Effect of H-7 and Lat-B on retinal physiology.

PURPOSE: To investigate the effects of H-7 and Latrunculin B (Lat-B) on retinal vascular permeability and electrophysiology at concentrations that increase outflow facility in monkeys. METHODS: One eye of 1 rhesus and 22 cynomolgus monkeys received an intravitreal bolus injection of H-7 or Lat-B; the opposite eye received vehicle. Multifocal electroretinograms (mfERGs), and photopic and scotopic full-field electroretinograms (ffERGs, sERGs) were recorded in subsets of monkeys at baseline and at multiple time-points post-H-7 or Lat-B. Vitreous fluorophotometry (VF) and fluorescein angiography (FA) were also performed. RESULTS: No differences between the H-7 or Lat-B treated and control eyes were found in ffERGs, mfERGs, sERGs, or in FAs in any monkey. No significant difference was found in vitreous fluorescein levels between H-7 treated or Lat-B treated vs. control eyes. CONCLUSIONS: No effect on retinal vascular permeability or retinal electrophysiology was apparent after intravitreal administration of H-7 or Lat-B at doses that increase outflow facility and lower IOP when given intracamerally.

Effect of age on outflow resistance washout during anterior chamber perfusion in rhesus and cynomolgus monkeys.

When aqueous humour outflow resistance is measured by two-level constant pressure perfusion in non-human primate eyes, a progressive decrease in outflow resistance, known as the 'washout effect' occurs with time. The effect of age on total outflow resistance washout (the reciprocal of outflow facility (OF)) was determined in rhesus and cynomolgus monkeys. In cynomolgus monkeys, the effect of time between exchange of the anterior chamber (AC) contents and post-exchange OF measurements on outflow resistance was also examined. Total OF was determined at baseline in one eye of 35 rhesus monkeys aged 4-29 yrs, and in 27 cynomolgus monkeys, aged 3-17 yrs, at baseline and after 10 min AC exchange with 1 or 2 ml Bárány's perfusand or in Bárány's containing 0.01-1% dimethyl sulfoxide (DMSO) or Tris base. Resistance washout did not differ with age at baseline in rhesus or cynomolgus monkeys. Similarly, no changes were found when comparing post-exchange resistance washout vs. age in cynomolgus monkeys that had undergone AC exchange with Bárány's perfusand only or Bárány's containing 0.01-1% DMSO or Tris base. Rate of resistance washout decreased with increased length of time between exchange of the AC contents and post-exchange outflow facility readings (-0.0004+/-0.0092 mmHg min(-1) microl(-1) yr(-1); p = 0.016). Several explanations for these findings are plausible.

Studies on the mechanism of action of timolol and on the effects of suppression and redirection of aqueous flow on outflow facility.

Long-term use of drugs that suppress aqueous humor formation, such as timolol and dorzolamide, or that redirect aqueous humor outflow from the trabecular meshwork, such as prostaglandin F2alpha analogues, could cause underperfusion of the trabecular meshwork and a secondary decrease in outflow facility. We investigated the mechanism of suppression of aqueous humor formation by timolol in monkey eyes by measuring aqueous humor ascorbate levels. We also determined whether suppression of aqueous humor formation with and without redirection of aqueous humor away from the trabecular meshwork could lead to a subsequent reduction in outflow facility, and whether this reduction was correlated with increased fibronectin levels in anterior chamber aqueous humor. In cynomolgus monkeys, unilateral dose/aqueous humor formation response curves were generated for timolol, dorzolamide, and a combination of timolol + dorzolamide. Aqueous humor formation and/or outflow facility were measured in both eyes after approximately four days, four weeks and seven weeks of twice daily treatment with 3.5 microg timolol + 1.0 mg dorzolamide to one eye and 30% DMSO to the other. In some monkeys, 5 microg prostaglandin F2alpha-isopropyl ester (PG) was added to timolol + dorzolamide for 4-week treatments. Intraocular pressure and corneal endothelial transfer coefficients (k(a)) were also measured at four weeks. Aqueous humor fibronectin levels were determined in four monkeys after approximately 9.5 weeks of timolol + dorzolamide treatment. Aqueous humor formation, intraocular pressure, and aqueous humor ascorbate levels were also determined in rhesus monkeys at baseline and after a single unilateral topical administration of 25 microg timolol. Compared to baseline for the same eye, aqueous humor formation was significantly decreased in treated eyes at all doses of timolol and at 1.8 and 4 mg dorzolamide. Compared to the opposite control eye, aqueous humor formation was lower in treated eyes after 3.5 and 5 microg timolol and after all doses of dorzolamide. Aqueous humor formation after treatment with 3.5 microg timolol + 1.0 mg dorzolamide was decreased in treated vs. control eyes, after four days and was suppressed in both treated and control eyes after four weeks of treatment, but not when PG was added. There was no difference in k(a) values with or without the addition of PG. Intraocular pressure was significantly lower in both treated and control eyes vs. baseline after approximately 6.5 weeks treatment with timolol + dorzolamide when taken 2 hr after the last dose and after approximately 3.5 weeks treatment with timolol + dorzolamide + PG when measured 6 hr after the last dose. Outflow facility after treatment with timolol + dorzolamide was unchanged after four days, tended to be lower in the treated vs. control eyes after four and seven weeks, and was significantly lower in treated vs. control eyes after four weeks treatment with timolol + dorzolamide + PG (0.352 +/- 0.052 vs. 0.515 +/- 0.096 microl min(-1) mmHg(-1), p < or = 0.02). Both treated vs. control eye aqueous humor fibronectin levels were below the level of detection for our assay (0.01 microg ml(-1)). The 25 microg timolol dose decreased ipsilateral, but not contralateral intraocular pressure (12.6 +/- 1.7 vs. 15.2 +/- 0.9; p < 0.05) and aqueous humor formation (1.40 +/- 0.08 vs. 2.03 +/- 0.09 microg ml(-1), p < or = 0.01). There was no difference in anterior chamber ascorbate levels in treated vs. control eyes or compared to their respective baselines. Our findings indicate that timolol affects neither ciliary epithelial transport of ascorbate nor aqueous fibronectin levels. Our data also indicate that decreasing aqueous humor formation over a period of time can lead to reduction in outflow facility, particularly when combined with therapy that redirects aqueous from the trabecular meshwork. Future intraocular pressure-lowering therapies for glaucoma may better be directed at enhancing flow through the trabecular pathway as opposed to decreasing aqueous humor formation or rerouting aqueous humor away from the trabecular meshwork.

Gene therapy using p21WAF-1/Cip-1 to modulate wound healing after glaucoma trabeculectomy surgery in a primate model of ocular hypertension.

Glaucoma is a common eye disease associated with elevated intraocular pressure (IOP). Lowering IOP is the only acceptable therapy for glaucoma and slows progression of the disease. Filtration surgery, which introduces a guarded ostomy through the sclera into the anterior chamber of the eye to allow the escape of aqueous humor, is the most reliable method for effective IOP lowering. Success of this surgery is limited by scarring of the ostomy, so this procedure is often accompanied by the use of antimetabolites, such as mitomycin C (MMC), to block the wound healing response. Although effective in preventing scarring, antimetabolites also yield unwanted side effects, such as hypotony and tissue degeneration due to cellular destruction. This study presents an alternative to antimetabolites by using gene therapy to introduce the human gene for p21(WAF-1/cip-1) (p21) to cause cell cycle arrest of surrounding cells rather than their destruction. In this procedure, p21 was delivered using a recombinant adenovirus to ocular hypertensive monkey eyes. These eyes then underwent filtration surgery. Results show that eyes treated with p21 exhibited open surgical ostomies by both functional and histological criteria, and did not display any side effects seen in control animals that were treated with MMC.

Effects of the marine macrolides swinholide A and jasplakinolide on outflow facility in monkeys.

PURPOSE: To determine effects of the marine macrolides swinholide A (Swin A) and jasplakinolide (Jas), alone or in conjunction with latrunculin B (Lat B) on outflow facility in monkeys. METHODS: Total outflow facility was measured by two-level constant-pressure perfusion of the anterior chamber before and after exchange with Swin A, Jas, or vehicles followed by continuous anterior chamber infusion of drug or vehicle, in opposite eyes of cynomolgus monkeys. The effect of a facility-ineffective dose of Jas plus a threshold or submaximal facility-effective dose of the actin depolymerizer Lat B on outflow facility was also determined. RESULTS: Ten or 100 nM Swin A or 20, 100, or 500 nM Jas had no significant effect on outflow facility. However, 500 nM Swin A and 2.5 microM Jas significantly increased facility by 80% +/- 21% and 157% +/- 57% (mean +/- SEM) respectively, adjusted for corresponding baselines and resistance washout in contralateral control eyes. The facility increase in the eye treated with 500 nM Jas with 60 or 200 nM Lat B was similar to that in the eye treated with 60 or 200 nM Lat B only. CONCLUSIONS: Swin A (which severs actin filaments and sequesters actin dimers) and Lat B (which sequesters actin monomers) similarly increase outflow facility. The potent inducer of actin polymerization Jas (500 nM) neither inhibits nor potentiates the facility increase induced by Lat B (60 or 200 nM). A higher dose of Jas increases rather than decreases outflow facility.

Effects of serotonergic compounds on aqueous humor dynamics in monkeys.

PURPOSE: The effects of several serotonergic agonists on aqueous humor formation (AHF), total outflow facility (OF) and intraocular pressure (IOP) were investigated in living cynomolgus monkeys. METHODS: We determined the effect of a single topical unilateral 300 microg or 3 mg dose of the 5-HT agonists serotonin, 5-carboxamidotryptamine (5-CT), sumatripan, gepirone, and 8-hydroxy-2(di-n-propylaminotetralin) (8-OH-DPAT) and a 450 microg dose of flesinoxan on IOP (Goldmann applanation tonometry), AHF (scanning ocular fluorophotometry) and total OF (8-OH-DPAT only, topically and intracamerally). RESULTS: Serotonin, 5-CT, sumatripan or gepirone had no significant effect on IOP or AHF. 8-OH-DPAT caused an AHF increase of approximately 70% over 6 hr in both ipsilateral drug- and contralateral vehicle-treated eyes, but no significant change in IOP compared with baseline measured on a separate occasion in the same animals. 8-OH-DPAT did not increase protein levels or rate of entry of systemically administered fluorescein in the anterior chamber aqueous humor compared to historic controls, and no difference was seen between ipsilateral and contralateral eyes. Flesinoxan had no effect on IOP and produced an insignificant 25% increase in flow in treated eyes compared to baseline. CONCLUSION: The results for 8-OH-DPAT and possibly flexinoxan indicate the presence of a secretion-stimulating 5-HT1A receptor in monkey ciliary epithelium that has little effect on IOP. OF was unchanged following 8-OH-DPAT administered topically or following intracameral exchange.

Low doses of pilocarpine do not significantly increase outflow facility in the cynomolgus monkey.

Low doses (10(-9)-10(-6) M) of pilocarpine reportedly increase outflow facility in the organ-cultured human eye, suggesting a direct action on the trabecular meshwork. M3 muscarinic receptors have been found in both cultured human trabecular meshwork cells and tissue. We determined whether low pilo doses would increase outflow facility in the living monkey. The anterior chambers of both eyes of 17 pentobarbital anesthetized cynomolgus monkeys were cannulated and outflow facility measured bilaterally by 2-level constant pressure perfusion after an initial 2 ml exchange with Bárány's perfusand containing 24.5 microM phenylephrine (PE). Two subsequent exchanges were performed with one eye receiving Bárány's + PE + 10(-10)-10(-4) M pilocarpine and the contralateral eye receiving only Bárány's + PE. Outflow facility was measured for 35-40 min following each exchange. Accommodation and pupil diameter were measured before each exchange and approximately every 10 min during facility measurements. Outflow facility was significantly increased by 154 and 313% in eyes treated with 10(-5) M and 10(-4) M pilocarpine, respectively, related to contralateral controls. Accommodation and miosis also were induced only at 10(-5) M (accommodation, 3.3 +/- 1.6 diopters, NS; miosis, -4.1 +/- 0.5 mm, P < or = 0.001) and 10(-4) M (accommodation, 10.6 +/- 0.0 diopters, P < or = 0.02; miosis, -3.4 +/- 1.0 mm, P < or = 0.025) pilocarpine. We conclude that low anterior chamber doses of pilocarpine do not increase outflow facility in the living monkey as reported in the organ-cultured human eye, nor do they induce miosis or accommodation. All three parameters respond to pilocarpine at similar doses, and there is no functional evidence of a meaningful outflow facility-relevant pilocarpine effect on the trabecular meshwork at doses lower than those which affect the ciliary muscle.

H-7 increases trabecular facility and facility after ciliary muscle disinsertion in monkeys.

PURPOSE: To determine the effects of the serine-threonine kinase inhibitor H-7 on total outflow facility in iridectomized + ciliary muscle (CM)- disinserted, and on trabecular facility in normal, monkey eyes. METHODS: Total outflow facility was determined by two-level constant pressure perfusion of the anterior chamber. Trabecular outflow facility was determined from accumulation in blood of intracamerally infused radioiodinated albumin at two intraocular pressure levels. RESULTS: Three-hundred micromoles of intracameral H-7 doubled facility in iridectomized + CM- disinserted monkey eyes and contralateral iridectomized-only eyes. Four 5-microl drops of 400 mM H-7 applied topically followed 2 hours later by anterior chamber exchange for 10 minutes and intracameral infusion for 90 minutes with 100 microM H-7 increased trabecular and total outflow facility by 135%+/-29% and 105%+/-35% (n 5, P < 0.01, P < 0.05), respectively, compared with contralateral vehicle-treated eyes. CONCLUSIONS: H-7 increases trabecular outflow facility in monkeys by a mechanism independent of the CM, presumably acting directly on the trabecular meshwork.

Endothelin-1 effects on aqueous humor dynamics in monkeys.

PURPOSE: To report data showing no effect of endothelin-1 on aqueous humor formation and total outflow facility in the cynomolgus monkey, in contrast to the enhancement of outflow facility in monkeys and the suppression of aqueous humor flow in rabbits previously reported by others. METHODS: Living monkeys received, unilaterally: (i) intracameral endothelin-1 (10 microl bolus, or 2ml or 4ml exchange; final intracameral concentration 1 or 10 nM) with total outflow facility measured for up to 1 hr post-treatment by two-level constant pressure perfusion, and (ii) intravitreal endothelin-1 (20 microl, final intravitreal concentration 0.1 or 1 microM) with aqueous humor flow measured by scanning fluorophotometry for 5 hr starting 12 hr post-treatment. Contralateral eyes received vehicle. RESULTS: Endothelin-1 had no effect on total outflow facility or aqueous humor flow with any of the methods described. CONCLUSION: Endothelin-1 seems have a variable effect on aqueous humor dynamics within and between species.

Atropine reduces but does not eliminate the age-related decline in perfusion outflow facility in monkeys.

Accommodative amplitude and outflow facility decline with age in rhesus monkeys and humans. In monkeys, there is an age-related reduction in ciliary muscle (CM) mobility due to stiffening of its posterior and outer attachments. Since the CM inserts into the trabecular meshwork (TM) and CM contraction deforms the TM so as to increase outflow facility, we asked whether the age-related decline in outflow facility observed in anesthetized monkeys is consequent to the age-related reduction in CM mobility. One eye of 19 pentobarbital-anesthetized rhesus monkeys aged 4-24 years underwent 2-level constant pressure perfusion of the anterior chamber (AC) for 40 min to measure baseline total outflow facility. Both eyes then received 100 micrograms of atropine, given topically to the central cornea. The AC of the second eye was cannulated 40 min after atropine treatment. Facility was measured simultaneously in both eyes for 40 min beginning 45 min after atropine treatment. Baseline facility declined with age by -0.0160 +/- 0.0059 microliters min-1 mmHg-1 yr-1 (P = 0.009). The average facility at baseline was approximately 50% higher in the youngest (ages 4-10 years) compared to the oldest (ages 21-25 years) animals (P < or = 0.03). Following atropine facility decreased by approximately 25% in the youngest monkeys (N.S.), but not at all in the oldest animals when compared to baseline. Thus, the age-related facility decline persisted after atropine in both eyes, but was not as dramatic as prior to atropine (-0.0092 +/- 0.0050 [P < or = 0.09] and -0.0078 +/- 0.0044 [P < or = 0.10] microliter min-1 mmHg-1 yr-1 respectively). Apparently, atropine inhibits facility-relevant anesthetic-induced ciliary muscle tone to a greater extent in younger than in older monkeys, presumably because stiffening of the posterior and outer attachments of the CM in the older animals has already reduced its ability to move and thereby deform the TM. The inability of atropine to completely eliminate the age-related facility decline indicates the presence of atropine-independent, facility-relevant age-dependent changes in the TM itself, such as loss of cells or build-up of extracellular material.

Effect of DMSO and exchange volume on outflow resistance washout and response to pilocarpine during anterior chamber perfusion in monkeys.

PURPOSE: To determine the dependence of outflow resistance washout on anterior chamber (AC) fluid exchange volume (EV); the minimum EV required for complete AC mixing, and the effect of 0.01-5% DMSO in the exchange solution on outflow resistance. METHODS: Total outflow facility was determined in 63 pentobarbital-anesthetized cynomolgus monkeys, before and after AC exchange, for 10 min, with 1, 2, and 4 ml of Bárány's perfusand, containing 0.01-5% DMSO or containing 1 microgram/ml or 5 micrograms/ml pilocarpine HCL (pilo), each volume. RESULTS: Post-exchange facility increased by 21% (p < 0.05) vs. baseline for both the 1 and 2 ml Bárány's volumes and by 50% (p < 0.001) for the 4-ml volume. Washout-corrected post-exchange facility was unchanged from baseline, following 1 or 2 ml exchange with 1 microgram/ml pilo; 5 micrograms/ml pilo increased facility by 130 +/- 41% and 174 +/- 40% respectively, relative to baseline. Exchange with 4 ml of 1 microgram/ml pilo increased facility 72% more than the 2 ml (p < 0.029) and 100% more than the 1 ml (p < 0.014) volumes. When corrected for their respective baselines, 2 ml of 5 micrograms/ml pilo increased facility 12.7 +/- 4.4% more than the 1 ml volume (p < 0.05). Post exchange facility was not significantly increased over baseline in eyes exchanged with 2 ml of 0.01-4% DMSO or in eyes exchanged with Bárány's alone. When corrected for their respective baselines, post-exchange facility was significantly lower in eyes exchanged with 5% DMSO vs. Bárány's (p = 0.022). CONCLUSIONS: One- and 2-ml EVs preserve outflow resistance equally well, and substantially better than 4 ml. Two ml is the minimum volume necessary for adequate mixing of AC drug solutions, to achieve the full facility effect. Concentrations of DMSO up to 4% in a 2-ml EV can solubilize compounds without additionally decreasing outflow resistance.

Intraocular pressure measurement in cynomolgus monkeys. Tono-Pen versus manometry.

PURPOSE: In living cynomolgus monkey eyes, the authors compared manometrically set and measured intraocular pressure (IOP) with simultaneous IOP readings obtained with the Tono-Pen (TP), a handheld applanation tonometer based on the Mackay-Marg principle. METHODS: In three pentobarbital-anesthetized cynomolgus monkeys, IOP was set and measured manometrically after anterior chamber cannulation through the peripheral cornea with a 26-gauge needle connected to a vertically adjustable reservoir and a pressure transducer. Intraocular pressure was raised in approximately 5 mm Hg steps from 5 mm Hg to 60 mm Hg and then lowered in 5 mm Hg steps to 5 mm Hg, with TP measurements taken at each increment and decrement in open and stopcock modes. RESULTS: Linear regression analysis of TP on manometric readings for grouped data from all six eyes, with each data point representing the average of all the TP readings from one eye at each manometric pressure setting, showed a slope 0.692 +/- 0.016 and 0.683 +/- 0.023 (both significantly different from 1; P < 0.001), intercept 1.21 +/- 0.60 and 1.64 +/- 0.82 mm Hg (both significantly different from 0.0, P < 0.05), and correlation coefficient 0.981 and 0.96 in open stopcock and closed stopcock mode, respectively. There were no striking differences when the data were analyzed for individual eyes or animals, for open versus closed stopcock manometry, or for increasing versus decreasing manometric IOP. CONCLUSIONS: The TP provides reproducible measurements of IOP in cynomolgus monkeys, with measurement accuracy dependent on the generation of an appropriate calibration curve.