Accommodative function in rhesus monkeys: effects of aging and calorie restriction.

Numerous degenerative changes in the visual system occur with age, including a loss of accommodative function possibly related to hardening of the lens or loss of ciliary muscle mobility. The rhesus monkey is a reliable animal model for studying age-related changes in ocular function, including loss of accommodation. Calorie restriction (CR) is the only consistent intervention to slow aging and extend lifespan in rodents, and more recently the beneficial effects of CR have been reported in nonhuman primates. The goal of the present study was to evaluate age-related changes in ocular accommodation and the potential effect of long-term (>8 years) CR on accommodation in male and female rhesus monkeys. Refraction, accommodation (Hartinger coincidence refractometer), and lens thickness (A-scan ultrasound) were measured in 97 male and female rhesus monkeys age 8-36 years under Telazol/acepromazine anesthesia. Refraction and accommodation measurements were taken before and after 40% carbachol corneal iontophoresis to induce maximum accommodation. Half the animals were in the control (CON) group and were fed ad libitum. The CR group received 30% fewer calories than age- and weight-matched controls. Males were on CR for 12 years and females for eight years. With increasing age, accommodative ability declined in both CON and CR monkeys by 1.03 ± 0.12 (P = 0.001) and 1.18 ± 0.12 (P = 0.001) diopters/year, respectively. The age-related decline did not differ significantly between the groups (P = 0.374). Baseline lens thickness increased with age in both groups by 0.03 ± 0.005 mm/year (P = 0.001) and 0.02 ± 0.005 mm/year (P = 0.001) for the CON and CR groups, respectively. The tendency for the for the lens to thicken with age occurred at a slower rate in the CR group vs. the CON group but the difference was not statistically significant (P = 0.086). Baseline refraction was -2.8 ± 0.55 and -3.0 ± 0.62 diopters for CON and CR, respectively. Baseline refraction tended to become slightly more negative with age (P = 0.070), but this trend did not differ significantly between the groups (P = 0.587). In summary, there was no difference in the slope of the age-related changes in accommodation, lens thickness, or refraction in the carbachol-treated eyes due to diet. These data are consistent with previous findings of decreased accommodative ability in aging rhesus monkeys, comparable to the age-dependent decrease in accommodative ability in humans. This study is the first to indicate that the accommodative system may not benefit from calorie restriction.

Ultrasound biomicroscopy of the aging rhesus monkey ciliary region.

Ultrasound biomicroscopy of the living rhesus monkey ocular ciliary region was undertaken to identify age-dependent changes that might relate to the progression of presbyopia. Monkeys were anesthetized and pharmacologically cyclopleged, the eyelids were held open with a lid speculum, and sutures were placed beneath the medial and lateral rectus muscles. Ultrasound biomicroscopy imaging of the nasal and temporal quadrants of the eye were performed, and the live images were recorded to videotape. Subsequent image analysis was performed to obtain objective morphometric measurements of the ciliary body region. The ciliary body inner radius of curvature, outer radius of curvature, inner arc length, area, thickness, perimeter, zonular fiber length, and circumlental space were measured. Zonular space was calculated. The circumlental space decreased with increasing age in the temporal quadrant. The other morphologic measurements were not significantly correlated with age or body weight. Most morphologic measurements were significantly different comparing temporal vs. nasal quadrants. Bifurcation of the posterior zonular fibers was frequently observed. Although temporal circumlental space was the only measurement found to change with age, ultrasound biomicroscopy of the living rhesus ciliary region did identify distinct nasal vs. temporal asymmetries, which may reflect anatomical requirements for convergence-associated accommodation.

Enthacrynic and acid effects on inner wall pores in living monkeys.

PURPOSE: The influence of the inner wall of Schlemm's canal on aqueous outflow facility remains poorly understood. We examined the relationship between inner wall pore characteristics and outflow facility in living primate eyes in which facility had been pharmacologically increased by ethacrynic acid (ECA) infusion and in contralateral control eyes. METHODS: Outflow facility (two-level constant pressure perfusion) was measured in eight pairs of living monkey eyes before and after administration of a bolus dose of either 0.125 mM ECA or vehicle. After exsanguination, eyes were fixed in situ under constant-pressure conditions (mean fixation pressure approximately 19 mm Hg). The density and diameter of inner wall pores and the number and area of platelet aggregates on the inner wall of Schlemm's canal were measured by scanning electron microscopy. RESULTS: In ECA-treated eyes, outflow facility increased 63% (P < 0.0001), intracellular pore density decreased 46% (P = 0.0094), intracellular pore size increased 27% (P = 0.049), platelet aggregate density increased 158% (P < 0.0001), and area covered by platelets increased 210% (P = 0.012) relative to contralateral controls. Although the average density and size of intercellular pores were essentially unaffected by ECA, an increased density of large (> or = 1.90 microm) intercellular pores was seen in ECA-treated eyes. The density of intracellular pores increased with the duration of fixative perfusion. Other than a weak negative correlation between outflow facility and intracellular pore density in ECA-treated eyes (P = 0.052), facility was not correlated with inner wall pore features. CONCLUSIONS: Our data are most consistent with a scenario in which ECA promotes formation of large intercellular pores in the inner wall of Schlemm's canal, which are then masked by platelet aggregates. Masking of intercellular pores, combined with fixation-induced alteration of inner wall pore density, greatly complicates attempts to relate facility to inner wall structure and suggests that in vivo pore density is smaller than in fixed tissue. Additionally, facility-influencing effects of ECA on the juxtacanalicular tissue cannot be excluded.

Accommodation dynamics in aging rhesus monkeys.

Accommodation, the mechanism by which the eye focuses on near objects, is lost with increasing age in humans and monkeys. This pathophysiology, called presbyopia, is poorly understood. We studied aging-related changes in the dynamics of accommodation in rhesus monkeys aged 4-24 yr after total iridectomy and midbrain implantation of an electrode to permit visualization and stimulation, respectively, of the eye's accommodative apparatus. Real-time video techniques were used to capture and quantify images of the ciliary body and lens. During accommodation in youth, ciliary body movement was biphasic, lens movement was monophasic, and both slowed as the structures approached their new steady-state positions. Disaccommodation occurred more rapidly for both ciliary body and lens, but with longer latent period, and slowed near the end point. With increasing age, the amplitude of lens and ciliary body movement during accommodation declined, as did their velocities. The latent period of lens and ciliary body movements increased, and ciliary body movement became monophasic. The latent period of lens and ciliary body movement during disaccommodation was not significantly correlated with age, but their velocity declined significantly. The age-dependent decline in amplitude and velocity of ciliary body movements during accommodation suggests that ciliary body dysfunction plays a role in presbyopia. The age changes in lens movement could be a consequence of increasing inelasticity or hardening of the lens, or of age changes in ciliary body motility.

Effect of ticrynafen on aqueous humor dynamics in monkeys.

OBJECTIVE: To determine the effect of ticrynafen, a nonsulfhydryl-reactive compound similar to ethacrynic acid, on outflow facility in normotensive monkey eyes and on intraocular pressure (IOP) in monkey eyes with laser-induced glaucoma. METHODS: In normotensive eyes, facility (perfusion) was measured shortly before and after bolus or exchange intracameral infusion of ticrynafen or vehicle in opposite eyes, and 3.5 to 4.5 hours after 5 days of twice-daily 2% ticrynafen or vehicle ointment. In glaucomatous eyes, baseline and vehicle diurnal IOP curves were established, 2% ticrynafen ointment was given twice daily for 5 days, and IOP was measured immediately before and 0.5 to 6 hours after each morning treatment. RESULTS: In normotensive eyes, exchange 2-mL influsion of 0.2-, 1-, or 4-mmol/L ticrynafen increased facility by 33% +/- 6% (mean +/- SEM), 73% +/- 18%, and 60% +/- 11%, respectively. Day 5 posttreatment facility was higher in the ticrynafen group than in controls by 28% +/- 9%. In glaucomatous eyes, maximum IOP decline, from approximately 35 mm Hg, was 7.5 +/- 2.0 mm Hg on day 4 and 9.8 +/- 2.4 mm Hg on day 5 of twice-daily ticrynafen treatment. CONCLUSION: The facility-increasing, IOP-lowering action of ticrynafen, ethacrynic acid, and derivatives may not depend entirely on sulfhydryl reactivity. CLINICAL RELEVANCE: Whether such drugs as ethacrynic acid and ticrynafen prove valuable for glaucoma therapy, at the least they are useful probes to study aqueous outflow mechanisms.

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.

Aging effects on accommodation and outflow facility responses to pilocarpine in humans.

OBJECTIVE: To determine the relationships among age, outflow facility, and refractive and facility responses to pilocarpine in humans. METHODS: Refraction, intraocular pressure, and outflow facility were determined in 30 normal volunteers aged 20 to 75 years, by coincidence refractometry, applanation tonometry, and Schiøtz tonography, respectively, before and 1 hour after a 30-microL drop of 2% or 6% pilocarpine. Simple regression of baseline facility, postpilocarpine facility, and facility change, on age and refractive change singly and jointly, was performed. Stepwise regression models and graphic conditioning plots were used to determine, for each facility variable, its relationship to age or refractive change specifically. RESULTS: Baseline outflow facility and maximum pilocarpine-induced refractive change (ie, accommodation) declined with age, but the decrease in intraocular pressure and the facility response to pilocarpine did not. After adjusting for age, for baseline facility, there was no further relationship to 6% pilocarpine-induced accommodation, and a slight residual relationship to 2% pilocarpine-induced accommodation. After adjusting for both 2% or 6% pilocarpine-induced accommodation, the relationship to age was still significant. The facility increase after 2% or 6% pilocarpine did not depend on age and/or accommodative amplitude. CONCLUSIONS: In humans, as previously described in rhesus monkeys, an age-related loss of ciliary muscle mobility may compromise the basal function of the trabecular meshwork. However, unlike monkeys, humans exhibit no loss of the intraocular pressure or outflow facility response to pilocarpine with age.

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.

Effect of daily topical ethacrynic acid on aqueous humor dynamics in monkeys.

We determined the effect of 1.5% ethacrynic acid (ECA) in ointment on intraocular pressure (IOP) and outflow facility following 5 days of topical treatment in ocular normotensive cynomolgus monkeys. Twelve monkeys received a 1 cm strip of ointment containing ECA in one eye but without ECA in the other once daily for 5 days. On Day 1 and Day 5 IOP was measured immediately before and 1 and 3 h after treatment. Outflow facility (perfusion) was determined 3.5 h after treatment on Day 5. The ECA-vehicle IOP differences averaged -2.8 +/- 0.8 (S.E.M) (p < 0.01), -1.7 +/- 0.6 (p < 0.02) and -3.7 +/- 0.7 mm Hg (p < 0.001), equivalent to IOP reductions of 21 +/- 6% (p < 0.01), 13 +/- 5% (p < 0.02) and 26 +/- 5% (p < 0.002) respectively, at 0, 1 and 3 h respectively after treatment on Day 5. Facility averaged 40 +/- 15% higher (p < 0.03) in the ECA-treated compared to the vehicle-treated eyes 4 h after treatment on Day 5.

epsilon-Aminocaproic acid does not inhibit outflow resistance washout in monkeys.

PURPOSE: To determine whether the anti-fibrinolytic agent epsilon-aminocaproic acid (EACA) inhibits the washout of resistance to aqueous humor outflow during anterior chamber perfusion in cynomolgus monkeys, as it does in rabbits. METHODS: Nine adult ocular normotensive cynomolgus monkeys underwent bilateral anterior chamber perfusion with Bárány solution, containing 3.8 mM EACA unilaterally. Total outflow facility was determined in both eyes simultaneously for approximately 4 hours by the two-level constant pressure method. The data were analyzed using a linear regression model that tested treated versus control eye differences over time against a slope and intercept of 0.0. RESULTS: Outflow facility increased and resistance decreased significantly over time similarly in both EACA-treated and control eyes; i.e., neither the slopes nor the intercepts for facility or resistance, respectively, differed between the eyes over the entire 4-hour measurement period or for the initial 90 minutes considered separately. The facility increase and resistance decrease as functions of perfusion volume also were similar in EACA-treated and control eyes. CONCLUSIONS: EACA at this dose does not prevent resistance washout in the cynomolgus monkey, in contrast to the rabbit. This species difference may relate to the vastly different anatomy and physiology of their outflow pathways.

Intraocular pressure measurement after penetrating keratoplasty: minified Goldmann applanation tonometer, pneumatonometer, and Tono-Pen versus manometry.

The accuracy of intraocular pressure measurement with the minified Goldmann applanation tonometer, the pneumatonometer, and the Tono-Pen tonometer were compared in post-mortem human eyes which had undergone penetrating keratoplasty. Enucleated post-mortem human eyes underwent same sized (7.75 mm) or 0.5 mm oversized (8.25 mm) autologous penetrating keratoplasty. Intraocular pressure was then set and measured manometrically while being determined successively with each tonometer over the range of 0-65 mm Hg. Linear regression analysis comparing tonometric and manometric readings showed: (1) minified Goldmann applanation tonometer-slope 0.985 and 0.944, intercept 1.64 and 2.55 mm Hg, correlation coefficient 0.99 and 0.99 in same sized and oversized grafted eyes respectively; (2) pneumatonometer-slope 1.008 and 0.990, intercept 3.37 and 3.69 mm Hg, correlation coefficient 0.99 and 0.98; (3) Tono-Pen-slope 1.061 and 1.002, intercept 5.01 and 4.06 mm Hg, correlation coefficient 0.97 and 0.98. We concluded that the minified Goldmann applanation tonometer is as accurate or more accurate than the pneumatonometer and the Tono-Pen in post-mortem post-keratoplasty human eyes, and may be an economical, convenient alternative to the latter two instruments in clinical practice.

Effect of ethacrynic acid on aqueous outflow dynamics in monkeys.

PURPOSE: To determine the long-term effect of ethacrynic acid (ECA) on aqueous outflow dynamics in ocular normotensive monkeys. METHODS: (1) Twelve cynomolgus monkeys received 10 microliters of 2.5 mM (= 7.5 micrograms) ECA intracamerally in one eye, vehicle in the other; outflow facility (perfusion) was determined at 1 hour, 24 hours, or 1 week, and intraocular pressure (IOP; applanation) at 24 hours, 1 week, and 6 to 7 weeks later. Six other cynomolgi received 10 microliters of 0.13 or 1.3 mM phalloidin in one eye 45 minutes before receiving ECA OU; facility was measured 1 hour after ECA. (2) Groups of five rhesus monkeys underwent intracameral injection of 2.5, 5.0, or 10 micrograms ECA in one eye, vehicle in the other, with IOP measured hours to weeks thereafter. (3) Five rhesus monkeys received 540 micrograms of ECA unilaterally as a 30-microliter topical drop once daily for 4 days. On the first and fourth treatment days, baseline IOP, pupil diameter, and refraction were measured immediately before and again at 2, 4, and 7 hours after topical treatment. RESULTS: (1) ECA divided by vehicle facility averaged 1.83 +/- 0.23 (SEM) (P < 0.02, n = 6), 1.50 +/- 0.27 (P < 0.11, n = 7), and 1.05 +/- 0.10 (n = 7) at 1 hour, 24 hours, and 1 week, respectively. IOP was 1 to 2 mm Hg lower in ECA eyes 24 hours (P < 0.05, n = 5) and 6 to 7 weeks (P < 0.05, n = 7) after treatment. Phalloidin did not diminish the 1-hour ECA facility effect. (2) Five (but not 2.5 or 10) micrograms of ECA lowered IOP 1 to 3 mm Hg, starting at 2 hours and lasting up to 48 hours. The maximum ECA effect (-2.6 +/- 0.25 mm Hg; P < 0.001, n = 5) occurred at 4 hours. IOP, corneal thickness and endothelial cell count, and anterior chamber depth were not significantly different 8 weeks after 5 micrograms ECA or vehicle. (3) Once-daily unilateral topical application of 540 micrograms of ECA induced no change in IOP, refraction, or pupil diameter compared to contralateral vehicle-treated control eyes. There were no significant ECA-related ocular bio-microscopic abnormalities. CONCLUSIONS: ECA may lower IOP and increase outflow facility longer than previously thought, but not by affecting meshwork actin filaments.

Age-related loss of ciliary muscle mobility in the rhesus monkey. Role of the choroid.

Ciliary muscle topography was studied in rhesus monkey eyes (aged 6 to 29 years) bisected meridionally through cornea and optic nerve head. Half of each eye was incubated in atropine sulfate, the other in pilocarpine hydrochloride, and both were then processed for histologic study. Several ciliary muscle sections from the original cut margin and the middle of the half eyes were traced and compared quantitatively. In sections from the middle, where the attachments of the muscle were presumably intact, the pilocarpine effect on ciliary muscle topography was lost with age. In sections near the cut margin, where some of the posterior attachments were disrupted and the choroid had detached from the sclera, the pilocarpine effect persisted with age. These findings suggest that loss of ciliary muscle movement with age is caused by decreased compliance of its posterior attachment.

Accommodation and ciliary muscle muscarinic receptors after echothiophate.

Twice daily topical administration of echothiophate for 2 weeks to the eyes of living cynomolgus monkeys produced profound subsensitivity of the accommodative response to pilocarpine and an approximately 50% decrease in the number of specific binding sites for 3H-quinuclidinyl benzilate (3H-QNB) in the ciliary muscle without a change in their affinity. When echothiophate treatment was discontinued, functional cholinergic sensitivity and the number of QNB binding sites both returned to normal over a similar 4-8 week period. Most animals had a modest overshoot of both functional sensitivity and number of binding sites for at least several weeks thereafter. The treated to control eye ratios for the number of binding sites and accommodative response to pilocarpine were correlated and the plot of log treated to control binding site ratio versus treated to control accommodation ratio resembled a dose-response curve. Similarly, the treated versus control eye differences for the two parameters were correlated, with the regression line passing through the 0-0 axis intercept. Collectively, these findings suggest that agonist-induced modulation of functional cholinergic sensitivity in the parasympathetically innervated (as opposed to denervated) ciliary muscle occurs by a muscarinic receptor-mediated mechanism. This system appears to provide a useful model to study the regulation of ciliary muscle cholinergic sensitivity.

Accuracy of formulae for calculating left ventricular volumes of the equine heart.

Echocardiography may be an accurate method of measuring left ventricular (LV) volumes and mass of the horse's heart. If so, studies of the heart size and hypertrophy would be possible. This study evaluated geometric models of the external and internal LV shapes, to determine which could be applied to echocardiographic measurements. We preserved 30 horses' hearts and measured their dimensions and cross sectional areas. These measurements were entered into seven formulae representing different geometric models of the ventricle and its chamber. We derived a correction factor to estimate the long axis as a fixed proportion of the external diameter, so that volumes could be determined from an M-mode or a cross sectional echocardiogram. Statistical analysis of the regressions of known volumes against calculated volumes measured by water displacement, demonstrated that the ellipsoid formula using cross sectional areas was very accurate in representing the external shape of the left ventricle (slope = 1.01 r2 = 96.3) and its chamber (slope = 0.83, r2 = 94.3). Myocardial volume, measured by subtracting internal (chamber) volume from external volume, was also calculated accurately (slope = 1.01, r2 = 96.5). The ellipsoid formula using directly measured diameter was only slightly less accurate. LV mass could be calculated by applying the specific gravity of equine myocardium, 1.05, to the myocardial volume. Formulae recommended for evaluating are, with M-mode echocardiography: [equation: see text] and with 2D echocardiography [equation: see text] where De is the external diameter, Di is the internal diameter, Ae the external area and Ai the internal area.

13-cis-retinoic acid: effect on urinary bladder carcinogenesis by N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide in Fischer rats.

The failure of 13-cis-retinoic acid to inhibit either the incidence or severity of bladder carcinoma in female Fischer rate initiated with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) suggests that inhibition of bladder carcinogenesis by natural and synthetic retinoids is carcinogen-class specific, and adds an element of complexity to approaches in chemoprevention.

Synthetic retinamides: effect on urinary bladder carcinogenesis by FANFT in Fischer rats.

The failure of N-ethylretinamide and N-(2-hydroxyethyl)retinamide to inhibit the incidence or severity of bladder carcinoma in female Fischer rats initiated with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide supports the concept that the inhibition of bladder carcinogenesis by natural and synthetic retinoids is carcinogen-class specific.