Cervical sympathetic stimulation in monkeys and the effects on outflow facility and intraocular volume

Transcription

1 Cervical sympathetic stimulation in monkeys and the effects on outflow facility and intraocular volume A study in the East African vervet (Cercopithecus aethiops) William J. Casey The effects of cervical sympathetic stimulation have been studied in monkeys with the use of supramaximal 1 msec, rectangular pulses at 10 per second. Stimulation resulted in a mean decrease in intraocular volume of 5.0 pi (presumably due to intraocular vasoconstriction). The two-level constant pressure infusion method has been used to estimate facility of outflow. A mean decrease in facility of ± fil minr 1 per millimeter Hg was observed on the stimulated side, representing a decrease of 10 to 20 per cent. The effect was not modified by a large systemic dose of atropine (1.0 mg. per kilogram) and could not be blocked by 10 mg. per kilogram of the alpha-adrenergic blocking agent, phentolamine, which blocked the pupillary response and the intraocular vasoconstriction. Experiments with the beta-adrenergic blocking agent, propranolol, were inconclusive. Facility was not significantly changed by section of the preganglionic sympathetic nerve. It has been postulated that the facility change is either due to an effect on the trabecular meshwork or on specialized adrenergic ciliary muscle fibers. A,. drenergic effects on outflow facility have been extensively studied in the rabbit eye. Following superior cervical ganglionectomy, there is a transient, but very large, facility increase. 1 ' 2> 3 This has been From the Department of Physiology, Makerere University College, Kampala, Uganda, and the Department of Pharmacology, University of Uppsala, Sweden. Work done during leave of absence from the Department of Ophthalmology, University of California Medical Center, San Francisco (permanent address of author). This study was supported by United States Public Health Service Grants NB and NB (postdoctoral fellowship). 33 attributed to release of adrenergic transmitter from degenerating nerve endings.- 3 The introduction of adrenergic substances into the rabbit eye has also been shown to result in a substantial facility increase.' 1 ' " G At the present time, the location and the exact nature of the receptor or receptors remain unclear. Little work on adrenergic mechanisms has been done on primates. Clinical studies have indicated that facility is increased in patients treated chronically with topical epinephrine. 7 - s '!) It has been reported that topical noradrenaline and isoprenaline produce a reduction in intraocular pressure similar to that of epinephrine. 10 No facility

2 34 Casey Investigative Ophthalmology February 1966 measurements were made in this study. Since so little is known about adrenergic effects on outflow facility in man, it seemed worthwhile to undertake a study of sympathetic stimulation in monkeys. The present work describes the changes in outflow facility and intraocular volume on unilateral stimulation of the cervical sympathetic. The two-level constant pressure infusion method has been used Materials and methods East African vervet monkeys (Cercopithecus aethiops) of both sexes weighing between 2.0 and 6.3 kilograms were used. The monkeys were placed in a squeeze cage, anesthetized with intravenous pentobarbital (Veterinary Nembutal), 20 to 30 mg. per kilogram body weight, and kept warm with an electric heating pad. Nerve stimulation. A 3 cm. portion of the preganglionic cervical sympathetic nerve was carefully dissected free of adjacent structures, cut caudally, and pulled through an insulated silver wire electrode (interelectrode distance 3 mm.). With the electrode in place, the neck musculature was folded over the electrode and the wound closed with several sutures. Supramaximal square-wave stimuli were used. The stimulus frequency was 10 per second, the stimulus duration was 1 msec. The voltage employed was well above that necessary to give maximal pupillary dilatation. The left sympathetic nerve was stimulated in all experiments except one where the preparation was unsatisfactory on the left and the right side was used (one propranolol experiment). Three parameters were used to verify an intact nerve preparation: (1) Pupillary dilatation. A definite homolateral response was noted on stimulation and at the end of stimulation there was a definite reduction in pupil size. (2) Pilomotor activity. The erection of homolateral temporal scalp hairs was noted on stimulation and in most cases relaxation could be seen on cessation of stimulation. (3) Intraocular volume change. A definite volume decrease could be observed in the stimulated eye and, where studied, a corresponding increase was seen at the end of stimulation. These three parameters were noted in all experiments except those in which the alpha-blocking agent phentolamine was given. This point is discussed below. Infusion method. The two-level constant pressure method used was virtually the same as that described by Barany. 11 ' 12 However, in the present experiments, two needles were inserted into each eye and the infusion and pressure pathways were kept independent. This technique permits neglect of needle resistance in the calculations. A full discussion of this point may be found elsewhere. 13 The anterior chamber of each eye was cannulated with two 27 gauge unbranched needles fired from a needle gun. 1 ' 1 No topical anesthesia was used. One needle was connected by narrow polyethylene tubing to a manifold leading to a strain gauge pressure transducer. The other needle was connected by similar tubing to a manifold leading to a 1 ml. polyethylene container suspended on a force transducer which could be set at different levels above the eye. The weight of the container with its content of infusion fluid was recorded and facility estimated from the difference in rate of inflow between two pressure levels. The lower pressure was set at 2,5 mm. Hg above intraocular pressure. The higher level was 9.4 mm. Hg above the lower level (11.9 mm. Hg above intraocular pressure). The duration of each infusion period was 4 or 5 minutes, alternating between each of the two pressure levels. The infusion fluid used was that described by Barany. 12 Pupil measurements. Measurements of pupil size were made with the use of a millimeter rule held close to the animal's eye and viewed at a distance of 20 cm. No attempt was made to standardize the illumination. Pupil diameters of both the experimental and the control eye were recorded just before the beginning of stimulation, after the stimulator was turned on, just before it was turned off, and immediately after it was turned off. Drug administration. Various drugs were given in an attempt to modify the stimulation effect. These were administered parenterally 25 to 35 minutes before the beginning of stimulation. The drugs given were atropine sulfate, pilocarpine hydrochloride, phentolamine (Regitine, Ciba), and propranolol (Inderal, ICI). Pronethalol (Alderlin, ICI) was first tried as a beta-adrenergic blocking agent but was found to be too toxic in the dosage used (10 mg. per kilogram body weight). Propranolol, a recently introduced betablocking agent, 15 said to be more potent than pronethalol, was used instead and found to be well tolerated in the same dosage. Calculations. Facility is defined as AF/AP. If F is the rate of flow of infusion fluid (in microliters per minute) out of the reservoir and into the eye at any given infusion pressure level, then AF is the difference in rate of flow between the two infusion pressure levels (one set at 2.5 mm. Hg above intraocular pressure and the other at 11.9 mm. Hg above). AP, then, is the difference between the two pressures (9.4 mm. Hg). In the figures, the first average facility value during the stimulation period is at about 12 minutes after stimulation has begun. The second point is 4 to 5 minutes thereafter. The period of stimulation was

3 Volume 5 Number 1 Cervical sympathetic stimulation in monkeys to 25 minutes. The time course for the poststimulation period was the same. Intraocular volume change. Early in the course of this investigation it became apparent that on stimulation there was a sudden influx of fluid into the eye from the infusion fluid reservoir. The experiments were designed to permit quantitation of this change in volume. The reservoir containing the infusion fluid was always left in its upper position when the stimulator was turned on. The record of the continuously falling reservoir weight (calibrated in microliters of infusion fluid) then showed an abrupt downward displacement at the beginning of stimulation. From this displacement, the volume change was calculated. A similar treatment of the change at the point where the stimulator was turned olf gave a measure of the volume increase observed at that time. However, this volume increase was not determined in all experiments. Results Fig. 1 shows average values for outflow facility before and during sympathetic stimulation in 13 monkeys. It can be seen that facility decreased in the experimental eye but not in the control eye after the beginning of stimulation. The mean facility change (AC) in the experimental eye was ± /A min.- 1 per millimeter Hg (P < 0.01), for the control eye ± /x\ min.- 1 per millimeter Hg (mean ± standard error of the mean, n = 13). The AC was determined separately for each eye by calculating the difference between the mean of the two points during stimulation and the mean of the two points before stimulation. The poststlmulation values are not shown in the figure because a poststimulation period was not performed in all of the experiments. In those where it was studied (6 animals), the experimental eye returned to near control level. There was a moderate rise in facility above preperiod levels in both eyes following stimulation. Drug effects. In 11 animals, atropine sulfate, 1.0 mg. per kilogram, was given intramuscularly (nine experiments) or intravenously (two experiments) prior to the beginning of infusion. It was felt worthwhile to see if the stimulation effect could be modified in any way by the elimination of cholinergic tone. It can be seen from Fig. 2 that the effect is quite similar to that in the nonatropinized animals. The mean AC for the experimental eye was ± ^,1 min.- 1 per millimeter Hg (P < 0.01), for the control ± /J min.- 1 per millimeter Hg (mean ± standard error of the mean, n = 11). In four experiments, a study was made of stimulation in animals pretreated with the alpha-adrenergic blocking agent phen- CONTROLO o STIM MIN Fig. 1. Effect of cervical sympathetic stimulation on facility, C. Abscissa: time in minutes counted from beginning of stimulus. n= 13 vervets. Fig. 2. Effect of cervical sympathetic stimulation on facility, C, after 1 mg. per kilogram atropine. Abscissa: time in minutes counted from beginning of stimulus. n= 11 vervets.

4 36 Casey litocstigatioe Ophthalmology February 1966 tolamine (Regitine) 10 mg. per kilogram body weight given intravenously (two experiments ) or intramuscularly (two experiments ). Since the parameters used to judge the nerve preparation (see Methods) are known to be mediated by alpha-adrenergic receptors, a normal stimulation period was run in the animals before phentolamine was given in order to assure that the nerve was intact. After the end of the first stimulation, the drug was given intra- Fig. 3. Effect of cervical sympathetic stimulation on facility, C, after 10 mg. per kilogram phentolamine. Abscissa: time in minutes counted from beginning of stimulus, n = 4 vervets. venously or intramuscularly about 30 minutes before the second stimulation, another preperiod was run, and then stimulation was begun again. The pooled results from the four experiments are shown in Fig. 3. The facility decrease persisted despite alpha blockade (P < 0.05). The mean AC for the experimental eye was ± [x\ min." 1 per millimeter Hg and for the control ± /J min.- 1 per millimeter Hg (mean ± standard error of the mean, n = 4). Fig. 4 shows the pupil and intraocular volume effects of the first stimulation without phentolamine and of the second stimulation after phentolamine. The blockade is evidently effective since both responses have virtually been abolished. The inability of phentolamine to block the facility response* in doses which caused marked block of the pupillary dilatation and the intraocular volume change does not necessarily prove that the facility change is not an alpha effect. It could be that an inhibitorif alpha effect on ciliary muscle tone is more difficult to block than an excitatory effect. The matter has, therefore, been approached in a different way. A beta-adrenergic blocking agent was given and in some cases a background of cho- BEFORE PHENTOLAMINE PUPILS BEFORE DURING VOL CHANGE STIM STIM ON STIM _ * ft r, 1 1 i HJ m^. jul or mm 8 6 A AFTER PHENTOLAMINE PUPILS BEFORE DURING VOL CHANGE STIM STIM ON STIM ft T T _L_, to I Fig. 4. Effects of cervical sympathetic stimulation on pupil size and intraocular volume before and after 10 mg. per kilogram phentolamine. The dark bar is the stimulated eye and the clear bar is the control. Ordinates: pupil size in millimeters and volume change in microliters. A decrease in volume is plotted upward, an increase is downward. The standard error of the mean is indicated for each, n = 4 vervets.

5 Volume 5 Number.1 Cervical sympathetic stimulation in monkeys 37 linergic tone was supplied by a moderate systemic dose of pilocarpine. It was expected that an inhibitory alpha effect would be more easily demonstrable under these conditions. Unfortunately, the results were inconclusive because of a large facility rise in both eyes following the drugs. Fig. 5 shows the results of three experiments where the monkeys were pretreated with the beta-blocking agent propranolol (Inderal), 10 mg. per kilogram body weight given intramuscularly. Facility in the experimental eye did not decrease on stimulation either with respect to the preperiod or with respect to the control. This may be interpreted as a blockade of the effect. However, in the poststimulation period, there was a large rise in facility in both eyes and the experimental eye rose to a level above the control. This could be interpreted to mean that facility in the experimental eye would have been higher in the stimulation period but for the stimulation effect. In other words, stimulation may have had a facility-decreasing effect. Pretreatment with the combination of propranolol, 10 mg. per kilogram body weight intramuscularly, and pilocarpine, 0.5 mg. per kilogram intramuscularly, was performed in another five experiments and the results are shown in Fig. 6. Again, there is no facility drop during stimulation but rather an enormous facility increase in both eyes continuing through both the stimulation and poststimulation periods. Again, the stimulated eye ends up with a facility higher than the control and again the same interpretation could be made regarding the possibility of a masked stimulation effect. Reliability of prolonged stimulation. The question arises as to how effective the nerve preparation was over the course of a continuous stimulation prolonged for 20 to 25 minutes. Fig. 7 shows the effect of stimulation on pupil size for all except the phentolamine experiments (shown in Fig. 4). It can be seen that there is a mydriatic response at the beginning of stimulation and a corresponding reduction MIN Fig. 5. EfFect of cervical sympathetic stimulation on facility, C, after 10 mg. per kilogram propranolol. Abscissa: time in minutes counted from beginning of stimulus, n = 3 velvets.

6 38 Casey Investigative Ophthalmology February MIN Fig. G. Effect of cervical sympathetic stimulation on facility, C, after 0.5 mg. per kilogram pilocarpine and 10 mg. per kilogram propranolol. Abscissa: time in minutes counted from beginning of stimulus, n = 5 velvets. ( STIMULATION i l l ft ft 1^ t 1 ft n = 13 ATROPINE n = 11 PROPRANOLOL Gnd : n M ft ft J. n - T ^H T i ^H T. PILOCARPINE it ftir PROPRANOLOL Fig. 7. Effect of cervical sympathetic stimulation on pupil size. The dark bar is the stimulated eye and the clear bar is the control. The standard error of the mean is indicated for each.

7 Volume 5 Number 1 Cervical sympathetic stimulation in monkeys 39 in pupil size at the end. This indicates the persistence of the stimulation effect throughout. Since observations of pupil changes involve a certain amount of subjectivity, an illustration of the intraocular volume change is shown in Fig. 8. A consistent homolateral volume decrease was noted in all experiments at the beginning of stimulation (except phentolamine treated, see Fig. 4). There is no significant difference among them and the mean volume decrease for the group as a whole was 5.0 jul. The volume change at the end of stimulation was studied in the eleven atropine experiments. A consistent volume increase was noted at that point and Table I shows a comparison between the volume decrease observed at the beginning of stimulation and the volume increase at the end. As an additional check, a brief stimulation was made to assure that the pupil effect was still present at the end of the experiment. The mydriatic effect at the end was observed in all but the phentolamine-treated animals. The effect of preganglionic section. Since the nerve preparation entails preganglionic section, a comparison has been made between the facility in the experimental eye and that in the control eye (mean of two prestimulation values). The mean AC (C of experimental eye - C of control eye) was found to be not significant in either the untreated animals (AC = ± 0.119, n = 13) or in the atropinized animals (AC = ± 0.083, n = 11). Discussion The apparent decrease in intraocular contents consistently observed on stimulation is most likely a blood volume change. However, the contraction of orbital smooth muscle may influence intraocular volume by distorting the globe. An initial abrupt rise in intraocular pressure has been shown to occur in the dog and cat following stimulation of the cervical sympathetic and contraction of orbital smooth muscle. 10 There, the smooth muscle contraction may A V NO DRUG n = 13 ATROPINE n = 11 PROPRANOLOL and PILOCARPINE n = 5 i PROPRANOLOL n=3 1 A- Fig. 8. Effect of cervical sympathetic stimulation on intraocular volume. A decrease in volume is plotted upward, an increase is downward. The standard error of the mean is indicated for each. Table I. Intraocular volume change (AV) occurring at the beginning and at the end of stimulation. Animals pretreated with atropine (n = 11) Stimulated eye Control eye Stimulator on \ Stimulator off ± Volume in microliters expressed as the mean - the standard error of the mean. A negative sign means that fluid passed from the infusion reservoir into the eye. A positive sign means that fluid passed from the eye into the reservoir. have brought about a decrease in the volume of the scleral envelope. It is possible that the orbital smooth muscle effect might work in the other direction depending on the shape of the eye. If the globe were slightly elliptical and the muscle contraction were to move the eye in the direction of a more nearly spherical configuration, an increase in the volume of the scleral envelope would result. Very probably, the orbital muscle effect contributes little to the observed volume change. However, it is worth considering that the net change observed could be increased or decreased by these extraocular influences. It is evident that on stimulation of the eervical sympathetic there is in the homolateral eye a moderate decrease in outflow facility which is statistically significant.

8 40 Casey Investigative Ophthalmology February 1966 What is the nature of this effect and how is it mediated? Three possible sites come to mind. It could be an effect on vasomotor tone in the episcleral and conjunctival vessels, an action on the ciliary muscle, or an effect on the trabecular meshwork. An attempt has been made through pharmacological means to distinguish between the various possibilities. Intense vasoconstriction of the episcleral and conjunctival vessels could conceivably decrease facility of outflow. Such an effect would almost certainly be mediated by alpha receptors and phentolamine would be expected to block it. The facility effect was not blocked by phentolamine while the intraocular volume decrease which must be largely vascular in origin was almost completely blocked. Thus, it does not seem likely that the facility change is due to an effect on vessels. A double innervation of the ciliary muscle has long been postulated. 17 A relaxing effect of sympathomimetic agents on the monkey ciliary muscle has been reported. 1 s Recent histochemical studies on the monkey ciliary muscle, with the use of the fluorescence technique for demonstration of monoaminergic fibers, have confirmed the presence of adrenergic nerves around muscle cells. 19 Sympathetic inhibition of the ciliary muscle could account for a facility decrease. Current concepts of adrenergic mechanisms describe alpha-receptors as innervated and beta-receptors as uninnervated. If it were an inhibitory alpha effect mediated by the release of adrenergic transmitter (noradrenaline) at nerve endings in the muscle, it ought to have been blocked by phentolamine. However, it is conceivable that the dose was not sufficient to block an inhibitory alpha effect. It is possible, of course, that it is an inhibitory beta effect mediated by some agent, as yet unidentified, released from nerve endings. One piece of evidence which strongly points against the acceptance of ciliary muscle inhibition, be it alpha or beta, as an explanation of the stimulation effect is the persistence of the effect after such a large dose of atropine (1.0 mg. per kilogram). Atropine in this dosage, given under identical experimental conditions to monkeys of the same species, has been shown to decrease facility presumably by reducing cholinergic tone.- 0 In the atropinized animals, where ciliary muscle tone should be minimal, an inhibitory mechanism must be relatively ineffectual in increasing resistance. However, the effect was greater, if anything, after atropine. Since an inhibitory effect on ciliary muscle can now reasonably be excluded, the only remaining possible muscle effect might be through the activation of specialized adrenergic ciliary muscle fibers (presumably beta) with a facility-decreasing action. The other alternative is that it acts directly on the trabecular meshwork. At the present time, there are no data to support a choice between these two possibilities. It is difficult to say what part, if any, adrenergic mechanisms play in the regulation of outflow facility. No significant difference was noted between the control eye and the eye with preganglionic section. A similar lack of effect of preganglionic section has been reported in the rabbit eye. 21 It is possible that the situation might be different in the conscious animal. It is quite probable that there are striking species differences in the mechanisms of aqueous outflow regulation. In the rabbit, sympathomimetics increase outflow facility. In the monkey, stimulation of the nerve causes a decrease in facility. How this can be reconciled with the reported facility improvement of patients on chronic epinephrine therapy, 7 ' s - is difficult to say at this time. More work needs to be done on adrenergic mechanisms in the primate. I wish to thank Professor Peter Wright of the Department of Physiology at Makerere for his hospitality, Professor Ernst Barany for invaluable criticism and encouragement, and the Wellcome Trust, London, for animal facilities donated to Professor Wright's department, without which these studies could not have been undertaken.

9 Volume 5 Ninn bar 1 Cervical sympathetic stimulation in monkeys 41 REFERENCES 1. Langham, M. E., and Taylor, C. B.: The influence of superior cervical ganglionectomy on intraocular dynamics, J. Physiol. 152:447, Sears, M. L., and Barany, E. H.: Outflow resistance and adrenergic mechanisms, Arch. Ophth. 64:839, Barany, E. H.: Transient increase in outflow facility after superior cervical ganglionectomy in rabbits, Arch. Ophth. 67:303, Eakins, K. E.: The effect of intravitreous injections of norepinephiine, epinephrine and isoproterenol on the intraocular pressure and aqueous humor dynamics of rabbit eyes, j. Pharmacol. & Exper. Therap. 140:79, Sears, M. L., and Sherk, T. E.: The trabecular effect of noradrenaline in the rabbit eye, INVEST. OPHTH. 3:157, Gnadinger, M., and Barany, E.: Die Wirkung der /3-adrenergischen Substanz Isoprenlin auf die AusHuss-Fazilitat des Kaninchenauges, von Craefes Arch. Ophth. 167:483, Garner, L. L., Johnstone, W. VV., Ballintine, E. J., and Carrol, M. E.: Effects of 2 per cent levo-rotatoiy epinephrine on the intraocular pressure of the glaucomatous eye, Arch. Ophth. 62:230, Ballintine, E. J.: The anticholinesterases in glaucoma, in Newell, F. W., editor: Transactions of the Fifth Conference, New York, 1960, Josiah Macy, Jr. Foundation Publications, p Becker, B., Pettit, T. H., and Cay, A. J.: Topical epinephrine therapy of open-angle glaucoma, Arch. Ophth. 66:219, Weekers, R., Delmarcelle, Y., and Custin, J.: Treatment of ocular hypertension by adrenalin and diverse sympathomimetic amines, Am. J. Ophth. 40:666, Barany, E.: The mode of action of pilocarpine on outflow resistance in the eye of a primate (Cercopithecus aethiops), INVEST. OPHTH. 1: 712, Barany, E. H.: Simultaneous measurement of changing intraocular pressure and outflow facility in the vervet monkey by constant pressure infusion, INVEST. OPHTH. 3:135, Barany, E.: To be published. 14. Sears, M. L.: Miosis and intraocular pressure changes during manometry, Arch. Ophth. 63:707, Black, J. W., Crowther, A. F., Shanks, R. C, Smith, L. H., and Dornhorst, A. C: A new adrenergic beta-receptor antagonist, Lancet 1:1080, Henderson, E. E., and Starling, E. H.: The influence of changes in the intraocular circulation on the intraocular pressure, }. Physiol. 31:305, Duke-Elder, S., and Wybar, K. C: System of ophthalmology': The anatomy of the visual system, London, 1951, Kimpton, vol. II, p van Alphen, G. \V\, Robinette, S. L. 3 and Macri, F. J.: Drug effects on ciliary muscle and choroid preparations in vitro, Arch. Ophth. 68:81, Ehinger, B.: In press. 20. Barany, E.: Proceedings of Glaucoma Research Conference, Am. J. Ophth. 56:470, Linner, E., and Prijot, E.: Cervical sympa thetic ganglionectomy and aqueous flow, Arch. Ophth. 54:831, 1955.