Prosfanoids in Rabbit Aqueous Humor: Effect of Loser Photocoagulation of the Iris

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1 Prosfanoids in Rabbit Aqueous Humor: Effect of Loser Photocoagulation of the Iris Robert N. Weinreb, David Weaver, and Murray D. Mitchell The authors measured concentrations of prostanoids (prostaglandin-like substances) in aqueous humor from normal pigmented rabbit eyes and from those subjected to argon laser photocoagulation of the iris. The predominant prostanoids quantitatively were prostaglandin E 2 (PGE 2 ), PGF 2a, and PGD 2 with minor amounts of 6-keto-PGF lo and thromboxane B 2. In all cases, concentrations of prostanoids in laser-treated eyes were substantially greater than those in normal eyes. This finding was particularly striking in the case of PGE 2 which increased 60-fold from 87 pg/ml to 5.5 ng/ml after irradiation. Concentrations of prostanoids following photocoagulation were related to the number of administered laser lesions and prostanoid release was associated with an initial hypertensive response and disruption of the blood-aqueous barrier. Invest Ophthalmol Vis Sci 26: , 1985 When traumatized, the rabbit eye undergoes a pattern of changes consisting of miosis, conjunctival and iris hyperemia, increased intraocular pressure and breakdown of the blood-aqueous barrier. The pattern of ocular changes and the severity of the response depend on the species 1 and type of injury. 2 " 4 An acute ocular response occurs when the laser is used to photocoagulate the iris. Since prostaglandins (PGs) and neural elements are both involved in the mediation of this response, photocoagulation of the iris has provided a useful model to study the injury response. 5 " 9 PG-like activity in aqueous humor has been previously measured using only a crude bioassay. 6 After ruby laser photocoagulation of the pigmented rabbit iris, PG-like activity in aqueous extracts appeared to be of the E-type and chromatography of partially purified extracts of iris tissue indicated a predominance of prostaglandin E 2 (PGE 2 ). However, it is now recognized that many prostanoids (PG-like substances) have similar actions and may not be separated by the bioassay system. Hence, it is important to determine whether the prostanoids released in response to photocoagulation are PGE 2 or a combination of other prostanoids with PGE-like activity. In the current investigation, we measured concentrations of specific prostanoids in aqueous humor From the Department of Ophthalmology, University of California, San Diego, San Diego, California. Submitted for publication: August 28, Reprint requests: Robert N. Weinreb, MD, UCSD Medical Center/Ophthalmology, 225 Dickinson Street (H-898), San Diego, CA from normal rabbit eyes and from those subjected to laser photocoagulation of the iris and have correlated their levels with the number of administered laser lesions. Materials and Methods Animals and Experimental Protocol We used adult pigmented (Dutch) rabbits of either sex which weighed at least 2.5 kg. All eyes were initially examined with a slit lamp and only animals without signs of ocular inflammation were included in the study. We performed anterior chamber paracentesis to obtain samples of aqueous humor for the measurement of prostanoids and protein in normal rabbits and at timed intervals after iris photocoagulation in treated rabbits. These animal investigations conform to the ARVO Resolution on the Use of Animals in Research. In the first series of experiments, we evaluated the prostanoids in aqueous humor pooled from six photocoagulated eyes, 60 min after 16 laser applications, and from six eyes of three normal rabbits. After identifying PGE 2 as quantitatively the major prostanoid in the aqueous humor samples from laserirradiated eyes, we examined the influence of two, four, eight, and 16 burns on its release 15 or 60 min after laser treatment in individual aqueous samples. PGE 2, PGD 2, and PGF 2a were then measured in individual samples of aqueous humor of laser-treated (both four and 16 burns) and contralateral untreated eyes at 15, 30, 90, 120, and 360 min after treatment. In the final series of experiments, we attempted to correlate the concentrations of PGE 2 in individual 1087

2 1088 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / August 1985 Vol. 26 Table 1. Prostanoids in aqueous humor Prostanoid Prostaglandin E 2 Prostaglandin F 2a Prostaglandin D 2 Thromboxane B 2 6-keto-prostaglandin F, a Prostaglandin F metabolitef Prostaglandin E metabolite! Concentration of Prostanoid (pg/ml) Treated* 5,549 1,580 1, Control * Treated eyes had 16 argon laser lesions administered to the iris and aqueous humor withdrawn after 1 hr and pooled, t 13,l4-dihydro-l5-keto-PGF 2o. % 1 l-deoxy-l3,14-dihydro-15-keto-l l,16-cyclo-pge 2. aqueous samples with intraocular pressure and aqueous protein. Laser Photocoagulation In all experiments, an argon (488 nm) laser was used to photocoagulate the surface of the iris of one eye of each rabbit. Under local anesthesia (0.5% proparacaine), a contact lens with a +66D planoconvex button was placed over the laser-treated eye to focus the laser light (100 ^m diameter, 1 watt and 0.2 sec). Laser applications were 1 mm from the pupillary margin and spaced circumferentially at regular intervals in a counterclockwise direction over 360 deg. When 16 applications were used, eight were placed in each of two adjacent concentric circles. After treatment, the contact lens was removed. Paracentesis At designated time after irradiation about 100 ix\ of aqueous humor was withdrawn from both the treated and untreated eyes of each rabbit with a 27- gauge needle and a tuberculin syringe. Ten minutes prior to paracentesis, ketamine hydrochloride (20 mg/kg) and acepromazine (0.4 mg/kg) were administered intramuscularly for anesthesia. Anterior chamber paracentesis was performed only once in each eye and corresponded to a specific number of laser applications and a specific time interval. Intraocular Pressure Using a calibrated pneumatonometer, intraocular pressure was measured just prior to photocoagulation (baseline) and then at 15, 30, 60, 120, and 360 min post-treatment. The change in intraocular pressure in the laser-treated and control eye was calculated at each time interval by subtracting the baseline intraocular pressure from the measured intraocular pressure. The corrected change in intraocular pressure was then obtained by subtracting the change in intraocular pressure in the control eye from that in the laser-treated eye. Throughout the 6-hr postoperative period, the rabbits had access to food and water. Prostanoid Assays We measured PGE 2, PGF 2a, PGD 2, thromboxane B 2 (TXB 2 ), 6-keto-PGF la (the major degradation product of prostacyclin) and products of PGF 2a and PGE 2 metabolism (13, 14-dihydro-15-keto-prostaglandin F 2a and ll-deoxy-13, 14-dihydro-15-keto-l 1, 16-cyclo-prostaglandin E 2, respectively) by sensitive and specific radioimmunoassays. The radioimmunoassays employed have been described and validated in detail elsewhere. 10 " 14 The cross-reactivities of the antisera with other major prostanoids are almost exclusively less than 1%. Protein A modification of the method of Lowry 15 was used to assay for protein from aliquots of the same aqueous samples used for prostanoid analysis. Results The predominant prostanoids found in the aqueous humor 60 min after iris photocoagulation were PGE 2, PGF 2a, and PGD 2 with minor amounts of 6-keto- PGF la and TXB 2 (Table 1). The concentrations of prostanoids in the laser-treated eyes were substantially greater than those in the control eyes. This finding was particularly striking in the case of PGE 2 (5.5 ng/ ml after treatment), the concentration of which was increased by more than 60-fold after laser irradiation. This difference was less pronounced for PGF 2a (1.6 ng/ml) and PGD 2 (1.4 ng/ml), the concentrations of which increased by about four-fold and three-fold, respectively. There were also clear differences between the treated and control eyes in concentrations of 6- keto-pgf, a (two-fold) and TXB 2 (four-fold); however, only low concentrations of these prostanoids were found. Although substantial amounts of products of both PGE 2 and PGF 2a metabolism were detected, differences between laser-treated and control eyes were minimal. After ascertaining that PGE 2 was the major prostanoid in the aqueous humor following iris photocoagulation, we evaluated the relationship between the number of laser lesions and PGE 2 release after 15 min and 60 min (Fig. 1). The concentration of PGE 2

3 No. 8 PROSTANOIDS IN RABBIT AQUEOUS HUMOR / Weinreb er ol min _ O 60 min PGE, PGD, APGF. 2a (VJ 8, 6000 T3 C 4000 o o cil Time ( minutes ) Number of Burns Fig. 1. Concentrations of PGE 2 (ng/ml) in aqueous humor 15 ( ) and 60 min (O) after photocoagulating the pigmented rabbit iris with two, four, eight, or 16 lesions. Each point represents the concentration of PGE 2 in one aqueous sample of one rabbit eye. in aqueous humor of eyes treated with 16 lesions was more than three-fold greater than that in the aqueous humor of eyes with four lesions at both 16 and 60 min after irradiation. There were no apparent differences in the PGE 2 levels in aqueous humor obtained from eyes with two and four lesions or eight and 16 lesions. To further evaluate this relationship, we then assayed PGE 2, PGD 2 and PGF 2a, the three major PG products released after iris photocoagulation, at given times after either 16 (Fig. 2) or four (Fig. 3) applications of laser light. At all time points examined, the levels of PGE 2 exceeded those of PGD 2 and PGF 2a. PG concentrations were maximal within 30 min of laser treatment in most cases and within 90 min of treatment in all cases. The concentrations of prostanoids released after 16 lesions were greater than those released after four lesions at all measured time intervals. Small amounts of PGF 2a were detected after 16 lesions and none could be detected after four laser lesions. We then attempted to relate the amount of PGE 2 released following the administration of either four or 16 laser lesions with the change in intraocular pressure and aqueous protein. Fig. 2. Concentrations of PGE 2 ( ), PGD 2 ( ), and PGF 2n (A) (pg/ml) 15, 30, 120, and 360 min after photocoagulating the pigmented rabbit iris with 16 lesions. Each point represents the concentration of prostaglandin in one aqueous sample of one rabbit eye. Intraocular Pressure Within 15 min after photocoagulation, the intraocular pressure was increased in both groups of lasertreated eyes. Intraocular pressure reached a peak within 30 min and then decreased during the subsequent experimental period. In general, the corrected change in intraocular pressure was greater in the eyes with the greater number of laser lesions (Fig. 4). By two hours after treatment, intraocular pressure in the laser-treated eyes was reduced below baseline Time ( minutes) Fig. 3. Concentrations of PGE 2 ( ) and PGD 2 ( ) (pg/ml) 15, 30, 90, 120, and 360 min after photocoagulating the pigmented rabbit iris with four lesions. Each point represents the concentration of prostaglandin in one aqueous sample of one rabbit eye.

4 1090 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / August 1985 Vol r Treated 16 Burns O Control Treated 4 Burns A Control LJ ~ Time (minutes) Time (minutes) 360 Fig. 4. The effect of laser photocoagulation of the pigmented rabbit iris on corrected change in intraocular pressure. The corrected change in intraocular pressure (mmhg) is calculated by subtracting the change in intraocular pressure from baseline in the contralateral untreated eye from that in the laser-treated eye. Intraocular pressure (mean ± SE) was determined for laser-treated and contralateral eyes at 15 (n = 15), 30 (n = 12), 60 (n = 9), 120 (n = 6), and 360 (n = 3) min, respectively, for 16 ( ) and four (A) laser lesions Fig. 6. Concentrations of PGE 2 (pg/ml) in the aqueous humor samples of eyes treated with 16 laser lesions ( ) and contralateral untreated eyes (O) as well as eyes treated with four lesions (A) and contralateral untreated eyes (A). Each PGE 2 value (mean ± SE) represents the results from three aqueous samples. Protein Compared with contralateral untreated eyes, the aqueous humor protein increased in eyes with either four or 16 laser lesions (Fig. 5). The concentration of protein began to decrease in laser-treated eyes after 2 hr and was generally higher in the aqueous humor of eyes with 16 laser lesions. Also, it remained elevated in these eyes for a longer period than in eyes with fewer lesions. Even after 6 hr, the concentration of protein was elevated in the aqueous humor of lasertreated eyes compared with that of untreated eyes Time (minutes) Fig. 5. The effect of laser photocoagulation of the pigmented rabbit iris on the concentration of protein in aqueous humor. Protein (mg/dl) was measured in samples of aqueous humor from eyes treated with 16 laser lesions ( ) and contralateral untreated eyes (O) as well as eyes treated with four laser lesions (A) and contralateral untreated eyes (A). Each protein value (mean ± SE) represents the results from three aqueous samples. PGE 2 In the eyes receiving either four or 16 laser lesions, the concentrations of PGE 2 in the aqueous humor were increased at 15 min compared with the contralateral untreated eyes (Fig. 6). In general, PGE 2 levels in the aqueous humor began decreasing within 1 hr, although they were still raised after 6 hr. Discussion Previously, investigators used only a crude bioassay to measure PG-like activity in aqueous humor after ruby laser photocoagulation of the pigmented rabbit iris. 6 In those studies, PG-like activity in aqueous extracts appeared to be of the E-type and chromatography of partially purified extracts of iris tissue indicated a predominance of PGE 2. In the current investigation, we employed sensitive and specific radioim-

No. 8 PROSTANOIDS IN RABBIT AQUEOUS HUMOR / Weinreb er ol. 1091 munoassays to quantitate the prostanoids released into the aqueous humor after iris photocoagulation with an argon laser.

5 No. 8 PROSTANOIDS IN RABBIT AQUEOUS HUMOR / Weinreb er ol munoassays to quantitate the prostanoids released into the aqueous humor after iris photocoagulation with an argon laser. PGE 2 was the major prostanoid found and its concentration increased rapidly following laser irradiation. Further, the magnitude of this increase was related to the number of administered laser lesions. Besides PGE 2, other prostanoids were identified in the aqueous humor samples including PGD 2 and PGF 2a. To our knowledge, these prostanoids have not been measured previously in such experimental circumstances. Their effects in the normal rabbit eye or in mediating the laser-induced injury response may well be different from those of PGE 2 since these prostanoids have different and often opposing actions in other physiological and biochemical systems. For example, these three PGs have qualitatively and quantitatively differing actions on adenylate cyclase activity, guanylate cyclase activity and calcium mobilization and may have specific receptors. 16 Our finding of PG metabolites in the aqueous humor of both photocoagulated and normal rabbit eyes may also be important. 15-Hydroxyprostaglandindehydrogenase (PGDH) is the main enzyme in prostanoid catabolism in most, if not all, tissues studied and its activity is very low in ocular tissues Removal of endogenously released PGs from the intraocular fluids is now considered to occur by facilitated transport across the blood-ocular barrier maintained by the epithelium of the ciliary processes and possibly also the posterior iris and retinal pigment epithelium Therefore, biologically effective amounts of PGs can be eliminated from the anterior chamber by specific uptake as well as bulk outflow of aqueous humor. The possibility that PGs can be metabolized by intraocular tissues, as has been shown in other systems, 20 may influence the response of the eye to injury as well as the fate of exogenously administered PGs. Extrapolation of this laser-induced response in the rabbit eye to the primate eye should be done with caution since responses to ocular trauma within different vertebrate species are known to vary considerably. 1 For example, whereas rabbits exhibit an indomethacin-sensitive response to many forms of irritation which is presumably PG-mediated, responses to similar irritants in owl monkeys are generally indomethacin-resistant and manifested only by pupillary constriction. 13 The explanations for these species differences have not been clearly ascertained although it is possible that anatomic differences in the tissues which comprise the blood-aqueous barrier play a role. Biochemical differences between species are also undoubtedly contributing. For example, cyclooxygenase activity is greater in the anterior uvea of the albino rabbit eye than in that of the human. 21 In this investigation, we have demonstrated that there is substantial PG release following photocoagulation of the pigmented rabbit iris associated with an initial hypertensive response and an accompanying disruption of the blood-aqueous barrier. The tissue source of these PGs is likely to be the iris, although other tissues, particularly the ciliary body, cannot be excluded. We measured more that 13 ng/ml of PGE 2 in the aqueous humor 30 min after the iris received 16 argon laser lesions. Introduction of 5 ng of PGE 2 into the anterior chamber of rabbits also can induce a disruption of the blood-aqueous barrier. 22 The concentration of PGE 2 in the aqueous humor following such an injection is similar to the maximal concentration of endogenous PGE 2 after laser irradiation assuming an aqueous humor volume of 300 y\. This reproducible experimental procedure, in which the response to the localized laser lesion is readily quantifiable, can be used to further evaluate the biosynthesis and action of prostanoids. Key words: prostanoid, prostaglandin, laser, iris, photocoagulation Acknowledgments The authors thank Jo Corbin for excellent technical assistance, Donna Chesnut for expert editorial assistance, and Professor MJNC Keirse (Leiden University, The Netherlands) for the kind gift of PGE 2 antiserum. References 1. Klein EM and Bito LZ: Species variations in the pathophysiologic responses of vertebrate eyes to a chemical irritant, nitrogen mustard. Invest Ophthalmol Vis Sci 24:184, Jampol LM, Neufeld AH, and Sears ML: Pathways for the response of the eye to injury. Invest Ophthalmol 14:184, Camras CB and Bito LZ: The pathophysiological effects of nitrogen mustard on the rabbit eye. I. The biphasic intraocular pressure response and the role of prostaglandins. Exp Eye Res 30:41, Camras CB and Bito LZ: The pathophysiological effects of nitrogen mustard on the rabbit eye. II. The inhibition of the initial hypertensive phase by capsaicin and the apparent role of substance P. Invest Ophthalmol Vis Sci 19:423, Neufeld AH, Jampol LM, and Sears ML: Aspirin prevents the disruption of the blood-aqueous barrier in the rabbit eye. Nature 238:158, Unger WG, Perkins ES, and Bass MS: The response of the rabbit eye to laser irradiation of the iris. Exp Eye Res 19:367, Unger WG, Cole DF, and Bass MS: Prostaglandin and neurogenically mediated ocular response to laser irradiation of the rabbit iris. Exp Eye Res 27:209, Butler JM, Unger WG, and Cole DF: Axon reflex in ocular

1092 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / Augusr 1985 Vol. 26 injury: sensory mediation of the response of the rabbit eye to laser irradiation of the iris. Quart J Exp Physiol 65:261, 1980.

6 1092 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / Augusr 1985 Vol. 26 injury: sensory mediation of the response of the rabbit eye to laser irradiation of the iris. Quart J Exp Physiol 65:261, Unger WG, Butler JM, and Cole DF: Prostaglandin and an increased sensitivity of the sympathetically denervated rabbit eye to laser-induced irritation of the iris. Exp Eye Res 32:99, Mitchell MD and Hint APF: Prostaglandin production by intrauterine tissues from periparturient sheep: use of a superfusion technique. J Endocrinol 76:111, Mitchell MD: A sensitive radioimmunoassay for 6-keto-prostaglandin F, o : preliminary observations on circulating concentrations. Prostaglandins Med 1:13, Mitchell MD, Bibby JG, Hicks BR, Redman CWG, Anderson ABM, and Turnbull AC: Thromboxane B 2 and human parturition: concentrations in the plasma and production in vitro. J Endocrinol 78:435, Mitchell MD, Kraemer DL, and Strickland DM: The human placenta: a major source of prostaglandin D 2. Prostaglandins Leukotrienes Med 8:383, Mitchell MD, Ebenhack K, Kraemer DL, Cox K, Cutrer S, and Strickland DM: A sensitive radioimmunoassay for 11- deoxy-13, 14-dihydro-15-keto-11, 16-cyclo-prostaglandin E 2 : application as an index of prostaglandin E 2 biosynthesis during human pregnancy and parturition. Prostaglandins Leukotrienes Med 9:549, Lowry OH, Rosebrough NL, Farr AL, and Randall RJ: Determination of protein content with the folin-phenol reagent. J BiolChem 193:265, Samuelson B, Goldyne M, Granstrom E, Hamberg M, Hammerstrom S, and Maltnsten C: Prostaglandins and thromboxanes. Ann Rev Biochem 47:997, Eakins KE, Atwal M, and Bhattacherjee P: Inactivation of prostaglandin E by ocular tissues in vitro. Exp Eye Res 19: 141, ^ 18. Bito LZ and Baroody R: Concentrative accumulation of 3 H- prostaglandins by some rabbit tissues in vitro: the chemical nature of the accumulated 3 H-labelled substances. Prostaglandins 7:131, Bito LZ and Wallenstein MC: Transport of prostaglandins across the blood-brain and blood-aqueous barriers and the physiological significance of these absorptive transport processes. Exp Eye Res (Suppl)25:229, Flower RJ: The role of prostaglandins in parturition with special reference to the rat. In The Fetus and Birth, Knight J and O'Connor M, editors. Amsterdam, Elsevier Science Publishers BV, 1977, pp Kulkarni PS, Fleisher L, and Srinivasan BD: The synthesis of cyclooxygenase products in ocular tissues of various species. Curr Eye Res 3:447, Beitch BR and Eakins KE: The effects of prostaglandins on the intraocular pressure of the rabbit. Br J Pharmacol 37:158, 1972.

Prostaglandins E 3 and D 3 Lower Intraocular Pressure. (P-Pf)/(A) References INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / August 1985 Vol.

Prostaglandins E 3 and D 3 Lower Intraocular Pressure. (P-Pf)/(A) References INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / August 1985 Vol. 1178 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / August 1985 Vol. 26 In eyes with the disrupted blood-ocular barriers, however, the fluorescence of fluorescein will be quenched by it binding to plasma