prostaglandin a2 increases uveoscleral outflow and trabecular outflow facility in the cat

Exp. Eye Res. (1995) 61, 649-657

Prostaglandin A 2 Increases Uveoscleral O u t f l o w and Trabecular O u t f l o w Facil i ty in the Cat

C A R O L B. TORIS*, M I C H A E L E. Y A B L O N S K I , Y U N - L I A N G W A N G AND M I E K O H A Y A S H I

Department of Ophthalmology, University of Nebraska Medical Center, Omaha, NE 68198-5540, U.S.A.

(Received Columbia 6 February 1995 and accepted in revised form 13 July 1995)

Prostaglandins (PG) are very effective ocular hypotensive agents. It is generally agreed that these drugs reduce intraocular pressure primarily by increasing uveoscleral outflow. They may also increase trabecular outflow facility though available evidence is less convincing. It has been hypothesized that PGs may increase facility of uveoscleral outflow in addition to their other mechanisms, but this has not ye been tested. To help clarify the ocular hypotensive mechanism of action of a derived PG of the A type cats were treated twice daily for one week with PGA 2 (0'01%) to one eye and vehicle to the other Measurements were made of aqueous flow and outflow facility with fiuorophotometry and of intraoculal pressure with pneumatonometry. From these values, uveoscleral outflow was calculated. In addition total outflow facility, uveoscleral outflow, and uveoscleral outflow facility were determined with invasiv, methods.

PGA 2 significantly reduced IOP by a mean of at least 4.7 mmHg in all experiments with all P-value r less than 0"01. Compared with contralateral vehicle-treated control eyes, uveoscleral outflow in tho treated eye was significantly (P < 0"05) increased by at least 50% using two different methods of measurement. Compared with baseline day, PGA 2 significantly (P E 0.05) increased aqueous flow by l ' 8 # l m i n -1, fluorophotometric outflow facility by 0 . 3 6 / d m i n l mmHg 1 and fluorophotometric uveoscleral outflow by 2.0 #1 min 1. Total outflow facility was not significantly different comparing treated with contralateral control eyes. Facility of uveoscleral outflow was ~< 0 '02 /d rain 1 mmHg -1 for both control and treated eyes.

It is concluded that PGA 2 decreases IOP in cats by increasing uveoscleral outflow and trabecular outflow facility as measured with fluorophotometry. A significant increase in aqueous flow reduces the ocular hypotensive effect. © 1995 Academic Press Limited

Key words: prostaglandins; cats; aqueous; uveoscleral; trabecular ; outflow; ocular: pressure; facility.

1. Introduction

Pros tag land ins (PGs) are a pa r t i cu la r ly exci t ing class of ocu la r hypotens ive drug because they signif icant ly lower i n t r aocu la r pressure (IOP) at very small concen- t ra t ions wi th few side-effects in a var ie ty of m a m m a l s (Bito et al., 1 9 8 9 : Camras , 1995) . The p r imary and least amb iguous m e c h a n i s m of ac t ion of PGs is an increase in uveosclera l outf low wh ich has been found across species in monkeys , rabbi ts and h u m a n s (Kaufman and Crawford, 1 9 8 9 ; Gabelt and Kaufman, 1 9 8 9 : Nilsson et al., 1 9 8 9 : Poyer. Gabelt and Kaufman, 1 9 9 2 ; Toris, Camras and Yablonski, 1993). Less s t r a igh t fo rward are the effects of PGs on total outf low facility and t r abecu la r outf low facility. For these pa ramete rs , results r ange from no effect to signif icant increases (Hayashi , Yablonski and Bito, 1987 ; Kaufman et al., 1 9 8 9 ; Gabelt and Kaufman, 1 9 9 0 : Poyer et al., 1992). One possible exp lana t ion for an increase in to ta l outf low facility following PG t r e a t m e n t m a y be an increase in uveosclera l outflow facility wh ich is inc luded in the m e a s u r e m e n t (Kaufman, 1992) .

* For correspondence at: Department of Ophthalmology, 600 South 42nd Street, University of Nebraska Medical Center, Omaha, NE 68198-5540, U.S.A.

0 0 1 4 4 8 3 5 / 9 5 / 1 2 0 6 4 9 + 0 9 $12.00/0

The present s tudy was designed to clarify the ocula r hypotens ive m e c h a n i s m of ac t ion of PGA 2 inc luding its effects on uveosclera l outf low facility. In addit ion, the s tudy compares invasive versus non invas ive me thods of m e a s u r i n g uveosclera l outflow and t o t a l / t r abecu la r outf low facility. Cats were chosen over o ther m a m m a l s because of thei r large eyes, thei r res is tance to b l o o d - a q u e o u s barr ier b r e a k d o w n (relative to rabbits) and thei r ease of hand l ing and cost (relative to pr imates) . PGAa is a more effective ocula r hypotens ive agen t in cats then either PGE 2 or PGF2= (Bito, Baroody and Miranda , 1987) so PGA 2 t r e a t m e n t was used in all studies described herein.

2. Materials and Methods

Adul t cats of e i ther sex weighing 2 -5 -4 .0 kg were used in these studies. All an ima l exper iments described here in were approved by the Ins t i tu t ional An ima l Care and Use Commit tee of the Universi ty of Nebraska Medical Center.

Fluorophotometry

Cornea and anterior chamber volumes. Mathemat i ca l ca lcu la t ion of the cornea and anter ior c h a m b e r

© 1995 Academic Press Limited

650 C. B. TORIS ET AL.

TABLE I

Measurements and abbreviations

Az Acetazolamide d Anterior chamber depth Cr~ Trabecular outflow facility determined with

fluorophotometry : Cf~ = Ctrab ~ f lu Ct . Uveoscleral outflow facility C~ Pseudofacility Cto t Total outflow facility: Cto I = Ct,.ab~-Cfu-~Cps C,r~b Trabecular outflow facility Fa Aqueous flow Fa' Aqueous flow following acetazolamide

administration Fur~ Uveoscleral outflow determined with

fluorophotometry Fu,,, Uveoscleral outflow determined with intracameral

infusion of FlTC-dextran IOP Intraocular pressure IOP' Intraocular pressure following acetazolamide

administration PG Prostaglandin Pv Intrascleral venous pressure t Cornea thickness V~,. Volume of the anterior chamber V~ Volume of a segment of a sphere bordered by the

anterior surface of the cornea and an imaginary plane across the anterior surface of the lens

E~,2 Volume of a segment of a sphere bordered by the posterior surface of the cornea and an imaginary plane across the anterior surface of the lens

V,. Volume of the cornea y Cornea diameter

volumes were made in each cat using the following equations.

V.~,~ = n / 6 ( d + t ) [ ( d + t ) 2 + 3 / 4 f l (1)

V~s2 = n / 6 d 2 [ 3 / 4 y 2 / ( d + t ) + d - 3t] (2)

g. = va.~,- v~.~ (3 )

Vae ~- V.as 2 X 1"8 ( 4 )

where y = cornea diameter, t = cornea thickness, d = anterior chamber depth, Vas ~ = volume of a segment of a sphere bordered by the anterior surface of the cornea and an imaginary plane at the anterior surface of the lens, V~s2 = volume of a segment of a sphere bordered by the posterior surface of the cornea and an imaginary plane at the anterior surface of the lens, V~. = volume of the cornea, V~c = volume of the anterior chamber. These and all other abbreviations are listed in Table I.

Cornea diameter was measured in the nasotemporal plane of the eye with calipers. To confirm the accuracy of the measurement, four eyes were enucleated, bisected at the equator and y was measured at both the inner and outer limbal surfaces using calipers and a dissecting microscope. The resulting measurements were virtually identical to those measured in sltu, hence y was judged to be accurate.

Central cornea thickness and anterior chamber axial depth were measured with Haag-Streit pachy-

meters. These measurements used a corneal radius of curvature of 8'8 based on the average value of 8-8 + 0"3 mm (mean + S.E.M.) determined in ten eyes of five cats by measurement with a keratometer.

The calculated cornea volume [eqns (1)-(3)] was checked for accuracy by comparison with a direct method of measurement. Eight corneas from the globes of four cats were submerged in phosphate buffered saline (0"0IM, pH7 '4) in a graduated cylinder. The total displacement of the buffer by all eight corneas was measured and divided by the number of corneas. The cornea volumes using this method averaged 188 /d (n = 8), compared with 165/~1 using eqns (1)-(3) (n = 44, data pooled from several studies). The mathematically determined volume may be slightly underestimated because the assumption was made that the cornea is of uniform thickness, whereas in actuality it is thicker near the limbus. Despite this, the mathematically calculated cornea volume was used in this study because individual measurements could be obtained.

The equation for V~ is the product of the volume of a segment of a sphere times 1-8. The latter factor takes into account the bowing of the iridial boundary of the anterior chamber in the far periphery in the cat. This was estimated to be about 45% of total aqueous volume based on gross and microscopic examination. This calculation was tested for accuracy by comparison with the effective anterior chamber volume determined with a tracer dilution technique (Bill, 1966a). In six cats, two 23 gauge needles were inserted into the anterior chamber through the cornea, one at the temporal and the other at the nasal limbus. Each needle was attached to PE-50 tubing and a 1 cm 3 syringe connected to an infusion-withdrawal system which coupled both syringes. This enabled simultaneous infusion and withdrawal of small volumes of fluid. Fluorescein isothiocyanate dextran (FITC- dextran, 70 kDa; 10-15/zl of 1 × 10 ~ M) was injected through one needle into the anterior chamber. After mixing the contents of the anterior chamber for 1 min with the infusion and withdrawal system, a small volume of aqueous was collected and its fluorescence along with the fluorescence of the perfusate were measured with a Fluorotron Master fluorophotometer (Coherent, Palo Alto, CA, U.S.A.). The volume of fluid required to dilute the perfusate to the new concentration approximates the original volume of the anterior chamber. The mean volume with the dye dilution technique was 820_+ 137 #l (mean _+ s.D.) compared with 8 0 5 + 8 7 calculated with eqn (4). Because individual cats vary greatly in anterior chamber volume, the individual mathematically calculated volumes were used in this study.

Intraocular pressure. All IOP measurements were made with a pneumatonometer (Digilab Modular One, BioRad, Santa Anna, CA, U.S.A.) which was checked before each use with a calibration verifier supplied

PGA2 AND AQUEOUS HUMOR DYNAMICS

with the instrument. The tip and membrane of the pneumatonometer were cleaned and adjusted until the reading was within 1 mmHg of the standard 25 mmHg pressure. Acceptable readings were those in which the standard deviation was less than 1 mmHg.

Fluorophot.ometric determination of aqueous flow (Fa), outflow facility (Ciz) and uveoscleral outflow (Fuji). Into each eye of 11 cats, ten drops of 2% fluorescein sodium solution were instilled one at a time, at 5 min intervals starting at around 2000 hr. Twelve hours later, cats were anesthetized with subcutaneous xylazine, (2 mg kg -1) and intramuscular ketamine (10 mg kg !). Supplemental doses of ketamine (5 mg kg 1) were given as needed to maintain a light level of anesthesia throughout the day (four or five additional injections).

Fluorophotometric scans of the cornea and anterior chamber were made in duplicate at one hour intervals for 4 hr. Duplicate scans in each eye were averaged for each time period. From t h e rate of decay of fluorescence in the anterior chamber and cornea, baseline aqueous flow (Fa) was determined (Hayashi, Yablonski and Mindel, 1990; Yablonski et al., 1978).

Immediately following the fourth set of fluorophotometric scans, IOP was measured and then acetazolamide, 20 mg kg -1, was injected intravenously through the cephalic vein. Acetazolamide decreases IOP by reducing aqueous flow thus enabling calculation of fiuorophotometric outflow facility [Cf~; eqn (5) below]. After a 1 hr wait, fluorophotometric measurements were resumed at 45 min intervals until three additional sets of duplicate scans were collected (2-25 hr). The aqueous flow during the last 45 min period after the administration of acetazolamide was designated Fa'. Within 3 min of the last fluorophotometric measurement, IOP was measured again (lOP'). Cfl was calculated by dividing the change in aqueous flow by the corresponding change in IOP after the administration of acetazolamide (Yablonski et al., 1987)

Cr, = (Fa--Fa ' ) / ( IOP--IOP' ) (5)

Uveoscleral ou t fow (FUrl) was obtained with the equation (Yablonski et al., 1987)

Fu z = Fa -- Ct~(IOP-- Pv) (6)

where Pv is the pressure in the aqueous drainage vessels (intrascleral veins). This pressure was estimated using a equation describing the relationship between Pv and IOP in the cat (Bill, 1963)

Pv = ( lOP- 9.4)/1-66 (7)

With a mean IOP of 17 mmHg in all control eyes, Pv was calculated to be 5 mmHg. This value was used for the Pv of all cats both before and after PGA 2 treatment.

Following baseline measurements, one randomly assigned eye was treated twice daily for 1 week and on

651

the morning of the eighth day with one drop of PGA 2 made up in ethanol (Sigma Chemical Co., St. Louis, MO, U.S.A.) and diluted to 0 '01% with vehicle (balanced salt solution, BSS). The fellow eye was similarly treated with vehicle. Fluorescein was administered at 2 0 0 0 h r on day 7 and fluorophotometry was repeated starting 1 hr after the final PGA 2 dose on day 8. Values for lOP, Cfl, FUrl, and Fa in treated eyes were compared with contralateral control eyes and with baseline day using a two-tailed paired t-test. With a two-sided test of significance at a significance level of 0.05, our sample size of 11 gives a probability of 0-80 for detecting a 25 % change in Fa, an 80% change in C~.1 and a doubling of Fuf~.

Invasive Measurements

Total outflow facility (Cto,). Eight of the 11 cats from the fluorophotometry studies were used for this measurement. One month or more after fluorophotometry, again one randomly assigned eye was treated with PGA 2 twice a day for a week and on the morning of the eighth day. The fellow eye was similarly treated with vehicle. One hour after the last dose, Ctot was measured with a two-level constant pressure infusion method (B~irany, 1964) under ketamine and xylazine anesthesia, The anterior chambers were perfused with a solution of Normosol- R (pH 7.4, containing heparin 1Uml-1 ; Abbot Laboratories, North Chicago, IL, U.S.A.) at either 2.5 or 10-0 mmHg above the spontaneous lOP. After a 10 min stabilization period, the rate of flow of perfusate required to maintain the predetermined IOP, was measured. Flow was the average of three consecutive 4 min readings. Cto , was the ratio of the differences in flow at each of the two pressures divided by the pressure difference (7"5 mmHg). Treated eyes were compared with contralateral control eyes with a two- tailed paired t-test. With a significance level of 0.05, our sample size of eight, gives a probability of 0"80 for detecting a 55 % increase in Cto t.

Uveoscleral outflow (Fui,,,). The techniques described below are modifed from a previously reported method (Suguro, Toris and Pederson, 1985).

(a) PG treated cats. Ten cats were used for this procedure, four from the fluorophotometry and Cto t

studies, and six which were previously untreated and untested. Two to three months after any previous study, the same eye previously treated with PGA 2 was treated again twice daily for 3 days and on the morning of the fourth day and the fellow eye was similarly treated with vehicle. Remodeling of the extracellular matrix of the ciliary body occurs by 4 days of PGF2~ treatment to monkeys (Lfitjen-Drecoll and Tamm, 1988), so by 4 days of PGA 2 treatment to our cats, a steady-state condition was assumed. Eyes from formerly untested cats were randomly assigned to PGA 2 or vehicle treatment.


TABLE II

lOP, fluorophotometricall~t determined aqueous flow, trabecular outflow facility and uveoscleral outflow before and after PGA 2 treatment

IOP Fa C n Furl Day Eye (mmHg) (,ul min -~) (/d min -t mmHg 1) (#1 min 1)

Baseline Control 14.0 + 3"5 6'0 + 1"4 0'48 + 0-36 2"1 + 2"2 Treated 13.5 + 3"4 5"0 _+ 1' 7 0- 39 ± 0.21 2.0 + 1.5

After PGA 2 Control 12.9 + 2.5 uS" 7 ± 0-9 0.49 ± 0.44 1.9 + 2.5 Treated 8.8 ± 2'6* 6-8 ± 1"8"[" 0" 75 ± 0-545 4.0 ± 1-6§

Values are mean+s.D. , n = 11. One eye (Treated) was treated twice daily for 1 week and on the morning of the eighth day with O.01% PGA~, and the fellow eye (Control)

was similarly treated with vehicle, lOP. intraocular pressure ; Fa. aqueous flow; Cfr fluorophotometric outflow facility; Furl, fluorophotometric uveoscleral outflow.

All comparisons were made using Student 's two-tailed paired t-test. * P < 0.OO1 compared with baseline day and P < 0-02 compared with contralateral control eye.

P = 0"05 compared with baseline day. :[: P < 0"05 compared with baseline day. § P ~< 0.05 compared with both baseline day and contralateral control eye.

One hour after the final dose, animals were anesthetized and both anterior chambers were cannu- lated with a 22 gauge needle connected with PE-50 tubing to a strain gauge pressure transducer and a reservoir containing 10 ~ M FITC-dextran in Normosol-R. The reservoir was set at a height equivalent to 18 to 20 mmHg pressure as measured by the pressure transducer. A second cannula was placed in the anterior chamber to drain the anterior chamber fluid. This fluid was collected for the last 25 min and saved. The eyes were perfused with the FITC-dextran solution for 30 minutes, after which the cats were killed by an overdose of intravenous sodium pento- barbital. The eyes were immediately enucleated, the corneas removed and the surface of the iris and chamber angle were thoroughly washed with phosphate buffered saline (0.01 M, pH 7"4) to remove any intracameral tracer. The eyes were then transferred to a clean Petri dish and dissected into iris, anterior uvea, posterior urea, anterior sclera, posterior sclera, retina and extraocular tissue. The dissected tissues were homogenized and centrifuged in a standard volume of buffer, and the tracer concentrations of the super- natant were measured with the fluorophotometer. The tracer concentration from the aqueous collected for 25 min was also measured.

Uveoscleral outflow was calculated by dividing the sum of all the tissue tracer values, expressed as equivalent volumes of aqueous, by the infusion time. Treated eyes were compared with control eyes with a two-tailed paired t-test. With a sample size of 10, this method can detec~ a 45% change in Fuin,. at a significance level of 0.05 and power of 0.80.

(b) Acetazolamide treated cats. Because acetazol- am±de was used in the fluorophotometric studies to calculate C. and estimate uveoscleral outflow, it was important to verify that this drug, by itself, had no effects on uveoscleral outflow. Under anesthesia, five cats were pretreated intravenously with 20 mg kg -1 of

acetazolamide 1 hr before intracameral FITC-dextran infusion. Uveoscleral outflow was measured in all ten eyes (as described above for Fuin,. ). Similar measurements were made in seven eyes from four untreated cats to serve as controls. Differences between the means were tested for significance with random effects analysis of variance.

Facility of uveoscleral outflow ( C~,). In 11 previously untested cats, both eyes were treated with PGA 2 (0-01%) twice a day for 3 days and on the morning of the fourth day, 1 hr before tracer infusion into the anterior chamber. Both eyes were perfused with FITC- dextran for 30min, one at 10 and the other at 20 mmHg pressure. FUin ,, w a s calculated and values were compared in fellow eyes with a two-tailed paired t-test.

In a separate group of six previously untested cats, one eye was treated with PGA 2 twice a day for 3 days and on the morning of the fourth day. One hour later, in three of these animals, FITC-dextran was infused into the anterior chamber of both eyes at 10 mmHg pressure while both eyes of the remaining three animals were infused at 20 mmHg pressure. The eyes were dissected and FUin ,. w a s calculated. Values in the low pressure infusion group were compared with the high pressure infusion group with unpaired t-test.

3. Results

All values are reported as mean+s.D, unless otherwise noted.

The results of the fluorophotometric studies are summarized in Table lI. Four hours after the final dose of PGA 2, IOP was significantly (P < 0-001) decreased in treated eyes compared with both baseline day (4 .7+3.2) and contralateral control eyes (4.0 +3-9 mmHg). Aqueous flow was significantly (P = 0-05) increased by 36% in treated eyes compared

PGA2AND AQUEOUS HUMOR DYNAMICS

TABLE II I

Intraocular pressure and aqueous flow before and after systemic acetazolamide

653

Intraocular pressure (mmHg) Aqueous flow (/d m i n -1)

Day Eye Before AZ After AZ P* Before AZ After A2 P*

Baseline Con~ol 14'0±3.5 9.7±3.1 0.0001 6.0±1.4 4.4±1.3 0.005 Treated 13-5±3'4 9.5±2"7 0"0004 5.0±1-7 3"7±1"5 0.01

AfterPGA~ Control 12.9±2-5 7'8±1"2 0.0002 5"7±0"9 3.2±1"7 0'003 Treated 8"8±2'6 6.4±1"4 0"004 6.8±1"8 4.1±2'1 0"0003

Values are mean±s .D, n = 11. One eye (Treated) was treated twice daily for one week and on the morning of the eighth day with 0.01% PGA 2, and the fellow eye (Control)

was similarly treated with vehicle. Intraocular pressure and aqueous flow measurements were taken immediately before and 2.3 hr after administration of acetazolamide (AZ).

* Probability for difference between before and after AZ treatment based on Student's two-tailed paired t-test.

TABLE IV

Intraocular pressure and invasively determined uveoscleral outflow in PGA2-treated and control eyes

Intraocular pressure Uveoscleral outflow Eye (mmHg) (#1 min 1)

Control 19.7+ 5"7 1"5 + 0.6 PGA 2 13.1 ± 5'0* 2.3 + 1.1"

Values are mean±s.o . , n = lO. One eye (PGA2) was treated twice daily for 3 days and on the

morning of the fourth day with 0.01% PGA 2, and the fellow eye (Control) was similarly treated with vehicle.

*P < 0.005 compared with control eye; (Student's two-tailed, paired t-test).

TABLE V

Total outflow facility and intraocular pressure in PGA 2- treated and control eyes

Intraocular Total outflow pressure facility

Eye (mmHg) (/~1 min 1 mrnHg 1)

Control 18-5 + 6-4 1"2 + 1.1 PGA~ 10"9 + 6,5* 1.6 ± 1'0

Values represent mean±s.D., n = 8. One eye (PGA.2) was treated twice daily for one week and on the

morning of the eighth day with 0.01% PGA 2, and the fellow eye (Control) was similarly treated with vehicle. Total outflow facility was measured with a two-level constant pressure infusion method.

*P < 0.01 compared with control eye (Student's two-tailed, paired t-test).

with baseline day but not with contralateral control eyes. Uveoscleral outflow was significantly (P ~< 0.05) increased two-fold in treated eyes compared with contralateral control eyes and with baseline values. Fluorophotometric outflow facility was significantly (P < 0"05) increased by 0"36 #1 min -1 mmHg -1, or almost two-fold, in treated eyes compared with baseline day but not with contralateral control eyes.

The effectiveness of acetazolamide at reducing IOP and aqueous flow [to calculate C, with eqn (5)] is

TABLE VI

Uveoscleral outflow at two different infusion pressures in PGA2-treated eyes

Infusion lOP before Uveoscleral pressure infusion outflow (mmHg) (mmHg) (/d min 1)

10 10"3±3'0 0"9±0'9 20 10"6±2"5 1"1±0"9

Values represent mean + S . D . , n = 11. Cats were pretreated bilaterally twice daily for 3 days and on the

morning of the fourth day with 0"01% PGA. 2.

summarized in Table III. Acetazolamide significantly (P < 0-02) reduced IOP and Fa by a mean of at least 2 . 4 _ 2-1 mmHg and 1-3 ± 1.4/A min -1, respectively, in all comparisons.

Using invasive methods, uveoscleral outflow was significantly (P < 0.005) increased 50% in treated eyes compared with contralateral control eyes 1 hr after the final dose of PGA 2 to one eye (Table IV).

Total outflow facility measured with two-level constant pressure infusion, was not significantly different in treated eyes compared with control eyes measured 1 hr after the final dose of PGA2 to one eye (Table V).

Following bilateral t reatment with PGA 2, the mean uveoscleral outflow in eyes perfused with tracer at 20 mmHg was not significantly different from that in contralateral eyes perfused at 10 mmHg (Table VI). Cfu was calculated to be 0.02 #1 min 1 mmHg-L

Following unilateral t reatment with PGA v the mean uveoscleral outflow in PGA~-treated eyes perfused with tracer at 10 mmHg pressure was not significantly different from the mean uveoscleral outflow in similarly treated eyes infused at 20 mmHg pressure nor in contralateral vehicle-treated control eyes infused either at pressures of 10 or 20 mmHg (Table VII). The uveoscleral outflow of the treated eye at both infusion pressures was higher than the untreated eye; however, the sample size was too small for

654 C. B. TORIS E T A L .

TABLE VII

Uveoscleral outflow at two different infusion pressures in PGA2-treated and control eyes

10 mmHg infusion pressure 20 mmHg infusion pressure

IOP before infusion Uveoscleral outflow IOP before infusion Uveoscleral outflow Eye (mmHg) (/d min 1) (mmHg) (/d rnin -1)

Control 18.1 + 6" 3 1-4 _+ 0"8 20" 5 _+ 6.1 1.4 + O. 3 PGA. 2 10"6 + 1"4 2'3 + 1"2 13"1 + 4'4 2"2 + 0"6

Values represent mean_+ S.D., n = 3. One eye (PGA2) was treated twice daily for 3 days and on the morning of the fourth day with 0.01% PGA 2. and the fellow eye (Control)

was similarly treated with vehicle.

statistical significance. A larger sample size does show a statistically significant PGA2-induced increase in uveoscleral outflow magnitude (Table IV).

Mean uveoscleral outflow in cats pretreated with intravenous acetazolamide (1.8 + 0-2 /A min 1) w a s the same as that of untreated cats (1.8 + 0-8/d min -1, mean + S.E.M.).

4. Discussion

The experiments reported here demonstrate that the primary mechanism by which 1 week of twice daily PGA 2 treatment reduces IOP in cats is an increase in uveoscleral outflow. This is consistent with studies in which humans, monkeys and rabbits were treated with PGF2~, its analogs or other prostaglandins for varying lengths of time (Kaufman et al., 1989; Nilsson et al., 1989: Gabelt et al., 1989; Poyer et al., 1992; Toris et al., 1993). An increase in uveoscleral outflow in prostaglandin-treated cats had not been reported previously, however, one study in cats treated with either PGA 2 or PGF2~ suggested such a mechanism (Hayashi et al., 1987).

Prostaglandins are the first ocular hypotensive agents found to function primarily by increasing uveoscleral outflow (Kaufman et al., 1989; Gabelt et al., 1989; Nilsson et al., 1989; Poyer et al., 1992: Toris et al., 1993). Other agents such as ~-adrenergic agonists (Serle et al., 1991 : Wang et al., 1993 ; Toris, Gleason et al., 1995) and ~l-adrenergic antagonists (Wang et al., 1992: 2han et al., 1993) have also been found to increase uveoscleral outflow. These findings stress the importance of measuring uveoscleral outflow in studying the actions of pharmacological agents which may be used to treat glaucoma.

Two methods were employed in this study to assess uveoscleral outflow. The standard tracer infusion method (Fu.l,.) has been used for decades and measures uveoscleral outflow directly. However, it is invasive, precluding its use in humans. In our hands, it is sufficiently sensitive to detect a 45% increase in uveoscleral outflow with our sample size and statistical analysis criteria. The fluorophotometric method (Fun) was developed more recently and is a mathematic calculation making it an indirect determination. This

technique has been used to evaluate glaucoma treatments in humans (Yablonski, Cook and Gray, 1985; Hayashi, Yablonski and Novak, 1989; Toris et al., 1993; Toris, Tafoya et al., 1995; Toris, Gleason et al., 1995). This method is advantageous in that it is non-invasive, repeatable and allows for measurement of other parameters of aqueous humor dynamics in the same eye at the same time. Because Fur, is calculated using several terms each with their own variabilities, predictably large standard deviations are noted. The coefficient of variation for fluorophotometric uveoscleral outflow is 127% (compared with 51% for uveoscleral outflow determined with tracers) which means it is much less sensitive than Fui., and only large differences in Fu. will be detectable with our selected sample size. The sample size was chosen to minimize animal numbers and because only large changes in uveoscleral outflow are likely to be of clinical significance. Despite the two fundamentally different ways to access uveoscleral outflow in this study, the means were similar, and both methods detected a significant increase in uveoscleral outflow following PGA 2 treatment.

The calculation of fluorophotometric uveoscleral outflow requires the determination of intrascleral venous pressure as well as aqueous flow, fluorophotometric outflow facility and IOP [eqn (6)]. In the cat the intrascleral veins drain aqueous humor instead of the episcleral veins as found in primates (Bill, 1962). Unlike episcleral veins, the intrascleral veins are not accessible to noninvasive measurement (venomanometry) and were not measured in our study, Instead, we utilized an equation describing the relationship between IOP and intrascleral venous pressure in the cat (Bill, 1963). We calculated a mean intrascleral venous pressure in control conditions and then made the assumption that the pressure in the aqueous drainage vessels was not changed following PGA 2 treatment, This assumption is based on a study in cats in which no change in episcleral venous pressure was reported after PGA 2 treatment (Hayashi et al., 1987). It is reasonable to assume that intrascleral venous pressure is also unaffected by such treatment. Therefore, the same value for intrascleral venous pressure was used for all calculations of

PGA2AND AQUEOUS HUMOR D Y N A M I C S

fluorophotometric uveoscleral outflow regardless of IOP or drug treatment.

Uveoscleral outflow in the present study was determined invasively in several separate experiments with mean values ranging from 0-9 to 2-3 #1 min ~ in treated eyes. The reason for this variability is not known. Possibilities include animal age, size of globe and month of measurement. None of these variables was controlled for in this study. One or several of these variables may help to explain the differences in uveoscleral outflow from that reported previously in cat (Bill, 1966a).

Following 1 week of PGA 2 treatment to our cats, aqueous flow was 36% (1.8/zl min -~) higher in the treated eye compared with baseline day (P = 0.05). This increase is approximately equal in magnitude to the observed increase in uveoscleral outflow and if it were not for the concomitant increase in fluorophotometric outflow facility, no IOP reduction would have been observed. An increase in aqueous flow has also been reported in cats treated with PGF2~ (Hayashi et al., 1987). In that study as in ours, there was no significant difference in aqueous flow between treated eyes and contralateral control eyes. In the isolated rabbit iris-ciliary body, PGF2~ increased short- circuit current in the presence of HCQ- which could be interpreted to mean that PGF2~ increases aqueous humor secretion (Chu, Candia and Iizuka, 1986). Several in vivo studies in rabbits, monkeys and humans, have reported that prostaglandins either increase or have no effect on aqueous flow (cf. Kaufman et al., 1989; Camras, 1995).

The measurement of facility of outflow through the trabecular meshwork is difficult to accomplish. The most widely used techniques, tonography, two-level constant rate infusion and two-level constant pressure infusion (Bill, 1977) measure total outflow facility (Ctot) which includes pseudofacility (Cps) and uveoscleral outflow facility (Cfu) as well as trabecular outflow facility (Ctrab) (Kaufman et al., 1985)

C, ot = C~rab + Cf, + C~ (8)

Recently, two studies have measured the effect of prostaglandins on trabecular outflow facility by deter- mining the amount of an intracameral tracer reaching the systemic circulation (Gabelt et al., 1990; Poyer et al., 1992). The fluorophotometric technique of the current study measures outflow facility which equals trabecular outflow facility plus uveoscleral outflow facility

Cf, = Ct,,,, b + Cf,, (9)

but does not include pseudofacility. Pseudofacility is not a component of Crx since the change in aqueous flow needed to make the determination is directly measured and not estimated, as is the case with tonography and the two-level constant-pressure infusion methods. Thus any changes in pseudofacility which would result in a change in aqueous flow are

655

negated because the change in aqueous flow is measured directly by fluorophotometry. Uveoscleral outflow facility is included in the determination because the decrease in aqueous flow (from acetazolamide treatment) might affect flow of aqueous humor through both the trabecular and the uveoscleral outflow routes. Therefore, this measurement is called ' fluorophotometric outflow facility' instead of ' trabecular outflow facility'.

Uveoscleral outflow facility is small in monkeys (about 0'02 F lmin 1 mmHg-l; Bill, 1966b) and in cats (current study). This would contribute less than 5% to fluorophotometric out fow facility. Hence, fluorophotometric outflow facility should be a reasonable estimation of trabecular outflow facility as long as uveoscleral outflow facility is negligible in comparison.

It has been hypothesized that prostaglandins may alter uveoscleral outflow facility (Kaufman, 1992). Prostaglandins appear to modify the interstitial tissue of the ciliary body (Lfitjen-Drecoll et al., 1988) which could theoretically increase the facility for outflow through this tissue (Kaufman, 1992). An increase in CN as a result of an increase in Cr, may be incorrectly interpreted as an increase in trabecular outflow facility [see eqn (9)]. Because this is an important issue, separate studies were conducted to evaluate the effect of PGA 2 on uveoscleral outflow facility alone under controlled IOP conditions (Tables VI and VII). Changes in IOP minimally affected the uveoscleral drainage rate in eyes treated with either PGA 2 or vehicle indicating that PGA,, does not affect uveoscleral outflow facility in cats. Therefore, the increase in fluorophotometric outflow facility that was found in the treated eyes compared with baseline measurements can be interpreted as an increase in trabecular outflow facility.

The baseline values of fluorophotometric outflow facility in cats (0 .38-0-48/d min-1 mmHg 1) are similar to trabecular outflow facility values in monkeys ( 0 . 3 5 # l m i n - l m m H g 1; Gabelt et al., 1990) and rabbits (0"34/d rain 1 mmHg-X: Poyer et al., 1992), yet are substantially smaller than total outflow facility values in cats (1.1-1.6/t l min -1 mmHg, Bill, 1966a; Hayashi et al., 1987; current study). The large differences between fluorophotometric outflow facility and total outflow facility in cats may be explained by a large pseudofacility [eqn (8) minus eqn (9) yielding eqn (10) below]

f r o t - - e l l = Cos ( 1 0 )

Another explanation for the large differences is a substantial washout phenomenon found in the two- level constant pressure infusion method to determine Cto t (Erickson and Kaufman, 1981; Kaufman, True- Gabelt and Erickson-Lamy, 1988).

In the current study, both methods of measuring outflow facility detected increases of similar magnitude after PGA 2 treatment. The increase of 0.36 ,ul min 1

mmHg 1 using the fluorophotometric method (C~1)


represented a 9 0 % change which was s tat is t ical ly significant. The increase of 0 . 4 0 / d m i n i m m H g 1 (C, ot) us ing the t racer me thod represented a 33% change wh ich was not significant. It is possible tha t the change in C,o t is real bu t the power of the test was not sufficient to detect it. This m a y be an example of a type II s tat is t ical error.

To ca lcula te Crl [eqn (5)], aqueous flow and IOP were decreased wi th in t ravenous ace tazo lamide (Table [II). The ca lcu la t ion is based on the a s sumpt ion tha t ace tazolamide does not affect uveosclera l outflow, t r abecu la r outf low facility and in t rasc lera l venous pressure. This is true for t r abecu la r outf low facility (Kupfer, 1973) and uveosclera l outf low (current s tudy) but not necessar i ly for in t rasc lera l venous pressure. An ear ly s tudy in cats repor ted an ace tazolamide- induced increase in the pressure in the in t rasc lera l veins and a reduced pressure head for aqueous d ra inage (Bill, 1963) . In this event, fluoro- pho tomet r ic uveoscleral outf low (Fu,) repor ted in the cu r ren t s tudy would be underes t imated . However , uveosclera l outf low measu red wi th invasive pro- cedures (FUin,) wi thout the need for ace tazolamide adminis t ra t ion , is actual ly smal ler t h a n FUrl, ind ica t ing tha t the effect of acetazolamide on in t rasc le ra l venous pressure does not induce a signif icant e r ror in the ca lcu la t ion of Fu , . In addi t ion to acetazolamide, t imolol was tr ied as ano ther pha rmaco log ica l m e a n s to the same end bu t it did not re l iably reduce IOP in cats (da ta not shown).

In conclusion, PGA 2 t r e a t m e n t in cats causes a reduc t ion in IOP associated wi th an increase in uveosclera l outf low and t r abecu la r outf low facility. Uveoscleral outf low facility is no t altered. A signif icant increase in aqueous flow s o m e w h a t d iminishes the ocu la r hypotens ive effect.

Acknowledgements

Technical assistance was provided by Brandon Madson and Doris Velasquez.

Supported by NIH grant EYO7836 (Dr Yablonski), Gifford Laboratory funds and an unrestricted grant from Research to Prevent Blindness.

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