Cytoskeletal drugs prevent posterior capsular opacification in human lens capsule in vitro.

BACKGROUND: To determine whether the cytoskeletal drugs H-7 and Latrunculin B (LAT-B) inhibit posterior capsular opacification (PCO) in the cultured human lens capsular bag. METHODS: Following extracapsular cataract (lens) extraction in human donor eyes, the capsular bag was prepared and cultured by standard techniques. Forty-eight capsular bags were studied, of which 13 were treated with H-7 (50, 100 or 300 µM), 12 with 1% BSS (vehicle of H-7), 11 with LAT-B (2, 5 or 10 µM), and 12 with 0.25% DMSO (vehicle of LAT-B). Forty out of the 48 capsular bags were from paired eyes of 20 donors, with one bag being treated with H-7/LAT-B and the other with BSS/DMSO for each pair, including 20 for the H-7-BSS protocol and 20 for the LAT-B-DMSO protocol. The medium with the cytoskeletal drug/vehicle was replaced every 3-4 days for 4 weeks. PCO was assessed daily using inverted phase-contrast microscopy, and scored on a 4-point scale. RESULTS: In all cultures with BSS or DMSO, residual lens epithelial cells (LECs) on the anterior capsule migrated to and proliferated on the posterior capsule by 3-7 days, and apparent LEC growth on the posterior capsule with severe capsular wrinkling (PCO Grade 3) was seen by 2-3 weeks. When treated continuously with H-7 or LAT-B, the migration and proliferation of LECs and the capsular wrinkling were inhibited in a dose-dependent manner, with the inhibition being complete (PCO Grade 0) in the 300 µM H-7 (n = 8, p < 0.001) or 10 µM LAT-B culture (n = 3, p = 0.002). CONCLUSION: H-7 and LAT-B dose-dependently inhibited PCO formation in the cultured human lens capsular bags, suggesting that cytoskeletal drugs might prevent PCO formation after surgery in the human eye.

SAR and molecular mechanism study of novel acylhydrazone compounds targeting HIV-1 CA.

We synthesized a series of acylhydrazone compounds bearing naturally occurring amino acids' side chains as HIV assembly inhibitors. Biological evaluation indicated that the compounds had anti-SIV and capsid assembly inhibitory activities. The structure-activity relationship (SAR) study showed that compounds bearing proper aromatic side chains had potential antiviral activities. The molecular modeling experiments revealed the molecular mechanism that they could bind to CA in the same manner as CAP-1 and occupy two more grooves.

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.

Synthesis and antiviral activities of novel acylhydrazone derivatives targeting HIV-1 capsid protein.

HIV-1 capsid protein (CA) plays important roles in the viral replication cycle. A number of acylhydrazone derivatives that act as inhibitors of HIV-1 CA assembly, were designed and synthesized. The synthesized compounds were tested for their antiviral activities and cytotoxicities using CEM cells. Some derivatives also were assayed for their ability to inhibit HIV-1 CA assembly in vitro. Among them, compounds 14f and 14i display the most promising potency with EC(50) values of 0.21 and 0.17 microM respectively.

Discovery of dual inhibitors targeting both HIV-1 capsid and human cyclophilin A to inhibit the assembly and uncoating of the viral capsid.

HIV-1 assembly and disassembly (uncoating) processes are critical for the HIV-1 replication. HIV-1 capsid (CA) and human cyclophilin A (CypA) play essential roles in these processes. We designed and synthesized a series of thiourea compounds as HIV-1 assembly and disassembly dual inhibitors targeting both HIV-1 CA protein and human CypA. The SIV-induced syncytium antiviral evaluation indicated that all of the inhibitors displayed antiviral activities in SIV-infected CEM cells at the concentration of 0.6-15.8 microM for 50% of maximum effective rate. Their abilities to bind CA and CypA were determined by ultraviolet spectroscopic analysis, fluorescence binding affinity and PPIase inhibition assay. Assembly studies in vitro demonstrated that the compounds could potently disrupt CA assembly with a dose-dependent manner. All of these molecules could bind CypA with binding affinities (Kd values) of 51.0-512.8 microM. Fifteen of the CypA binding compounds showed potent PPIase inhibitory activities (IC(50) values<1 microM) while they could not bind either to HIV-1 Protease or to HIV-1 Integrase in the enzyme assays. These results suggested that 15 compounds could block HIV-1 replication by inhibiting the PPIase activity of CypA to interfere with capsid disassembly and disrupting CA assembly.

Combined effects of H7 and pilocarpine on anterior segment physiology in monkey eyes.

PURPOSE: To determine, in monkey eyes in vivo if low doses of the cholinergic agonist pilocarpine (PILO) can enhance the outflow facility responses to a maximal dose of the serine-threonine kinase inhibitor H7 without producing apparent miosis and/or excessive accommodation. METHODS: Outflow facility was determined by two-level constant pressure perfusion in living monkeys after 24.5 microM phenylephrine (PE) bilaterally (stimulates the iris dilator without influencing the iris sphincter, ciliary muscle, or outflow facility, and facilitates the measurement of miosis and accommodation), 24.5 microM PE + 300 microM H7 (maximal outflow facility-effective dose) bilaterally, or 24.5 microM PE + 300 microM H7 +/- 2 or 10 microM PILO (2 microM PILO alone does not significantly increase outflow facility or accommodation but moderately constricts the pupil; 10 microM PILO is a threshold outflow facility-effective dose) in opposite eyes. Pupil diameter (Vernier calipers) and accommodation (coincidence refractometer) were measured essentially concurrently. RESULTS: Outflow facility in the PE + H7 + 10 microM PILO eyes was 73% higher than that in the PE + H7 eyes (n = 6; p < 0.05). Accommodation was greater (n = 4; 2.6 vs. 0.6 D; p < 0.05) and pupil diameter was smaller (n = 6; 3.4 vs. 7.6 mm; p < 0.02) in the former than in the latter. No significant difference in outflow facility, accommodation or pupil diameter was observed between the PE + H7 + 2 microM PILO eyes and the PE + H7 eyes. CONCLUSIONS: In living monkeys, 10 microM PILO, but not 2 microM PILO, enhances the 300 microM H7-induced increase in outflow facility with only approximately 3 diopters of accommodation, whereas 300 microM H7 partially inhibits the 2 microM PILO-induced, but not the 10 microM PILO-induced miosis. This suggests that, although the miosis following the threshold facility-effective dose of PILO cannot be reduced by combination with the maximal facility-effective dose of H7, the combination may at least benefit young glaucoma or ocular hypertension patients who are bothered by PILO-induced myopia more than by miosis.

Effects of Vitrectomy and Lensectomy on Older Rhesus Macaques: Oxygen Distribution, Antioxidant Status, and Aqueous Humor Dynamics.

Purpose: The purpose of this study is to evaluate effects of vitrectomy (PPV) and lens extraction with intraocular lens implantation (PE/IOL) on molecular oxygen (pO2) distribution, aqueous humor antioxidant-oxidant balance, aqueous humor dynamics, and histopathologic changes in the trabecular meshwork (TM) in the older macaque monkey. Methods: Six rhesus monkeys underwent PPV followed by PE/IOL. pO2, outflow facility, and intraocular pressure (IOP) were measured. Aqueous and vitreous humor specimens were analyzed for antioxidant status and 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative damage. TM specimens were obtained for immunohistochemical and quantitative PCR analysis. Results: pO2 at baseline revealed steep gradients in the anterior chamber and low levels in the posterior chamber (PC) and around the lens. Following PPV and PE/IOL, pO2 significantly increased in the PC, around the IOL, and angle. IOP increased following both surgical interventions, with no change in outflow facility. Histopathologic analysis did not show changes in TM cell quantification, but there was an increase in 8-OHdG. Quantitative PCR did not reveal significant differences in glaucoma-related gene expression. Aqueous and vitreous humor analysis revealed decreased ascorbate and total reactive antioxidant potential and increased 8-OHdG in the aqueous humor only in the surgical eyes. Conclusions: Oxygen distribution in the older rhesus monkey is similar to humans at baseline and following surgical interventions. Our findings of histopathologic changes of TM oxidative damage and alterations in the oxidant-antioxidant balance suggest a potential correlation of increased oxygen exposure with oxidative stress/damage and the development of open angle glaucoma.

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.

Effect of low-dose latrunculin B on anterior segment physiologic features in the monkey eye.

OBJECTIVES: To determine if low doses of topical latrunculin B (LAT-B) will increase outflow facility and decrease intraocular pressure without damaging the cornea and if they will inhibit miotic and accommodative responses to pilocarpine in monkeys. METHODS: We measured intraocular pressure (Goldmann tonometry) before and after 1 and 9 doses of 0.005% and 0.01% topical LAT-B and vehicle given twice daily on successive weeks; outflow facility (perfusion) following 15 doses; central corneal thickness (ultrasonic pachymetry) before and after 1 and 9 doses of 0.01% LAT-B and vehicle; pupillary diameter (calipers); and accommodation (refractometry) before and after 1 dose of 0.005% and 0.02% LAT-B. RESULTS: Latrunculin-B dose-dependently decreased intraocular pressure, multiple doses more than a single dose. Maximal mean +/- SEM hypotension after 1 dose was 2.5 +/- 0.3 mm Hg (0.005% LAT-B; n = 8; P<.001) or 2.7 +/- 0.6 mm Hg (0.01% LAT-B; n = 8; P<.005); maximal mean +/- SEM hypotension after 9 doses was 3.2 +/- 0.5 mm Hg (0.005% LAT-B; n = 8; P<.001) or 4.4 +/- 0.6 mm Hg (0.01% LAT-B; n = 8; P<.001). Outflow facility was increased by mean +/- SEM 75% +/- 13% (n = 7; P<.005). Central corneal thickness was not changed after 1 or 9 doses of 0.01% LAT-B. Miotic and accommodative responses to intramuscular pilocarpine were dose-dependently inhibited. With 0.02% LAT-B, inhibition of miosis was substantial, whereas the inhibition of accommodation was only about 25%. With 0.005% LAT-B, the effects were trivial. CONCLUSIONS: In ocular normotensive monkeys, 0.005% and 0.01% LAT-B administered topically increases outflow facility and/or decreases intraocular pressure without corneal effects. Multiple doses reduce intraocular pressure more than a single dose. Latrunculin-B dose-dependently relaxes the iris sphincter and ciliary muscle, with some separation of miotic and accommodative effects. Clinical Relevance Multiple treatments with low topical doses of LAT-B may substantially reduce outflow resistance in eyes with glaucoma without adversely affecting the cornea.

Effects of topical H-7 on outflow facility, intraocular pressure, and corneal thickness in monkeys.

OBJECTIVES: To determine if low concentrations of H-7 (1-[5-isoquinoline sulfonyl]-2-methyl piperazine) topically applied to the eye increases outflow facility and decreases intraocular pressure (IOP) without affecting the cornea in monkeys, and to evaluate if the effect of H-7 on IOP is pressure dependent. METHODS: Single or multiple doses of 5% H-7 or vehicle (20 micro L) were administered topically to opposite eyes of normal monkeys. A single dose of 2% H-7 or vehicle (50 micro L) was administered to the glaucomatous eye of monkeys with laser-induced unilateral glaucoma, with vehicle on day 1 and H-7 on day 2. RESULTS: In normotensive eyes, 1 dose of 5% H-7 maximally decreased IOP by a mean +/- SEM of 2.5 +/- 1.0 mm Hg (-16.7% +/- 5.5%) at 3 hours. Higher baseline IOP and repeated dosing were associated with greater IOP reduction. Outflow facility was increased, but central corneal thickness was not affected. In glaucomatous eyes, 1 dose of 2% H-7 maximally decreased IOP by a mean +/- SEM of 5.8 +/- 0.6 mm Hg (-16.9% +/- 1.6%) at 2 hours. CONCLUSIONS: Five percent H-7 increases outflow facility and decreases IOP, but does not affect corneal thickness. Multiple doses of H-7 induce greater reduction of IOP than a single dose. The effect of H-7 on IOP may be pressure dependent. Clinical Relevance Multiple topical treatments with low doses of H-7 or analogues may substantially reduce outflow resistance in the hypertensive eye without meaningfully affecting the cornea.

Effects of latrunculin B on outflow facility, intraocular pressure, corneal thickness, and miotic and accommodative responses to pilocarpine in monkeys.

PURPOSE: To determine if low doses of topical latrunculin B (LAT-B) will increase outflow facility and decrease intraocular pressure (IOP) without adversely affecting the cornea, and inhibit miotic and accommodative responses to pilocarpine, in ocular normotensive monkeys. METHODS: Intraocular pressure was measured by Goldmann tonometry before and after one and nine dose(s) of 0.005% and 0.01% topical LAT-B/vehicle given twice daily on successive weeks. Outflow facility was then measured by perfusion following 15 doses. Central corneal thickness was measured by ultrasonic pachymetry before and after one and nine dose(s) of 0.01% LAT-B/vehicle. Pupillary diameter (calipers) and accommodation (refractometry) before and after one dose of 0.005% and 0.02% LAT-B were determined. RESULTS: LAT-B dose-dependently decreased IOP, multiple doses more than a single dose. Maximal hypotension after one dose was 2.5 +/- 0.3 mm Hg (0.005% LAT-B; n = 8; P < .001) or 2.7 +/- 0.6 mm Hg (0.01% LAT-B; n = 8; P < .005); maximal hypotension after nine doses was 3.2 +/- 0.5 mm Hg (0.005% LAT-B; n = 8; P < .001) or 4.4 +/- 0.6 mm Hg (0.01% LAT-B; n = 8; P < .001). Outflow facility was increased by 75 +/- 13% (n = 7; P < .005). Central corneal thickness was not changed after one or nine dose(s) of 0.01% LAT-B. The miotic and accommodative responses to intramuscular pilocarpine were dose-dependently inhibited. At 0.02% LAT-B, the inhibition of miosis was essentially complete when compared with the pre-LAT-B value, whereas the inhibition of accommodation was only about 25%. At 0.005% LAT-B, the effects were trivial. CONCLUSIONS: In ocular normotensive monkeys, 0.005/0.01% LAT-B administered topically increases outflow facility and/or decreases IOP, but does not affect the cornea. Multiple doses reduce IOP more than a single dose. LAT-B dose-dependently relaxes the iris sphincter and ciliary muscle, with some separation of the miotic and accommodative effects.

Application of canaloplasty in glaucoma gene therapy: where are we?

PURPOSE: Schlemm's canal (SC) inner wall is adjacent to the juxtacanalicular trabecular meshwork (TM) over their entire circumference. We seek to transfer reporter and therapeutic genes to these outflow-modulating tissues via canaloplasty surgery in live monkeys. METHODS: A standard canaloplasty surgical approach was performed in cynomolgus monkeys using flexible canaloplasty catheters, modified for monkey eyes with a 175-µm outer diameter and an LED-lighted tip. A 6-0 prolene suture was used for the exact localization of SC. Trypan blue was injected during catheter withdrawal to document catheter placement within SC and to determine ease of injecting fluid into SC. Before, during, and after the injection, the position of the catheter and the anatomic details were video-captured with an externally positioned noncontact endoscopic imaging system and 50 mHz ultrasound biomicroscopy (UBM). RESULTS: A 360° catheterization and injection of dye into SC was achieved. Suture, catheter, and trypan blue were imaged with the endoscope camera system and the catheter was also visualized with UBM. Trypan blue was seen in the SC over 5 clock hours after a 1 clock-hour insertion of the catheter. CONCLUSIONS: A modified canaloplasty catheter device might be used for gene delivery to the SC/TM area without circumferential catheterization. Further studies comparing different delivery methods of the vector/transgene into the SC using canaloplasty are needed.

A Potential Application of Canaloplasty in Glaucoma Gene Therapy.

Canaloplasty, a recently developed non-penetrating glaucoma surgical approach, may restore physiological outflow routes in primary open-angle glaucoma with less risk of severe postoperative complications than trabeculectomy. Since the inner wall of Schlemm's canal (SC) is directly in contact with the trabecular meshwork (TM) for 360 degrees and the catheter device used in canaloplasty allows viscoelastic to be injected into the entire length of SC, canaloplasty might also be used to perform SC/TM-targeted delivery of transgene vectors for glaucoma gene therapy. This hypothesized new method for transgene delivery may give the transgene access to the entire inner wall of SC and the whole juxtacanalicular region of the TM and allow the transgene to be expressed in both the TM and SC without affecting the cornea, iris and ciliary body. Further, this strategy might have a greater trabecular outflow resistance-decreasing effect than either the genetic or surgical approach alone.

Comparisons of actin filament disruptors and Rho kinase inhibitors as potential antiglaucoma medications.

Dynamics of the actin cytoskeleton in the trabecular meshwork play a crucial role in the regulation of trabecular outflow resistance. The actin filament disruptors and Rho kinase inhibitors affect the dynamics of the actomyosin system by either disrupting the actin filaments or inhibiting the Rho kinase-activated cellular contractility. Both approaches induce similar morphological changes and resistance decreases in the trabecular outflow pathway, and thus both have potential as antiglaucoma medications. Although the drugs might induce detrimental changes in the cornea following topical administration, lower drug concentrations in larger volumes as used clinically, but not higher drug concentrations in smaller volumes as used experimentally, could minimize corneal toxicity. Additionally, developments of trabecular meshwork-specific actin filament disruptors or Rho kinase inhibitors, prodrugs and new drug-delivery methods might avoid the drugs' toxicity to the cornea. Gene therapies with cytoskeleton-modulating proteins may mimic the effects of the cytoskeleton-modulating agents and have the potential to permanently decrease trabecular outflow resistance.

Synthesis and antiviral evaluation of new N-acylhydrazones containing glycine residue.

N-acylhydrazones containing glycine residue 3a-j and 8a-h were synthesized as HIV-1 capsid protein assembly inhibitors. The structures of the novel N-acylhydrazone derivatives were characterized using different spectroscopic methods. Antiviral activity demonstrated that compound 8c bearing 4-methylphenyl moiety was the most active with low cytotoxicity.

Effect of H-7 on secondary cataract after phacoemulsification in the live rabbit eye.

PURPOSE: This study is aimed to determine if the serine-threonine kinase inhibitor H-7 inhibits secondary cataract after phacoemulsification in the live rabbit eye. METHODS: Eighteen rabbits underwent extracapsular lens extraction by phacoemulsification in 1 eye. The eye was treated with intravitreal H-7 (300 or 1,200 µM; n = 6 or 5) or balanced salt solution (BSS) (n = 7) immediately after the surgery and twice weekly for 10 weeks. Each eye received slit lamp biomicroscopy once a week, during which posterior capsule opacification (PCO) was evaluated. The eye was then enucleated and the lens capsule was prepared, fixed, and imaged. PCO was evaluated again on the isolated lens capsule under a phase microscope. Soemmering's ring area (SRA) and the entire lens capsule area were measured from capsule images on a computer and the percentage of SRA (PSRA) in the entire capsule area was calculated. Wet weight of the capsule (WW) was determined on a balance. RESULTS: No significant difference in PCO was observed in any comparison. No significant differences in SRA, PSRA, and WW were observed between the 300 µM H-7-treated eye and the BSS-treated eye. However, SRA, PSRA, and WW in the 1,200 µM H-7-treated eye were significantly smaller than those in the BSS-treated eye [28.3 ± 16.2 vs. 61.4 ± 8.86 mm(2) (P = 0.001), 33% ± 20% vs. 65% ± 15% (P = 0.01), and 65.6 ± 27.9 vs. 127.0 ±37.3 mg (P = 0.01)]. CONCLUSIONS: Intravitreal H-7 (1,200 µM) significantly inhibits Soemmering's ring formation in the live rabbit eye, suggesting that agents that inhibit the actomyosin system in cells may prevent secondary cataract after phacoemulsification.

Effect of daily prolonged ketamine anesthesia on intraocular pressure in monkeys.

PURPOSE: To determine if repeated intramuscular ketamine in monkeys on consecutive days affects intraocular pressure (IOP) and if the ketamine-induced IOP change has any relationship to systemic dehydration and/or changes in mean arterial pressure (MAP) of the animals. METHODS: Nine monkeys were studied per four protocols. IOP was determined hourly for 6 hr by Goldmann tonometry under ketamine anesthesia on 3 (protocol 1) or 5 (protocols 2 and 3) consecutive days, or on alternating days 1, 3, and 5 (protocol 4). Monkeys in protocols 3 and 4, but not in protocols 1 and 2, received subcutaneous Ringer's fluids at the end of each 6-hr session on days 1-4 or days 1, 3, and 5; monkeys in protocols 2 and 3 received intravenous fluid infusion throughout the experiment on day 5. In protocols 2-4, MAP was measured hourly following each IOP measurement. RESULTS: Monkeys receiving ketamine but no Ringer's fluids in protocol 1 or 2 showed significant IOP declines on days 2-3 or 2-4. The IOP declines were greater in magnitude in protocol 1 than in protocol 2. Daily subcutaneous Ringer's fluids appeared to delay IOP declines in protocol 3. Continuous intravenous fluid infusion on day 5 variably prevented IOP declines in protocols 2 and 3. Monkeys receiving ketamine and subcutaneous fluids on alternate days in protocol 4 showed no decline in IOP. No significant relationship between IOP and MAP was observed. CONCLUSIONS: Anesthesia induced by repeated intramuscular ketamine on consecutive days may produce significant IOP declines. Systemic dehydration during the anesthesia seems to be the predominant factor contributing to the IOP reduction. However, inter-individual differences in monkeys indicate that multiple factors may be involved. This study also suggests that fluid supplementation plus alternating anesthesia with recovery days may prevent IOP reduction in monkeys resulting from daily prolonged ketamine anesthesia.

Latrunculin B effects on trabecular meshwork and corneal endothelial morphology in monkeys.

To determine the mechanism of latrunculin B (LAT-B)-induced decrease in outflow resistance and the effect of LAT-B on the cornea, structural changes of the trabecular meshwork (TM) and the corneal endothelium following LAT-B were studied in the live monkey eye. LAT-B (0.5 microM) and vehicle were administered by anterior chamber exchange and infusion with cationized and non-cationized gold solution in opposite eyes. The eyes were fixed by infusing Ito's solution and enucleated. Anterior segments were quadrisected and embedded in Epon-Embed 812. Morphology of the TM and the corneal endothelium was studied by light and electron microscopy. LAT-B-induced morphological changes in the TM included: (1) loss of microfilament integrity in cells, especially in TM cells on the collagen beams; (2) development of numerous cytoplasmic projections of the sub-canalicular cells (SUB); (3) reorganization of intermediate filaments in Schlemm's canal inner wall (IW) cells; (4) massive 'ballooning' of the juxtacanalicular (JXT) region, leading to a substantial expansion of the space between the IW of Schlemm's canal and the trabecular collagen beams; and (5) retention of extracellular matrix (ECM), trapped between the SUB cell layer and IW cells. No detrimental effects on tight junctions, giant vacuoles, and cell-cell and cell-ECM adhesions were observed. Endocytosis of gold particles was not affected. Morphology of the corneal endothelium of the LAT-B-treated eye was unchanged. In conclusion, TM changes in the LAT-B-treated eye suggest that the expansion of the JXT space may account for the decrease in outflow resistance induced by latrunculins. The outflow-effective concentration of LAT-B administered intracamerally does not significantly affect the corneal endothelium.

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.

Functional and structural reversibility of H-7 effects on the conventional aqueous outflow pathway in monkeys.

To determine the mechanism of H-7-induced outflow resistance decrease, the reversibility of H-7 effects on outflow pathway was studied physiologically and morphologically in live monkey eyes. Total outflow facility was measured by two-level constant pressure perfusion before (baseline measurement) and after (post-drug measurement) anterior chamber (AC) exchange with 300microM H-7 or vehicle in opposite eyes of eight monkeys. H-7 was then removed by AC exchange with drug-free vehicle in both eyes, followed by a 2.5hr waiting period, after which outflow facility was measured again with (Group 2; n=4) or without (Group 1; n=4) another preceding drug-free AC exchange. For morphological study, five monkeys were initially perfused similarly to Group 1 in physiology, but the facility measurement beginning 2.5hr after drug removal was either omitted or replaced by gold solution infusion. Following baseline measurement, two of the five monkeys received H-7 or vehicle in opposite eyes, while three monkeys received H-7 in both eyes 2.5hr apart, contributing one H-7-treated 'recovery' eye and one H-7-treated 'acute' eye. After perfusion, both eyes of all five monkeys were studied by light and electron microscopy. Outflow facility during post-drug measurement in the H-7-treated eye was significantly increased by two-fold. However, the facility increase was reduced when measured beginning 2.5hr after drug removal, with the reduction being greater in Group 1. 'Recovered' outflow facility after drug removal gradually increased again under continuous AC infusion with drug-free vehicle. Morphologically, major changes in and around Schlemm's canal (SC) in the H-7-treated 'acute' eye included protrusion of the entire inner wall (IW) into SC, relaxation of the IW cells and reorganization of the IW cytoskeleton. The changes in IW cells and juxtacanalicular region of the H-7-treated 'recovery' eye were non-uniform, with areas resembling the vehicle-treated eye ('contracted areas') and areas resembling the H-7-treated 'acute' eye ('relaxed areas'). The average junction-to-junction distances in the IW cells of the H-7-treated 'recovery' eye were intermediate between the vehicle-treated eye and the H-7-treated 'acute' eye. In conclusion, H-7's effect on outflow facility seems reversible, but AC exchange or continuous infusion with drug-free vehicle can re-elevate the 'recovered' outflow facility. Major morphological changes in the TM immediately after H-7 include IW protrusion, cellular relaxation and cytoskeleton reorganization. The decrease in 'relaxed areas' in the TM, in conjunction with the reversed outflow facility, 2.5hr after drug removal suggests that cellular relaxation in the TM is the structural basis for H-7-induced increase in outflow facility.