0.19 mm after zonular transection. Stretching decreased the lens power by 13.0 +/- 6.5 D before and 10.6 +/- 8.0 D after zonular transection. The load required to change the diameter of the lens by 1 mm decreased from 18.8 +/- 10.7 g before to 15.0 +/- 7.8 g after zonular transection. The absolute change in power per gram of loading decreased from 2.5 +/- 1.1 before to 2.0 +/- 1.2 D after zonular transection. The cynomolgus monkey lens retains a significant fraction of its accommodative ability after transection of the anterior zonules in simulated accommodation experiments.

Cabergoline: Pharmacology, ocular hypotensive studies in multiple species, and aqueous humor dynamic modulation in the Cynomolgus monkey eyes

The aims of the current studies were to determine the in vitro and in vivo ocular and non-ocular pharmacological properties of cabergoline using well documented receptor binding, cell-based functional assays, and in vivo models. Cabergoline bound to native and/or human cloned serotonin-2A/B/C (5HT2A/B/C), 5HT1A, 5HT7, α2B, and dopamine-2/3 (D2/3) receptor subtypes with nanomolar affinity. Cabergoline was an agonist at human recombinant 5HT2, 5HT1A and D2/3 receptors but an antagonist at 5HT7 and α2 receptors. In primary human ciliary muscle (h-CM) and trabecular meshwork (h-TM) cells, cabergoline stimulated phosphoinositide (PI) hydrolysis (EC50=19±7nM in TM; 76nM in h-CM) and intracellular Ca2+ ([Ca2+]i) mobilization (EC50=570±83nM in h-TM; EC50=900±320nM in h-CM). Cabergoline-induced [Ca2+]i mobilization in h-TM and h-CM cells was potently antagonized by a 5HT2A-selective antagonist (M-100907, Ki=0.29–0.53nM). Cabergoline also stimulated [Ca2+]i mobilization more potently via human cloned 5HT2A (EC50=63.4±10.3nM) than via 5HT2B and 5HT2C receptors. In h-CM cells, cabergoline (1μM) stimulated production of pro-matrix metalloproteinases-1 and -3 and synergized with forskolin to enhance cAMP production. Cabergoline (1μM) perfused through anterior segments of porcine eyes caused a significant (27%) increase in outflow facility. Topically administered cabergoline (300–500μg) in Dutch-belted rabbit eyes yielded 4.5μM and 1.97μM levels in the aqueous humor 30min and 90min post-dose but failed to modulate intraocular pressure (IOP). However, cabergoline was an efficacious IOP-lowering agent in normotensive Brown Norway rats (25% IOP decrease with 6μg at 4h post-dose) and in conscious ocular hypertensive cynomolgus monkeys (peak reduction of 30.6±3.6% with 50μg at 3h post-dose; 30.4±4.5% with 500μg at 7h post-dose). In ketamine-sedated monkeys, IOP was significantly lowered at 2.5h after the second topical ocular dose (300μg) of cabergoline by 23% (p<0.02) and 35% (p<0.004) in normotensive and ocular hypertensive eyes, respectively. In normotensive eyes, cabergoline increased uveoscleral outflow (0.69±0.7μL/min–1.61±0.97μL/min, n=13; p<0.01). However, only seven of the eleven ocular hypertensive monkeys showed significantly increased uveoscleral outflow. These data indicate that cabergoline's most prominent agonist activity involves activation of 5HT2, 5HT1A, and D2/3 receptors. Since 5HT1A agonists, 5HT7 antagonists, and α2 antagonists do not lower IOP in conscious ocular hypertensive monkeys, the 5HT2 and dopaminergic agonist activities of cabergoline probably mediated the IOP reduction observed with this compound in this species.

Histologic Processing of the Eye for Obtaining the Macular Region of the Retina of the Cynomolgus Monkey

Microscopic evaluation of the macular region of the retina is essential in preclinical drug safety studies in cynomolgus monkeys. A number of therapeutic agents are suspected of causing damage to the macula in humans; therefore, the macular region of the retina should be evaluated in preclinical drug safety studies in monkeys. Histopathologic examination of the fovea may be required for preclinical drug safety evaluation of certain ocular drugs for human risk assessment. Using the inferior oblique muscle as a landmark, the macula including the fovea was obtained in 100% of 20 eye sections. It is easier to obtain just the macular region of the retina than the fovea in the section. Measurements for the inferior oblique muscle (point of insertion), fovea, macula, and the optic disc for 20 eyes of untreated cynomolgus monkeys were taken. In the cynomolgus monkey, the macula was 2.01 mm in diameter and the fovea was 0.53 mm in diameter. The distance from the center of the optic disc to the center of the fovea was 3.23 mm. In the fixed eyes, the point of insertion of the inferior oblique muscle to the sclera was 5.96 mm. (The J Histotechnol 31:113, 2008)Submitted January 7, 2008; accepted with revisions June 22, 2008

Identification of the Active Site in the Heparin II Domain of Fibronectin that Increases Outflow Facility in Cultured Monkey Anterior Segments

To determine the active site in the Heparin II (HepII) domain of fibronectin that regulates outflow facility in cultured anterior segments and disrupts the actin cytoskeleton in transformed human trabecular meshwork (TM-1) cells. Outflow facility was determined by two-level, constant-pressure perfusion in cultured anterior segments of rhesus and cynomolgus monkey eyes. One segment from each pair was exchanged with either the HepII domain or an integrin/syndecan binding peptide (IDAPS or PPRARI) from the HepII domain. To assay changes in the actin cytoskeleton, TM-1 cells were incubated for 24 hours with or without the HepII domain, PPRARI, or IDAPS. Changes were monitored with phase and immunofluorescence microscopy. HepII domain (100 microg/mL) and PPRARI (500 microg/mL) increased outflow facility by 31% +/- 13% (n = 9, P < 0.05) and 24% +/- 9% (n = 8, P < 0.05), respectively in cultured anterior segments after an overnight infusion. Perfusion with IDAPS (500 microg/mL) had no effect on outflow facility. In TM-1 cultures, 250 microg/mL of the HepII domain or 4 mg/mL of PPRARI disrupted the assembly of actin filaments. A lower concentration of PPRARI (2 mg/mL) disrupted the actin cytoskeleton when used in combination with a nondisrupting concentration of the HepII domain (30-60 microg/mL). In contrast, IDAPS did not disrupt the actin cytoskeleton under any condition tested. The active site in the HepII domain that regulates outflow facility in cultured anterior segments and disrupts the actin cytoskeleton in TM-1 cells is the syndecan/integrin binding sequence, PPRARI.

Novel benzodifuran analogs as potent 5-HT 2A receptor agonists with ocular hypotensive activity

A series of 8-substituted benzodifuran analogs was prepared and evaluated for 5-HT 2A receptor binding and activation. Several compounds containing ether and ester functionality were found to be potent agonists. Topical ocular administration of 5, 18, and 25 effectively reduced intra-ocular pressure in the hypertensive cynomolgus monkey eye in the range of 25–37%.

AL-34662: A Potent, Selective, And Efficacious Ocular Hypotensive Serotonin-2 Receptor Agonist

The aim of this study was to determine the ocular pharmacological characteristics of AL-34662 (1-((S)-2-aminopropyl)-1H-indazole-6-ol), a new synthetic serotonin-2 (5-HT2) receptor-agonist ocular hypotensive agent. A variety of well-documented in vitro and in vivo procedures were utilized to study the pharmacological attributes of AL-34662. AL-34662 exhibited a high affinity for the rat and human 5-HT2 receptor (IC50=0.8-1.5 nM) and for cloned human 5-HT2A-C receptors (IC50=3-14.5 nM). AL-34662 stimulated phosphoinositide turnover in human ciliary muscle (h-CM; EC50=289+/-80 nM) and in human trabecular meshwork (h-TM; EC50=254+/-50 nM) cells. AL-34662 also mobilized intracellular Ca2+ ([Ca2+]i) in h-CM (EC50=140+/-23 nM) and h-TM (EC50=38+/-8 nM) cells, being a full agonist like 5-HT itself. AL-34662's effects in the h-CM (and h-TM) cells were potently antagonized by 5-HT2A-antagonist M-100907 (IC50=1.8+/-0.7 nM), but weakly by 5-HT2B-antagonist (RS-127445 IC50>10 microM), 5-HT2B/C- antagonist (SB-242084 IC50=2.08 microM) and 5-HT2C antagonist (RS-102221 IC50>1 microM). AL-34662 caused relatively minimal ocular discomfort and hyperemia in rabbit and guinea pig eyes. It efficaciously lowered intraocular pressure (IOP) in the conscious ocular hypertensive monkey eyes (33% at 300 microg). The (R)-enantiomer (AL-34707) and the racemate (AL-34497) were less potent and/or efficacious than AL-34662 in all of these assays. AL-34662 is a high-affinity 5-HT2 receptor agonist that potently mobilizes [Ca2+]i in h-CM and h-TM cells, and which efficaciously lowers IOP in conscious ocular hypertensive cynomolgus monkey eyes through a local effect with minimal side-effects.

Anastomotic Vessels Remain Viable after Photodynamic Therapy in Primate Models of Choroidal Neovascularization

Anastomotic vessels in exudative age-related macular degeneration (AMD) represent a serious clinical feature that reportedly does not respond well to either photocoagulation or photodynamic therapy (PDT). Anastomoses also occur in various animal models of choroidal neovascularization (CNV). In the present study, anastomotic vessels and their patency were evaluated in two primate CNV laser-trauma models after PDT, by using two novel photosensitizers. In cynomolgus (Macaca fascicularis) and squirrel (Saimiri sciureus) monkey eyes (n = 20), matrix placement of laser photocoagulation sites elicited CNV as a component of the development of fibrovascular tissue (FVT). FVT sites received PDT according to specific drug infusion and laser light treatment parameters. FVTs and anastomoses were evaluated by fundus photography, fluorescein angiography, and histologic examination. Anastomoses averaged approximately 48% of FVT sites, with greatest occurrence in the macaque. Although PDT with each photosensitizer effectively produced FVT closure, both retinal vessels and anastomoses remained patent. Although PDT is effective in closing the choroidal neovascularization in FVT, this technique was ineffective in occluding anastomotic vessels and their associated tributaries within the mid- to proximal retina. Various factors (vascular diameter, composition, blood flow, orientation) may contribute to continued anastomotic patency. By convention, such vessels would typically be defined as chorioretinal anastomoses (CRAs); however, continuing studies suggest the possibility that these neovessels constitute dual-origin hybrids. Regardless of origin, viable anastomoses provide one potential mechanism for revascularization to occur after PDT and may help to explain why CRAs are considered a poor prognostic sign in patients with AMD.

Safety and Efficacy of Intravitreal Injection of Ranibizumab in Combination With Verteporfin PDT on Experimental Choroidal Neovascularization in the Monkey

To study the safety and efficacy of intravitreal injections of anti-vascular endothelial growth factor antibody fragment (ranibizumab [formerly known as rhuFabV2], Lucentis; Genentech, South San Francisco, Calif) in combination with intravenous verteporfin (Visudyne; Novartis, East Hanover, NJ) photodynamic therapy (PDT) on experimental choroidal neovascularization in the monkey eye. Choroidal neovascularization was induced by laser injury in both eyes of cynomolgus monkeys and followed with weekly fundus photography and fluorescein angiography. Two weeks after induction, weekly treatments were initiated. These treatments included using either an intravitreal injection of ranibizumab (previously known as rhuFabV2) in combination with verteporfin PDT or a ranibizumab vehicle (placebo) in combination with verteporfin PDT (PDT only). Six animals (group 1) initially received intravitreal injections followed 1 week later by PDT. Four animals (group 2) initially received PDT followed 1 week later by intravitreal injection. Two animals (group 3) received injections and PDT on the same day at 2-week intervals. Photodynamic therapy was applied in all 3 groups every 2 weeks for 3 treatments with follow-up through 2 weeks after the last PDT treatment. Fluorescein angiograms were graded using a masked standardized protocol. The data were analyzed using the McNemar chi(2) test for matched pairs. No choroidal neovascularization leakage was observed in the eyes of animals treated with ranibizumab and PDT at day 21 or 42 after the start of the first treatment. Leakage persisted in eyes treated with PDT alone at 21 days (3 of 12 eyes) and 42 days (2 of 12 eyes). At all time points studied, the ranibizumab and PDT-treated eyes experienced better angiographic outcomes than the eyes receiving PDT alone. These preliminary data indicate that an intravitreal ranibizumab injection in combination with verteporfin PDT (ranibizumab and PDT) causes a greater reduction in angiographic leakage than PDT and intravitreal vehicle injection (PDT only) in experimental choroidal neovascularization. This combination therapy can potentially offer a new treatment modality for choroidal neovascularization in patients with macular degeneration and other diseases.

Effects of the Rho kinase inhibitor Y-27632 and the phosphatase inhibitor calyculin A on outflow facility in monkeys

Previous studies have shown that the inhibition of Rho kinase is involved in the regulation of outflow facility in the live rabbit eye and the enucleated porcine eye. However, it is unknown whether the Rho kinase inhibition will do the same in non-human primates. To determine if the Rho kinase inhibitor Y-27632 will reduce outflow resistance in the live monkey eye, if Y-27632 and the phosphatase inhibitor calyculin A (Caly-A which antagonises Y-27632-induced MLC dephosphorylation) will affect outflow facility differently, and if the latter will inhibit effect of the former on facility, we studied effects of Y-27632 and Caly-A on outflow facility in living monkeys separately and concurrently. Total outflow facility was measured by 2-level constant pressure perfusion of the anterior chamber (AC) before and after exchange with different doses of Y-27632 (1, 10 and 100 microM) or Caly-A (10, 50 and 100 nM), or vehicles, followed by continuous AC infusion of corresponding drug/vehicle solution, in opposite eyes of cynomolgus or rhesus monkeys. The effect of 100 microM Y-27632 or 100 nM Caly-A vs vehicle and the effect of 100 microM Y-27632+100 nM Caly-A vs 100 microM Y-27632 alone on outflow facility were also determined in monkeys pre-treated topically with 10 microl of 1% atropine in both eyes 1 hr before perfusion. Both Y-27632 and Caly-A dose-dependently increased outflow facility by up to 2-3 fold in monkeys, adjusted for baseline and contralateral control eye washout. Pre-treatment with 1% topical atropine partially inhibited the effect of 100 nM Caly-A, but not 100 microM Y-27632, on outflow facility. 100 nM Caly-A gradually and partially inhibited the Y-27632-induced facility increase. In conclusion, Y-27632 increases outflow facility in monkeys presumably by inhibiting cellular contractility in the TM. Caly-A increases outflow facility by complicated mechanisms perhaps including drug-induced ciliary muscle contraction and cytoskeletal reorganisation in TM cells. The partial inhibitory effect of Caly-A on the Y-27632-induced increase in outflow facility may reflect the former partially inhibiting the latter's relaxation of cells in the TM.

Hyperspectral imaging for measurement of oxygen saturation in the optic nerve head.

To evaluate a hyperspectral imaging technique for monitoring relative spatial changes in retinal oxygen saturation. The optic nerve head (ONH) and overlying vessels in cynomolgus monkey eyes were imaged with a fundus camera attached to a hyperspectral imaging system. Images were acquired with inspiration of room air and pure oxygen and at controlled intraocular pressures (IOP) of 15 mm Hg (normal) and 60 mm Hg (sustained for up to 5 minutes). Changes in relative blood oxygen saturation in the vessels and ONH were assessed from reflectance spectra. Saturation maps were derived from contributions of oxygenated and deoxygenated hemoglobin spectral signatures extracted from hyperspectral images. The results obtained with hyperspectral imaging were compared with known experimental outcomes. Pure oxygen markedly increased oxygen saturation in veins. Increases in arteries and the ONH were smaller. The results obtained with hyperspectral image analysis agreed with known changes in oxygen saturation from breathing experiments. Raising IOP reduced saturation in all structures and resulted in profound desaturation of arteries. During sustained high IOP, a rebound in saturation was observed in the ONH. Spatial maps clearly showed the saturation changes in arteries, veins, and surrounding tissues. Hyperspectral imaging can be adapted to measure and map relative oxygen saturation in retinal structures and the ONH in nonhuman primate eyes.

Functional damage to inner and outer retinal cells in experimental glaucoma.

To investigate the cellular sources underlying the functional damage observed by multifocal electroretinography (mfERG) responses of glaucomatous eyes of monkeys. First- and second-order (K1 and K2, respectively) mfERG responses of three normal and three experimentally induced glaucomatous eyes of cynomolgus monkeys were measured at two different levels of luminance. Retinal contributors to the responses were isolated by intravitreal injections of pharmacological agents that suppress specific retinal cells. gamma-Aminobutyric acid (GABA) and glycine were administered to block inner retinal function, followed by 2-amino-4-phosphonobutyric acid (APB), to block ON-bipolar cells. An inner retinal component removed by GABA and glycine was found in both the normal and glaucomatous eyes. However, it was attenuated in the latter, correlating with changes observed in the baseline K1 responses. Delays in the latency of outer retinal components were found in the responses of the glaucomatous eyes. K2 responses were dominated by an inner retinal contribution and were diminished in the responses of glaucomatous eyes. The outer retina responded to increased luminance with a shorter implicit time. A distinct wave part of the inner retinal component responded to increased luminance with increased amplitudes. The integration of the retinal sources forming the mfERG response was compared between normal and glaucomatous monkey eyes. Both inner and outer retinal functions were aberrant in the responses of the glaucomatous eyes, with the attenuation of the inner retinal function more conspicuous. Nevertheless, glaucomatous eyes retained certain inner retinal activity, despite the advanced stage of disease. K2 responses were more sensitive to glaucomatous changes than were K1 responses.

Nonvascular smooth muscle a-actin positive cells in the choroid of higher primates

Purpose. As an elaborated network of non-vascular smooth muscle a-actin positive cells (NVSMC) is present in the human eye, the purpose of this investigation was to describe the presence of NVSMC in the sclera and choroid of different mammals and primates. Methods. Whole mounts and tangential serial sections through the sclera and choroid of human donor eyes, of cynomolgus and rhesus monkey, mangabey, owl monkey, tree shrew, pig, cow, rabbit, cat, dog, and rat eyes were stained with a mouse-anti-smooth muscle a-actin antibody. The mean number/mm 2 of NVSMC was calculated in the central region of each quadrant. Results. In cynomolgus and rhesus monkey eyes, numerous NVSMC were located in the suprachoroidal and inner scleral region. In the temporal quadrant, the density of NVSMC was highest. NVSMC were semicircular arranged around the entering long ciliary artery and nerve. In the mangabey and owl monkey choroid, the number of NVSMC was about ten-fold less than in macaque monkeys. Most of the NVSMC were located parallel to large vessels and only some cells were seen in the suprachoroid and inner sclera. In the tree shrew monkey and in all other mammal eyes, no NVSMC could be detected. In the human eyes, in addition to the suprachoroidal and inner scleral NVSMC numerous cells were located in the choroid proper. The regional distribution and the orientation of the NVSMC was comparable in human and macaque monkey eyes. Conclusions. Comparing these findings with the literature, the presence of NVSMC correlates with the development of a central fovea and an elaborated accommodative system. Its functional significance remains to be determined.

Pathologic features of vascular endothelial growth factor-induced retinopathy in the nonhuman primate

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent ischemia-upregulated angiogenic protein that has been implicated in diabetic retinopathy. Intravitreal VEGF injections have not previously been shown to produce preretinal neovascularization. The purpose of this study was to further characterize the angiopathic changes that occur after intravitreal injections in a nonhuman primate and determine if preretinal neovascularization develops. DESIGN: Experimental animal study. METHODS: Vascular endothelial growth factor 165 was injected into the eyes of normal cynomolgus monkeys at regular intervals. As a control, normal eyes were injected with phosphate buffered saline. Color photography and fluorescein angiography were performed at regular intervals. The retinas were incubated for adenosine diphosphatase (ADPase) activity to visualize retinal vessels. The retinas were flat-embedded and areas of potential preretinal neovascularization were identified en bloc and serially sectioned. RESULTS: Areas of capillary nonperfusion and vessel dilation and tortuousity were seen by angiography. In serial sections, the nonperfused areas were found to be associated with endothelial cell hyperplasia in vessel lumens. Preretinal neovascularization originating only from superficial veins and venules was observed throughout peripheral retina, but was not seen in the posterior pole. Lacunae-like veins were subdivided by the process of intussusception and endothelial cell bridging. Arterioles demonstrated endothelial cell hyperplasia and microaneurysms. CONCLUSION: Intraocular injections of VEGF were sufficient to produce preretinal neovascularization in the nonhuman primate. Most vasculopathic structures were associated with endothelial cell hyperplasia. These results demonstrate that VEGF alone can produce many features of both nonproliferative and proliferative diabetic retinopathy including the previously undescribed development of preretinal neovascularization. This well-characterized VEGF-induced primate model of retinal neovascularization may be useful as a means of testing new treatments for retinal neovascularization.

The effects of several vasopressin receptor antagonists on normal intraocular pressure and the intraocular distribution of vasopressin receptor subtypes.

The aim of the present paper is to study the relation between vasopressin antagonism and the regulation of intraocular pressure (IOP). From the studies on the effect of several vasopressin receptor antagonists, VP-343, OPC-21268, YM087, OPC-31260 and SR121463, on normal IOP and the effect of VP-343 on pupil diameter in rabbit, it was shown that some vasopressin antagonists decreased normal IOP and VP-343 had no influence on pupil diameter. A vasopressin receptor mapping study in normal cynomolgus monkey eye revealed a high density binding site for a [H3]vasopressin V1 antagonist in the region of iris. These findings suggest that a vasopressin antagonist should decrease normal IOP without miosis and that vasopressin V1 receptors are present in iris.

Optic disc, foveal, and extrafoveal damage due to surgical separation of the vitreous.

To evaluate the morphologic outcomes resulting from surgical vitreoretinal separation in young adult primates. Vitrectomy and mechanical separation of the vitreous from the internal limiting lamina (ILL) of the posterior retina and surface of the optic disc were performed on 25 young adult cynomolgus monkey eyes in vivo. Lectin histochemical studies were used to evaluate the vitreoretinal interface. Morphologic outcomes were tabulated. In 11 of 25 eye regions, residual vitreous remained attached to the ILL in some of the regions. Localized ILL breaks or separation of the ILL from the neural retina was noted in 9 eyes. Retinal tissue loss, including avulsion of the ganglion cell, inner plexiform, or inner nuclear layers, was observed in 7 eyes. Avulsion of axon bundles in the optic disc was noted in 9 eyes. Significantly, partial- or full-thickness foveal tears were noted in 11 eyes. Based on the surgeons' intraoperative observations, small superficial optic disc or retinal hemorrhages were observed in 3 of 25 eyes. None of the eyes on which a vitrectomy alone was performed showed ILL damage, or retinal or optic disc tissue loss. Damage may occur to the optic disc, fovea, and extrafoveal retina as a result of surgical separation of the vitreous from the retina in young adult primates. These data support the contention that surgically induced damage at the level of the vitreoretinal interface may help explain the visual field defects noted after surgery to close full-thickness macular holes. These data also support the need for developing additional modalities to assist in vitreous separation, thereby reducing the risk of traumatic complications associated with purely mechanical procedures.