Exp. Eye Res. (1981) 33, 239-245

Drug Effects on Aqueous Humor Formation and Pseudofacility in Normal Rabbit Eyes

K E I T H GREE:N* t AND DAVID E L I J A H *

Departments of Ophthalmology* and Physiology,t Medical College of Georgia, Augusta, Georgia 30912, U.S.A.

(Received 28 September 1979 and accepted 11 February 1981, New York)

The effects of a series of drugs have been evaluated on aqueous humor formation rate (AHFR) and pseudofacility (Cps) in the living rabbit eye. Control values, determined 6 months apart, indicate the reproducibility of the techniques. Bradykinin, dopamine and a-MSH (the latter in both fasted normal or pigmented rabbits) increased AHFR and Cps while reducing intraocular pressure (IOP). Atropine increased AHFR and Cps while increasing IOP. Sotalol increased AHFR and Cps, while phenoxybenzamine decreased AHFR and had no effect on Cps, yet both drugs slightly increased IOP. Timolol slightly increased AHFR, but doubled Cps, with a slight fall in IOP. The steroids, prednisolone and dexamethasone both increased Cp8 and caused a slight fall in IOP while dexamethas0ne did not alter AHFR but prednisolone caused a slight increase. Data from previous in vitro studies on drug effects on isolated ciliary epithelial permeability and other in vivo studies were correlated to the present findings. The experimental data provide evidence for the complex mechanisms of action of these drugs.

Key words: aqueous humor dynamics ; rabbit; aqueous humor formation rate ; pseudofacility ; adrenergic drugs; steroids.

1. Introduction

Previous s tudies (Green and Padge t t , 1979) ind ica ted t h a t the adrenergic agonists , norepinephr ine , i soproterenol and phenylephr ine , reduced the in t r aocu la r pressure (IOP) of the no rma l eye b y va ry ing amounts . These drugs also induced changes in bo th aqueous humor fo rma t ion ra te ( A H F R ) and pseudofac i l i ty (Cps), a l though the ca lcu la ted va lue for x c [ the pressure index of the capi l lar ies (Bs 1963)] a p p e a r e d to be re la t ive ly una l te red , except by phenylephr ine . The in v ivo d a t a were c o m p a r e d to the in v i t ro effects of the drugs on the pass ive p e r m e a b i l i t y and ac t ive secret ion

of the c i l iary ep i the l ium .(Green and Griffin, 1978). Other agents known to a l te r I O P have p rev ious ly been eva lua t ed for the i r effects

on secret ion and Passive fluid p e r m e a b i l i t y of the i sola ted c i l iary ep i the l ium (Green, Griffin and Hensley, 1978). The presen t s t u d y was made to fur ther de l inea te the effects of these agents as t hey affect IOP , A H F R and Cps. The in v i t ro and in vivo effects of these drugs were cor re la ted with the i r known effects e i ther on I O P or t o t a l outf low

faci l i ty.

2. Materials and M e t h o d s

Adult albino rabbits (2-3 kg) were anesthetized with intravenous urethane (25 % w/v 0-9 ~o saline) and the anterior chamber cannulated with two 22-gauge needles after placement of the head in a stereotactic device, as described previously (Green and Padgett , 1979). One needle was connected to a transducer and also connected to a capillary tube which was used to collect samples and regulate IOP. The other needle was connected to the infusion syringe which was driven by a motorized infusion pump. The needles were placed at opposite sides of the eye with the bevels facing away from each other.

Following insertion of the needles, the steady-state IOP was determined with the stopcocks closed to the perfusion/collection system. After stabilization of the IOP, the capillary tube was adjusted to a height above the eye equal to the IOP and the stopcock opened. Minor

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240 K. GREEN AND D. ELIJAH

adjustments of • 1 mmHg to restore the IOP to the original steady-state levels were made at this time. The [3H]-inulin dilution technique of Macri (1967) was used to determine AHFR and Cps (Green and Padgett, 1979). Inulin infusion was at 0"103 ml/min for the first 15 min and 0"0206 ml/min thereafter; at this rate 4 min was required for fluid to pass from the perfusion syringe to the collection vial. Fifteen minutes after the reduction in infusion rate, during which time minor adjustments were made to equalize the outflow and inflow rates, two consecutive 15 rain samples of fluid effluent (numbers 1 and 2) were collected from the capillary tube into tared vials. The IOP was then raised by 5 mmHg, by adjusting the capillary tube and allowed to stabilize for 15 min at the new pressure. Two further consecutive 15 rain samples (numbers 3 and 4) were taken. All samples were weighed and 100 #l samples were taken of both perfusate solutions and of the perfusion solution. Any eye showing blood in the perfusate solution was discarded. Inulin samples were counted using a Nalgene filmware liquid scintillation system.

The data were analyzed according to the method of Macri (1967). Given that the inflow and outflow rates of the anterior chamber are equal then the degree of dilution of newly formed aqueous humor can be caculated as F a = F v (Cv/C a - 1 ) where F a is aqueous humor formation rate (/zl/min), Cp is the specific activity of the tJerfusate solution and C a is the specific activity of collected aqueous humor. Pseudofacflity was calculated for each series from the values obtained during the period of elevated IOP, according to the method of Macri (1967). The slope of the relationship between IOP and rate of aqueous humor formation gave the percentage change in the rate of aqueous humor production per mmHg rise in IOP. Although two samples (1 and 2) were collected at normal IOP and two collected (3 and 4) at an IOP 5 mmHg greater than the steady-state value, only collections 2 and 3 were used in subsequent calculations of AHFR and Cps. This was performed since the data indicated an average decrease from collection 1 to 2 of 7 % or 0"2/zl/min of AHFR. Should this have continued throughout collections 3 and 4 the values of Cvs would be overestimates : to reduce this to a minimum collection 2 was used for AHFR and 2 and 3 for the C~s calculation.

Drugs, and their concentrations, used were as follows: bradykinin, 10 -9 ~ perfused through the eye in the inulin solution and collections begun after 15 min of perfusion and after acute rise in IOP had subsided; ouabain, 15/tl of a solution containing 10#g/ml injected intravitreously 2 days prior to use; dexamethasone, 0-1% solution applied topically twice daily in young (8-week-old) and a 1% topical drop twice daily in older (2-3 kg) rabbits for at least 2 weeks; prednisolone was given twice daily for 7 days as a topical 1% solution; a-melanocyte stimulating hormone (10/tg/kg) given subcutaneously in fasted (29-48 hr) albino and pigmented rabbits about 1 hr prior to study; atropine, one 50 #l topical 2 % drop 30 rain before inulin perfusion; dopamine, a solution containing 5 mg of drug per 10 ml of heparinized saline was infused at 0"051 ml/min for 20 rain during the equilibration period; phenoxybenzamine, 0'5 mg/kg intravenously 30 rain before ocular perfusion; sotalol, 0"5 mg/kg intravenously 45 min before perfusion, and timolol, 0-5 %, as a 50/zl topical drop 1 hr before perfusion of the eye. Since studies on all of these drugs took approximately 8-10 months to complete, a control series was performed initially and further control series, as a check on technique, were performed at about 6 and 10 months.

3. Resu l t s

The control series in normal eyes showed an average aqueous format ion ra te of 2"83 t t l /min with only minor differences found a t different t imes, wi th similar pseudofaci l i t ies (Cps) (Table I). The d a t a indicate the reproduc ib i l i ty of the technique and the re l iab i l i ty of the drug effect de terminat ions .

The d a t a on drug effects are also given in Table I. B radyk in in grea t ly increased A H F R and Cps even a t a t ime when the I O P was below normal. Dopamine increased A H F R , and t r ip led Cps, while causing a significant fall in IOP. The effects of a -me lanocy te s t imula t ing hormone were different in a lbino and p igmented r abb i t s and in fas ted versus fed rabbi t s . In the albino, fas ted animals the effect was a sl ight increase in A H F R and a doubl ing of Cps, while in the p igmented rabb i t s both pa rame te r s

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increased greatly, together with a pupil dilation in 7 of 8 experiments. In both series, however, the result was a fall in IOP. In fed rabbits similar effects were observed in only about 50 ~o of the experiments. Atropine increased A H F R by about 30 % and doubled Cvs, despite the slight increase in IOP. Intravitreat ouabain caused a fall in IOP but no change in A H F R while Cvs doubled.

Dexamethasone in older rabbits had little effect on IOP or A H F R but caused an increase in Cvs. In young rabbits no change in IOP was found even after 3-~ weeks of treatment, save in one animal which does not allow statistical analysis. Prednisolone slightly increased A H F R and increased Cvs fivefold despite an almost negligible decrease in IOP.

Sotalol and phenoxybenzamine had almost no effect on IOP at the concentrations used in these experiments, yet each induced different effects. Sotalol increased A H F R by 30 % and doubled Cps, while phenoxybenzamine decreased A H F R and caused only a slight increase in Cps. Timolol caused a small ( + 25 %) increase in A H F R yet doubled Cw, despite a slight fall in IOP.

4. D i s c u s s i o n

The drug effects on IOP (Table I) are similar to those noted by previous authors. The present study reveals more concerning the mechanism of action of these drugs, especially when combined with in vitro studies on the permeability effects on the ciliary epithelium of those agents. Due to the experimental design it may be that drug concentrations from topical application at the time of inulin perfusion in the present experiments are reduced compared to those in the untouched eye because of anterior chamber perfusion, thus the effects may be larger than those found here. Previous determinations (Pederson and Green, 1973b) have shown that ocular perfusion with saline solutions allows the accurate determination of IOP in a cannula/transdueer system as used in the present experiments.

The observed drug-induced change in AHFR does not reflect the full drug effect, since total aqueous humor flow rate changes in response to lOP and the subsequent changes in pressure gradients (Kupfer, Gaasterland and Ross, 1971). The observed change in aqueous flow rate has been subjected to a correction factor to provide the real change (see Table I). Where lOP is changed from control values, the correction is given by (Cvs x change in IOP) ; usually the measured change in aqueous flow rate is an underestimate of the real change.

Since each drug evokes its own typical response, it is more informative to discuss each drug in relation to either previous in vitro examinations of their effects on passive fluid permeability, LpA, of the isolated ciliary epithelium (Green and Griffin, 1978; Green, Griffin and Hensley, t978; Green, Hensley and Lollis, 1979) or other indications of a change in permeability (e.g. aqueous protein concentration changes). Since Cvs =LpAx c (Pederson and Green, 1973a; B~r~ny, 1963) it is possible to obtain an estimate of the direction but not quantity, of the change in x c which is induced by drugs. Such estimates may aid in an evaluation of the site of drug action. In vitro measurements determine Lp of a fixed area of membrane, which does not represent the entire area available for filtration in the eye. Nevertheless, use of the above equation can indicate qualitative changes.

Bradykinin is known to acutely increase IOP and aqueous humor protein levels (Cole, 1974; Cole and Unger, 1974), as well as increasing the passive permeability (LpA) of the isolated ciliary epithelium (Green, Griffin and Hensley, 1978). The

DRUG EFFECTS ON AQUEOUS DYNAMICS 243

increase in A H F R and Cps, therefore, is not surprising since this reflects an increased permeabili ty of the blood-aqueous barrier, even though the present determinations were made after the acute rise in IOP, when the IOP had fallen below normal but was steady. The calculated pressure index of the capillaries, Xc, was increased, suggesting a vascular effect of the drug, a result which correlates with in vivo findings {Cole, 1974).

Dopamine decreased IOP when given intravenously (Shannon, Mead and Sears, 1976), a finding verified by the present experiments, and also increased LpA of the ciliary epithelium by about 50 % at concentrations used here (Green, Hensley and Lollis, 1979). The increased A H F R and increased Cps are counter to the observed decrease in IOP, without proposing an increase in outflow facility which has, indeed, been a documented response to this drug (Shannon, Mead and Sears, 1976).

a-MSH has been found to induce an acutely increased IOP similar to tha t following minor t rauma (Dyster-Aas and Krakau, 1964), with a concomitant increase in aqueous protein concentration. The effect on A H F R and Cps was a pronounced rise in pigmented rabbits but a quanti tat ively smaller change in these parameters in albinos; the effect was found more consistently in fasted rather than fed rabbits as noted earlier (Dyster-Aas and Krakau, 1964). The present data indicate that a breakdown of the blood-aqueous barrier occurs in pigmented and albino rabbits, as indicated by the increased Cps and AHFR. Despite the quanti tat ive disparity in responses between albino and pigmented rabbits, the IOP in both groups decreased; such an IOP fall is frequently associated with an inflammatory reaction. In pigmented animals, a-MSH appears to cause hemodynamic effects even if an increased LpA is assumed (not an unreasonable assumption since protein enters the aqueous after in vivo treatment).

Atropine has no effect on LpA of the isolated ciliary epithelium except at dose levels of 10 -~ M (Green, Griffin and Hensley, 1978) and also has little or no effect on IOP (Table I). The increase in A H F R probably reflects the passive change in Cps , which further reflects an effect on Xc, since the latter is calculated to increase, indicating a possible effect on blood flow rather than a membrane effect at the dose levels employed here.

Dcxamethasone applied daily to weanling rabbits has been repo~ted to cause an increase in IOP after 2 weeks but no effect on older rabbits (Knepper, Breen, Weinstein and Blacik, 1978). The present data in older rabbits shows no change in A H F R but a 3- to 4-fold increase in Cps although the IOP is unchanged; the drug appears, therefore, to be affecting x c (to change Cps ) and, therefore, blood flow through the anterior uvea. In young animals we found no effect after 3~ weeks of t reatment, except in one animal out of 12 which showed an increase in IOP to a maximum of 32 mmHg. This contrasts with the effect seen by Knepper et al. (1978) where consistent changes in glycosaminoglycans of the trabecular meshwork were found leading to a rise in IOP. Prednisolone had no effect on IOP although slightly increasing A H F R while Cps was quintupled (Table I), implying an effect on x c and blood flow changes (either in terms of perfused area, A, or as a vasodilator).

Intravi t real ouabain rapidly decreases the IOP (Becker, 1963) and while it decreased active secretion across the isolated ciliary body it also increased LpA by 85 %, 0"14 to 0"27/~l/mmHg/min (Green and Pederson, 1972). A H F R is unchanged and Cps doubled after in vivo treatment. The latter must reflect the increased LpA and a change in blood flow, since x c appears to be increased, indicating both a membrane and a vasoactive effect of ouabain.

The adrenergic antagonists, sotalol (/?-blocker) and phenoxybenzamine (a-blocker)

244 K. GREEN AND D. ELIJAH

induce different effects on AHFR and Cps. These agents are both known, however, to reduce lOP (Green and Kim, 1976a, b). Phenoxybenzamine reduced A H F R and had little effect on Cps, which indicates an increased Xc, since no effect on LpA was found earlier (Green, Hensley and Lollis, 1979). Sotalol, on the other hand, increased A H F R which is also accompanied by a doubling of C~8. There appears to be an increase in Xc, perhaps indicating some hemodynamic change. Timolol, a •-adrenergic antagonist, has been found to be effective in reducing IOP and is thought to suppress AHFR in man (Zimmerman and Kaufman, 1977a, b). I t has been found to reduce lOP in rabbits for 4 hr, beginning 1 hr after administration (Radius, Diamond, Pollack and Langham, 1978); the present experiments were designed to fall within this time range. Our data indicate that timolol, 0"5 % slightly increases A H F R and doubles Cps. Although timolol has not been studied thus far for an effect on LpA, it would be anticipated to have some membrane effect, since the assumption of normal LpA results in an increased x c (indicating vasodilation).

In summary, several drugs have been evaluated for their effects on A H F R and Cps in the living rabbit eye. The data indicate that few drugs act in a simple manner within the eye; rather, effects are seen on several characteristics which influence IOP. The complex behavior of drugs on the eye makes it difficult to ascribe simple functions to these drugs, which have only been revealed by a multifaceted approach using in vitro and in vivo techniques. Recently reported studies in man, for example, have indicated that epinephrine induces a complex series of events, including a decrease in IOP, with concurrent increases in aqueous humor flow and outflow facility; epinephrine was postulated to increase the rate of outflow through effects on the uveovortex outflow pathway (Townsend and Brubaker, 1980).

A C K N O W L E D G M E N T S

Supported in part by Public Health Service Resea~rch Grant EY 01329 and in part by the American Health Assistance Foundation Glaucoma Research Fund. We thank Mrs I. Prior for her valuable secretarial assistance. A Wang 2200 Computer, used for statistical data evaluation, was provided through a Research to Prevent Blindness, Inc., grant. We wish to thank Professor Ernst Bdrs for his generous help in resolving the calculations of pseudo facility.

R E F E R E N C E S

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DRUG EFFECTS ON AQUEOUS DYNAMICS 245

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