ATP Is Released Into the Rabbit Eye by Antidromic Stimulation of the Trigeminal Nerve

ATP is released into the rabbit eye by antidromic stimulation of the trigeminal nerve

Eugenio Maul and Marvin Sears

Antidromic stimulation of the trigeminal nerve produces an irritative response in the rabbit eye characterized by ipsilateral miosis, hyperemia, elevated intraocular pressure, and a disruption of the blood-aqueous barrier. The latter is a bilateral effect. The mediator or mediators involved in this response of the eye are unknown. Increased ATP levels in aqueous humor could be found after trigeminal stimulation. Treatment of rabbits with dipyridamole further increased ATP levels in aqueous humor after stimulation, confirming the findings of Holton that stimulation of sensory nerves causes a release of ATP. Intravitreal injections of ATP could not reproduce the ocular irritative response; however, an iridial hyperemia of long latency and an increase in aqueous humor protein levels were produced. The mechanism of this part of the reaction requires further study.

Key words: adenosine triphosphate, trigeminal nerve, aqueous humor, antidromic stimulation, irritative response, ocular hyperemia, dipyridamole

Antidromic stimulation of the trigeminal mitter receptor mechanism.4 The substance nerve produces an irritative response in the or substances released by trigeminal nerve rabbit eye characterized by ipsilateral miosis, stimulation have not been identified. The hyperemia of the iris and conjunctiva, in- purpose of this study was to learn whether traocular hypertension, and a disruption of adenosine triphosphate (ATP) is released into the blood-aqueous, barrier.u 2 This last effect the rabbit eye by trigeminal nerve endings is also present in the contralateral eye.3 after antidromic stimulation and whether The irritative response produced by stimu- ATP could stimulate an irritative response. lation of this neural pathway suggested that the effects were the consequence of a trans- Material and methods Animals. New Zealand, male, albino rabbits From the Department of Ophthalmology and Visual Sci- weighing 2 to 3 kg were used. When required, ence, Yale University School of Medicine, New Ha- general anesthesia was induced with sodium pen- ven, Conn. tobarbital, 15 mg/kg intravenously. This study was supported in part by U.S. Public Health Experimental groups. The trigeminal nerve was Service EY-00237, the Connecticut Lions Eye Re- exposed and mechanically stimulated as previ- search Foundation, Inc., and New Eyes for the ously described.3 One, 5, 10, and 20 min after Needy, Inc. Dr. Maul received support from New stimulation, the aqueous humor of both eyes was Eyes for the Needy, Inc., during the tenure of his obtained by paracentesis and set on crushed ice. glaucoma fellowship. Submitted for publication Jan. 16, 1978. Animals with exposure of the trigeminal nerve but Reprint requests: Dr. Marvin Sears, Department of not stimulated served as controls. Aqueous humor Ophthalmology and Visual Science, Yale University of anesthetized unstimulated rabbits and normal School of Medicine, 333 Cedar St., New Haven, rabbits under topical anesthesia was obtained at Conn. 06510. the same time. ATP concentration was deter-

Downloaded from iovs.arvojournals.org on 09/30/2021 Volume 18 Number 3 ATP released by antidromic stimulation 257

mined in all aqueous humor samples within min- final concentrations of 10"8M, 10"6M, and 10-*M. utes of finishing the experiment. The pupillary diameter was measured with a Six rabbits were pretreated with 250 £ig/kg in- caliper after addition of ATP to the bath. At the travenous dipyridamole 30 minutes before trigem- end of the experiment acetylcholine was added to inal stimulation. Three rabbits were treated with two and norepinephrine to the remaining two intraperitoneal dipyridamole, 1 mg/kg, divided preparations, and pupil diameter was again re- into two doses, administered 3 and 1 hr before corded. the nerve stimulation. (Dipyridamole was kindly Intravitreal administration of nucleotides. Ten supplied by Boehringer Ingelheim, N. Y.) Aque- microliters of a solution containing 28 nmol ATP, ous humor of these last two groups was obtained adenosine diphosphate (ADP), or adenosine mon- by paracentesis 1 min after stimulation and as- ophosphate (AMP) were administered to three sayed for ATP concentration. groups of rabbits. The solution was injected with a ATP measurement in neural tissue. Normal 30-gauge needle through the pars plana into the rabbits were sacrificed with an overdose of sodium vitreous under topical anesthesia. Ten microliters pentobarbital. The jugular veins were sectioned of saline were injected to the contralateral eye, and the head of the animal was then washed out of which served as a control. The animals were ob- blood with ice-cold saline perfusion through the served in a single-blind fashion for a 36 hr period; carotid arteries. The trigeminal nerve and Gasse- the pupil diameter and iris conjunctival hyperemia rian ganglion were excised and wet weighed sepa- were clinically recorded. Aqueous humor was ob- rately. The tissue pieces were mechanically ho- tained at 30 min and 8, 18, 24, and 36 hr after drug mogenized in ice-cold water, boiled for 10 min, administration, and protein concentration deter- then centrifuged for 15 min at — 10° C and mined with refractometry of a 50 fx\ sample.4 800 X g, in a Sorvall centrifuge. The supernatant Rabbits after paracentesis were eliminated from was assayed for ATP concentration. the group under observation. ATP assay. ATP concentration in the test sam- Statistical analysis. The significance of the re- ples was determined with the firefly (Photinus sults was calculated with the t test for sample spiralis) luciferin-lucinferase system. The samples means or, where specified, with the modified were boiled for the recovery of ATP because the Wilcoxon-White test for samples of different size.7 enzyme is inhibited by a number of substances, All data are given as the mean ± standard error of including various quenching agents.1 The light the mean. rate was measured with a Packard Tricarb liquid scintillation spectrometer at tritium setting, 65% Results gain, 3 sec after mixture of the test sample with ATP in aqueous humor of normal rabbits. the crude luciferase preparation. A standard curve Fourteen samples were assayed: the ATP for ATP from 2 to 50 pM was obtained with each present was 4.50 ± 0.7(14) nmol/L. There ATP assay. was no significant difference in ATP between In addition, internal standards were used, the test sample was divided into two halves, a known paired samples of aqueous humor nor in that amount of ATP was added to one half before boil- of normal aqueous humor obtained from rab- ing, and the same amount was added to the other bits under topical or general anesthesia. half after boiling. ATP in aqueous humor of rabbits with uni- ATP concentration in whole blood was deter- lateral stimulation of the trigeminal nerve. mined in eight rabbits. ATP measurements were When the trigeminal nerve of one side was also done in aqueous humor of four animals 1 min stimulated the irritative response previously after disruption of the lens with a needle through reported could be observed. ATP concentra- the pars plana. tion in aqueous humor measured 1 min after Effect of ATP on the iris preparation. Four stimulation was higher than in controls. In- irises of normal rabbits were mounted between terestingly, ATP of the contralateral side was two plexiglass rings like a diaphragm and set into a 10 ml chamber at 25° C filled with buffered solu- also higher than in controls. The mean for tion according to the method of Farnebo and controls was 3.8 ± 0.7 nmoles/L (11). One Hamberger,6 and oxygen 95% was bubbled into minute after stimulation the aqueous humor the solution. The pupil was allowed to stabilize for of the stimulated side was 16.2 ± 3 nmol/L a 30 min period, then ATP was added to reach (15) in the treated side (p < 0.01) and 14.7 ±

Downloaded from iovs.arvojournals.org on 09/30/2021 Invest. Ophthalmol. Visual Sci. 258 Maul and Sears March 1979

n=l5 Table I. ATP content of neural tissue 20 n=l5 Trigeminal nerve Gasserian ganglion E n=6 (fig/gm-) 5 10 37.7 31 28.5 27.7 C ST CL ST CL ST CL ST CL 37.7 19.4 I 5 10 20 MIN. AFTER STIMULATION Fig. 1. ATP concentration in aqueous humor 3 (15) in the contralateral side. The ATP of (nmol/L) of the control (C), stimulated side (ST), aqueous humor 5, 10, and 20 min after stimu- and contralateral side (CL) 1, 5, 10, and 20 min- lation was significantly lower (Fig. 1). utes after unilateral stimulation of the trigeminal ATP in aqueous humor of rabbits pre- nerve. Mean ± S.E.M. treated with dipyridamole after unilateral n«3 stimulation of the trigeminal nerve. When in- travenous dipyridamole, 250 /u-g/kg, was

n=3 administered 30 min before stimulation, the 40 i- ATP level in aqueous humor of the stimulated side was 10.4 ± 5 nmol/L (6), and that 30 of the contralateral side was 8.85 ± 3.7 (6).

E n°l5 These measurements were not different from n=l5 .5 20 n = 6 those in the stimulated group that did not a. T n«6 < receive the drug. When dipyridamole was 10 administered intraperitoneally, 1 mg/kg, ATP 1 min after stimulation was 36.9 ± 12 nmol/L (3) in the stimulated side and 24.8 ± ST CL 3T CL ST CL 14 (3) in the contralateral side, values sig- C (DIP250>ifl/kg) (DIP lOOO^fl/kg) nificantly higher than in the stimulated group TRIGEMINAL STIMULATION (Fig. 2) without dipyridamole treatment Fig. 2. ATP concentration in aqueous humor of (p < 0.01, Wilcoxon-White test). stimulated (ST) and of contralateral (CL) side 1 ATP content of trigeminal nerve and gan- min after stimulation of the trigeminal nerve in glion. In four nerve pieces and in two animals without treatment (DIP 0) and in animals Gasserian ganglions the ATP content was de- pretreated with dipyridamole (DIP) 250 and 1000 termined approximately 30 min after the /u,g/kg. Mean ± S.E.M. animal was sacrificed. The values are given in Table I in micrograms of ATP per gram wet

6 weighed tissue. NE IO" M Effect of various ATP doses on the iris diaphragm preparation. ATP had no effect in vitro on the pupil diameter. In four iris prep- 4 6 arations ATP concentration reached 10~ M %1-Ach IO" M without producing a change in the pupillary diameter. The preparation responded appro- priately to 10~6M acetylcholine and norepinephrine at the end of the experiment (Fig. 3). Fig. 3. The pupillary diameter of four iris dia- Effect of ATP, ADP, and AMP on the phragm preparations after adding ATP to reach rabbit eye after intravitreal injection. Thirty 10"8M, 10"6M, and 10~4M, and after adding nor- minutes after intravitreal injection of ATP no epinephrine (NE) and acetylcholine (Ach) to the significant change in the clinical aspect of the bath. eye could be observed. The protein contents

Downloaded from iovs.arvojournals.org on 09/30/2021 Volume 18 Number 3 ATP released by antidromic stimulation 259

600

2 400 n=5 n=5 200 n = 5 n=5 i h 18 24 36 TIME ( hrs) Fig. 4. Protein concentration in aqueous humor (mg/dl) at 30 min and 8, 18, 24, and 36 hr after intravitreal administration of 28 nm of ATP to the treated side (A) and saline to the control side (•). Mean ± S.E.M.

n=6

1200 - i

1000 -

15 800 z n = 6 Z o 600

X 6 i L 400 •{ 200 n = 6

~i 3 1 • * 18 24 36 TIME (hrs) Fig. 5. Protein concentration in aqueous humor (mg/dl) at 30 min and 8, 18, and 24 hr after intravitreal administration of 28 nmol ADP to the treated side (A) and saline to the control side (•). Mean ± S.E.M.

of aqueous humor of the treated and un- protein in the aqueous humor diminished treated sides were 130 ± 48 mg/dl (5) and progressively over the 36 hr period of obser- 130 ± 27 (5), respectively. Eight hours after vation (Fig. 4). The pupil remained un- injection no change could be observed; the changed at all times. protein content of the treated and untreated After ADP was injected into the vitreous, a sides was 50 mg/dl. Eighteen hours after in- similar picture could be observed: no change jection a mild hyperemia of the treated iris in pupil diameter, mild hyperemia at 18 hr, could be observed and protein measure- and a more pronounced increase in aque- ments revealed 523 ± 9 mg/dl (11) and ous humor protein concentration (p < 0.01) 66 ± 13 (9), respectively, in the treated and (Fig. 5). untreated sides (p < 0.01). A trend toward When AMP was injected into the vitreous, normalization of the disruption of the blood- no clinical effect could be detected on the aqueous barrier could be observed, in that blood-aqueous barrier. Protein measure-

Downloaded from iovs.arvojournals.org on 09/30/2021 Invest. Ophthalmol. Visual Sci. 260 Maul and Sears March 1979

ments 30 min after injection were 162 ± 67 unknown. Evidence has been presented that mg/dl (4) and 72.6 ± 36 (4), respectively, in vasodilatation in the rabbit ear after anti- the treated and untreated sides. Eighteen dromic nerve stimulation is mimicked by hours after, these measurements were ATP injections, and it has been shown that 58 ± 2.6 mg/dl (6) and 60 ± 2.5 (5). ATP is liberated when the great auricular ATP measurements in blood and in aque- nerve of the rabbit is stimulated.8 ous humor after lens disruption. ATP in There is speculation about the possible whole rabbit blood was 1.06 ± 0.15 nmol/L role of ATP in nervous tissue for the mainte- (8). These values coincide well with previ- nance of membrane permeability8 and as a ously reported measurements.8 ATP in aque- cofactor for the vesicular storage of norepi- ous humor after lens disruption was 61.0 ± nephrine.16 ATP has been demonstrated to 3.4 nmol/L (4) as opposed to the contralateral be released from motor nerve terminals dur- side, 7.4 ± 2.5 (4) (p < 0.01). ing stimulation, and the trophic function of motor fibers has been related to this re- Discussion lease.17 A nonadrenergic inhibitory nerve The effects of trigeminal nerve stimulation system has been described in the gut. These on the rabbit eye are well established in the fibers release ATP upon stimulation and have literature.2' 3' 9 A consensual effect on the been named purinergic nerves.18 Indirect blood-aqueous barrier also characterizes the evidence for the existence of an ATP-releas- effect of this nerve stimulation.3 Chemical ing nerve fiber system has been presented in irritants such as nitrogen mustard have been the eye.19 Whether this sort of system might demonstrated to exert their effect upon the be involved in controls for ordinary levels of eye by way of the trigeminal nerve in the intraocular pressure or involved in the irrita- rabbit.10 tive ocular response is not known. Dose-response curves of iridial conjuncti- Considerable ATP was present in the val hyperemia, miosis, intraocular hyperten- trigeminal nerve and in the Gasserian gan- sion, and disruption of the blood-aqueous glion in the present work. It was less than barrier that follow stimulation of this neural that reported for other nerves.16 Perhaps the pathway with nitrogen mustard strongly sug- difference might represent losses during the gest the effects to be the consequence of a postmortem delay. Nevertheless, ATP mea- transmitter receptor mechanism.4 The sub- sured represents strictly that of neural tissue, stance or substances released by trigeminal since blood contamination was avoided. The stimulation are not known. Sympathetic de- purpose of these measurements was to find nervation does not prevent the effects of out whether there was a difference in the trigeminal stimulation nor that of nitrogen ATP content of ganglion and nerve that might ls 10 mustard applied topically to the eye. Ex- allow speculation about the origin of ATP re- periments in the atropinized animal or with leased. The results presented do not allow degenerated third nerve have shown that such determination, and investigation of this these do not eliminate the effects of trigemi- part will need to be conducted at a subcellu- 12 nal stimulation."' Inhibition of prosta- lar level. The trigeminal nerve provides glandin synthesis by indomethacin does not fibers for the sensation of the cornea, and it prevent the effect of topical nitrogen mustard 3 10 supplies the iris with an important myeli- or trigeminal stimulation. ' Prostaglandins nated nerve fiber network.20' 21 When the have not been demonstrated in aqueous 13 nerve is stimulated, a significant increase in humor after nerve stimulation. Analogy be- the content of ATP in the aqueous humor tween the mechanism of trigeminal irritation occurs. Aqueous humor is an extracellular and axon reflex skin vasodilatation has been fluid bathing the anterior segment of the eye discussed. "Pain" fibers might be involved in 10 14> 13 and is essentially free of cells. After stimula- both reactions, ' but the mediator is tion, cells did not increase significantly and

Downloaded from iovs.arvojournals.org on 09/30/2021 Volume 18 Number 3 AT? released by antidromic stimulation 261

could not constitute a source of the increased leased into the eye by antidromic stimulation ATP. The increase in ATP content of aqueous and that an iridial hyperemia and disruption humor was bilateral. The crossed fibers of the of the blood-aqueous barrier develop after in- trigeminal nerve innervating the contralat- jection of ATP into the vitreous. 2 3 eral eye ' are probably the origin of the ATP The secretarial assistance of Susan Fleischmann and detected in the contralateral aqueous. ATP in Elena Maria Korzenszky is gratefully acknowledged. blood is in the red cells; plasma is essentially free of ATP. Thus the disruption of the blood-aqueous barrier that follows trigeminal REFERENCES stimulation cannot account for the increase 1. Maurice, D. M.: Constriction of the pupil in the observed. Treatment of animals with di- rabbit by antidromic stimulation of the trigeminal pyridamole, 1 mg/kg, further increased ATP nerve, J. Physiol. 123:45P, 1954. 2. Perkins, E. S.: Influence of the fifth cranial nerve on levels in aqueous humor after stimulation. the intraocular pressure of the rabbit eye, Br. J. Dipyridamole, a coronary vasodilator, inhib- Ophthalmol. 41:257, 1957. 22 its 3'5'-AMP phosphodiesterase. It also in- 3. Maul, E., and Sears, M. L.: The contralateral effect hibits ATP uptake from blood by heart and of antidromic stimulation of the trigeminal nerve on lung23 and blocks the reuptake of ATP by the rabbit eye, INVEST. OPHTHALMOL. 15:564, 1976. 18 4. Maul, E., and Sears, M. L.: Objective evaluation of purinergic nerve endings. experimental ocular irritation, INVEST. OPHTHAL- The increase in ATP detected after stimu- MOL. 15:308, 1976. lation in animals treated with dipyridamole 5. Strehler, B. L.: Bioluminiscence assay: principles strengthens the possibility that the actual and practice, Methods Biochem. Anal. 16:99, 1968. 6. Famebo, L.-O., and Hamberger, B.: Release of source of ATP increase is from neural tissue. norepinephrine from isolated rat iris by field stimu- When ATP or related nucleotides were in- lation, J. Pharmacol. Exp. Ther. 172:332, 1970. jected into the vitreous, the physiopathologi- 7. Mainland, D.: Elementary Medical Statistics, Phil- cal effect of trigeminal stimulation could not adelphia, 1963, W. B. Saunders Co., chap. XIV. be mimicked, although an iridial hyperemia 8. Holton, P.: The liberation of adenosine triphosphate on antidromic stimulation of sensory nerves, J. of long latency and an increase in aqueous Physiol. 145:494, 1959. humor protein levels was elicited. Twenty- 9. Magendie, F.: De 1'influence de la sinquieme paire eight nanomoles injected into the vitreous de nerfs sur la nutrition et les fonctions de l'oeil, J. represent roughly a final concentration of Physiol. Exp. Pathol. 4:176, 1824. 10~5M. This represents about 103 times the 10. Jampol, L. M., Neufeld, A. H., and Sears, M. L.: ATP measured in the aqueous humor 1 min Pathway for the response of the eye to injury, IN- VEST. OPHTHALMOL. 14:184, 1975. after stimulation. Pharmacological matching 11. Bernard, C.: Lepons sur la Physiologie et al of the effect observed after nerve stimulation Pathologie du Systeme Nerveux, Paris, 1858, J. B. has been a problem in the classic works on Bailliere, vol. 2, p. 205. neurotransmitter substances. 12. Huhtala, A., Johansson, G., and Saari, M.: Myeli- 8 5 nated nerves of guinea pig after denervation of the Holton reported a discrepancy of 10 be- ophthalmic division of the trigeminal nerve, Oph- tween the ATP collected and the ATP thalmol. Res. 7:354, 1975. needed to match vasodilatation of the great 13. Cole, D. F., and Unger, W. C: Prostaglandins as auricular nerve stimulation. Brown et al.24 mediators of the eye to trauma, Exp. Eye Res. 17: reported a discrepancy of 100 to imitate a 357, 1973. 14. Duke-Elder, P. M., and Duke-Elder, W. S.: The twitch in the case of acetylcholine at the neu- vascular response of the eye, Proc. R. Soc. Lond. romuscular junction. Further studies are re- 109B:19, 1931. quired relating doses of ATP to the mild but 15. Celander, O., and Folkow, B.: The nature and dis- genuine iridial hyperemia found. tribution of afferent fibers provided with the axon From our experiments it cannot be con- reflex arrangement, Acta Physiol. Scand. 29:359, 1953. cluded that ATP is responsible for the irrita- 16. Lagercrantz, H., Fried, G., and Dahlin, I.: An at- tive response to trigeminal nerve stimula- tempt to estimate the in vivo concentrations of tion. It is clear, however, that ATP is re- noradrenaline and ATP in sympathetic large dense

Downloaded from iovs.arvojournals.org on 09/30/2021 Invest. Ophthalmol. Visual Sci. 262 Maul and Sears March 1979

core nerve vesicles, Acta Physiol. Scand. 94:136, tion of the myelinated nerves of the cat iris after 1975. denervation of the ophthalmic division of the 17. Selinsky, E. M., and Hubbard, J. I.: Release of ATP trigeminal nerve: an electron microscopic study. from rat motor nerve terminals, Nature 243:404, Exp. Eye Res. 17:281, 1973. 1973. 22. Vigdahl, R. L., Mongin, J., Jr., and Marquis, R.: 18. Burnstock, G.: Purinergic nerves, Pharmacol. Rev. Platelet phosphodiesterase and its inhibition by 24:509, 1973. vasodilators, Biochem. Biophys. Res. Commun. 42: 19. Burnstock, G., Gannon, B. J., Malmfors, I., and 1088, 1971. Rogers, D. C.: Changes in the physiology and fine 23. Kolassa, N., Pfleger, K., and Tram, M.: Species dif- structure of the taenia of the guinea pig caecum fol- ferences in action and elimination of adenosine after lowing transplantation into the anterior eye cham- dipyridamole and hexobendine, Eur. J. Pharmacol. ber, J. Physiol. 219:319, 1971. 13:320, 1971.

20. Phillis, J. W.: The Pharmacology of Synapses, Ox- 24. Brown, G. L., Dale, H. H., and Feldberg, W.: ford, 1970, Pergamon Press, p. 6. Reaction of the normal mammalian muscle to acetyl- 21. Saari, M., and Kiviniemi, P.: Wallerian degenera- choline and to eserine, J. Physiol. 87:394, 1936.

Copyright information The appearance of a code at the bottom of the first page of an original article in this journal indicates the copyright owner's consent that copies of the article may be made for personal or internal use, or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc., P.O. Box 765, Schenectady, N.Y. 12301, /518/374- 4430, for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale.

Downloaded from iovs.arvojournals.org on 09/30/2021