Changes in outflow facility and content of norepinephrine in iris and ciliary processes of albino rabbits after cervical ganglionectomy

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1 Changes in outflow facility and content of norepinephrine in iris and ciliary processes of albino rabbits after cervical ganglionectomy M. Sears, K. Mizuno, C. Cintron, A. Alter, and T. Sherk Changes in outflow facility and in the content of norepinephrine in the iris and ciliary processes of the rabbit eye have been followed at early and late intervals after cervical ganglionectomy. Outflow facility was measured in vivo with a constant rate infusion technique. Norepinephrine tissue levels ivere determined by fluorescence spectrometry. The results indicate that the "early" increase in outflow facility after ganglionectomy is associated with a rapid decrease in the norepinephrine content of the iris and ciliary processes. The "late" decrease in outflow facility persists as long as the iris and ciliary processes are depleted of norepinephrine. A return to more normal levels of outflow facility occurs as norepinephrine levels begin to reappear in the tissues. The relationship between these later-than-2-hour changes in facility and the changes in tissue concentration of norepinephrine is discussed. Some support for this relationship was derived from experiments xoith bilateral ganglionectomy. Bilateral ganglionectomy prolongs the duration of the facility decrease and similarly induces a depletion of tissue norepinephrine of greater duration than that accompanying unilateral denervation. 0,ne approach to the study of the influence of the sympathetic nervous system upon intraocular pressure has been the technique of denervation. It is known that 2 hours after cervical ganglionectomy in rabbits the intraocular pressure falls on the From the Department of Surgery, Yale University School of Medicine, New Haven, Conn. This work was presented in part at the National Institute of Health sponsored Glaucoma Research Conference, Del Monte, Calif., September, This work was supported in part by grants from the United States Public Health Service NB 0381, Research to Prevent Blindness, Inc., and Connecticut Lions Eye Research Foundation, Inc. 312 ipsilateral side. 1 The decrease in intraocular pressure was related to an increase in outflow facility. 2 ' 3 A study of this increase in outflow facility was made with adrenergic blocking agents 3 and its transient nature described and discussed.' 1 Several days after ganglionectomy the outflow facility was found to be supersensitive to adrenergic substances, 5 and, for the first time, a pharmacologic agent, norepinephrine, could be shown to act on outflow facility via the anterior chamber. 0 Further investigations correlated alterations in outflow facility after sympathetic denervation with changes in the content of norepinephrine in iris and ciliary processes. ' s The current work extends these observations.

2 Volume 5 Number 3 Norepinephrine changes after cervical ganglionectomy 313 Methods Intraocular pressure and outflow facility. Albino rabbits, mostly males, weighing between 2 and 3 kilograms, were maintained on a diet of pellets and water ad lib. Under anesthesia with intravenous thiopental sodium, more than 0 animals were subjected to unilateral right cervical ganglionectomy and about 50 to bilateral ganglionectomy. Intraocular pressure and outflow facility were measured in the manner previously described in detail, 9 after unilateral ganglionectomy, at these intervals: 12, 16, 20, and 2 hours, 3 days, 1, 2, and weeks, and, after bilateral ganglionectomy, at 1 week, and 1, 2, and 3 months. The rabbits were anesthetized with intravenous Urethane (ethyl carbamate, Merck) and both eyes cannulated by means of a spring and trigger device. Intraocular pressures were recorded with a Sanborn electromanometer system and facilities of aqueous outflow determined by measuring the steady state increase in intraocular pressure caused by infusion of isotonic saline at 2 y\ per minute simultaneously into the anterior chamber of both eyes. Preparation of tissue extracts. Two comparable groups of rabbits were similarly ganglionectomized and, at appropriate intervals, anesthetized and bilaterally enucleated. The eyes were immediately placed on ice, and blood and excess tissue removed. A scleral incision was made 3 mm. posterior to the limbus, and the irises and ciliary processes teased from the eye at the iris root, blotted on filter paper, and weighed on a torsion balance. Individual tissue specimens (about 50 mg.) were then minced with scissors, transferred to a glass homogenizer containing 1 ml. of cold 0.N perchloric acid, and homogenized at 0 C. The homogenate was allowed to stand for 30 minutes in ice with occasional stirring and then centrifuged at about 9,000 g for minutes. The supernatant solution was then poured into a ml. graduated centrifuge tube. The residue were re-extracted twice with additional perchloric acid and added to the supernatant fluid, which was stored overnight at -20 C. After the extracts were thawed, 5 ml. of 1 per cent ethylene diamine tetraacetate (EDTA) was added. Then N potassium carbonate was added dropwise with constant stirring to bring the ph to about.5. The precipitated potassium perchlorate was spun down at 0 C. and washed twice with 1 ml. and 5 ml. of cold demineralized distilled water and the washings added to the extract. The supernatant solution from the potassium perchlorate precipitation was added to the washings and the final volume adjusted to ml. with distilled water. None of the tissue samples was ever pooled except in the case of the three day and one week denervated eyes when no norepinephrine could be detected in individual tissue specimens. Analyses for catecholamines. Norepinephrine and epinephrine in tissue extracts were determined fluorimetrically after oxidation and subsequent rearrangement to the noradrenolutin and adrenolutin by a slight modification of the method of Bertler and associates. 11 To 2 ml. of the extract at about ph 6.5, 1.5 ml. of 0.1M phosphate buffer ph 6.5, 0.1 ml. of zinc sulfate solution (0.25 per cent), and 2 ml. of distilled water were added. Oxidation was accomplished by addition of 0.1 ml. of 0.25 per cent potassium ferricyanide. After 2 minutes, 1.0 ml. of a mixture of 9 parts of 5N sodium hydroxide containing EDTA 2.5 Gm. per 0 ml. and 1 part of a 2 per cent solution of ascorbic acid was added, and the total volume was finally adjusted to ml. with distilled water. Ten minutes later samples were pipetted into cuvettes, 2. by 0.25 inches in size, and examined in the high sensitivity conversion kit of the Turner Fluorometer No. 1. Samples were activated first at 05 mi* and then at 36 m/i, and readings of fluorescence taken at 530 m/i. The relative intensities of fluorescence per unit concentration were determined with standards in preliminary experiments and were found to be: adrenolutin on 05 rriju activation 1.0; adrenolutin on 36 m/i activation 0.8; noradrenolutin on 05 m/i activation 1.0; noradrenolutin on 36 mfi activation 0.2. From the intensity of fluorescence for each of these substances and the total fluoresence of the mixture at both activation wavelengths, it was possible to calculate the concentration of each in a mixture using simultaneous equations. In no instance of preliminary runs with control tissue specimens did the epinephrine content exceed per cent of the total catecholamine value. Canglionectomy did not appear to affect epinephrine levels, a finding noted for other tissues as well. 12 In some instances, however, normal values for epinephrine content approached the level of sensitivity of the method, i.e., 5 to utig amine. Difficulties inherent in analyses for small amounts of epinephrine in the presence of norepinephrine have been discussed elsewhere. 13 Therefore, importance was not attached to the results obtained for epinephrine analyses of the extracts and these have not been reported, although account was taken of these results for calculation of the final norepinephrine concentrations. Tissue blanks and reagent blanks were treated in the same way as the extracts. Standards of norepinephrine and epinephrine (1-arterenolbitartrate and 1-epinephrine, Calbiochem, Inc., Los Angeles, Calif.) as well as reagent blanks were run in parallel with the samples. In order to determine the presence of interfering impurities, a known amount of norepinephrine was added to one portion of the extract and treated in the same way as the sample (internal standard). The tissue

3 31 Sears, Mizuno, Cintron, Alter, and Sherk Investigative Ophthalmology June 1966 Table I. Intraocular pressure and outflow facility after unilateral cervical ganglionectomy* Time ajter RCGf Controls 12 1 hours clays 1 week 2 No. of rabbits Pressure 1.1 ± ± ± ± ± ± ± 0, ± ± ± ± ± ± ± ± ± ± ± 0.21 ± 0.19 ± 0.56 ± 0.63 ± 9 ± 0.15 ± 0.16 ± 0.19 ± Facility O ± 0.2 ± 0.1 ± 0.20 ± 0.20 ± 0.16 ± 0.23 ± 0.21 ± 0.19 ±, Facility ratio, /t 0.99 ± ± ± ± ± ± O ± ± ± 6 "All values are expressed as mean ± s.e.m., pressure in mm. Hg, facility in /i\ per min. mm. Hg. ] Right cervical ganglionectomy. tmean of the individual ratios of the facility value for the right eye divided by the facility value for the left eye. Probability, p, that a ratio is significantly different from unity, was determined by using the Student t-test. V Table II. Content of norepinephrine in iris and ciliaiy processes after cervical ganglionectomy Time Controls 8 hoursf days 1 week 2 12 weeks f 8 12 No. of Eyes (W/G) 2.8 ± ± ± ± ± ± ± ± ± ± 0.2 (W/G) 2.6 ± ± ± 0, ± ± ± ± ± ± 0. 2,30 ± ± 0.2 ± 0.2 Mean of individual differences (R-L) (ng/g) -0., ± ± ± ± ± ± 0.3-2,1 ± ± ± 0. - ± 0.8 /* 1.15 ± ± ± ± ± ± ± ± 0. V < < < < < <5 Mean of the individual ratios of the norepinephrine content in the right iris and ciliary processes divided by the left. Probability, \>, that a given ratio is significantly different from unity, was determined by the Student t test. All values recorded as mean ± s.c.m. f After right cervical ganglionectomy. Bilateral cervical ganglionectomy. Table III. Outflow facility during chronic sympathetic denervation* Time Controls () 1 week () weeks (2) 8 weeks (12) 12 weeks (8) P 16.1 it dt it it it 0.95 C 0.21 ± ± 9 ± ± 0.21 ± 3 Bilateral cervical ganglionectomy. Units as in Table I. P 16,5 ± ± ± ± 18.5 ± c 0.20 ± ± 0.11 ± ± 0.20 ±

4 Volume 5 Number 3 Norepinephrine changes after cervical ganglionectomy NORADNAUN R /j_ ± sem A OUTFLOW FACILITY R /L±sem RIGHT CERVICAL GANGLIONECTOMY hrs 3 days I week 2 wks wks I2wk» Fig. J. Outflow facility and iridial norepinephrine after unilateral cervical ganglionectomy. concentrations of norepinephrine are expressed in terms of the free base. Results Mean values for intraocular pressure and outflow facility after unilateral cervical ganglionectomy are shown in Table I. When 20 hours had elapsed, a reduction in intraocular pressure associated with significant increase in outflow facility occurred. At 2 hours a further slight increase in outflow facility was noted. Three days after unilateral ganglionectomy, outflow facility was significantly reduced on the denervated side, and a significant reduction persisted at one week. Two weeks after ganglionectomy a slight decrease in outflow facility was found, and by four weeks outflow facility became equal on right and left sides. Fig. 1 shows the relationship between norepinephrine content of iris and ciliary processes and outflow facility before, and at several intervals after, right cervical ganglionectomy. For convenient comparison, the average ratio of the facility and norepinephrine content of the right side to the left side obtained for each animal has been taken from Table I, and plotted on the ordinate. Twenty and 2 hours after denervation a significant decrease in tissue norepinephrine occurred. At three days and one week only trace amounts of norepinephrine were found in the iris preparations. The time course of this decrease is compatible with that described by several investigators for other organs 12-1I " 1G and for the rabbit iris. s Two and four weeks later small amounts of norepinephrine were found in the tissues, although differences between the normally innervated and denervated sides appeared to persist (Table II). Table III shows the effect of bilateral ganglionectomy on outflow facility and intraocular pressure. Marked reductions in outflow facility were found after one week and were noted to persist for at least two months after denervation. At three months the outflow facility appeared to return to normal in the bilateral ganglionectomized animals, although at this time iridial nor-

5 316 Sears, Mizuno, Cintron, Alter, and Sherk Investigative Ophthalmology June 1966 epinephrine levels had still not reached 25 per cent of normal values (Table II). Similar prolonged decreases have been obtained after complete postsynaptic denervation of other organs. ' 18 Discussion Barany has proposed that the transient increase in outflow facility occurring after ganglionectomy is the result of the action upon the trabecular meshwork of an adrenergic agent released from the iris or ciliary processes into the aqueous humor. This hypothesis was based on blocking experiments with intracameral phenoxybenzamine and on the observation that systemic pretreatment with Reserpine prevented the facility increase. Barany observed that ganglionectomy had no early effects, i.e., within 5 minutes, three hours, or six hours, on intraocular pressure or facility. The present study indicates that no drop in iridial norepinephrine occurs until between 16 and 20 hours after ganglionectomy. In a few experiments a transient reduction in norepinephrine content at 30 minutes was found and may be related to mechanical stimulation of the sympathetic chain during ganglionectomy. At intervals within 8 hours after manipulation, repletion of norepinephrine stores may still occur, 13 and at this time no significant difference between normally innervated and denervated sides was found. At 20 hours the first significant reduction in norepinephrine content was noted and increases in outflow facility paralleled this change (Fig. 1). The presence of normal levels of iridial norepinephrine during the early hours after ganglionectomy correlates with the absence of any effect on outflow facility at this time, as the later (20 hours) decrease in (release of) iridial norepinephrine does with the demonstrated increase in facility. Evidence for actual release of catecholamines into the aqueous humor after cervical ganglionectomy has been obtained in studies employing tritiated norepinephrine (unpublished observations). Another sign of the release of iridial catecholamines into the aqueous humor was noted upon observation of the pupils. A rapid, almost immediate, miosis occurred after ganglionectomy. Then, between 16 and 20 hours, 90 per cent of the animals exhibited an ipsilateral mydriasis which lasted for an hour or two and then disappeared. Miosis again supervened and lasted for weeks. The transient mydriasis, like the transient ganglionectomy effect, occurs at a time when catecholamines are released from iris and ciliary processes, and now act via the aqueous humor. The early "ganglionectomy effect" related to the release of norepinephrine contrasts with the later effect of denervation when no iridial norepinephrine is available for release and subsequent action on the trabecular meshwork (Fig. 1). Now outflow facility is reduced (Tables I and III). This finding, taken together with the restoration of normal values for outflow facility as iridial norepinephrine begins to reappear (Fig. 1), would imply that, under circumstances of normal innervation, a continuous liberation of tissue catecholamines into the aqueous humor acts to keep outflow facility up. The observation that dibenamine decreases outflow facility under conditions of normal adrenergic innervation 2 also supports this conclusion. Finally, this hypothesis about aqueous outflow and adrenergic mechanisms fits with current concepts for many autonomic effector organs, which are influenced by tonic activity under resting conditions. 19 There is the possibility that ganglionectomy induces changes in the chemical composition of the aqueous humor which are responsible for the late facility decrease. Excess protein related to an increase in permeability after ganglionectomy might diminish outflow facility. To examine this hypothesis, several animals were subjected to unilateral paracentesis. Then, a few days and one week later, outflow facility in both eyes was determined. No difference could be found between facility values for right and left eyes of individual animals. Although it is still possible that chemical

6 Volume 5 Number 3 Norepinephrine changes after cervical ganglionectomy 31 changes in the aqueous humor, other than an absence of norepinephrine, may in some way explain the late decrease in outflow facility after unilateral ganglionectomy, it is interesting to consider the more pronounced and prolonged reduction of outflow facility found after bilateral ganglionectomy (Table III). This procedure must denervate the rabbit eye more completely than unilateral removal of the cervical ganglion (Table II). The possibility of bilateral crossed sympathetic innervation to the rabbit eye, either directly or via intermediate accessoiy ganglia, was previously considered in light of the demonstration of enhanced outflow supersensitivity to norepinephrine after bilateral, as compared with unilateral, ganglionectomy. 0 In Holland's histologic studies of nerve regeneration six weeks after unilateral ganglionectomy, although the iris was not included, a study of the trabeculum revealed a relative preservation of the overall density of trabecular innervation.' 0 The role of accessory ganglia in the innervation of the rabbit eye was offered as an explanation for the finding of a relatively intact innervation. Similar reasoning may explain the more prolonged decrease in outflow facility noted after bilateral ganglionectomy as compared with unilateral ganglionectomy. Under conditions of persisting accessory ganglia (supplied by crossed innervation), an early postganglionic pathway could be established if functional contact with these cells is made by collateral sprouts. 21 This mechanism may account for the more rapid recovery of outflow facility after unilateral as compared with bilateral denervation. It probably also explains the effect of nicotine in producing mydriasis after unilateral cervical ganglionectomy in the cat. 22 The inference from these studies of a small bilateral contribution to the sympathetic innervation of the rabbit eye and from observations of the restoration of a tonic facility effect is not incompatible with the observed lack of a contralateral decrease in iridial norepinephrine after unilateral ganglionectomy (Table II). In this regard, our findings are in agreement with other rabbit studies, s but are not in accord with Burn and Rand's 23 observations for the cat. Maximal supersensitivity is apparently related directly to the totality of denervation. Similarly, the reappearance of tonic activity associated with low levels of norepinephrine is compatible with partial reinnervation and the persistence of some degree of supersensitivity denervation. Hence small numbers of regenerating or persistent fibers may influence these properties significantly, but contribute only slightly to the total content of norepinephrine in a tissue with a considerable concentration. FENCES 1. Linner, E., and Prijot, E.: Cervical sympathetic ganglionectomy and aqueous flow, Arch. Ophth. 5: 831, Sears, M. L., and Barany, E. H.: Outflow resistance and adrenergic mechanisms, Arch. Ophth. 6: 839, Langham, M. E., and Taylor, C. B.: The influence of superior ganglionectomy on the intraocular dynamics, J. Physiol. 152:, Barany, E. H.: Transient increase in outflow facility after superior cervical ganglionectomy in rabbits, Arch. Ophth. 6: 303, Sears, M. L., and Sherk, T. E.: Supersensitivity of aqueous outflow resistance in rabbits after sympathetic denervation, Nature 19: 38, Sears, M. L., and Sherk, T. E.: The trabecular eflect of noradrenalin in. the rabbit eye. INVEST. OPHTH. 3: 15, Sears, M. L.: Contribution to glaucoma research conference, National Institutes of Neurological Diseases and Blindness, Del Monte, Calif. Am. J. Ophth. 55: 821, Eakins, K. E., and Eakins, H. M. T.: Adrenergic mechanisms and the outflow of aqueous humor from the rabbit eye, J. Pharmacol. & Exper. Therap. 1: 60, Sears, M. L.: Outflow resistance of the rabbit eye: technique and effects of acetazolamide, Arch. Ophth. 6: 823, Sears, M. L.: Miosis and intraocular pressure changes during manometry in mechanically irritated rabbit eyes studied with improved manometric technique, Arch. Ophth. 63: 0, Bertler, A., Carlsson, A., and Rosengren, E.: A method for the fluorimetric determination

7 318 Sears, Mizuno, Cintron, Alter, and Sherk Investigative Ophthalmology June 1966 of adrenalin and noradrenalin in tissues, Acta physiol. scandinav. : 23, Benmiloud, M., and Euler, U.S. v.: Effects of Bretylium, Reserpine, Guanethidine, and Sympathetic denervation on the noradrenaline content of the rat submaxillary gland, Acta physiol. Scandinav. 59: 3, Euler, U. S. v., and Ryd, C: Effect of sympathetic denervation and adrenalectomy of the catecholamine content of the rat submaxillary gland, Acta physiol. scandinav. 59: 62, Weiner, N., Perkins, M., and Sidman, R. L.: Effect of Reserpine on noradrenaline content of innervated and denervated brown adipose tissue of the rat, Nature (Lond.) 193: 13, Falck, B.: Observations on the possibilities of the cellular localization of monoamines by a fluorescence method, Acta physiol. scandinav. 56: Suppl Kirpekar, S. M., Cervoni, P., and Furchgott, R. F.: Catecholamine content of the cat nictitating membrane following procedures sensitizing it to norepinephrine, J. Pharmacol. & Exper. Therap. 135: 180, Euler, U.S.v., and Purkhold, A.: Effect of sympathetic denervation on the noradrenaline and adrenaline content of the spleen, kidney, and salivary glands in the sheep, Acta physiol. scandinav. 2: 212, Stromblad, B. C. R., and Nickerson, M.: Accumulation of epinephrine and norepinephrine by some rat tissues, J. Pharmacol. 13: 15, Hillarp, N.-A.: Peripheral autonomic mechanisms, in Handbook of physiology, Sect. I, Vol. 2, Washington, D. C, 1960, American Physiological Society. 20. Holland, M. G., von Sallman, L., and Collins, E. M.: A study of the innervation of the chamber angle; the origin of trabecular axons revealed by degeneration experiments, Am. J. Ophth. (Part II): 206, Murray, J. G., and Thompson, J. W.: The occurrence and function of collateral sprouting in the sympathetic nervous system of the cat, J. Physiol. 135: 133, Atkinson, R. A., Witt, D., and Long, J. D.: Mechanism of the sympathomimetic response of the cat's iris to nicotine, T. Pharmacol. & Exper. Therap. 1: 12, Burn, J. H., and Rand, M. J.: The cause of the supersensitivity of the smooth muscle to noradrenaline after sympathetic degeneration, J. Physiol. 1: 135, 1959.