REINNERVATION OF SUBMAXILLARY GLANDS AFTER PARTIAL. mainly in sympathetic ganglia, in which marked sprouting has been shown

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1 Q. Jl exp. Phy8iol. (1968) 53, 1-18 REINNERVATION OF SUBMAXILLARY GLANDS AFTER PARTIAL POSTGANGLIONIC DENERVATION. By N. EMMELIN and C. PEREC*. From the Institute of Physiology, University of Lund, Sweden. (Received for publication 8th March 1967) Supersensitivity following section of the chorda tympani within the hilum of the submaxillary gland of the cat, producing decentralization and partial postganglionic denervation, was found not to diminish during six months provided that preganglionic regeneration was prevented. When this was allowed to occur there was a very marked reduction of the sensitivity, a pronounced increase of the choline acetyltransferase activity, a lively secretion in response to chorda stimulation: the secretory responses to eserine injected into the gland through the salivary duct, which were very small shortly after the operation, increased in spite of decreasing sensitivity. These observations are taken as evidence that collateral regeneration occurred from the remaining postganglionic neurones but only when contact with the central nervous system had been re-established by preganglionic regeneration. IN the autonomic nervous system collateral regeneration has been studied mainly in sympathetic ganglia, in which marked sprouting has been shown to take place [Murray and Thompson, 1957]. There is some indirect evidence that sprouting may occur from parasympathetic nerves in the submaxillary gland of the cat; Nordenfelt 11965] found anincreased choline acetyltransferase (E.C ) activity in the gland after sympathetic ganglionectomy and suggested that degeneration of sympathetic fibres might induce sprouting from the cholinergic glandular nerves. This gland seems to offer a preparation well suited for experiments on collateral regeneration in the parasympathetic nervous system. It has a rich supply of parasympathetic fibres, and it is possible to carry out a partial postganglionic parasympathetic denervation, leaving some parasympathetic neurones within the gland intact. In our first experiments the sensitivity of the submaxillary gland to secretory agents was studied on the assumption that reinnervation ought to reduce the supersensitivity caused by the partial postganglionic denervation. The supersensitivity to stimulating drugs which develops after this operation arises as a consequence of loss of some action of acetylcholine on the gland, and this acetylcholine originates from two sources: it is released from the postganglionic parasympathetic endings by the secretory impulse from the central nervous system, and it leaks continuously from these endings in amounts which are normally subthreshold as far as secretion is concerned; this occurs even in the absence of secretory impulses, for instance after preganglionic section [see Emmelin, 1961; 1965]. Section of the chorda tympani within the hilum of the gland to produce a partial postganglionic parasympathetic denervation eliminates the first source of acetylcholine, thus producing the supersensitivity characteristic of preganglionic denervation. At the same time, however, a great number of postganglionic fibres are cut and leakage of acetylcholine from their endings may cease when they have * Fellow of the Consejo Nacional de Investigaciones Cientificas y Tecnicas, Buenos Aires, Argentina. 1

2 Sprouting in Submaxillary Glands 11 degenerated; the sensitivity of the gland cells therefore rises above the level reached after decentralization. A subsequent extension of the postganglionic field of innervation by collateral sprouting from the remaining postganglionic parasympathetic neurones could be supposed to reduce the degree of supersensitivity. In these experiments there was, however, very little fall in sensitivity during the months following the denervation. For obvious rcasons it was necessary to prevent the connection with the central nervous system being re-established; this would certainly have lowered the supersensitivity. The preganglionic chorda fibres were therefore cut at intevals. This precaution involves, however, at the same time a theoretical complication. It may be that little sprouting, or none at all, occurs from a postganglionic neurone inactivated by disconnection from the central nervous system. In a second series of experiments the preganglionic fibres were allowed to regenerate after the partial postganglionic denervation. In order to find out whether postganglionic sprouting occurred, the secretory effect of a standard dose of eserine was estimated, assuming that sprouting increased the number of cholinergic endings and thereby the amounts of acetylcholine leaking in the gland. At the end of the observation period an acute experiment was carried out. The secretory effects of stimulation of the normal and the regenerated chorda, and of injection of eserine and pilocarpine were examined and finally the submaxillary glands were weighed and their choline acetyltransferase activity estimated. METHODS All the observations were made on submaxillary glands of cats. The animals were examined repeatedly, anaesthetized with a short-acting barbiturate, at intervals varying between four days and some weeks over a period which in two cases amounted to more than one year and a half (Table I). In the first series the cats were anaesthetized with hexobarbitone given intracardially after induction with ether. The two submaxillary ducts were cannulated from the mouth using fine glass cannulae. Standard doses of adrenaline ( 5, 1, 2, a and sometimes 1 pg./kg.) were injected intracardially and the drops of saliva secreted were counted as a measure of the level of sensitivity [see Emmelin, 1964 a]. The chorda-lingual nerve was cut. About three weeks later, when the supersensitivity due to this decentralization had reached a maximum, the partial postganglionic parasympathetic denervation was made by cutting the chorda within the hilum of the gland [Emmelin, 196]. The sensitivity was then studied repeatedly and the chordalingual nerve recut about every six weeks. In some of these cats the chorda was finally given a chance to regenerate after a period of 5-14 months, and some of these cats were included in the next group. In this second series the sensitivity of the two submaxillary glands towards adrenaline was also studied repeatedly, but in addition the secretory effect of eserine was then estimated each time. For this purpose a cannula supplied with a threeway tap was inserted into the submaxillary duct. Eserine sulphate, 2,ug. in 2 ml. saline solution (or in some few cases 1,pg. in 1 ml.), was injected into the gland during five seconds; five seconds later the tap was turned so as to allow saliva to flow out, and the drops of saliva were recorded on a smoked drum. Since hexobarbitone has an atropine-like effect it was in these experiments replaced by another short-acting barbiturate. Baytinal (Bayer). To prevent reflex salivation, which often

3 12 Emmelin and Perec occurs with this anaesthetic, hexamethonium (5 mg./kg.) was given intracardially [see Emmelin, 1964 a]. The chordalingual nerve was not cut in a separate operation bit the pre- and the partial postganglionic sections were made at the same time. Acute experiments were made under chloralose anaesthesia (6-8 mg./kg. intravenously). The submaxillary ducts were exposed and cannulated in the neck. Secretory responses were recorded on two drums; on one the drops of saliva were marked using an electromagnetic lever, on the other the secretory rate was recorded using an ordinate writer. Secretion was induced by supramaximal stimulation of the chorda-lingual nerve; when the nerve had been cut many times it was sometimes difficult to find, and the stimulating electrode was then applied to the region of the submaxillary duct. The chorda was then stimulated after intravenous injection of hexamethonium. Secretion was also evoked by injections of eserine through the ducts into the glands, and by pilocarpine hydrochloride given either intravenously or by way of the ducts. The submaxillary glands were excised, cleaned and weighed and their choline acetyltransferase activity was estimated according to the method of Hebb [see Nordenfelt, 1965]. RESULTS Fig. 1 summarizes the results obtained in nine cats of the first series observed over a period of eight months. Three weeks after the parasympathetic decentralization when the supersensitivity according to previous experience had reached a plateau the partial postganglionic denervation was carried out. This was followed by a temporary lowering of the sensitivity, described earlier and attributed to an evanescent increase in the leakage from the degenerating fibres causing the 'degeneration secretion' [Emmelin, 1964 b]. This effect on the sensitivity is maximal after three days. In the present series the sensitivity was estimated four days after the operation, when the sensitivity had probably started to rise, and the dip in the curve therefore appears smaller than in the earlier investigation. The sensitivity gradually increased to a level above that reached three weeks after centralization. During nearly six months the sensitivity was then found to remain practically unchanged. The chorda-lingual nerve was recut several times in each cat during this period. In the experiments of fig. 2, belonging to the second series, the preganglionic chorda was left to regenerate. The figure gives mean values obtained on six cats. Four days after the combined preganglionic and partial postganglionic denervation the response to adrenaline was found to be increased. Preganglionic denervation is known to increase the sensitivity noticeably of within two or three days [Emmelin and Muren, 1952]. After having reached a maximum the sensitivity started to decline. That preganglionic reinnervation occurred at this stage was evidenced by the fact that saliva flowed from the cannula in the duct of the anaesthetized animal until hexamethonium had been administered. Eserine injected into the gland prior to the decentralization produced a lively salivation. The flow was due mainly or exclusively to acetylcholine accumulated in the gland and not in the general circulation, for no secretion was elicited from the contralateral gland. This acetylcholine was not released by nerve impulses from the central nervous system or elicited in the postganglionic cell body by the

4 Sprouting in Submaxillary Glands 13 eserine, for hexamethonium had been given. It is reasonable to assume the secretion to be due to acetylcholine leaking from the postganglionic para- 2 + ADR 15 t 5 X/K X / MONTHS FIG. 1. Sensitivity of the submaxillary glands of nine cats before and after section of the chorda-lingual nerves (1 ) and after partial postganglionic parasympathetic denervation ( i ). Abscissa: time in months. Ordinate: drops of saliva secreted in response to 5 pg. adrenaline/kg. After the postganglionic denervation the chorda-lingual nerve was recut at intervals of about six weeks. ADR 15 6 ESERINE MONTHS FIG. 2. Responses of the submaxillary glands of six cats to 5 jg. adrenaline/kg intracardially (o - o) and 2,ug. eserine sulphate injected through the duct (x- x). At the arrow the chorda was cut within the hilum of the gland. Abscissa: time in months. Ordinate to the left: number of drops of saliva after adrenaline; to the right: number of drops secreted during 15 minutes after injection of eserine. sympathetic endings and accumulating to effective concentrations in the presence of the cholinesterase inhibitor. Four days after the operation the response to eserine was markedly decreased, in spite of the sensitization of

5 14 Emmelin and Perec the gland. Control experiments showed that no decrease of the effect of eserine occurred four days after section of the preganglionic fibres only. It can be concluded that in the experiments of fig. 2 a great number of postganglionic fibres had been destroyed, but that some neurones were intact. The responses to eserine then increased markedly. This can be explained by the pronounced supersensitivity which appeared at the same time. When the sensitivity decreased the secretory effect of eserine also diminished. Gradually, however, - - FiG. 3. Secretory effects of supramaximal stimnulation of the chorda-lingual nerves of two cats. The upper record is from a cat in which partial postganglionic denervation had been made about II months earlier. In the cat of the lower record this operation was made 19 months before the acute experiment and during the first six months the chorda-lingual nerve was recut five times. Time marks (uppermost in both records) is in minutes and underneath is shown secretion from the normal gland (to the left) and from the gland operated on (to the right). the response to eserine started to increase again, in spite of decreasing sensitivity. This occurred in all the six cats of this series. Responses larger than those obtained at the beginning of the experiment were eventually seen; this was very likely due to the fact that some supersensitivity remained. In the acute experiments on these cats stimulation of the chorda tympani was found to evoke a lively secretion, which was abolished by hexamethonium, showing that preganglionic fibres had reinnervated ganglion cells and not directly the glandular cells. This possibility had to be taken into account as a source of error. It has proved feasible to reinnervate the secretory cells of the parotid gland from the hypoglossal nerve without any intercalated synapse [Emmelin, Malm and Strdmblad, 196]; on the other hand, we have earlier in different connections made numerous altogether unsuccessful attempts to supply the submaxillary gland cells directly with preganglionic chorda fibres or with hypoglossal fibres. Fig. 3 demonstrates the secretory responses to

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7 16 Emnmelin and Perec stimulation of the normal and the regenerated chorda. From the figure it can be seen that restitution was marked but not complete. The maximal secretory rate was lower on the operated than on the normal side, as shown in the figure and confirmed when an ordinate recorder was used to estimate the flow rate. This applies to all the experiments of this series. For comparison an experiment is also included in fig. 3 in which the chordalingual nerve had been recut five times in about six months; the sensitivity of the gland remained high during this period and in the course of the following 13 months, when no operation on the nerve was made, it was only slightly lowered. The figure shows that the response to chorda stimulation was small in this case. The choline acetyltransferase activity, expressed as amount of acetylcholine formed per gland per hour, was estimated in submaxillary glands from 16 cats. The results are summarized in Table I. Nine of the cats belong to the group in which the chorda-lingual nerve was not recut. A few weeks after partial postganglionic parasympathetic denervation of submaxillary glands of cats Nordenfelt [1963] found a choline acetyltransferase activity in the operated gland which was only per cent of that of the contralateral gland. In the present experiments, in which preganglionic reinnervation had occurred and the glands were studied 4-1- months after the postganglionic denervation, the enzyme activity was much higher (45-86 per cent of that of the contralateral gland), indicating an extensive reinnervation of the gland. From the Table it can also be seen that the gland weight had been restored in proportion to the degree of reinnervation, as judged by the acetyltransferase activity. In the cats of the second group of Table I, comprising seven animals, the chorda-lingual nerve had been cut repeatedly after the postganglionic denervation. No reduction in the supersensitivity had been observed during this time. In the course of the following months, when no attempts were made to prevent regeneration of the chorda, the sensitivity was lowered to a varying degree, and in the acute experiments some responses to chorda stimulation were obtained. Thus, for instance, the lower record of fig. 3 originates from cat no. 11 of Table I, whereas the upper record corresponds to cat no. 8. The Table suggests that in the second group of cats very little extension of the field of innervation had occurred in cats nos. 1, 11 and 12, more in the remaining four cats. DIsCUSSION Previous work indicates that numerous postganglionic parasympathetic neurones are destroyed when the chorda is dissected and cut within the hilum of the submaxillary gland [Emmelin, 196, 1964 b; Nordenfelt, 1963]. This is supported by the present finding that the secretory responses to eserine are greatly diminished some days after the operation. During the months following partial postganglionic parasympathetic denervation, when the sensitivity of the gland cells is decreasing, there is a pronounced increase in

8 Sprouting in Submaxillary Glands 17 the secretory responses to a standard dose of a cholinesterase inhibitor, applied locally in the gland after injection of a ganglion blocking agent. This observation strongly suggests that at that period the remaining postganglionic neurones extend their field of innervation by collateral regeneration. This conclusion is supported by other observations: the very marked reduction in the denerv-ation hypersensitivity; the pronounced secretory effect of chorda stimulation; and a considerable rise in the choline acetyltransferase activity, from 14 to as much as 86 per cent of that of the control gland. The experiments also suggest that sprouting from the postganglionic neurone occurs only provided the neurone is connected with the central nervous system; flow of secretory impulses in the parasympathetic pathway may be a condition for the collateral regeneration to take place. In this connection it is interesting to note that Hoffman [1952] observed that sprouting from intact axons in partially denervated muscles could be accelerated by electrical stimulation of the spinal cord or the nerve trunks. Of interest is also the finding of Nordenfelt [1965] that removal of the superior cervical ganglion causes an increase in the choline acetyltransferase activity in the submaxillary gland before, but not after, section of the preganglionic chorda fibres. The present experiments are complicated by the facts that on the one hand decentralization is unavoidable for technical reasons while, on the other hand, connection with the central nervous system seems necessary for the postganglionic sprouting to take place. Reinnervation of the remaining postganglionic neurones must thus occur before sprouting can be expected. The experiments have thus to be extended over a long time period. Complete regeneration after decentralization of the submaxillary gland of the cat may require between four months and a year [Nordenfelt and Perec, 1967]. Unfortunately some at least of the indicators of postganglionic sprouting used in the present investigation are affected by preganglionic regeneration also: the supersensitivity is lowered, and the acetyltransferase activity increased; according to Nordenfelt [1963] the enzyme is somewhat reduced by decentralization. The conclusion that postganglionic sprouting has occurred in the present experiments is based on the facts that, in spite of the loss of numerous postganglionic neurones, the glands have to such a great extent returned towards a normal condition with regard to sensitivity, acetyltransferase activity and secretory responses to nerve stimulation. Even stronger support for the idea that sprouting occurs is given by the finding that the secretory effects of eserine markedly and gradually increase when the appearance of reflex secretion signals that the ganglia have been reinnervated. The possibility that the presence of regenerated preganglionic cholinergic fibres within the gland could in itself explain the increased responses to eserine can be rejected since in control experiments decentralization did not diminish the secretory effects of eserine. In his extensive light and electron microscopical investigations on the innervation of the salivary glands Garrett [1966 a and b] has recently used our method of partial postganglionic parasympathetic denervation of the submaxillary gland of the cat. It seemed for several reasons, difficult to VOL. LIII, NO

9 18 Emmelin and Perec ascertain whether collateral regeneration took place in these preparations, and in view of our finding that preganglionic regeneration has first to occur it is unlikely that there was any marked sprouting since the glands were examined within 64 days after the denervation. In the parotid gland, however, there were signs of sprouting already days after parasympathetic denervation. A reasonable source of these sprouts seemed to be nerves which according to numerous reports reach the parotid gland through anatomically unknown channels [see Burgen and Emmelin, 1961; Garrett, 1966 a; Emm;elin, 1967; Emmelin and Holmberg, 1967]. Since the connection between these fibres in the gland and the central nervous system was not interrupted by the operation, sprouting could be assumed to start early. ACKNOWLEDGMENTS This work was supported by the Swedish Medical Research Council. REFERENCES BURGEN, A. S. V. and EMMELIN, N. G. (1961). 'Physiology of the salivary glands', Monographs of the Physiol. Soc. No. 8. EMMELIN, N. (196). 'Supersensitivity of the submaxillary gland following exclusion of the postganglionic parasympathetic neurone', Brit. J. Pharmacol. 15, EMMELIN, N. (1961). 'Supersensitivity following "pharmacological denervation"', Pharmacol. Rev. 13, EMMELIN, N. (1964 a). 'Collecting saliva intermittently over long time periods in anaesthetized animals', In Salivary glands and their secretions. Ed. by Sreebny and Meyer. Oxford: Pergamon. EMMELIN, N. (1964 b). 'Influence of degenerating nerve fibres on the responsiveness of salivary-gland cells', J. Physiol. 171, EMMELIN, N. (1965). 'Action of transmitters on the responsiveness of effector cells', Experientia, 21, EMMELIN, N. (1967). 'Nervous control of the salivary glands'. In Handbook of Physiology: Alimentary Canal I. [In the press.] EMMELIN, N. and HOLMBERG, J. (1967). 'Impulse frequency in secretory nerves of salivary glands', J. Physiol. 191, EMMELIN, N., MALM, L. and STROMBLAD, R. (196). 'Functional union between hypoglossal and postganglionic parasympathetic nerve fibres', Experientia 17, 282. EMMELIN, N. and MUREN, A. (1952). 'The sensitivity of submaxillary glands to chemical agents studied in cats under various conditions over long periods', Acta physiol. scand. 26, GARRETT, J. R. (1966 a). 'The innervation of salivary glands. III. The effects of certain experimental procedures on cholinesterase-positive nerves in glands of the cat', Jl R. microsc. Soc. 86, GARRETT, J. R. (1966 b). 'The innervation of salivary glands. IV. The effects of certain experimental procedures on the ultra-structure of nerves in glands of the cat', Jl R. microsc. Soc. 86, HOFFMAN, H. (1952). 'Accleration and retardation of the process of axon-sprouting in partially denervated muscles', Austral. J. exp. Biol. 3, MURRAY, J. G. and THOMPSON, J. W. (1957). 'The occurrence and function of collateral sprouting in the sympathetic nervous system of the cat', J. Physiol. 135, NORDENFELT, I. (1963). 'Choline acetylase in normal and denervated salivary glands', Q. Jl exp. Physiol. 48, NORDENFELT, I. (1965). 'Choline acetylase in salivary glands of the cat after sympathetic denervation', Q. Jl exp. Physiol. 5, NORDENFELT, I. and PEREC, C. (1967). 'Acetylcholine synthesis in reinnervated salivary glands', Q. Ji exp. Physiol. 52,