Reports. Ciliary ganglion stimulation, I. Effects on aqueous humor inflow and outflow.

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1 28 Investigative Ophthalmology January 1975 Reports Fig. 1. Photomicrograph showing lesions from one animal bilaterally in supraoptic nucleus {dark arrow) above optic tracts (OT, outlined in dash). A portion of the electrode tract also strains on the right section (ET), Because each section was cut on the bias, the lesions are present in successive sections which have been mounted side by side. Discussion. The animal model with unilateral optic nerve section resulting in ipsilateral alteration of IOP responses to water drinking and various drugs2' 1 suggests a control mechanism mediated through the optic nerves. The hypothesis of a central control mechanism for IOP is supported by this preliminary data which suggests that electrical lesions in the supraoptic nucleus can produce a loss of the IOP response to water drinking in the eye with an intact optic nerve. Both animals receiving the lesions developed loss of IOP response to water drinking. Histologic examination of the site of the lesion one week and three months after it was applied showed damage in the supraoptic nucleus confirming that the current passed was in the site of experimental design. From the Departments of Ophthalmology and Neuroscience, College of Medicine, University of Florida, Gainesville, Fla Supported in part by United States Public Health Service Grant EY00033 from the National Eye Institute, Training Grant MH from the Center for Neurobiological Sciences, and a grant from National Society for the Prevention of Blindness, Inc. Submitted for publication June 28, Reprint requests: Dr. Constance R. Fitzgerald, Department of Ophthalmology, College of Medicine, University of Florida, Box 733, Gainesville, Fla REFERENCES 1. Riise, D.; and Simonsen, S. E.: Intraocular pressure in unilateral optic nerve lesion, Acta Ophthalmol. 47: 750, Krupin, T., Podos, S. M., and Becker, B.: The effect of optic nerve transection on osmotic alterations of intraocular pressure, Am. J. Ophthalmol. 70: 214, Podos, S. M., Krupin, T., and Becker, B.: Optic nerve transection and intraocular pressure response to various drugs, INVEST. OPHTHALMOL. 9: 492, Von Sallman, L., Macri, F. J., Wanko, T., et a!.: Some mechanisms of centrally induced eye pressure responses, Am. T. Ophthalmol. 42 (pt. 2): 130, Schmerl, E., and Steinberg, B.: Central control of intraocular pressure by active principles, Am. J. Ophthalmol. 31: 1097, Schmerl, E., and Steinberg, B.: Separation of diencephalic centers concerned with pupillary motility and ocular tension, Am. J. Ophthalmol. 33: 1379, Gloster, J., and Greaves, D. P.: Effect of diencephalic stimulation upon intraocular pressure, Br, J. Ophthalmol. 41: 513, Sawyer, C. H,, Everett, J. W., and Green, J. D.: The Rabbit diencephalon in stereotaxic coordinates, J. Comp. Neurol. 101: 801, Ciliary ganglion stimulation, I. Effects on aqueous humor inflow and outflow. FRANK J. MACRI AND STANLEY J. CEVARIO. Stimulation of the ciliary ganglion in an enucleated, arteriauy perfused cat eye preparation produced a sustained increase in aqueous humor formation and an increase in the rate of aqueous

2 ... Nmnhcr Reports 29 30r 10 P nn (mmhg) _ 1700 Perfusate Flow 1610 (pl/min) 1520 T,!t I t Stimulation L U.LLU Fig. 1. Effects of ciliary g. stimulation on IOP and arterial perfusate flow. Time: 20 seconds between heavy vertical lines. _ i i rr. humor outflow. The increased aqueous humor formation imiuced by ciliary ganglion stimulation has been found to be pressuredependent and therefore suggests that ultra filtration may be the underlying mechanism of action. No change in capillary permeability of the ciliary body could be demonstrated. The possible presence of neiirogenie mechanisms which could inlluence aqueous humor dynamics has been studied for many years. Brain stimulation experiments 1 ' as well as in situ ciliary ganglion stimulation have produced equivocal results. '' ' A procedure has been developed in which the enucleated cat eye, with its ciliary ganglion attached, has been perfused intraarterially through the ophthalmic artery.' The preparation is viable as manifested by pupillary and intraocular pressure changes upon stimulation of either the sympathetic or psympathelic nerves. The preparation also responds to drug administration by both pupillary and intraocular pressure (IOP) changes as well as by changes of aqueous humor inflow and outllow. This report will describe an increase in aqueous humor formation brought about by sustained stimulation of the preganglionic fibers of the ciliary ganglion in this isolated eye preparation. Some aspects on the mechanism of action for this response are also reported. Methods. Eyes obtained from adult cats of both sexes were used in these experiments. The procedure for enucleation and arterial perfusion was identical to that previously reported/' IOP was measured manometrically using a Sanbom Model 267B pressure transducer. Arterial perfusate How rate was monitored by the use of a dillerentialpressure type Howmeler" modified to have a How capacity of approximately 2 ml. per minute, at the hydrostatic pressure utilized. Turnover of aqueous humor was determined by measuring the decay of intracamerally injected '''Itagged serum albumin, using a gamma probe." In the experiments in which the eye pressure was to be maintained constant, the open end of an outlet tube communicating with the aqueous humor via a No. 24 hypodermic needle shaft was set at the desired height. Aqueous humor formation in these eyes was determined by a dilution technique utilizing inulinc" which was continuously perfused through the anterior chamber. 8 Oxygenated (95 per cent O=, 5 per cent CO.) Eagle Basal Media No. 1 (EBM), ph 7.3, was used as the arterial perfusate medium and, where appropriate, as the anterior chamber perfusate solution. The perfusate solution and the eye were maintained at a temperature of 37 C. The extraocularly located ciliary ganglion was not perfused; the perfusion solution was directed only into the intraocular blood vessels. The ganglion with its pre and postganglionic fibers were kept moist by covering them with a thin layer of cotton wetted with EBM. Preganglionic fiber stimulation was performed by utilizing stainlesssteel electrodes. A Crass, Model S4 stimulator with an isolation unit Model SIU4a delivered squarewave biphasic pulses at 30 Hz. and 6 V. Pulse duration was 1 msec. All of the eyes were perfused with eserine (1.0 pg per milliliter) for the time period of 15 minutes before and continuously during the period of ganglionic stimulation. Results. Effects of ciliary ganglion stimulation (11 eyes). Electrical stimulation of the preganglionic fibers of eseritiized eyes produced pupillary constriction and an immediate rise of IOP as well as a de

3 30 Reports Jtmiwnj V 26 c E o Control I ^Stimulation TIME (minutes) Fig. 2. Time course of ciliary ganglion stimulation effects on aqueous humor formation. Table I. Effects of ciliary ganglion stimulation Eserine (11) Eserine + stimulation A Mean + S.li. ( ) Number of experiments. Inflow (til minr') IOP (mm. Hg) \"C" (til min.> mm. Hg') ± 0.72 e ± i ± t ± ± ±0.12 P < 0.02 P < 0.05 P < 0.02 crease in the rate of arterial perlusate How (Fig. 1). These responses became maximal usually within 15 minutes. The rate of formation of aqueous humor was increased and paralleled the time course of the IOP rise (Fig. 2). During the interval of 15 to 30 minutes after onset of stimulation, aqueous humor production had increased by 18 ia min.' and a mean increase in IOP of 7.5 mm. Hg was observed (Table I). Calculation of facility of outflow "C demonstrated a 0.64 fi\ min. 1 mm. Hg 1 increase at this time period. A single topical application of a 1:100 dilution of nicotine to the ciliary ganglion in three experiments caused an almost immediate partial reversal of the nerve stimulation effects (Fig. 3). Within 15 minutes the mean rate of aqueous humor formation in these experiments had decreased from 37 jul min. 1 to 26 /ul min." 1. Dependency of arpicous humor formation on arterial perjusate pressure. In five experiments, the IOP was maintained constant at 15 mm. Hg. Alter maximal ellects of ganglionic stimulation had been obtained, the perfusate head pressure was altered in two decrements. As may be seen in Fig. 4, an excellent linear correlation was observed in each eye for the rate of aqueous humor formation versus arterial perfusate head pressure. The average relationship could be expressed by the form: inflow (A»1 min. 1 ) = arterial perfusion pressure (mm. Hg) Stimulation effects on the bloodaqueous humor harrier. 1 r> I tagged human serum albumin (RISA) was placed in the arterial perfusate

4 Volume II Number 1 Reports 31 IOP (mmhg) 30r 20 10H 1700 r Perfusate Flovy 1610 (pl/min) Nicotine Fig. 3. Blocking action of nicotine on responses induced by ciliary g. stimulation. Nicotine applied directly on the ciliary g. reservoir and allowed to continuously perfuse through the eye. No needles were placed in the eyes nor was any surgery perlormed on the globe in order that trauma would be minimized. The accumulation of KISA in the aqueous humor was monitored by a gamma probe placed in Iront ol the cornea. RISA accumulated in the aqueous humor in a linear fashion (Fig. 5). The data was expressed as the slope of this function (C.P.M. min.'). Stimulation of the pieganglionic fibers of the ciliary g. produced rapid sustained pupillary constriction. In each eye after stimulation there was an increase in the rate of RISA accumulation (Table II) which averages 2.71 times control values. Discussion. The data recorded in these experiments allows for the calculation of the resistance (R) of aqueous humor outflow from the eye. "R" is usually expressed as the reciprocal, or "facility of outllow" (C) and calculated from the equation C = where F = rate of aqueous humor inflow, P, = intraocular pressure, and P,. = episeleral venous pressure. It is assumed that P P in these experiments equals zero. Because the assumption that P«equals zero or does not change may not be accurate, the data reported in the results must be viewed with this reservation. It is included to illustrate qualitative, if not quantitative, changes in this parameter. The continuous arterial peitusion of eserine, in the concentrations used, had no marked elfects on aqueous humor production or on IOP. The use ol eserine was intended to maximize the responses produced by ganglionic stimulation by Table II. Accumulation rale of I 1 * albumin in aqueous humor Experiment No fi Average + S.E". C.P.M. min.' K',,.,. C.P.M. miiir K.I..M. Kriln<r,.l S inhibiting the enzymatic hydrolysis ol endogcnouslv released Ach. Stimulation of the preganglionic fibers of the ciliary g. in these experiments has been loiincl to increase the rate of aqueous humor formation by 18 /'I min." 1. The IOP was increased by 7.5 mm. Hg as would be anticipated by the increased aqueous humor volume. The resistance of the aqueous humor drainage channels from the 1 " eye was decreased. "C"( ) was found to increase from 0.80 fi\ min. 1 mm. Hg" 1 to 1.44 /'I min." 1 mm. Hg" 1. These resistive changes confirm similar findings of Arnialy. 1 The certainty that the response is directly mediated by the ciliary g. and not indirectly by possible central re Ilexes is obtained in these experiments which were perlornied on enucleated eyes. The possibility (hut the aqueous humor responses could have been mediated bv stimulation of fibers which

5 32 Reports Investigative Ophthalmology January T 25 E 20 1 o 15 E '.' /o 1^ Arterial Perfusate Pressure (mmhg) Fig. 4. Arterial pressure dependency of the elevated aqueous humor formation rate induced by ciliary ganglion stimulation. Fine lines represent individual experiments. Heavy line is the average of the five experiments. pass through the ciliary g. could be discounted by the rinding of an inhibition of the response by topical application of nicotine to the ganglion. Nicotine blocks synaptic transmission by persistent depolarization. The elevated rate of aqueous humor production was found to be linearly dependent on the arterial perfusate pressure. This is rather strong evidence to indicate that the rise in aqueous humor formation brought about by ciliary g. stimulation may well be due to an increase in an ultrafiltration process. An attempt was made to determine whether ciliary g. stimulation produced an increased leakiness of the bloodaqueous humor barrier to account, in part, for the increased ultrafiltration. Tracer amounts of I'"'albumin in the arterial perfusate were found to accumulate in the aqueous humor at a constant rate. Stimulation of the ciliary g. caused the rate of accumulation to increase 2.71fold. Although it is tempting to ascribe the increased protein accumulation in the aqueous humor to membrane changes, it must be noted that ganglionic stimulation also increased the rate of aqueous humor production 2.75fold ( fi\ min." 1 to MI min.'). Because of the very close similarity in the multiples of the protein and aqueous humor accumulations it would appear more likely that the increase in protein movement is due to the increase of aqueous humor formation in which the protein remains in an unchanged concentration. It may be, therefore, that the increase in aqueous humor production brought about by ciliary g. stimulation is due solely to hydrostatic pressure changes. The data presented here in regard to changes of aqueous humor inflow, IOP, and "C" and the arterial pressure dependency of the inflow, very closely resemble similar findings obtained in the enucleated eye where acetylcholine was used as the agonist.' 1 ' ' ' From the Clinical Branch, National Eye Institute, National Institutes of Health, United States Department of Health, Education, and Welfare,

6 Volume 14 Numhi'r 1 Reports 33 o K=50 20 Control t 4C St imulation TIME (minutes) = Fig. 5. Accumulation of '"'Iserum albumin in the anterior chamber. Tracer amounts ol 1 '' i ' 1 1 serum albumin were placed in the arterial perfusate. Ciliary g. stimulation produces an almost immediate increase in the rate of accumulation. Bethesda, Md Submitted for publication Aug. 19, Reprint requests: Dr. F. J. Maori, Room 2D04, National Eye Institute, Bethesda, Md REFERENCES 1. Greaves, D. P., and Perkins, E. S.: Inlluence of the third cranial nerve on intraocular pressure, Br. f. Ophthalmol. 37: 54, von Sallmann, L., and Lowenstein, O.: Responses of intraocular pressure and cutaneous vessels to electric stimulation in the Diencephalon, the Ninth Francis I. Proctor Lecture, Am. J. Ophthalmol. 39: 11, Schmerl, E., and Steinberg, B.: The role of ciliary and superior cervical ganglia in ocular tension, Am. J. Ophthalmol. 32: 947, Arrnaly, M. F.: Studies on intraocular effects of the orbital parasympathetic pathway. III. Effects on steadystate dynamics, Arch. Ophthalmol. 62: 817, Macri, F. J., and Cevario, S. J.: The induction of aqueous humor formation by the use of Ach + eserine, INVEST. OPHTHALMOL. 12: 910, Macri, F. J., and Brown, J. G.: The constrictive action of aeetazolaniicle on the iris arteries of the cat, Arch. Ophthalmol. GG: 570, Macri, F. (., and O'Rourke, ).: Measurements of aqueous humor turnover rates using a gamma probe, Arch. Ophthalmol. S3: 731, l'97o. 8. Oppelt, VV. W., Patlak, C. S., and Rail, D. P.: Effect of certain drugs on cerebrospinal Huid production in the dog, Am. J. Physiol. 20G: 247, Macri, F. J., Cevario, S. J., and Ballintine, E. J.: The arterial pressure dependency of the increased aqueous humor formation induced by Ach + eserine. INVEST. OPHTHALMOL. 13: 153, Morphology of the breakdown of the bloodaqueous barrier in the ciliary processes of the rabbit eye after prostaglandin E... TORCEIR VECCE, ARTHUR II. NEUFELD, AND MARVIN L. SEARS. Prostaglandin E f, administered topically to the rabbit eye, causes disruption of the bloodaqueous barrier resulting in a large increase in the protein content of the aqueous humor. The route of plasma proteins into the aqueous humor was studied with the electron microscope, using horseradish peroxidase as a protein tracer. The tracer penetrated the tight junctions of the nonpigmentcd layer of the ciliary epithelium, filling the lateral intercellular elcfts and staining the internal limiting membrane. These morphological studies confirm the prior physiologic demonstration that, in response to prostaglandin, plasma proteins enter the posterior chamber via the intercellular clefts of the nonpigmentcd epithelium. The purpose of this work is to establish possible mutes by which plasma proteins enter the aqueous humor upon disruption of the bloodaqueous barrier. This barrier, which maintains the noimally low protein content of the aqueous humor, has been defined morphologically in the ciliary processes using the protein tracer, horseradish peroxidase. 1 ' Using inhibitors of the synthesis of prostaglandins, it was shown that the disruption of the barrier that occurs alter certain types of trauma to the eye is, in part, caused by the action ol endogeiiously released prostaglandins. 1 Neufeld and Sears' did experiments studying the site ol the disruption by comparing the relative increases of protein in the anterior and posterior chambers. The findings supported the conclusion that, in the rabbit eye, one site of breakdown of the bloodaqueous barrier in response to prostaglandin Ej is in the epithelium of the ciliary processes. In this paper we wish to report our observations on the route ol horseradish peroxidase through the disrupted barrier after topical administration of prostaglandin E.. to the eyes of rabbits. Methods. Eight male, albino rabbits of two to three kilograms body weight were anesthetized with sodium pentobarbital and given 600 mg. of aspirin rectallv. Five of these were experimental animals, tlire2 were controls. One hour later the animals received 250 nig. of horseradish peroxidase