The effect of topical /-epinephrine on regional ocular blood flow in monkeys

The effect of topical /-epinephrine on regional ocular blood flow in monkeys Albert Aim A 25 ixl volume of a 1-% \-epinephrine borate solution applied on the cornea of one eye in 12 monkeys reduced blood flow through the iris and the ciliary body by 59% and 20%, respectively, compared to the untreated control eyes. In the ciliary body the main effect was in the ciliary processes. Blood flow was determined, with the labeled microsphere method 1, 4, and. 6 hr after application of the drug. The average blood flow reductions were of the same order in the three groups, indicating a duration of at least 6 hr. No effect on blood flow through the retina or the choroid was observed. Key words: /-epinephrine eye drops, regional ocular blood flow, monkeys, labeled microspheres I n previous studies sympathetic stimulation caused marked blood flow reduction in the uvea in cats 1 and monkeys. 2 In monkeys the largest effect was found in the iris where blood flow was reduced by 52%, whereas for the other parts of the uvea reductions of 22% to 30% were obtained. This study was performed to determine the effect on ocular blood flow of a single application of the adrenergic drug /-epinephrine on the cornea in monkeys. Ocular blood flow was determined by means of intracardiac injections of radioactively labeled microspheres. Methods Twelve monkeys, nine cynomolgus (Macaca ints) and three vervet (Cercopithecus ethiops) of either sex weighing between 2.0 and 3.3 kg were From the Departments of Physiology and Ophthalmology, University Hospital of Uppsala, Uppsala, Sweden. This study was supported by a grant (EY 00475-09) from the National Eye Institute, P.H.S. and by a grant (B76-14X-147) from the Swedish Medical Research Council. Submitted for publication Nov. 20, 1978. Albert Aim, M.D., Department of Ophthalmology, University Hospital, S-750 14 Uppsala 14, Sweden. used. Anesthesia was induced by sodium methohexital, 50 to 100 ing intramuscularly (Brietal Sodium; Eli Lilly & Co., Indianapolis, Ind.), and was maintained by intravenous injections of pentobarbital sodium (Penthotalsodiuni; Abbott Laboratories, North Chicago, 111.). The animals were tracheotomized and artificially ventilated. Arterial blood pressure was measured. As a rule, arterial Po 2, PCO 2, and ph were determined, and ventilation was adjusted to cause normal values for* blood gases. Heparin, 500 IU/kg body weight, was used to prevent clotting of blood. A 25 /JL\ volume of a 1% solution of /-epinephrine borate (Eppy; Pharmacia Fine Chemicals, Uppsala, Sweden) was applied with a constriction pipette into the conjunctival sac of one eye. In the other eye a placebo solution (Eppy without /-epinephrine supplied by the manufacturer) was similarly applied. The pupil diameter on both sides was determined in daylight with a slide ruler prior to administration of the drugs and then at varying intervals until blood flow was determined, which was made after 1 hr in six monkeys, after 4 hr in three, and after 6 hr in another three monkeys. Labeled microsphere method. Blood flow was determined by injections of radioactively labeled microspheres into the left ventricle of the heart. The heart was exposed through a thoracotomy, and the microspheres were injected through a needle pushed through the wall of the left ventri- 0146-0404/80/050487+05$00.50/0 1980 Assoc. for Res. in Vis. and Ophthal., Inc. 487

488 Aim Invest. Ophthalmol. Vis. Sci. May 1980 6 hr Time after application of l-epinephrine Fig. 1. Blood flow in eyes treated with /-epinephrine expressed as percent of blood flow through the untreated control eyes. The values 1, 4, and 6 hr after application of the drug are the means ± S.E. of six, three, and three monkeys, respectively. cle. The injection took 10 to 20 sec. The microspheres act as a nonrecirculating blood flow indicator, and blood flow to various tissues can be calculated if the radioactivity of a known reference bloodflowis known. Since an additional aim of the experiments was to compare bloodflowvalues obtained with spheres of two different sizes, a mixture of 15 ± 5 and 35 ± 5 /xm spheres (mean and S.D.), labeled with 85 Sr and 141 Ce, respectively (3M Co., St. Paul, Minn.) was injected in nine monkeys, and only 15 fxm spheres were used in the remaining three monkeys. Of each sphere size, 1.0 ml of a 1% suspension (sp. act. 10 mci/ gm) was used. The reference blood flow was obtained by sampling the spontaneous flow from a cannulated femoral artery during 60 sec starting at the time of the microsphere injection. The flow rate varied bewteen 1 and 2 ml/min. At the end of this period the animals were killed by intravenous injections of KG. Both eyes were enucleated and dissected. The iris, ciliary body, retina, and choroid were taken as separate samples. Samples of ciliary muscle and ciliary processes were obtained by dissecting the ciliary body after glutaraldehyde fixation and dehydration. Reference blood was weighed, and the radioactivity for all tissue and blood samples was determined by gamma spectrometry. Blood flow estimates for the various tissues were obtained by dividing the activity of the tissue sample by the activity per milligram of blood flow per minute calculated for the reference flow. In the present report only results obtained with 15 fxm spheres will be presented. Data comparingflowvalues obtained with the two sphere sizes have already been published. 3 Results At the time of injection of the microspheres the following data were obtained (mean ± S.E.): mean arterial blood pressure: 135 ± 8 cm H 2 O (n = 11); arterial PO 2, PCO 2, and ph: 80 ± 4 mm Hg (n = 9), 32 ± 2 mm Hg (n = 9), and 7.53 ± 0.02 U (n = 11), respectively. The placebo solution had no significant effect on the pupil diameter, whereas all eyes treated with Z-epinephrine became mydriatic. The difference in pupil size between control and treated eyes ranged from 0.5 to 5.5 mm. In the six monkeys where blood flow determinations were made 4 or 6 hr after application of the drug, the mydriatic response was maximal at 1 hr in only one animal, whereas in the other five animals a further increase in pupil size was obtained with a maximum between 1 and 2 hr after drug application. Table I presents the blood flow values obtained in all eyes, and the differences in blood flow through the untreated eye minus blood flow through the treated eye for the various ocular tissues. Significant reductions in the treated eyes were observed for iris and ciliary body, corresponding to 59% and 20% of blood flow through the untreated eyes, respectively. For the parts of the ciliary body a significant reduction in blood flow was observed in the ciliary processes only, corresponding to 28% of blood flow through the untreated eyes. Fig. 1 presents blood flow through the iris and ciliary body of the treated eyes expressed as percent of blood flow through untreated eyes. The mean reduction in blood flow through the iris and ciliary body was of the same order 1, 4, and 6 hr after application of/-epinephrine. Discussion Labeled microsphere method. The accuracy of the method largely depends on the amount of spheres trapped in each tissue

Volume 19 Number 5 Topical l-epinephrine effect on blood flow 489 Table I. Blood flow through untreated and treated eyes and the difference (blood flow through untreated eye minus blood flow through treated eye). Tissue Untreated eye Treated eye Difference p values Blood flow in mg/min/whole tissue: Retina Iris Ciliary body Choroid Blood flow in gm/min/100 gm of tissue: Ciliary processes* Ciliary muscle 32.1 ± 3.4 7.9 ± 1.4 73.0 ± 6.8 505 ± 75 163 ±32 155 ± 28 30.4 ± 3.9 3.2 ± 0.5 58.7 ± 6.7 525 ± 79 117 ± 28 127 ± 20 1.7 ± 2.0 4.7 ± 1.2 14.3 ± 4.1-20 ± 33 46 ± 13 28 ± 16 <0.005 <0.01 <0.01 Weights for the ciliary processes and muscle are calculated from the dry weight on the assumption that the dry weight/wet weight ratio is 0.20. Mean ± S.E., n = 12. Significance levels indicate the possibility that no difference existed. Student's t test. sample. Chance variation in the distribution of the spheres can be expected to follow a Poisson distribution. Thus, to obtain blood flow values within 10% of the true value in 95% of experiments, one must have about 400 spheres in the sample. 4 In the present study about 4.4 X 10 (i 15 /im spheres were injected. Cardiac output was not determined, but if we assume a similar cardiac output as in rheses monkeys, about 250 ml/min/kg body weight, 5 the mean cardiac output for the monkeys in the present study would be about 625 ml/min. It is then possible to make an estimate of the number of spheres in the various tissue samples by multiplying the observed mean blood flow by 7, since 4.4 X 10 6 spheres distributed in 625 ml corresponds to 7 spheres//^ blood flow. For ciliary processes and muscle the mean values of blood flow per tissue sample were 10 and 15 /ul/min, respectively. Thus the smallest number of spheres can be expected to become trapped in the iris, about 25 and 50 in treated and control eyes, respectively, and ciliary processes and muscle can be assumed to contain 75 to 100 spheres. To obtain 400 spheres in these samples, at least in the control eyes, five to 10 times as many spheres have to be injected. Injection of such a large amount of spheres should be avoided if possible, since it will have an effect on the systemic circulation and increase the arterial blood pressure. Thus, for these tissues, the amount of spheres used in the present study cannot be expected to demonstrate the significance of small changes in blood flow. It should be stressed, however, that even in these tissues it should be possible to demonstrate large changes in blood flow and that observed significant differences cannot be explained by an inadequate number of spheres in the samples. Reliable results can be obtained only if the amount of spheres that escape through the capillary bed is negligible. Since no reference method is available for studies on regional ocular blood flow, the use of a mixture of spheres of two different sizes has been used to evaluate this possibility in regional ocular blood flow determinations. In nine of the monkeys of the present study a mixture of 15 and 35 ju,m spheres was injected. Flow values obtained with the two sphere sizes could then be compared. The details of this comparison have been published elsewhere.' A significant escape of 15 ftm spheres would have resulted in higher flow values for 35 /xm spheres than for 15 /xm spheres. No such difference was observed for any ocular tissue in either treated or untreated eyes, and it was concluded that 15 /xm spheres give reliable results in studies on regional ocular blood flow. Anterior uvea. /-Epinephrine caused a marked reduction in blood flow through the iris and ciliary body. The effects were of the same order as previously found in monkeys during intense sympathetic stimulation 2 where blood flow was reduced by 52% and 22% for iris and ciliary body, respectively.

490 Invest. Oplithalmol. Vis. Sci. May 1980 With sympathetic stimulation there was no obvious difference in effect on ciliary muscle and ciliary processes. The observed blood flow reductions were 29% and 23%, respectively. In the present study, the reduction was statistically significant onlyfor the ciliary processes. Blood flow determinations at various times after application of the drug revealed no diminishing of the effect, indicating a duration of at least 6 hr. This result is consistent with the observation that the concentration of /-epinephrine in iris and ciliary body in rabbits is very similar 1 and 6 hr after topical applications. 6 Choroid. Choroidal vessels have alphaadrenergic receptors, and supramaximal sympathetic stimulation reduces choroidal blood flow in monkeys by 30%. 2 In the present study, no significant effect on choroidal blood flow was observed, indicating that penetration of the drug into the choroid was poor. Retina. The intraocular part of the retinal vessels lack adrenergic nerves, 7 and sympathetic stimulation has no consistent effect on retinal blood flow in monkeys. 2 Intra-arterial injections of norepinephrine had no effect on retinal bloodflowin cats, which was assumed to be due to difficulties for the drug to penetrate the blood-retinal barrier. 8 In pigs intravenous infusion of norepinephrine has been reported to increase retinal blood flow by a factor of 4. 9 As suggested by the authors, this increase may have been caused by a marked increase in blood pressure. The fact that neither sympathetic stimulation nor intraarterial injection of norepinephrine have effects on retinal blood flow does not exclude the possibility that topical application may have an effect. A vasoconstriction may take place if the retinal vascular smooth muscles contain alpha-adrenergic receptors that although to a large extent protected from the influence of circulating catecholamines by the blood-retinal barrier, may be approached by topically applied /-epinephrine diffusing into the vitreous body. A vasodilatation is also conceivable by one of two mechanisms: either beta-adrenergic vasodilating receptors or secondary to a beta-adrenergic increase in retinal metabolism, as has been demonstrated in the brain. 10 However, no effect on retinal circulation was found in the present study. The dose of/-epinephrine that reaches the retina after application of a single drop to the cornea in phakic eyes may, however, be too small to have an effect. Higher dose levels are achieved in aphakic eyes. 11 Since repeated topical application of/-epinephrine in aphakic eyes may result in cystoid macular edema, 12 studies on the effect on retinal circulation of repeated applications of /-epinephrine to aphakic eyes should be of interest to evaluate the possibility that this is due to a vascular or metabolic disturbance. Thus a single drop of /-.epinephrine on the cornea in monkeys resulted in a vasoconstriction in the anterior uvea of at least 6 Induration. A long-term study to determine whether this is a lasting effect or not should be made before possible consequences of the use of /-epinephrine in glaucoma can be evaluated. REFERENCES 1. Aim A, and Bill A: The effect of stimulation of the cervical sympathetic chain on retinal oxygen tension and on uvea], retinal, and cerebral blood flow in cats. Acta Physiol Scand 88:84, 1973. 2. Aim A: The effect of sympathetic stimulation on blood flow through the uvea, retina and optic nerve in monkeys (Macaca irus). Exp Eye Res 25:19, 1977. 3. Aim A, Tornquist P, and Stjernschantz J: Radioactively labeled microspheres in regional ocular blood flow determinations. Bibl Anat 16:24, 1977. 4. Buckberg GD, Luck JC, Payne DB, Hoffman JIE, Archie JP, and Fixler DE: Some sources of error in measuring regional blood flow with radioactive microspheres. J Appl Physiol 31:59S, 1971. 5. Forsyth RP, Nies AS, VVyler F, Neutze J, and Melmon KL: Normal distribution of cardiac output in the unanesthetized, restrained rhesus monkey. J Appl Physiol 25:736, 1968. 6. Wei C, Anderson JA, and Leopold I: Ocular absorption and metabolism of topically applied epinephrine and a dipivalyl ester of epinephrine. INVEST OPHTHALMOL VISUAL SCI 17:315, 1978. 7. Ehinger B: Adrenergic nerves to the eye and to related structures in man and in the cynomolgus monkey (Macaca irus). Exp Eye Res 5:42, 1966. 8. Aim A: Effects of norepinephrine, angiotensin, dihydroergotamine, papaverine, isoproterenol, histamine, nicotinic acid, and xanthinol nicotinate on

Number 5 Topical \-epinephrine effect on blood flow 491 retinal oxygen tension in cats. Acta Ophthalmol epinephrine: relevance of the blood-brain barrier. 50:707, 1972. Am J Physiol 231:483, 1976. 9. Malik AB, van Heuven WAJ, and Satler LF: Effects 11. Kramer SG: Retinal uptake of topical epinephrine in of isoproterenol and norepinephrine on regional aphakia. In Symposium on Ocular Therapy, Leoocular blood flows. INVEST OPHTHALMOL 15:492, pold III, and Burns RP, editors. New York, John 1976. Wiley & Sons, Inc., vol. 9, pp. 73-86. 10. MacKenzie ET, McCulloch J, O'Keane M, Pickard 12. Kolker AE and Becker B: Epinephrine maculop- JD, and Harper AM: Cerebral circulation and nor- athy. Arch Ophthalmol 79:552, 1968. Information for authors Most of the provisions of the Copyright Act of 1976 became effective on January 1, 1978. Therefore, all manuscripts must be accompanied by the following written statement, signed by one author: "The undersigned author transfers all copyright ownership of the manuscript (title of article) to The Association for Research in Vision and Ophthalmology, Inc., in the event the work is published. The undersigned author warrants that the article is original, is not under consideration by another journal, and has not been previously published. I sign for and accept responsibility for releasing this material on behalf of any and all co-authors." Authors wall be consulted, when possible, regarding republication of their material.