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Effects of extrinsic denervation on net water transport and motility of the feline gallbladder in vivo
JOURNAL
OF SURGICAL
RESEARCH
36, 563-570 (1984)
Effects of Extrinsic Denervation on Net Water Transport Motility of the Feline Gallbladder in Vivo S. BJ~RCK, *Department
and
M.D., PH.D.,* A. DAHLSTR~~M, M.D., PH.D.,? AND H. AHLMAN, M.D., PH.D.*
of Surgery and tlnstitute
of Neurobiology,
University ofGc%eborg, Gijteborg, Sweden
Presented at the Annual Meeting of the Association for Academic Surgery, Syracuse, New York, November 2-5, 1983 The influence on the concentrating ability of the gallbladder after extrinsic denervation was studied in anesthetized cats, previously subjected to truncal vagotomy, and/or celiacectomy, and compared with sham-operatedcontrols.Net water absorption wasstudied by perfusion techniques.Acute experiments were performed under basal conditions and or-adrenoceptorstimulation (iv infusion of norepinephrine (NE), 1 pg/kg.min). Gallbladder biopsies were studied by fluorescence microscopy to visualize and quantitate catecholamines.Three weeks after celiacectomy basal absorption had decreasedsignificantly. In the short-term vagotomy group no changeswere demonstrated. However, in the long-term vagotomy group there was a fourfold increase in absorptive capacity, which decreasedto control levels after LYadrenoceptor blockade (phentolamine 1 mg/kg iv). Long-term vagotomy with subsequentceliacectomy caused no significant changes. Infusion of NE increased net water absorption by 70 + 16% in all experimental groups except in long-term vagotomized animals, where the high basal absorption was not further augmented. One hour after NE infusion controls returned to basal absorption rate, while denervated cats remained at stimulated levels. In long-term vagotomized gallbladders there were morphological signs of adrenergic proliferation (increased total number of nerve terminals, sprouting, and elevated levels of intraneuronal NE). In conclusion these results suggest that the adrenergic nervous system is important for full absorptive capacity of the gallbladder. The increased absorption after longterm vagotomy, abolishedafter cY-adrenoceptor blockade, might well be explained by the parallel adrenergic proliferation. This hypothesis was further corroborated in animals with long-term vagotomy, where subsequent surgical adrenergic denervation restored basal absorption to control levels. There was no motility responseto NE in controls or short-term vagotomy animals, but after previous celiacectomy a short relaxation was Seenindicating denervation supersensitivity of /3-adrenoceptors.After long-term vagotomy there was a contractile response indicating activation of a-adrenoceptors as outlined in the absorption study.
The sympathetic innervation of the gallbladder originates from adrenergic neurons in the celiac ganglion [3], while the parasympathetic innervation mainly comes from hepatic branches of the vagal trunks [ 171.However, both the vagal and splanchnic nerves contain numerous axons immunoreactive for several neuropeptides [ 16, 241. Within the gallbladder wall there are Substance P-, enkephalin-, and VIP-immunoreactive nerve fibers [26, 28, 311, and abundant nerve cells containing these peptides in support of an extrinsic as well as an intrinsic peptidergic innervation. We have earlier documented the adrenergic influence on net water transport and motility of the gallbladder in acute ex563
periments [5]. Electrical stimulation of the splanchnic nerves or cr-adrenoceptor stimulation by infusion of NE increased the rate of net water absorption from the gallbladder lumen, while adrenergic &receptor stimulation by infusion of isoproterenol relaxed the gallbladder without obvious influence on the concentrating ability [6]. Electrical stimulation of the vagal nerves or infusion of acetylcholine induced gallbladder contraction but had no effect on the concentrating ability. However, similar vagal stimulation after pretreatment with atropine decreasedthe net water transport by a nonadrenergic, noncholinergic mechanism, and induced gallbladder relaxation. Somatostatin was therefore tested as a candidate 0022-4804184 $1 SO Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
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JOURNAL OF SURGICAL RESEARCH: VOL. 36, NO. 6, JUNE 1984
hormone for these supposedly peptidergic effects. However, somatostatin was without effect on basal net water transport [7], but inhibited the expected decrease in transport during vagal activation after atropinization. On the other hand somatostatin did not interfere with the enhanced net water absorption at splanchnic nerve stimulation. The functional integrity of the gallbladder may be influenced by extrinsic denervations. Thus, Schein et al. [29] showed that implantation of gallstones into the canine gallbladder after truncal vagotomy resulted in cholecystitis but was without effect in animals with intact vagal nerves. Sympathectomy may even have a protective effect against experimental cholecystitis [ 181. Denervation procedures may induce marked changes in the receptor responsiveness in several organs; for example, Sundin and Dahlstrom [30] reported that parasympathetic denervation of the feline urinary bladder caused a compensatory increase of the adrenergic innervation with hypersensitivity of a-adrenoceptors. Similarly, Ekstrom [ 141reported a sensitization of cr-adrenoceptors after parasympathetic denervation of the rat parotid gland. Against this background, we were interested to study the chronic effects of various kinds of extrinsic denervations on the motor and secretory function of the feline gallbladder in vivo.
balance was continuously recorded by a pen recorder. A nonabsorbable marker, [‘4C]polyethylene glycol, was added to the perfusate. The net water transport was calculated from the perfusion rate and the change in concentration of the tracer during its passagethrough the gallbladder. Physiological studies were performed on animals divided into the following experimental groups. 1. Sham-operated controls (n = 9). 2. Tmnsthoracic truncal vagotomy 3 weeks prior to experiments (n = 6). 3. Truncal vagotomy 3 months prior to the experiments (n = 6). 4. Celiacectomy (celiac and superior mesenteric ganglia excised) 3 weeks prior to the experiments (n = 6). 5. Truncal vagotomy 3 months prior to celiacectomy, 3 weeks (n = 5).
Controls (group 1). Net water transport studies were performed in all animals during basal conditions for 1 hr, thereafter.during the infusion of NE (1 p/kg - min iv) for another hour, followed by a poststimulation period of 1 hr. In a separate group of three animals, vagotomized for 3 months, absorption studies were performed before and after blockade of the a-adrenoceptors with phentolamine given as a bolus dose (l-2 mg/kg iv). The weight changes on the balance reflect not only net water absorption in the gallbladder but also displacements of water to or from MATERIAL AND METHODS the gallbladder lumen. The time course made Experiments were performed on 40 cats, it possible to differ between displacements due anesthetized with chloralose (50-70 mg/kg to motility (fast changes) and changes in net body wt iv) after induction with ketamine (50 water absorption (slow changes) across the mg/kg body wt iv). The animals were fasted gallbladder wall [32]. For the determination for 24 hr but had free accessto water. The of the adrenergic innervation fluorescence experimental approach was similar to that de- histochemistry for biogenic amines according scribed in an earlier paper [32]. The method to the Hillarp-Falck technique was used [2]. includes a continuous perfusion of the gall- Biopsies (2 X 2 mm) were taken from the bladder (ligated cystic duct) with an electrolyte fundus, body, and neck of three control gallsolution through two catheters inserted into bladders and from four animals vagotomized the fundus and the neck of the gallbladder 3 months prior to the study. The biopsies were and connected to separate chambers of an placed on coded papers, frozen in liquid proelectromagnetic balance. The pylorus was li- pane, cooled by liquid nitrogen, and freeze gated. The total weight on the electromagnetic dried at -45°C for 3 days. After treatment
BJijRCK, DAHLSTRGM,
AND AHLMAN:
565
GALLBLADDER
In this group infusion of NE did not cause any motility response. Vagotomy for 3 weeks (group 2). In all six experiments the basal water transport was in the same range as in controls (group l), but the infusion of NE did not cause a significant change in the absorptive capacity. Neither did infusion of NE cause any motility response. Vagotomyfor 3 months (group 3). The basal net water transport was increased almost four times compared with controls. During the infusion of NE, the absorption was not further RESULTS augmented, and remained at high levels also after the infusion was stopped. I. Net Water Transport (Fig. 1) and In these animals infusion of NE induced a Gallbladder Motility (Table 1) prolonged contraction of the gallbladder after Controls (Group 1). In nine cats there was an initial brief relaxation (~1 min). an average increase in net water transport of Celiacectomyfor 3 weeks(group 4). During 72% in responseto the infusion of NE during resting conditions the net water transport was 60 min. The mean arterial blood pressure in- significantly lower than in controls. Infusion creasedby 2 1 k 3 mm Hg during the infusion. of NE caused a significant increase in water After cessationof infusion the water transport absorption, that persisted after the infusion decreasedto prestimulatory levels. was stopped. with paraformaldehyde gas at 80°C and paraffin embedding in vucuo, the tissue specimens were sectioned at 10 pm, placed on glassslides, and mounted as previously described [c.f. 121. The specimens were examined in a fluorescence microscope equipped for transillumination and with filter combinations as described earlier [ 121. Statistics. The calculations of physiological data on net water transport were performed with analysis of variance (Duncan’s method for multiple comparison).
m
q
BASAL
ABSORPTION
STIMULATED TION AFTER
GROUP IV ‘f GANGLIONi ECTOMY
ABSORP-
STIMULATION
GROUP V VAGOTOMY +GANGLIONECTOMY
n=9 n=6 n.6 n.6 t-t-5 FIG. 1. Net water absorption in gallbladders of denervated animals (groups 2-5) compared with shamoperated controls (group 1) during basal conditions, at NE infusion, and 1 hr poststimulation. **P < 0.005, *P < 0.05 denote significant changes within each group, while denotes significant difference from control group (P -c 0.05).
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JOURNAL OF SURGICAL RESEARCH: VOL. 36, NO. 6, JUNE 1984 TABLE 1
Vagotomyfor 3 months and a-adrenoceptor blockade (Fig. 2). The basal absorption was
MOTILITY RESPONSESOFTHEGALLBLADDER ATTHEINFUSIONOFNE
Controls (group 1): 0 Vagotomy 3 weeks (group 2): 0 Vagotomy 3 months (group 3): brief relaxation followed by prolonged contraction Celiacectomy (group 4): short relaxation Vagotomy + celiacectomy (group 5): short relaxation
n=6 n=6 n=6 n=6
high in all three animals and was decreased by the iv infusion of phentolamine (during 5 min) to about 80% of the initial value. These animals all relaxed their gallbladders during phentolamine injection. II. Histochemical
Studies (Figs. 3 and 4)
n=5
In control gallbladders the formaldehydeinduced green NE fluorescence was localized n=3 to abundant varicose adrenergic nerve fibers, distributed mainly to the muscle layer and to the blood vessels,but a few thin fibers were The motility response to NE was a short observed also in the submucosa. Long-term vagotomized gallbladders showed a prominent relaxation (t5 min). Vagotomy and celiacectomy (group 5). The thickening of all layers of the wall compared basal rate of absorption was in the same range with the controls (X2-3) and the mucosa was as controls, but differed from long-term va- also folded. There was a parallel increase in gotomized animals. The net water transport the total number of adrenergic nerve termiwas not significantly increased by the infusion nals, and the varicosities had in general a stronger fluorescence intensity, indicating of NE. The motility responseto NE was similar as higher concentration of NE in all locations studied. The nerve terminal net work of the in group 4-short relaxation.
Vagotomy 3 months and phentolamine (no NE infusion): relaxation
FIG. 2. Representative experiment in one long-term vagotomized animal studied during basal conditions. Note the fist change in gallbladder volume (= relaxation) at injection of phentolamine. Note also the change from rapid absorption initially in this animal to slow absorption (slow change in text) at blockade of ol-adrenoceptors.
BJijRCK, DAHLSTROM,
AND AHLMAN:
GALLBLADDER
567
times over controls at basal conditions. Thus, this increased absorption could not be further augmented by NE (Fig. 1). Three weeks after celiacectomy a significant decreasein net water transport, compared with controls, was seen. In animals with long-term vagotomy and subsequent celiacectomy the absorption of water was higher than in controls, but not as high as in animals with long-term vagotomy alone.
FIG. 3. Fluorescencemicrograph of transverse section of the gallbladder wall in a sham-operated animal. Numerous adrenergic nerve terminals (arrow indicates such terminals around a vessel) and adrenergic nerve fibers with specific blue-green fluorescencepresent throughout the entire wall. Nonspecific autofluorescence(due to bile salts) present on the luminal side on top of the mucosa (M). S = serosal side. X 160.
submucosa time was clearly denser than in control gallbladders. Growth cones, indicating proliferation of adrenergic nerve terminals, were observedin one of the vagotomized gallbladders. DISCUSSION
In the present study the concentrating function of the feline gallbladder was studied by a perfusion technique after various denervation procedures. The experimental setup also enabled the study of gallbladder motility as a function of volume changes within the gallbladder. Furthermore the study included visualization of the adrenergic nerves in the gallbladder wall by use of fluorescence histochemistry in controls and long-term vagotomized animals. Lu-Adrenoceptorstimulation caused an increase in net water transport in all groups except in long-term vagotomized animals. On the other hand in these animals the water transport was increased about four
FIG. 4. Fluorescencemicrographs of transverse section of the gallbladder wall in a long-term vagotomy animal. Note the increased wall thickness and the increased total number of adrenergic nerve fibers, especially in the submucosa. Most fibers are notably thicker and also the individual varicosities (arrows indicate a vessel) have an increased fluorescenceintensity indicating high intracellular levels of NE. M = mucosal and S = serosal side. X140. Lower right: Detail showing hypertrophy of adrenergic varicosities of arteries in the muscle layer and around submucosal vessels(the lower let?). X2 10.
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One hour following a-adrenoceptor stimulation with NE only controls (group 1) returned to basal absorption rate, while all denervated groups remained at stimulated levels (Fig. 1). Furthermore, in three long-term vagotomized animals a-adrenergic blockade with phentolamine caused an 80% reduction in net water transport (Fig. 2). These observations together indicate that adrenergic nerves participate in the increase of net water transport, probably via an a-adrenergic mechanism. It is noteworthy that, using fluorescence histochemistry, thicker and more strongly fluorescent adrenergic fibers were observed in an increased number in the gallbladder wall 3 months after vagotomy. Thus, in these animals the increasedabsorption, which could be markedly reduced by a-adrenoceptor blockade could be related to an increased number of adrenergic terminals, which in addition contained more NE than those of control gallbladders. An a-adrenoceptor-mediated increasein net water transport of the feline gallbladder has earlier been observedin acute experiments [5]. Similar adrenergic mechanisms are also operating in the small intestine and the kidney [4,8, 13, 15, 19,211. The precise site ofaction for such an cu-adrenoceptor-mediated mechanism in net water transport is not clearly understood. However, since the epithelial cells of the gallbladder are under direct adrenergic nervous control [22], a direct effect by NE on the epithelial cell is a possible mode of action. The observed increased adrenergic nerve supply to the mucosa after long-term vagotomy may explain the marked change in net water transport in these gallbladders. The results from our histochemical studies corroborate the findings of Tsurumi and Onda [34], who described a similar proliferation of adrenergic fibers in all layers of the gallbladder wall in patients who had undergone gastrectomy, a surgical procedure that probably had severed the vagal fibers to the gallbladder. An alteration in adrenergic innervation and in adrenoceptor function of the feline urinary bladder, similar to the findings in the present study, was reported by Sundin and Dahlstriim
[30]. The Badrenoceptor response to hypogastric nerve activation observed in controls was changed into an a-adrenoceptor response after long-term parasympathetic denervation, together with a marked increase in the adrenergic innervation of the detrusor muscle. In a study of the rat parotid gland, Ekstriim [ 141 found a sensitization of cu-adrenoceptorsfollowing parasympathetic denervation of the gland. Also in the CNS Crutcher and Davis [ 111 found noradrenergic sprouting in responseto choline@ denervation of the sympathohabenular connection. Thus, some evidence indicates that parasympathetic denervation in general will induce proliferation of the adrenergic nervous system changing the pharmacological adrenoceptormediated responses. In the present study a significant decreasein basal net water transport in the gallbladder was found 3 weeks after celiacectomy compared with controls. A complete sympathetic denervation of the gallbladder seems less likely after celiacectomy because of contribution of adrenergic fibers from the vagus [2,25], and perivascular fibers [23]. However, Baumgarten and Lange [3] found a convincing morphologic reduction in the biliary adrenergic innervation after celiacectomy. Studies of the kidney, where adrenergic nerve terminals make contact with the basement membrane of proximal and distal tubular cells [2], have shown a graded reduction in the reabsorption of sodium and water after surgical denervation [4]. The decreasein net water transport of the gallbladder after surgical celiacectomy agrees well with the assumption that adrenoceptors are important for the full absorptive capacity of the gallbladder epithelium. The gallbladder smooth muscle has a main population of B-adrenoceptors, and only a small population of cu-adrenoceptors[27]. The b-adrenoceptors mediate relaxation of the gallbladder, e.g., by isoproterenol or splanchnit activation [ 51.This would explain the lack of significant motor response to NE in controls and short-term vagotomized animals (Table 1).
BJijRCK, DAHLSTRtjM,
AND AHLMAN:
In animals with excision of the celiac ganglion or in animals with the combined celiacectomy and vagotomy the gallbladder initially relaxed in response to NE infusion. These results could be interpreted as an increasedsensitivity of the &adrenoceptors, to some degree also stimulated by NE, in the sympathetically denervatedtissue, asoriginally described by Cannon and Rosenbleuth [lo]. In long-term vagotomized animals, the a-adrenoceptor stimulation caused a brief relaxation of the gallbladder, followed by a prolonged contraction. The contractile response may indicate an increased population and/or increased sensitivity of a-adrenoceptors after long-term vagotomy as concluded from the absorption studies. Our prelimmary data indicate that prostaglandin E2 is released into the gallbladder lumen after splanchnic nerve stimulation in atropine-pretreated animals. Since infusion of PGE2has been shown to induce a contraction of the gallbladder recorded by similar technique [33], and since prostaglandins may participate in the local regulation of NE release [c.f. 351,prostaglandins may play a role in the adrenergic regulation of the function of the gallbladder. Interestingly indomethacin, an inhibitor of prostaglandin synthesis, has antagonistic effectson increased absorption and motility of the gallbladder [33,36], which may have clinical applications. We conclude that the adrenergic nervous system within the gallbladder wall undergoes significant changesin responseto extrinsic denervations. These changesinclude the content of noradrenergic transmitter and also the receptor responsiveness. An intact adrenergic nerve supply appears important for full absorptive capacity, while chronic parasympathetic denervation induces a compensatory increase in adrenergic innervation (and a-adrenoceptor sensitivity), leading to markedly increased absorption. ACKNOWLEDGMENT
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REFERENCES Ahlman, H., Larson, G. M., Bombeck, C. T., and Nyhus, L. M. Origin of the adrenergic nerve fibres in the subdiaphragmatic vagus in the dog. Amer. J. Surg. 137: 116, 1979. Barajas, L., and Muller, J. The innervation of the juxtaglomerular apparatus and surrounding tubules: A quantitative analysis by serial section electron microscopy. J. Ultrastruct. Rex 43: 107, 1973. Baumgarten, H. G., and Lange, W. Extrinsic adrenergic innervation of the extrahepatic biliary duct system in guinea-pigs, cats and rhesusmonkeys. 2. Zellforsch. 100: 606, 1969. 4. Bencsath, B., Asztalos, B., et al. Renal handling of sodium after chronic sympathectomy in the anaesthetized rat. Amer. J. Physiol. 236(6): F513, 1979. 5. Bjiirck, S., Jansson,R., and Svanvik, J. The adrenergic influence on concentrating function in the feline gallbladder. Guf 23: 1019, 1982. 6. Bj&ck, S., Jansson, R., and Svanvik, J. Influence of electrical vagal stimulation and acetylcholine on the function of the feline gallbladder. Stand. J. Gastroenterol. 18: 129, 1983. 7. Bjiirck, S., and Svanvik, J. The influence of somatostatin on gallbladder responseto intraduodenal and autonomic nerve stimulation in the cat. Stand. J. Gastroenterol.. in press, 1984. 8. Brunsson, I., Eklund, S., et al. The effect of vasodilatation and sympathetic nerve activation on net water absorption in the cat small intestine. Acta Physiol. Stand. 106: 61, 1979. 9. Bukhave, K., and Rask-Madsen, J. Prostaglandin E2 in jejunal fluids and its potential diagnostic value for selecting patients with indomethacin sensitive diarrhoea. Eur. J. Cfin. Invest. 11: 191, 1981. 10. Cannon, W. B., and Rosenbleuth, A. The supersensitivity of denervated structures. New York: Macmillan, 1949. 11. Crutcher, K. A., and Davis, J. N. Noradrenergic sprouting in responseto cholinergic denervation: The sympathohabenularconnection. Exp. Neural. 70: 187, 1980. 12. Dahlstram, A., and Fuxe, K. A method for demonstration of adrenergic nerve fibres in peripheral nerves. Z. Zellforsch 62: 602, 1964. 13. Durbin, T. L., Rosenthal, K., et al. Clonidine and lidamine (WHR-1142) stimulate sodium and chloride absorption in the rabbit intestine. Gustroenterol. 82: 1352, 1982. 14 Ekstrijm, J. Sensitization of the rat parotid gland to secretagoguesfollowing either parasympathetic or sympathetic denervation or decentralization. Acta Physiol. &and. 108: 253, 1980. Field, M., and McCall, J. Ion transport in rabbit ileal mucosa. Effects of catecholamines. Amer. J. Physiol. 225: 852, 1973. I+.
This work was supported by the Swedish Medical Research Council (5220, 2207, 4984).
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16. Fumess, J. B., and Costa, M. Types of nerves in the enteric nervous system. Neuroscience 5: 1, 1980. 17. Hirt, A. Handbuch der vergleichenden Anatomie der Wirbeltier, Band 2, Haft I, 735. L. Balk E. Goppert, E. KaIlius, W. Lubosch. Berlin, 1934. 18. Howard, J. M., ef al. The significance of the sympathetic nervous system in acute cholecystitis. Surgery 32: 251, 1952. 19. Hubel, K. A. Intestinal ion transport: Effect of norepinephrine, pilocarpine and atropine. Amer. J. Phys-
27. Persson,C. G. A. Adrenergic, cholecystokinetic and morphine-induced effectson extrahepatic biliary motility. Ada Physiol. Stand. (Suppl.) 383, 1972. 28. Polak, J. M., Bloom, S. R., Sullivan, S. N., and Facer, P. Enkephahn-like immunoreactivity in the human gastrointestinal tract. Lancet 1: 972, 1977. 29. Schein, C. J., and Gliedman, M. L. The influence of vagotomy in the normal and diseased gallbladders. Digestion 3: 243, 1970.
iol. 231: 252, 1976.
20. Jonsson,G. Quantification of fluorescenceof biogenic monoamines, demonstrated with the formaldehyde fluorescencemethod. Stuttgart: Gustav FischerVerlag, 1971. 21. Kreis, G. J., and Fordtran, J. S. Physiology and pathophysiology of ion and water movement on the human intestine. In M. H. Sleisengerand J. S. Fordtran (Eds.), Gastrointestinal Disease. Philadelphia/ London/Toronto: Saunders, 1978. Pp. 267. 22. Kyosola, K., and Penttilit, 0. Adrenergic innervation of the human gallbladder. Histochemistry 54: 209, 1977. 23. Larson, G., Ahlman, H., Bombeck, C. T., and Nyhus, L. M. Gastric acid secretion after chemical sympathectomy. Surgery 85(5): 534, 1979. 24. Lundbetg, J. M., H&felt, T., Nilsson, G., et al. Peptide neurons in the vagus, splanchnic and sciatic nerves. Acta Physiol. &and.
and mouse. In U. S. von Euler and B. Pemow (Eds.), Substance P. New York: Raven Press, 1977. P. 49.
104: 499, 1978.
25. Muryobayashi, T. J., Fujiwara, M., et al. Fhtorescence h&chemical demonstration of adrenergicnerve fibres in the vagus nerve of cats and dogs. Japan. J. Pharmacol. 18: 285, 1968.
26. Nilsson, G., and Brodin, E. Tissue distribution of substance P-like immunoreactivity in dog, cat, rat
30. Sundin, T., and Dahlstriim, A. The sympathetic innervation of the urinary bladder and urethra in the normal state and after parasympathetic denervation at the spinal root level. Stand. J. Ural. Nephrol. 7: 131, 1973. 31. Sundler, F., Alumets, J., H%ansson, R., et al. VIP innervation of the gallbladder. Gastroenterol. 72: 1375, 1977. 32. Svanvik, J., and Jansson,R. An experimental method for studying in vivo gallbladder absorption. Gasfroenterol. 72: 534, 1977.
33. Thornell, E., Svanvik, J., et al. Effect of intraarterial prostaglandin E2 on gallbladder fluid transport, motility and hepatic bile flow in the cat. Stand. J. Gastroenterol. 16: 1083, 1981.
34. Tsurumi, K., and Onda, M. A fluorescence histochemical study for the motility of the gallbladder. Gastroenterol. Japan. 44: 147, 1979. 35. Westfall, T. Local regulation of adrenergic neurotransmission. Physiol. Rev. 54(4), 1977. 36. Yamamoto, F. Contribution of prostaglandin to the contraction induced by catecholaminesin the isolated gallbladder of the guinea pig. Gastroenterol. Japan. 15: 433, 1980.
OF SURGICAL
RESEARCH
36, 563-570 (1984)
Effects of Extrinsic Denervation on Net Water Transport Motility of the Feline Gallbladder in Vivo S. BJ~RCK, *Department
and
M.D., PH.D.,* A. DAHLSTR~~M, M.D., PH.D.,? AND H. AHLMAN, M.D., PH.D.*
of Surgery and tlnstitute
of Neurobiology,
University ofGc%eborg, Gijteborg, Sweden
Presented at the Annual Meeting of the Association for Academic Surgery, Syracuse, New York, November 2-5, 1983 The influence on the concentrating ability of the gallbladder after extrinsic denervation was studied in anesthetized cats, previously subjected to truncal vagotomy, and/or celiacectomy, and compared with sham-operatedcontrols.Net water absorption wasstudied by perfusion techniques.Acute experiments were performed under basal conditions and or-adrenoceptorstimulation (iv infusion of norepinephrine (NE), 1 pg/kg.min). Gallbladder biopsies were studied by fluorescence microscopy to visualize and quantitate catecholamines.Three weeks after celiacectomy basal absorption had decreasedsignificantly. In the short-term vagotomy group no changeswere demonstrated. However, in the long-term vagotomy group there was a fourfold increase in absorptive capacity, which decreasedto control levels after LYadrenoceptor blockade (phentolamine 1 mg/kg iv). Long-term vagotomy with subsequentceliacectomy caused no significant changes. Infusion of NE increased net water absorption by 70 + 16% in all experimental groups except in long-term vagotomized animals, where the high basal absorption was not further augmented. One hour after NE infusion controls returned to basal absorption rate, while denervated cats remained at stimulated levels. In long-term vagotomized gallbladders there were morphological signs of adrenergic proliferation (increased total number of nerve terminals, sprouting, and elevated levels of intraneuronal NE). In conclusion these results suggest that the adrenergic nervous system is important for full absorptive capacity of the gallbladder. The increased absorption after longterm vagotomy, abolishedafter cY-adrenoceptor blockade, might well be explained by the parallel adrenergic proliferation. This hypothesis was further corroborated in animals with long-term vagotomy, where subsequent surgical adrenergic denervation restored basal absorption to control levels. There was no motility responseto NE in controls or short-term vagotomy animals, but after previous celiacectomy a short relaxation was Seenindicating denervation supersensitivity of /3-adrenoceptors.After long-term vagotomy there was a contractile response indicating activation of a-adrenoceptors as outlined in the absorption study.
The sympathetic innervation of the gallbladder originates from adrenergic neurons in the celiac ganglion [3], while the parasympathetic innervation mainly comes from hepatic branches of the vagal trunks [ 171.However, both the vagal and splanchnic nerves contain numerous axons immunoreactive for several neuropeptides [ 16, 241. Within the gallbladder wall there are Substance P-, enkephalin-, and VIP-immunoreactive nerve fibers [26, 28, 311, and abundant nerve cells containing these peptides in support of an extrinsic as well as an intrinsic peptidergic innervation. We have earlier documented the adrenergic influence on net water transport and motility of the gallbladder in acute ex563
periments [5]. Electrical stimulation of the splanchnic nerves or cr-adrenoceptor stimulation by infusion of NE increased the rate of net water absorption from the gallbladder lumen, while adrenergic &receptor stimulation by infusion of isoproterenol relaxed the gallbladder without obvious influence on the concentrating ability [6]. Electrical stimulation of the vagal nerves or infusion of acetylcholine induced gallbladder contraction but had no effect on the concentrating ability. However, similar vagal stimulation after pretreatment with atropine decreasedthe net water transport by a nonadrenergic, noncholinergic mechanism, and induced gallbladder relaxation. Somatostatin was therefore tested as a candidate 0022-4804184 $1 SO Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
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JOURNAL OF SURGICAL RESEARCH: VOL. 36, NO. 6, JUNE 1984
hormone for these supposedly peptidergic effects. However, somatostatin was without effect on basal net water transport [7], but inhibited the expected decrease in transport during vagal activation after atropinization. On the other hand somatostatin did not interfere with the enhanced net water absorption at splanchnic nerve stimulation. The functional integrity of the gallbladder may be influenced by extrinsic denervations. Thus, Schein et al. [29] showed that implantation of gallstones into the canine gallbladder after truncal vagotomy resulted in cholecystitis but was without effect in animals with intact vagal nerves. Sympathectomy may even have a protective effect against experimental cholecystitis [ 181. Denervation procedures may induce marked changes in the receptor responsiveness in several organs; for example, Sundin and Dahlstrom [30] reported that parasympathetic denervation of the feline urinary bladder caused a compensatory increase of the adrenergic innervation with hypersensitivity of a-adrenoceptors. Similarly, Ekstrom [ 141reported a sensitization of cr-adrenoceptors after parasympathetic denervation of the rat parotid gland. Against this background, we were interested to study the chronic effects of various kinds of extrinsic denervations on the motor and secretory function of the feline gallbladder in vivo.
balance was continuously recorded by a pen recorder. A nonabsorbable marker, [‘4C]polyethylene glycol, was added to the perfusate. The net water transport was calculated from the perfusion rate and the change in concentration of the tracer during its passagethrough the gallbladder. Physiological studies were performed on animals divided into the following experimental groups. 1. Sham-operated controls (n = 9). 2. Tmnsthoracic truncal vagotomy 3 weeks prior to experiments (n = 6). 3. Truncal vagotomy 3 months prior to the experiments (n = 6). 4. Celiacectomy (celiac and superior mesenteric ganglia excised) 3 weeks prior to the experiments (n = 6). 5. Truncal vagotomy 3 months prior to celiacectomy, 3 weeks (n = 5).
Controls (group 1). Net water transport studies were performed in all animals during basal conditions for 1 hr, thereafter.during the infusion of NE (1 p/kg - min iv) for another hour, followed by a poststimulation period of 1 hr. In a separate group of three animals, vagotomized for 3 months, absorption studies were performed before and after blockade of the a-adrenoceptors with phentolamine given as a bolus dose (l-2 mg/kg iv). The weight changes on the balance reflect not only net water absorption in the gallbladder but also displacements of water to or from MATERIAL AND METHODS the gallbladder lumen. The time course made Experiments were performed on 40 cats, it possible to differ between displacements due anesthetized with chloralose (50-70 mg/kg to motility (fast changes) and changes in net body wt iv) after induction with ketamine (50 water absorption (slow changes) across the mg/kg body wt iv). The animals were fasted gallbladder wall [32]. For the determination for 24 hr but had free accessto water. The of the adrenergic innervation fluorescence experimental approach was similar to that de- histochemistry for biogenic amines according scribed in an earlier paper [32]. The method to the Hillarp-Falck technique was used [2]. includes a continuous perfusion of the gall- Biopsies (2 X 2 mm) were taken from the bladder (ligated cystic duct) with an electrolyte fundus, body, and neck of three control gallsolution through two catheters inserted into bladders and from four animals vagotomized the fundus and the neck of the gallbladder 3 months prior to the study. The biopsies were and connected to separate chambers of an placed on coded papers, frozen in liquid proelectromagnetic balance. The pylorus was li- pane, cooled by liquid nitrogen, and freeze gated. The total weight on the electromagnetic dried at -45°C for 3 days. After treatment
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In this group infusion of NE did not cause any motility response. Vagotomy for 3 weeks (group 2). In all six experiments the basal water transport was in the same range as in controls (group l), but the infusion of NE did not cause a significant change in the absorptive capacity. Neither did infusion of NE cause any motility response. Vagotomyfor 3 months (group 3). The basal net water transport was increased almost four times compared with controls. During the infusion of NE, the absorption was not further RESULTS augmented, and remained at high levels also after the infusion was stopped. I. Net Water Transport (Fig. 1) and In these animals infusion of NE induced a Gallbladder Motility (Table 1) prolonged contraction of the gallbladder after Controls (Group 1). In nine cats there was an initial brief relaxation (~1 min). an average increase in net water transport of Celiacectomyfor 3 weeks(group 4). During 72% in responseto the infusion of NE during resting conditions the net water transport was 60 min. The mean arterial blood pressure in- significantly lower than in controls. Infusion creasedby 2 1 k 3 mm Hg during the infusion. of NE caused a significant increase in water After cessationof infusion the water transport absorption, that persisted after the infusion decreasedto prestimulatory levels. was stopped. with paraformaldehyde gas at 80°C and paraffin embedding in vucuo, the tissue specimens were sectioned at 10 pm, placed on glassslides, and mounted as previously described [c.f. 121. The specimens were examined in a fluorescence microscope equipped for transillumination and with filter combinations as described earlier [ 121. Statistics. The calculations of physiological data on net water transport were performed with analysis of variance (Duncan’s method for multiple comparison).
m
q
BASAL
ABSORPTION
STIMULATED TION AFTER
GROUP IV ‘f GANGLIONi ECTOMY
ABSORP-
STIMULATION
GROUP V VAGOTOMY +GANGLIONECTOMY
n=9 n=6 n.6 n.6 t-t-5 FIG. 1. Net water absorption in gallbladders of denervated animals (groups 2-5) compared with shamoperated controls (group 1) during basal conditions, at NE infusion, and 1 hr poststimulation. **P < 0.005, *P < 0.05 denote significant changes within each group, while denotes significant difference from control group (P -c 0.05).
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Vagotomyfor 3 months and a-adrenoceptor blockade (Fig. 2). The basal absorption was
MOTILITY RESPONSESOFTHEGALLBLADDER ATTHEINFUSIONOFNE
Controls (group 1): 0 Vagotomy 3 weeks (group 2): 0 Vagotomy 3 months (group 3): brief relaxation followed by prolonged contraction Celiacectomy (group 4): short relaxation Vagotomy + celiacectomy (group 5): short relaxation
n=6 n=6 n=6 n=6
high in all three animals and was decreased by the iv infusion of phentolamine (during 5 min) to about 80% of the initial value. These animals all relaxed their gallbladders during phentolamine injection. II. Histochemical
Studies (Figs. 3 and 4)
n=5
In control gallbladders the formaldehydeinduced green NE fluorescence was localized n=3 to abundant varicose adrenergic nerve fibers, distributed mainly to the muscle layer and to the blood vessels,but a few thin fibers were The motility response to NE was a short observed also in the submucosa. Long-term vagotomized gallbladders showed a prominent relaxation (t5 min). Vagotomy and celiacectomy (group 5). The thickening of all layers of the wall compared basal rate of absorption was in the same range with the controls (X2-3) and the mucosa was as controls, but differed from long-term va- also folded. There was a parallel increase in gotomized animals. The net water transport the total number of adrenergic nerve termiwas not significantly increased by the infusion nals, and the varicosities had in general a stronger fluorescence intensity, indicating of NE. The motility responseto NE was similar as higher concentration of NE in all locations studied. The nerve terminal net work of the in group 4-short relaxation.
Vagotomy 3 months and phentolamine (no NE infusion): relaxation
FIG. 2. Representative experiment in one long-term vagotomized animal studied during basal conditions. Note the fist change in gallbladder volume (= relaxation) at injection of phentolamine. Note also the change from rapid absorption initially in this animal to slow absorption (slow change in text) at blockade of ol-adrenoceptors.
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times over controls at basal conditions. Thus, this increased absorption could not be further augmented by NE (Fig. 1). Three weeks after celiacectomy a significant decreasein net water transport, compared with controls, was seen. In animals with long-term vagotomy and subsequent celiacectomy the absorption of water was higher than in controls, but not as high as in animals with long-term vagotomy alone.
FIG. 3. Fluorescencemicrograph of transverse section of the gallbladder wall in a sham-operated animal. Numerous adrenergic nerve terminals (arrow indicates such terminals around a vessel) and adrenergic nerve fibers with specific blue-green fluorescencepresent throughout the entire wall. Nonspecific autofluorescence(due to bile salts) present on the luminal side on top of the mucosa (M). S = serosal side. X 160.
submucosa time was clearly denser than in control gallbladders. Growth cones, indicating proliferation of adrenergic nerve terminals, were observedin one of the vagotomized gallbladders. DISCUSSION
In the present study the concentrating function of the feline gallbladder was studied by a perfusion technique after various denervation procedures. The experimental setup also enabled the study of gallbladder motility as a function of volume changes within the gallbladder. Furthermore the study included visualization of the adrenergic nerves in the gallbladder wall by use of fluorescence histochemistry in controls and long-term vagotomized animals. Lu-Adrenoceptorstimulation caused an increase in net water transport in all groups except in long-term vagotomized animals. On the other hand in these animals the water transport was increased about four
FIG. 4. Fluorescencemicrographs of transverse section of the gallbladder wall in a long-term vagotomy animal. Note the increased wall thickness and the increased total number of adrenergic nerve fibers, especially in the submucosa. Most fibers are notably thicker and also the individual varicosities (arrows indicate a vessel) have an increased fluorescenceintensity indicating high intracellular levels of NE. M = mucosal and S = serosal side. X140. Lower right: Detail showing hypertrophy of adrenergic varicosities of arteries in the muscle layer and around submucosal vessels(the lower let?). X2 10.
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One hour following a-adrenoceptor stimulation with NE only controls (group 1) returned to basal absorption rate, while all denervated groups remained at stimulated levels (Fig. 1). Furthermore, in three long-term vagotomized animals a-adrenergic blockade with phentolamine caused an 80% reduction in net water transport (Fig. 2). These observations together indicate that adrenergic nerves participate in the increase of net water transport, probably via an a-adrenergic mechanism. It is noteworthy that, using fluorescence histochemistry, thicker and more strongly fluorescent adrenergic fibers were observed in an increased number in the gallbladder wall 3 months after vagotomy. Thus, in these animals the increasedabsorption, which could be markedly reduced by a-adrenoceptor blockade could be related to an increased number of adrenergic terminals, which in addition contained more NE than those of control gallbladders. An a-adrenoceptor-mediated increasein net water transport of the feline gallbladder has earlier been observedin acute experiments [5]. Similar adrenergic mechanisms are also operating in the small intestine and the kidney [4,8, 13, 15, 19,211. The precise site ofaction for such an cu-adrenoceptor-mediated mechanism in net water transport is not clearly understood. However, since the epithelial cells of the gallbladder are under direct adrenergic nervous control [22], a direct effect by NE on the epithelial cell is a possible mode of action. The observed increased adrenergic nerve supply to the mucosa after long-term vagotomy may explain the marked change in net water transport in these gallbladders. The results from our histochemical studies corroborate the findings of Tsurumi and Onda [34], who described a similar proliferation of adrenergic fibers in all layers of the gallbladder wall in patients who had undergone gastrectomy, a surgical procedure that probably had severed the vagal fibers to the gallbladder. An alteration in adrenergic innervation and in adrenoceptor function of the feline urinary bladder, similar to the findings in the present study, was reported by Sundin and Dahlstriim
[30]. The Badrenoceptor response to hypogastric nerve activation observed in controls was changed into an a-adrenoceptor response after long-term parasympathetic denervation, together with a marked increase in the adrenergic innervation of the detrusor muscle. In a study of the rat parotid gland, Ekstriim [ 141 found a sensitization of cu-adrenoceptorsfollowing parasympathetic denervation of the gland. Also in the CNS Crutcher and Davis [ 111 found noradrenergic sprouting in responseto choline@ denervation of the sympathohabenular connection. Thus, some evidence indicates that parasympathetic denervation in general will induce proliferation of the adrenergic nervous system changing the pharmacological adrenoceptormediated responses. In the present study a significant decreasein basal net water transport in the gallbladder was found 3 weeks after celiacectomy compared with controls. A complete sympathetic denervation of the gallbladder seems less likely after celiacectomy because of contribution of adrenergic fibers from the vagus [2,25], and perivascular fibers [23]. However, Baumgarten and Lange [3] found a convincing morphologic reduction in the biliary adrenergic innervation after celiacectomy. Studies of the kidney, where adrenergic nerve terminals make contact with the basement membrane of proximal and distal tubular cells [2], have shown a graded reduction in the reabsorption of sodium and water after surgical denervation [4]. The decreasein net water transport of the gallbladder after surgical celiacectomy agrees well with the assumption that adrenoceptors are important for the full absorptive capacity of the gallbladder epithelium. The gallbladder smooth muscle has a main population of B-adrenoceptors, and only a small population of cu-adrenoceptors[27]. The b-adrenoceptors mediate relaxation of the gallbladder, e.g., by isoproterenol or splanchnit activation [ 51.This would explain the lack of significant motor response to NE in controls and short-term vagotomized animals (Table 1).
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In animals with excision of the celiac ganglion or in animals with the combined celiacectomy and vagotomy the gallbladder initially relaxed in response to NE infusion. These results could be interpreted as an increasedsensitivity of the &adrenoceptors, to some degree also stimulated by NE, in the sympathetically denervatedtissue, asoriginally described by Cannon and Rosenbleuth [lo]. In long-term vagotomized animals, the a-adrenoceptor stimulation caused a brief relaxation of the gallbladder, followed by a prolonged contraction. The contractile response may indicate an increased population and/or increased sensitivity of a-adrenoceptors after long-term vagotomy as concluded from the absorption studies. Our prelimmary data indicate that prostaglandin E2 is released into the gallbladder lumen after splanchnic nerve stimulation in atropine-pretreated animals. Since infusion of PGE2has been shown to induce a contraction of the gallbladder recorded by similar technique [33], and since prostaglandins may participate in the local regulation of NE release [c.f. 351,prostaglandins may play a role in the adrenergic regulation of the function of the gallbladder. Interestingly indomethacin, an inhibitor of prostaglandin synthesis, has antagonistic effectson increased absorption and motility of the gallbladder [33,36], which may have clinical applications. We conclude that the adrenergic nervous system within the gallbladder wall undergoes significant changesin responseto extrinsic denervations. These changesinclude the content of noradrenergic transmitter and also the receptor responsiveness. An intact adrenergic nerve supply appears important for full absorptive capacity, while chronic parasympathetic denervation induces a compensatory increase in adrenergic innervation (and a-adrenoceptor sensitivity), leading to markedly increased absorption. ACKNOWLEDGMENT
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This work was supported by the Swedish Medical Research Council (5220, 2207, 4984).
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