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36 Inocstigatioc Ophthalmology January 1975 Reports terior or iris processes and in the posterior ciliary processes. Discussion. Horseradish peroxidase is an acceptable tracer for plasma protein since it retains its molecular weight of about -40,000 daltons, corresponding to a molecular diameter of 45 A, after it is injected intravenously into laboratory animals.7 The results of our experiments, then, support the hypothesis, either directly or indirectly, that prostaglandin E.: causes changes in the tight junctions that seal the lateral intercellular clefts of the noiipigineiited epithelium of the ciliary processes in the rabbit eye."' We believe that the passage of tracer protein demonstrated in this study indicates that in response to prostaglandin E.., plasma proteins puss from the stroma of the ciliary processes into the posterior chamber via the intercellular clefts of the nonpigmeiited epithelium. Ellorts are in progress to determine whether the change in patency of the tight junctions is a direct action of prostaglandins or merely a result of increased hydrostatic pressure within the ciliary processes. While this manuscript was in press, Tamuras suggested that in response to prostaglandins, increased transport of Thorotrast occurred across the nonpigmeiited epithelium by membrane-bound vesicles and indicated this route for the entry ol protein into the posterior chamber. In our studies with peroxidase, a smaller molecule than Thorotrast, an insignificant number of peioxidase-containing vesicles was observed. We thank Barbara Brown, Eva Eugenes, and Robyn Rufner for technical assistance. From the Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Conn. This work was supported in part by United States Public Health Service Grants Nos. EY-00237 and EY-00785 and the John A. Hartford Foundation, Inc. Submitted for publication June 17, 1974. Reprint requests: Dr. A. H. Neufeld, Yale University School of Medicine, 333 Cedar St., New Haven, Conn. 06510. Key words: ciliary body, blood-aqueous barrier, prostaglandins, rabbit, horseradish peroxidase. REFERENCES 1. Shiose, Y.: Electron microscopic studies on blood-retinal and blood-aqueous barriers, Jap. ). Ophthalmol. 14: 73, 1970. 2. Vegge, T.: An epithelial blood-aqueous barrier to horseradish peroxidase in the ciliary processes of the vervet monkey (Ccrcopithccus aethiops), Z. Zellforsch. 144: 309, 1971. 3. Uusitalo, R., Palkama, A., and Stjernschantz, |.: An electron microscopical study of the blood-aqueous barrier in the ciliary body and iris of the rabbit, Exp. Eye Res. 17: 49, 1973. 4. Neufeld, A. H., Jampol, L. M., and Sears, M. L.: Aspirin prevents the disruption of the Downloaded From: http://iovs.arvojournals.org/ on 06/14/2017 5. 6. 7. 8. blood-aqueous barrier in the rabbit eye, Nature 238: 158, 1972. Neufeld, A. H., and Sears, VI. L.: The site of action of prostaglandin E; on the disruption of the blood-aqueous barrier in the rabbit eye, E.vp. Eye Res. 17: 445, 1973. dementi, F., and Palade, C. E.: Intestinal capillaries. I. Permeability to peroxidase and ferritin, |. Cell Biol. 41: 33, 1969. Vegge, T., Whither, F. O., and Olsen, B. R.: Horseradish peroxidase in plasma studied by gel infiltration, Histochemie 28: 16, 1971. Tanuira, T.: Effects of prostaglandin Ei and E.. on the ciliary body of albino rabbits: an electron microscopic study, fap. f. Ophthalmol. 18: 135, 1974. Pattern of ocular response to topical and systemic prostaglandin. KEITH GREEN AND KEUN KIM. Changes i)i hotli intraocular pressure and total outflow facility were determined after short- and long-term infusion and topical application of prostaglandin E, and E\. The intraocular pressure with both routes of administration increased within 15 minutes hy 10 to 15 mm. Hg; long-term infusion caused the intraocular pressure to he elevated for a longer time, although a fall in intraocular pressure occurred despite continued infusion. Total outflow facility did not increase until 30 minutes after initiation of treatment and thereafter increased further with time, irrespective of the route of drug application. The initial, increase, in intraocular pressure is suggested to he the result, of vascular changes, namely an increase in the leakiness of the iris vessels and the capillary pressure of the ciliary body vessels caused by the vasodilatory acrtions of prustaglandins. Prostaglandins (PC) are known to induce an e l e v a t i o n ol i n t i a o e u l a r pie.ssunj w h e n given inlra- caineially, 1 topically,-- :| or intravenously. 1 The ellects on total outflow facility, however, have not been well documented. Topical application ol PC-precursor, aiachidonic acid, or PCE, and PCE.. is known to increase outllow facility 30 minutes alter application ol the drug."1 lntracameral injection of PC produces a nonstatisticallv significant increase in total outllow facility.1 Inlravenous PC infusion has been reported not to increase total outllow laeility, 1 although the measurement was made at a time when the- intraocular pressure was at a peak, viz, within 10 minutes ol the initiation ol the infusion. The piesent .study was made to examine the time course ol cllect ol topical and systemic PC's on intraocular pressure and outllow lacility of the rabbit eye in order to clarify the initial phase of prostaglandin ellects in the eye. Reports 37 Volume 14 Nuwlx'r 1 Table F. Total outflow facility of I lie rabbit eye during intravenous infusion or topical application of prostaglandins Time (minutes) PC. Prc-ilnii PCE, 0.165 ±0.013 0.170 ±0.011 0.182 ±0.007 0.178 ±0.007 0.174 ±0.009 0.176 ±0.011 Route 60 minutes intravenous PCE, 10 minutes intravenous PCE, PCE.. PCE, Topical PCE,. 30 10 0.191" ±0.005 0.17211 ±0.010 0.1 S311 ±0.009 0.196 i; ±0.011 0.189" ±0.014 0.196' ±0.008 0.210 > ±0.019 0.222 : ' ±0.027 0.200"° ±0.012 — 0.315" ±0.020 0.2771 ±0.024 60 0.289±0.031 0.293±0.028 0.261' ±0.016 0.284' ±0.015 0.355 • ±0.030 0.366' 90 N — 6 — 6 0.361' ±0.017 0.368' ±0.028 — 12 6 6 6 ±0.021 T h e p i e - i l r u H p h a s e f o r t h e ( i ( ) - i i i n i u t e i n t i a v e n o i i s i n j e c t i o n of I ' G l i , a n d l ' G l i : w a s a 3 0 - i n i n u t e i n f u s i o n of v e h i c l e a l o n e w i t h C m d e t e i m i n e d d i n i n g t h e last t h r e e m i n u t e s of t h a t p e r i o d . I ' m o t h e r l i m e s a n i l m o d e s of a d m i n i s t r a t i o n , t h e p r e d n i n C i m w a s d e t e i m i n e d a l t e r t h e i n i t i a l s t a h i l i z a t i o n of i n t i a o e u l a i p r e s s u r e . S e e K i n s . I , 2 , a n d 3 f o r t h e t i m e s of d e t e r m i n a t i o n of C , , , , . " S i x d e t e r m i n a t i o n s of t o t a l o i i t l l o w l a c i l i t v w e r e m a d e , ' p > 0 . 0 0 1 ; - 0 . 0 0 5 ... p .;• 0 . 0 0 1 ; H').O25 > p 0 . 0 1 ; ' 0 . 0 5 > p ,> 0 . 0 2 5 ; ''(I.I > p > 0 . 0 5 ; " N o t s i ^ n i K c i i n t l v d i l f e r e n t . T h e t o t a l o i i t l l o w f a e i l i t \ a t v a r i o u s t i m e s is c o m p a r e d w i t h t h e i n i t i a l p r e - d r u y v a l u e f o r t h a t p r o s t a y l a n d i n r o u t e , a n d t i m e N = N u i n h e r of d e t e r m i n a t i o n s . Materials and methods. Solutions employed. Ten milligrams ol PCE, 01 I'CLV; wcic dissolved i n 1 n i l . <>l u t h a i i o l . T w o hundred inieioliters ol this solution was diluted with 1.8 ml. ol sodium carbonate solution (0.2 nig. per millilitcr). This solution was divided into 20 tubes each containing 0.1 ml. and the solutions dozen. For intravenous injections, one tube was taken and 0.9 ml. of 0.9 per cent i\aCI solution was added, giving a solution containing 0.1 ing. PC per inilliliter; a similar solution without PC was used in the control infusion periods. For topical applications, 0.4 ml. ol NaCI solution was added to a lube containing PC and 50 fi\ ol this solution (containing 10 /*g) was applied topically to the eye. In some control eyes a similar solution without PC was applied to the eye. Proveduiv. Adult albino rabbits, two to three kilograms, of either sex were anesthetized with 25 per cent methane in 0.9 per cent NaCI solution. Approximately 30 ml. gave a suitable depth of anesthesia. A 22-gaugc needle connected to a transducer, which also connected to a capillary tube by way ol a T-coiiiiection, was inserted into the anterior chamber by hand and the intiaocular pressure recorded. If the intraocular pressure was unstable or rising the eye was discarded: normally the intraocular pressure was stable within two to five minutes alter insertion of the needle. Total oiitllow facility ( d . . , ) was determined in one eye ol each pair using the method of Barany"1 using an increment of 5 mm. Ilg pressure. T h e other eye served either as a control (topical application) or as a check that the intraocular pressure response was unalleeted by the determination of facility (intravenous infusion). The following experiments were pertormed: inlu- Downloaded From: http://iovs.arvojournals.org/ on 06/14/2017 sion of prostaglandin vehicle for 30 minutes followed by 60 minute inlusion of 0.79 /ug per 7.9 /'I per minute I'CIL, OI PCE: or topical application of 10 Mg PCE, or PCE... For both the 10 minute infusion and topical application the only pre-drug period was that time prior to stabilization of the intraocular pressure. T h e intravenous inlusion ol this quantity of PC elevates intraocular pressure by 10 to 20 mm. Hg. Total outflow facility was determined at 10, 30, and 60 minutes after initiation of the infusion or topical application, and for the 10 minute intravenous infusion also at 90 minutes. II the intraocular pressure after any determination of Cm, was not within 2 mm. I l g of the pre-G\,,i pressure the eye was discarded. Results. Inlusion of vehicle alone for 30 minutes has no ellect on intraocular pressure ( F i g . 1) or Ci.,1 (Table 1). T h e repeated determination ol C,,,i in the same eye has no ellect on the course of the intraocular pressure, as illustrated by the pie- and post-facility pressures in all eyes ( Figs. 1, 2, and 3 ) . T h e pressures of the eyes in which lacilitv was determined exactly parallel that in the eyes in which no lacilitv determinations were made; the mean pressure difference between paired eyes during all infusion experiments is 0.4 mm, Ilg. Thus, neither the PC vehicle nor the measurement of C,,,, had any ellect on intraocular pressure. With all three modes of administration of PC there is a typical 10 to 15 mm. Hg intraocular pressure rise in the first 10 minutes. The 60-ininute inlusion causes an elevation of pressure over the baseline lor about 45 to 50 minutes and therealter, despite continued infusion, the values fall below the original intraocular pressure ( F i g . 1). Alter the cessation of the 10-minute infusion the 38 O)>litlialii\ologij ianunni 1975 Investi Reports 0 5 10 15 20 2b 30 0 5 TIME 10 15 20 25 (mm) 30 35 40 45 50 55 60 Fig. 1. Ellect of prostaglandin vehicle and 60-mimite infusion of PCE, and PCE.. on intraocular pressure. • • , PCE,; O O, PCE.; A A, PCE, control; A A, PCE. control. Total outflow facility was determined where indicated by C. Values are the mean t S.E.M. ot 12 determinations. 40 15 w 10 E 1 a 25 2 20 15 10 15 20 25 30 35 40 TIME 45 50 (mm) 55 60 65 70 75 80 85 90 Fig. 2. Ellect of 10-miniite infusion of PCE, and PCE, on intraocular pressure. • • , PCE,; O O, PCE... Total outflow facility was determined where indicated by C. Values are the: mean t S.E.M. of 24 determinations for PCE, and 12 determinations for PCE:. intraocular pressure tails rapidly below the original intraocular pressure and remains at this level lor up to 60 minutes (Fig. 2 ) . With topical application of 10 <ug of PCE, or PCE.. there is a substantial elevation of intraocular pressure which begins to fall at a b o u t 3 0 to 4 0 m i n u t u s , but is still a b o v e the original intraocular pressure at 60 minutes (Fig. 3 ) . The control eve shows an initial consensual response to PC in the treated eve, which is followed by a period where the intraocular pressure tails below baseline and is maintained at this level. The determinations of C,,,i show that the value has not increased significantly over the control at 10 minutes alter the addition of either systemic or topical PC. Indeed, the 10-iniiiute and pie-chug Ci.,1 values arc very similar, despite the fact that the intraocular pressure is almost at a maximum alter 10 minutes. With intravenous infusion C,..i has increased at 30 minutes only by a barely significant amount. The C,,,, value at 30 minutes following topical PC application, and at 00 and 90 minutes tor all routes of administration, is Downloaded From: http://iovs.arvojournals.org/ on 06/14/2017 significantly increased over the pie-drug values (Table I).' Discussion. The intravenous infusion of PCE, or PCE- lor 10 or 60 minutes induces an elevation of intraocular pressure, as found previously, 1 as does the topical application ol 10 Mg of PCE, or PCE-...-- •' Since C m is unchanged during a period when the intraocular pressure reaches a peak an explanation must be sought for the increase in intraocular pressure under these conditions. We have shown that aqueous inllow can be expressed as'1: L,,A [x, ( P , - P o ) - Pi] + S where L,, is the permeability of the ciliary epithelium, A is the area, x, is the "pressure index," 7 P. is capillary pressure, P,, is plasma colloid oncotic pressure, Pi is intraocular pressure, and S is secretion. P,, has been experimentally determined as 21 mm. Hg, s and under the conditions of the experiments reported here is constant. In order to cause an elevation in intraocular pressure during the initial phase of PC eflects in Reports Volume 1<1 Number 1 39 35 - E E 25 H 20 - T C i I C 10 i i 15 20 ,C 25 50 TIME (mm) i 55 T T * 1 1 10 45 i 1 50 55 T C | 60 Fig. 3. Efleet of topical application of 10 jug of PGE, and PGE; on intraocular pressure. • • , PGE,; O O, PCE.; A A, PCE,"control; A A, PCE, control. Total outflow facility was determined where indicated by C. Values are the mean i S.E.M. of 6 determinations. the absence of any change in Cn.i, none of the parameters L,,, A, or x,, must change, since L,,Ax, = CI.,I." There could, of course, be compensatory changes in each parameter which would be ollsetting and not upset the equality in the equation. It is known, however, that L,, is increased by PC. after 20 minutes exposure'1 and so is x., as evaluated by the increased perfusion of the ocular vessels 30 minutes alter PC.1" The other parameter, A, would, in face of increased vascular perfusion, also be expected to increase since the perfusion area would be enlarged. It is difficult to visualize that changes in these parameters before 30 minutes would be qualitatively different from the later responses, and thus one concludes that none of the parameters change at early times alter initiation of PC treatment. The doses of PC required to stimulate secretion, S, in vitro are well above those reported as causing ocular hypertension in vivo,'1 and it has been suggested" that the in vivo action may be ^^ to increased permeability of the blood-aqueous barrier rather than the stimulation of epithelial transport. Measurements of the permeability of the isolated ciliary epithelium after treatment with PC suggest that pseudofacility, or CMs, would be increased, in a dose-dependent manner, about 20 minutes after a specific drug concentration bathed the tissue.'1 Evidence has also been obtained in the whole animal to indicate that C,,s is increased and Citu,is unchanged following topical PC1-' :| but these measurements were also made 30 minutes after drug application. Intraocular pressure, Pi, is increased almost immediately after PC administration and with no change in any other parameter of the equation this would logically necessitate that there be a decrease in aqueous humor formation, if all the Downloaded From: http://iovs.arvojournals.org/ on 06/14/2017 parameters remained constant, and there is no evidence that any parameter should change except P,. Aqueous humor formation (aqueous inllow) is increased after PC application, however, and is responsible for the elevation in Pi and an increase in P,, or capillary pressure, would be responsible for this increase. It has been shown that topical PC causes a mailed vasodilation, particularly at the base of the iris, concurrent with an increased vascular permeability when measured 30 minutes after PC application.10 The increased vascular permeability ol the iris vessels changes the characteristics of the iris capillaries from a system where very little lluorescein emerges into the anterior chamber to a very leaky system capable of filling the anterior chamber with dye in less than 10 minutes.1" It would seem, therefore, that the initial intraocular pressure rise seen after intravenous or topical PC is caused by the vasodilatory effects of the drug. Vasodilation would cause a change primarily in capillary pressure and hence vascular permeability due to an increase in vessel diameter. The increase in P, would force more fluid out of the capillaries thereby increasing ultrafiltration. These ellects would be more pronounced if the vasodilation was primarily on the afferent ciliary blood vessels, and, in view of the marked ellect of PC's on intraocular pressure it is most probable that the initial effect would be confined to these vessels. Masuda and Mishima11 have, indeed, found that the mean capillary pressure is increased when measured 30 minutes after topical PC treatment even during maintenance of constant systemic blood pressure. The initial phase of PC-induced ocular hypertension, therefore, is caused by vascular changes,10 namely an increase in vascular permeability and vessel vasodilation. This phase is followed, as the PC concentration is locally elevated in the eye, 40 hwustigtititjc Ophthalmology January 1975 Reports by siii increase in the permeability of the ciliary (.•pithelium us well us possible changes in iris vascular permeability. These changes thereby result in an increase in the measured total outflow facility clue to the increase in Cp- alone, since C,,v is a measure of the permeability of the ciliary epithelium.'1 All the changes caused by PC, therefore, appear to be restricted to the aqueous inflow rather than having any involvement with trabecular, or true, outflow facility. From the Departments of Ophthalmology and Physiology, Medical College of Georgia, Augusta, Ca. 30902. This study was supported in part by United States Public Health Service Research Grant EY 00863 from the National Eye Institute. Submitted for publication Aug. 5, 1974. Reprint requests: K. Green, Ph.D., Room 3 D 11, R & E Building, Medical College of Georgia, Augusta, Ga. 30902. We thank Mrs. Debbie Graves for her assistance in the preparation of this manuscript. The prostaglandins were generously provided by Dr. |. E. Pike, The Upjohn Company, Kalamazoo, Mich. Key words: prostaglandins, rabbit, intraocular pressure, total outflow facility, aqueous formation, vasodilation, capillary pressure. REFERENCES 1. Waitzman, M. B., and King, C. D.: Prostaglandin influences on intraocular pressure and pupil size, Am. |. Physiol. 212: 329, 1967. 2. Kass, M. A., Podos.S. M., Moses, R. A., et al.: Prostaglandin E, and aqueous humor dynamics, INVEST. OPHTHALMOL, II: 1022, 1972. 3. Masuda, K., and Mishima, S.: Effects of prostaglandins on inflow and outflow of the aqueous humor in rabbits, Jap. J. Ophthalmol. 17: 300, 1973. 4. Chiang, T. S., and Thomas, R. P.: Ocular hypertension following intravenous infusion of prostaglandin E,, Arch. Ophthalmol. 88: 4 18, 1972". 5. Barany, E. H.: Simultaneous measurement of changing intraocular pressure and outflow lacility in the vervet monkey by constant pressure infusion, INVEST. OPHTHALMOL. 3: 135, 1964. 6. Pederson, J. E., and Green, K.: Aqueous humor dynamics: A mathematical approach to measurement of facility, pseudofacility, capillary pressure, active secretion, and x,., Exp. Eye Res. 15: 265, 1973. 7. Barany, E. H.: A mathematical formulation of intraocular pressure as dependent on secretion, ultiafiltration, bulk outflow, and osmotic reabsorption of fluid, INVEST. OPHTHALMOL. 2: 584, 1963. 8. Pederson, J. E., and Green, K.: Aqueous humor dynamics: Experimental studies, Exp. Eye Res. 15: 277, 1973. 9. Green, K.: Permeability properties of the Downloaded From: http://iovs.arvojournals.org/ on 06/14/2017 ciliary epithelium in response to prostaglandins, INVEST. OPHTHALMOL. 12: 752, 1973. 10. Whitelocke, R. A. F., and Eakins, K. E.: Vascular changes in the anterior uvea of the rabbit produced by prostaglandins, Arch. Ophthalmol. 89: 495, 1973. Adenosine 3',5'-monophosphate increases the outflow of aqueous humor from the rabbit eye. ARTHUR H. NEUFELD, DAVID K. DlJEKER, TORGEIR VECCE, AND MARVIN L. SEARS. Direct administration of cyclic-AMP into the. anterior chamber increases the outflow facility of the eye for aqueous humor. This is consistent with the hypothesis that catecholamines lower the intraocular pressure of the rabbit eye, at least in part, by a cyclic-AM P-mediated mechanism. This mechanism is active in the outflow channel? and increases the rate at which aqueous humor leaves the anterior chamber. Adrenergic agonists, administered to the eye, decrease intraocular pressure. The mechanism ol this response is important because catecholamines may have a role in the physiologic regulation of intraocular pressme and because the clinical use ol topical epinephrine is an ellective therapy for primary open-angle glaucoma. One of the means by which a decrease in intraocular pressure occurs is an increase in the outllow of aqueous humor. In the rabbit eye, the outflow of aqueous humor responds to catecholamine stimulation1; however, the site of action and the mechanism by which adrenergic agonists influence outflow are unknown. We wish to report what we believe is the next step in a series of events that leads to increased outflow of aqueous humor after an endogenous or exogenous adrenergic stimulus. We have demonstrated that adenosine 3',5'-monophosphate (cyclic-AMP), administered directly into the anterior chamber of the rabbit eye, increases the outflow facility of the eye for aqueous humor. Methods. Two to three kilogram male albino rabbits were anesthetized with intravenous urethane (25 per cent w/v) and given aspirin (600 mg.) rectally. As a control, three animals were not pretreatecl with aspirin. The anterior chamber of one eye was cannulated with two needles using the needle gun.1-' One cannula was used for delivery of the drug; the other cannula was connected by saline-filled polyethylene tubing to a transducer and a reservoir and was used to monitor intraocular pressure and to allow perfusion of the eye. Outflow facility was measured by perfusion at two levels of constant pressure, usually P, = 25 and P, = 30 mm. Ilg. The How from