Observations on the function of the gland after denervation have usually

Quarterly Journal of Experimental Phy8iology (1974) 59,1-9 REINNERVATION OF THE DENERVATED PAROTID GLAND OF THE CAT. By J. EKSTROM and N. EMMELIN. From the Institute of Physiology, University of Lund, Sweden. (Received for publication 24th May 1973) Functional recovery was studied in parotid glands of cats after cutting the auriculotemporal nerve. Denervation supersensitivity to methacholine started to decrease between 1 month and 2j months after the operation, and at the same time there was an increase in the secretory responses to eserine, injected through the salivary duct. Reflexly induced secretion increased and the activity of choline acetyltransferase, greatly reduced shortly after the denervation, rose gradually. However, even a year or more after the denervation the reinnervation was incomplete. In histochemical and electron microscopical investigations Garrett (1966a and b) demonstrated that the number of nerve fibres in the parotid gland of the cat was greatly reduced 6 days after section of the auriculo-temporal nerve. Some nerve fibres always remained, however, and this was the case even when the operation included sympathectomy. After 16 days a gradual reinnervation occurred and after 64 days the number of nerve fibres was roughly estimated to be 60% of that of the contralateral gland. Observations on the function of the gland after denervation have usually not covered such a long time period. During the first days following section of the auriculo-temporal nerve there is a period in which 'degeneration secretion' occurs [Emmelin and Str6mblad, 1958a; Ohlin, 1963; Emmelin, 1968]. The gland cells then acquire an increased sensitivity to chemical stimuli, as demonstrated with acetylcholine and adrenaline about 2 weeks [Str6mblad, 1955a and b, 1957] and with isoprenaline 3 weeks after the operation [Ohlin, 1964]. The secretory responses to cholinesterase inhibitors, studied 2-4 weeks after auriculo-temporal nerve section, are on the other hand greatly diminished [Emmelin and Str6mblad, 1958b]. The activity of various enzymes in the gland has been found to be reduced 2-5 weeks after section of the nerve [Str6mblad, 1955b, 1956, 1957; Nordenfelt, Ohlin and Str6mblad, 1960]. Of particular interest in this context is the finding that the activity of choline acetyltransferase is markedly diminished but not wholly abolished [Nordenfelt, 1963]. In the present investigation reinnervation of the parotid gland of the cat was studied over periods of several months following section of the auriculotemporal nerve and often excision of the superior cervical ganglion as well. METHODS Cats were anaesthetized with ether which was either continued for some time through a tracheal tube, or was replaced by a short-acting barbiturate, buthalitone (Baytinal Bayer), injected through a needle inserted into the left ventricle of the heart. A fine VOL. LIX, NO. 1-1974

2 Ekstr6m and Emmelin glass cannula was introduced into the right parotid duct from the opening in the mouth. The cannulae gave about 70 drops out of 1 ml. of distilled water. Some secretion usually occurred when the animal was under ether anaesthesia. Secretion was increased or evoked by pouring a solution of citric acid (10%) onto the tongue or by swabbing the pharyngeal mucosa with dry gauze tissue. The secretory response was recorded as drops on a smoked drum either under ether anaesthesia or after intracardial injection of a small dose of buthalitone (2-5 mg/kg). Reflexly induced secretion was then abolished by increasing the dose of buthalitone (to about 20 mg/kg) and injecting 5 mg hexamethonium/kg intracardially. The sensitivity of the secretory cells to methacholine was estimated by injecting doses of 0-5, 1 0 and 20,ug/kg through the needle in the heart. Between the injections of drugs saline solution was continuously given through the need]e to prevent clotting, and when required additional doses of buthalitone were administered. The parotid cannula was then replaced by another cannula of similar type supplied with a three-way tap to which a syringe could be attached. Eserine sulphate, 200,ug in 0-2 ml. saline solution, was injected into the gland during 5 sec; 5 sec later the tap was turned so as to allow saliva to flow out. and the drops of saliva were recorded on the smoked drum for a period of 15 min. Under continued buthalitone anaesthesia the right auriculo-temporal nerve was exposed as described by Burgen [1964] and cut. In many experiments the right superior cervical ganglion was extirpated as well. Five days later the secretory responses to reflex stimulation, methacholine and eserine were again estimated, and this was repeated at intervals of weeks or months, as shown in the text below. In the final experiment the cats were anaesthetized with chloralose (about 80 mg/kg intravenously after induction with ether) or with chloralose-urethane (50+500 mg/kg). The two parotid ducts were exposed and cannulated. The glossopharyngeal and in some cats the lingual nerves were dissected, cut and used for afferent stimulation, using supramaximal shocks of a frequency of 20-100/sec and a duration of 1-2 msec. Attempts were made to find a regenerated auriculo-temporal nerve and to stimulate it electrically, but this was not always possible, usually because of profuse bleeding from the region of the previous operation. Eventually the two parotid glands were excised, cleaned and weighed, and their choline acetyltransferase activity was estimated according to the method of Hebb as described in detail by Nordenfelt [1963, 1965a]. For statistical analysis Student's t-test was used; in the case of secretory responses paired comparisons were made. RESIuLTS 1. Sen8itivity to methacholine The sensitivity of the gland to methacholine was estimated at intervals. Lowering of the marked denervation supersensitivity was taken to show that the gland cells were increasingly exposed to transmitter released by secretory impulses and, in addition, leaking from the post-ganglionic nerves [see Emmelin, 1965]. Fig. 1 summarizes the results of sensitivity studies on 9 cats observed during 71 months. Following denervation the sensitivity rose rapidly and reached a maximum within 1 month. It then gradually fell, the first significant lowering being demonstrable between 24 and 31 months after the operation (P < 0.02). The pre-operative low sensitivity was, however, not reached within the observation period (P < 0.01). The dose of methacholine shown in Fig. 1 was 2,zg/kg.

Reinnervation of Parotid Gland Similar results were obtained with the standard doses 1 and 0 5,ug/kg. In these cats section of the auriculo-temporal nerve was combined with excision of the sympathetic ganglion. In another series, comprising 10 cats and observed during 31 months, the auriculo-temporal nerve was cut in 5 animals and in the other 5 the ganglion was removed as well. The supersensitivity produced was somewhat higher after the combined operation than when only the auriculotemporal nerve was out, but the course of the two sensitivity curves was about the same in the two groups. In both of them the decline between 1 and 22 months, suggested already in the series of Fig. 1, was statistically significant (P < 0.01). ESERINE 50 40 METHACHOLINE 30 15 201 t 10 3 0 5 14 30 45 75 105 165 225 DAYS FIG. 1. Secretory responses in drops of saliva to eserine, 200 ug, (white columns) and methacholine, 2 pg/kg, (dark columns) of nine cats, studied over a period of 71 months. Mean values±s.e.m. are given before and at intervals after section of the auriculo-temporal nerve and extirpation of the superior cervical ganglion. 2. Effect of eseritne The secretory response to eserine injected through the salivary duct under standard conditions was, likewise, recorded at intervals. The secretory effect was ascribed to acetylcholine leaking from post-ganglionic nerves and surpassing the secretory threshold when preserved by the cholinesterase inhibitor; the estimation was carried out in the presence of a ganglion-blocking agent, to prevent the release of acetylcholine by impulses from the central nervous system [Emmelin and Perec, 1968]. It can be seen in Fig. 1 that the secretory effect of a standard dose of eserine was greatly reduced 5 days after the operation, but a response was still obtained. During the following weeks it increased; it is reasonable to attribute this to the simultaneously rising sensitivity of the gland cells. However, when the sensitivity had reached a plateau and started to decline, the responses to eserine started to rise, to reach eventually about the pre-operative level. The first

4 Ekstrbm and Emmelin significant increase in the eserine response occurred between 1 and 2A months after the operation (P < 0.05). Similar results were obtained in the series of experiments where the parasympathetic nerve was cut in 5 cats and the superior cervical ganglion in addition extirpated in 5 other cats; and there were no differences in the secretory responses to eserine between the two groups of this series. 0 5 31 46 76 179 243 FiG. 2. Parotid secretion before and at intervals (given in days to the right of each test) after section of the auriculo-temporal nerve and extirpation of the superior cervical ganglion. Records from above: minute marks, signal, drops of saliva. The sections to the left show the secretory responses to citric acid solution poured on the tongue. In the right sections the first signal marks the injection into the left ventricle of methacholine 2 ftg/kg and the second signal the injection of eserine 200,ug into the salivary duct.

Reinnervation of Parotid Gland 5 3. Reflexly elicited secretion The flow of saliva which could be induced in ether or light barbiturate anaesthesia by pouring citric acid solution on the tongue was greatly reduced 5 days after section of the auriculo-temporal nerve. Some secretion could always be obtained, however, and this was true also when the operation was combined with sympathectomy. Fig. 2 shows a typical experiment, in which a cat was studied during a period of 243 days. The sensitivity to methacholine increased after the operation, then fell as described above; the responses to eserine increased from their low postoperative level and the flow which could be elicited reflexly increased more or less in parallel. Owing to the difficulty in standardizing the conditions for these experiments on reflex secretion, e.g. the depth of the anaesthesia, strictly quantitative comparisons cannot be made; but together these experiments suggested that the reflexly obtained secretory rates gradually increased almost but usually not quite to the level found before the operation. The salivation induced reflexly from the gland of the operated side was abolished by atropine (0.2 mg/kg) even in cases where the sympathetic innervation was intact. 4. Secretion due to afferent nerve stimulation In 25 cats kept for 31-15 months after section of the auriculo-temporal nerve acute experiments were carried out in which the two parotid ducts were cannulated and the afferent stumps of the glossopharyngeal nerves stimulated electrically. Excitation of the nerve of the operated side always produced secretion from the ipsilateral gland. Quantitative comparisons seemed difficult in these experiments also, but the general impression was that the flow obtained from the gland of the operated side was usually not quite as fast as that of the control side. In 3 cats the acute experiment was made already 3-4 weeks after the operation, and here the operated gland secreted much less than the control gland on afferent stimulation of the ipsilateral glossopharyngeal nerve. The secretion elicited from the operated gland could always be abolished with atropine. When a regenerated auriculo-temporal nerve could be found and stimulated, the secretory effect was smaller than on the control side. 5. Activity of choline acetyltransferase The choline acetyltransferase activity of the two parotid glands was studied in 38 cats. In 29 of them the whole glands were dissected out; in the remaining cats only a sample of glandular tissue was excised. The activity of the whole glands of the control side was 37-1 ±32,ug acetylcholine formed during 1 hr. Expressed per g of acetone powder per hour ('concentration') it was 168±10,ug acetylcholine in the whole group of animals. Section of the auriculo-temporal nerve, often combined with extirpation of the superior cervical ganglion, greatly diminished the acetylcholine synthesizing capacity; and since total amounts and concentrations were similarly affected, only concentrations,

6 Ekstrom and Emmelin which were examined in all the cats, will be considered. Fig. 3 shows the concentration of the enzyme, in percent of that of the contralateral gland, examined in the 38 cats, which were observed for periods of between 5 days and 25 months after the operation. After 5-7 days 135 ±4.8% of the activity remained. Examined after about 4 months, however, it was 42 8±5 3%; the increase from the low level is significant (P < 0.001). This increase continued, so that in the animals studied after 8-25 months the activity of the operated glands was 65-5±4'4% of that of the contralateral glands. No differences in enzyme concentration were obtained between glands deprived of the auriculo-temporal nerve only and glands in which the ganglion had been removed as well; this applies both to the group examined after about 4 months and to that studied later. The figure also includes a mean value obtained by Nordenfelt (1963) 2-5 weeks after auriculo-temporal nerve section. 110 0 100-._..._.. 900A v 80 A o 0 70 AA00 60-A A 0 50-AA A Av 40-0 A A o A 0 30 o0 (9 20 A 10 0 0 FIG. 3. 5 7 120 240 360 480 600 720 days Concentration of choline acetyltransferase of the cat's parotid gland after section of the auriculo-temporal nerve, open rings, or section of the auriculo-temporal nerve combined with extirpation of the superior cervical ganglion, solid triangles. The enzyme concentration of the operated glands is given as a percentage of that of the contralateral glands. The value after 5 weeks is a mean of 5 estimations (± S.E.M.) taken from Nordenfelt [1963]. DIscUSSION The experiments indicate that after section of the auriculo-temporal nerve gradual reinnervation of the parotid gland of the cat occurs. The methods used provide different types of information, which all point in this direction. The capacity to synthesize the transmitter increases; this suggests regeneration of nerves, since in salivary glands the choline acetyltransferase present seems to be confined to nerves [Nordenfelt, 1963]. The increased responses to eserine signal that more acetylcholine is released in the gland by physiological leakage from postganglionic nerves; acetylcholine set free by impulses from the central

Reinnervation of Parotid Gland nervous system is excluded in these experiments by deep anaesthesia and ganglion blocking drugs. It proved also possible to watch the functional recovery in each cat by recording at intervals the reflexly evoked secretion, and in an acute experiment the secretory response to afferent stimulation of oral nerves could be studied. Finally, repeated estimations of the sensitivity of the secretory cells to methacholine were useful. The gradual reduction of the denervation supersensitivity revealed that the gland cells were exposed to increasing amounts of transmitter; this was probably a combined action of acetylcholine liberated by secretory impulses and acetylcholine leaking from the postganglionic neurones [Emmelin, 1965]. In the morphological investigations by Garrett [1966a and b] signs of reinnervation were observed as early as 16 days after section of the auriculotemporal nerve (sometimes combined with sympathectomy). It seems reasonable to assume that function is re-established more slowly, and the present experiments indicate this to be the case. The supersensitivity was still at its maximum after 1 months and a decline was noted only one month later. It is true that 'desensitization' of the supersensitive glandular cells takes some time, but probably less than a week [Emmelin, 1965]. The eserine experiments might in fact hint that leakage of transmitter had begun to increase somewhat earlier, between 1 and 11 months after the operation, for after 1 months the eserine responses seemed enlarged in the face of unchanged sensitivity of the gland cells (Fig. 1). However, a significant increase in the eserine responses was not observed until after 21 months, i.e. at the time when a lowering of the sensitivity was first noticed. The acetylcholine synthesizing power remained at its lowest postoperative level during the first month, but it then increased rapidly. The reflexly elicited secretion increased, but the method scarcely allowed any conclusion as to the time at which functional recovery commenced. Morphologically, the reinnervation had proceeded to a level of about 60% of normal within a period of 64 days [Garrett, 1966a and b]. Functionally, it was apparently less advanced at that stage; the supersensitivity was not far from its maximum, and the recovery of the choline acetyltransferase had only reached 43% of normal about 4 months after the operation. The experiments with eserine might suggest that full recovery had taken place within about half a year. This is contradicted, however, by other observations. Even after 71 months a pronounced supersensitivity remained, and the choline acetyltransferase activity was only restored to 66% 8-25 months after operation. Furthermore, the reflexly induced secretion was usually not as rapid on the operated as on the normal side. The large eserine responses were no doubt due to the persistence of some supersensitivity. In the presence of eserine acetylcholine very likely reached still denervated and supersensitive gland cells; these cells were probably not, or to a smaller extent only, reached in the absence of the cholinesterase inhibitor in the experiments where acetylcholine was liberated reflexly. It may be pointed out that reinnervation of an organ following postganglionic denervation is usually incomplete [see Guth, 1965; Murray and Thompson, 1957; Nordenfelt and Perec, 1967]. The early reinnervation demonstrated histologically was attributed to 7

8 Ekstr6m and Emmelin sprouting from cholinergic fibres remaining in the gland after section of the auriculo-temporal nerve [Garrett, 1966a and b]. The existence of such fibres, connected to the central nervous system, is apparent from the present and other experiments [Ekstr6m and Emmelin, 1973]. Experiments on the submaxillary gland [Emmelin and Perec, 1968] suggested that when some of the postgangiolinic parasympathetic neurones had been destroyed there was a pronounced functional recovery by sprouting from the neurones that had been left; this occurred late, however, and only when these neurones had regained their contact with the central nervous system, all the preganglionic fibres being of necessity cut during the operation on the postganglionic neurones. The fairly early recovery in the present experiments may have been promoted by the fact that some cholinergic fibres, connected to the central nervous sytem, were present from the beginning so that sprouting could start soon. The gradual recovery taking part in the course of many months, on the other hand, was probably due to (incomplete) regeneration of classical type, possibly combined with sprouting. Simultaneous extirpation of the superior cervical ganglion did not greatly modify the effects of section of the auriculo-temporal nerve in the present experiments. There was no indication that more cholinergic fibres were removed in this way, nor that functional recovery in the reflexly elicited secretion was due in any sense to participation of adrenergic nerves. Nor was there any evidence that regeneration was promoted by the sympathetic ganglionectomy; this possibility was considered since ganglionectomy has been found to increase the choline acetyltransferase activity of otherwise normal parotid glands of cats, an effect which has been tentatively attributed to sprouting from the intact postganglionic parasympathetic neurones [Nordenfelt, 1965b]. ACKNOWLEDGMENT This work was supported by a grant (B72-14X-539) to N. E. from the Swedish Medical Research Council. REFERENCES BURG(EN, A. S. V. (1964). Techniques for stimulating the auriculotemporal nerve and recording the flow of saliva. In Salivary Glands and their Secretions, pp. 303-307. Ed. L. M. Sreebny and Julia Meyer. Pergamon Press, Oxford. EK:sTR6M, J. and EMMELIN, N. (1974). The secretory innervation of the parotid gland of the cat: an unexpected component. Quarterly Journal of Experimental Physiology, 59, 11-17. EMMELIN, N. (1965). Action of transmitters on the responsiveness of effector cells. Experientia, 21, 57-65. EMMELIN, N. (1968). Degeneration secretion from parotid glands after section of the auriculo-temporal nerves at different levels. Journal of Physiology, 195, 407-418. EMMELIN, N. and PEREC, C. (1968). Reinnervation of submaxillary glands after partial postganglionic denervation. Quarterly Journal of Experimental Physiology, 53, 10-18. EMMELIN, N. and STR6MBLAD, B. C. R. (1958a). A 'paroxysmal' secretion of saliva following parasympathetic denervation of the parotid gland. Journal of Physiology, 143, 506-514. EMMELIN, N. and STROMBLAD, B. C. R. (1958b). The effect of anticholinesterases on the parotid gland after parasympathetic decentralization or denervation. British Journal of Pharmacology, 13, 193-196.

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