Innervation of Extrahepatic Biliary Tract, With Special Reference to the Direct Bidirectional Neural Connections of the Gall Bladder, Sphincter of Oddi and Duodenum in Suncus murinus, in Whole-Mount Immunohistochemical Study.

Sphincter of Oddi dysfunction is one of the most important symptoms in post-cholecystectomy syndrome. Using either electrical or mechanical stimulation and retrogradely transported neuronal dyes, it has been demonstrated that there are direct neural pathways connecting gall bladder and the sphincter of Oddi in the Australian opossum and the golden hamster. In the present study, we employed whole-mount immunohistochemistry staining to observe and verify that there are two different plexuses of the extrahepatic biliary tract in Suncus murinus. One, named Pathway One, showed a fine, irregular but dense network plexus that ran adhesively and resided on/in the extrahepatic biliary tract wall, and the plexus extended into the intrahepatic area. On the other hand, named Pathway Two, exhibiting simple, thicker and straight neural bundles, ran parallel to the surface of the extrahepatic biliary tract and passed between the gall bladder and duodenum, but did not give off any branches to the liver. Pathway Two was considered to involve direct bidirectional neural connections between the duodenum and the biliary tract system. For the first time, morphologically, we demonstrated direct neural connections between gall bladder and duodenum in S. murinus. Malfunction of the sphincter of Oddi may be caused by injury of the direct neural pathways between gall bladder and duodenum by cholecystectomy. From the viewpoint of preserving the function of the major duodenal papilla and common bile duct, we emphasize the importance of avoiding kocherization of the common bile duct so as to preserve the direct neural connections between gall bladder and sphincter of Oddi.

Reconsideration of the histologic definitions used in the pathologic staging of extrahepatic bile duct carcinoma.

The histologic boundaries of the extrahepatic bile ducts (EBDs) are not well defined, despite the fact that pathologic staging of carcinomas arising in these structures requires the determination of extent of tumor invasion in this area. Perhaps in part, because the smooth muscle band in the EBD wall is not well formed throughout the length of these structures, a previous definition of the outer portion of the bile duct wall included "loose, richly vascularized connective tissue, interlaced with large nerve fibers." We have experienced difficulty in the application of these criteria in staging EBD carcinomas, which requires the histologic determination of the extent of the EBD wall. To systematically study the histologic features of EBD tissue boundaries, 34 EBD specimens obtained from autopsy were analyzed with attention to the distribution of blood vessels and nerve fibers along the length of this system. The EBD specimens were divided into lower, middle, and upper portions, and the locations of blood vessels and nerve fibers were then analyzed separately at each location. We defined the fibromuscular wall as the dense concentric arrangement of collagen and smooth muscle fibers surrounding the EBD mucosa. The location of blood vessels and nerve fibers was categorized as either (1) within, (2) junctional to, or (3) outside of the fibromuscular wall. Blood vessels and nerve fibers are located predominantly outside of the fibromuscular wall and are usually surrounded by adipose tissue throughout the entire EBD, however, their distribution in this location is not consistent. Because of these histologic features, we propose that the bile duct wall is more precisely defined as occurring between the mucosa and the outermost boundary of dense fibromuscular tissue, without consideration of the presence or absence of blood vessels and nerve fibers.

Innervation of the extrahepatic biliary tract.

The extrahepatic biliary tract is innervated by dense networks of extrinsic and intrinsic nerves that regulates smooth muscle tone and epithelial cell function of extrahepatic biliary tree. Although these ganglia are derived from the same set of precursor neural crest cells that colonize the gut, they exhibit structural, neurochemical, and physiological characteristics that are distinct from the neurons of the enteric nervous system. Gallbladder neurons are relatively inexcitable, and their output is driven by vagal inputs and modulated by hormones, peptides released from sensory fibers, and inflammatory mediators. Gallbladder neurons are cholinergic and they can express a number of other neural active compounds, including substance P, galanin, nitric oxide, and vasoactive intestinal peptide. Sphincter of Oddi (SO) ganglia, which are connected to ganglia of the duodenum, appear to be comprised of distinct populations of excitatory and inhibitory neurons, based on their expression of choline acetyltransferase and substance P or nitric oxide synthase, respectively. While SO neurons likely receive vagal input and their activity is modulated by release of neuropeptides from sensory fibers, a significant source of excitatory synaptic input to these cells arise from the duodenum. This duodenum-SO circuit is likely to play an important role in the coordination of SO tone with gallbladder motility in the process of gallbladder emptying. Now that we have gained a relatively thorough understanding of the innervation of the biliary tree under healthy conditions, the way is paved for future studies of altered neural function in biliary disease.

Responses of spinohypothalamic tract neurons in the thoracic spinal cord of rats to somatic stimuli and to graded distention of the bile duct.

Anatomical studies indicate that a relatively large percentage of spinohypothalamic tract (SHT) neurons are located within thoracic spinal segments. The aim of this study was to characterize the responses of SHT neurons in these segments of rats to innocuous and noxious stimulation of the skin and of a visceral structure, the bile duct. In addition, we attempted to determine the trajectories of the axons of the examined neurons within the diencephalon and brainstem. Fifty-three SHT neurons were recorded within segments T8-T13 in urethane anesthetized rats. Each cell was antidromically activated using current pulses < or = 30 microA delivered from the tip of an electrode located within the contralateral hypothalamus. The recording points were located in the superficial dorsal horn (9) and deep dorsal horn (44). All examined SHT neurons had receptive fields on the posterior thorax and anterior and ventral abdomen of the ipsilateral side. Ninety percent of the 41 SHT neurons responded exclusively (13) or preferentially (24) to noxious cutaneous stimuli. Thirteen of 27 (48%) examined units were activated by forceful distention of the bile duct. Response thresholds ranged from 30 to 40 mmHg. Responses incremented as pressures were increased to 50-80 mmHg. The axons of 22 of 28 (79%) examined SHT neurons appeared to cross the midline within the hypothalamus and terminate in the ipsilateral hypothalamus, thalamus or midbrain. The results indicate that SHT neurons in thoracic spinal cord of rats are capable of conveying somatic and visceral nociceptive information from the bile duct directly to targets at various levels of the brain bilaterally.

Effects of nitric oxide in the dorsal motor nucleus of the vagus on the extrahepatic biliary system in rabbits.

To investigate whether nitric oxide (NO) in the dorsal motor nucleus of the vagus (DMV) mediated an influence on the extrahepatic biliary system, we studied the effects of microinjection of NO-producing drugs into DMV on the motilities of the gallbladder (GB) and the sphincter of Oddi (SO) in anesthetized rabbits. Microinjection of the NO precursor L-arginine into the rostral DMV produced an increase in the GB and SO motilities, which can be counteracted by both NG-nitro-L-arginine-methyl (L-NAME), an inhibitor of NOS, and reduced hemoglobin (rHb), a scavenger of NO, and were eliminated by bilateral cervical vagotomy. On the other hand, the NO donor sodium nitroprusside (SNP) was able to mimic the excitatory effect of L-arginine. This effect can be antagonized by rHb, but not by L-NAME, for SNP supplied exogenous NO without activating NOS. These results indicate that NO in the DMV mediates an excitatory effect on the extrahepatic biliary system.

The innervation of rainbow trout (Oncorhynchus mykiss) liver: protein gene product 9.5 and neuronal nitric oxide synthase immunoreactivities.

We have explored the innervation of the rainbow trout (O. mykiss) liver using immunohistochemical procedures and light microscopy to detect in situ protein gene product 9.5 and neuronal nitric oxide synthase immunoreactivities (PGP-IR and NOS-IR). The results showed PGP-IR nerve fibres running with the extralobular biliary duct (EBD), hepatic artery (EHA) and portal vein (EPV) that form the hepatic hilum, as well as following the spatial distribution of the intrahepatic blood vessel and biliary channels. These nerve fibres appear as single varicose processes, thin bundles, or thick bundles depending on their diameter and location in the wall of the blood vessel or biliary duct. No PGP-IR fibres were detected in the liver parenchyma. NOS-IR nerve fibres were located only in the vessels and ducts that form the hepatic hilum (EBD, EHA, EPV); in addition, NOS-IR nerve cell bodies were found isolated or forming ganglionated plexuses in the peribiliary fibromuscular tissue of the EBD. No PGP-IR ganglionated plexuses were detected in the EBD. The location of the general (PGP-IR) and nitrergic (nNOS-IR) intrinsic nerves of the trout liver suggest a conserved evolutionary role of the nervous control of hepatic blood flow and hepatobiliary activity.

Close anatomical relationships between nerve fibers and MHC class II-expressing dendritic cells in the rat liver and extrahepatic bile duct.

The anatomical relationships between immunocytochemically identified nerve fibers and MHC class II-expressing antigen presenting dendritic cells were investigated in the rat hepatobiliary system using immunocytochemistry, confocal laser scanning, and electron microscopy. Close proximity of nerve fiber varicosities immunostained for PGP 9.5 and MHC class II-expressing dendritic cells was frequently observed in the wall of extrahepatic bile ducts, in Glisson's area, around central and hepatic veins, and in the liver capsule. Contacts between nerve fibers staining for substance P, calcitonin gene-related peptide, calretinin, and vasoactive intestinal polypeptide and dendritic cells were more often observed around extrahepatic bile ducts than in Glisson's area. Nerve fibers immunostaining for tyrosine hydroxylase and neuropeptide Y were numerous both in the wall of extrahepatic bile ducts and in Glisson's area and frequently contacted dendritic cells there. At the ultrastructural level, close membrane contacts between bare axolemmal areas of unmyelinated nerve fibers and processes of MHC class II-expressing cells were observed. These results demonstrate close anatomical relationships of nerve fibers from various sources with antigen presenting dendritic cells in the visceral domain and suggest modulation of antigen presentation by the autonomic nervous system.

Blood pressure reflexes following activation of capsaicin-sensitive afferent neurones in the biliopancreatic duct of rats.

1. Inflammatory diseases of the pancreas or diseases which cause obstruction within the biliary or within the biliary or pancreatic duct system are associated with severe pain. Although neuropeptides such as substance P are present in the biliary tree, only few capsaicin-sensitive, substance P-positive nerve fibres have been found in the ducts. In order to obtain functional evidence whether capsaicin-sensitive afferent neurones transmit nociceptive information arising from the biliopancreatic duct, blood pressure reflexes following electrical stimulation of the duct or increases in intraductal pressure were determined in barbiturate-anaesthetized rats. 2. Electrical stimulation of neurones in the biliopancreatic duct was carried out at 30 V, 3 ms, 50 Hz for 20s. In untreated animals the electrical stimulation resulted in rises in blood pressure by up to 25 mmHg, but in about a quarter of all animals tested this response was absent. Following the administration of phentolamine (7 mumol kg-1, i.p.) the blood pressure responses were changed to pronounced and reproducible depressor reflexes of -5 to -30 mmHg. Retrograde injections into the biliopancreatic duct of 300 microliters of a 154 mM sodium chloride solution produced increases in intraductal pressure of approximately 10 mmHg. This elicited shortlasting falls in blood pressure of 3-15 mmHg. Phentolamine significantly augmented the fall in blood pressure to 8-30 mmHg. 3. The depressor reflexes observed in both models after the administration of phentolamine were abolished by morphine (1 mumol kg-1, i.v.). The inhibition by morphine was reversed by naloxone (3 mumol kg-1, i.v.). Naloxone given before morphine did not affect the depressor reflex but prevented the inhibitory action of subsequently injected morphine.4. Acute s.c. injection of capsaicin (30 mg kg-1) abolished the depressor reflexes in response to both types of nociceptive stimulation in phentolamine-treated rats. The initial pressor effects of electrical stimulation were only partly inhibited by capsaicin whereas the basal depressor reflexes in response to elevation of intraductal pressure were abolished. In rats which had received capsaicin on the day before the experiment or had been treated with capsaicin as neonates, only minor rises in blood pressure were induced by electrical stimulation at the beginning of the experiment and no changes in blood pressure occurred after the administration of phentolamine. After adult or neonatal pretreatment with capsaicin the depressor reflexes in response to increased intraductal pressure were only small and were unchanged by phentolamine.5. The depressor reflexes following either electrical stimulation or increases in intraductal pressure were abolished by the unselective Beta-blocker, (-)-propranolol (3 micromol kg-1, i.p.), and greatly reduced by the Beta 1-blocker, metoprolol (6 micromol kg- 1, i.p.). The Beta2-preferring adrenoceptor antagonist, butoxamine(3 micromol kg-1, i.p.), had no effect on the depressor responses. The reflex falls in blood pressure were also abolished by hexamethonium (10 micromol kg-1, i.p.) but not by atropine (3 micromol kg-1, i.p.).6. Both models of stimulation of nociceptive afferents caused identical patterns of blood pressure responses following adrenalectomy or chemical sympathectomy. In adrenalectomized rats, the initial responses consisted of depressor reflexes which were not augmented but significantly reduced by phentolamine and further inhibited by metoprolol. In rats that had been pretreated with 6-hydroxydopamine(total dose 0.6 mmol kg-1) to accomplish chemical sympathectomy, nociceptive stimulation caused rises in blood pressure. Phentolamine treatment abolished these pressor effects but revealed only minor, if any, depressor responses that were unaffected by metoprolol.7. In summary, the hypotensive effects in both models constitute nociceptive reflexes since they are abolished by morphine and restored by naloxone. The afferent part of the reflex is mediated by nerve fibres sensitive to capsaicin. Both experimental procedures seem to elicit two, presumably separate, reflex mechanisms. Firstly, catecholamines released from the adrenal medulla elevate blood pressure or limit hypotensive responses via activation of vascular alpha receptors. Secondly, the reflex inhibition of the sympathetic nerve activity in the heart and the vasculature causes the nociceptive depressor reflexes.

Structure and innervation of the extrahepatic biliary system in the Australian possum, Trichosurus vulpecula.

The morphology, microanatomy and innervation of the biliary tree of the Australian possum, Trichosurus vulpecula, was examined. The gross morphology of the gallbladder, hepatic and cystic ducts, and the course of the common bile duct, conforms to those of other species. The sphincter of Oddi has an extraduodenal segment that extends 15mm from the duodenal wall; within this segment the pancreatic and common bile ducts are ensheathed together by sphincter muscle. Their lumens unite to form a common channel within the terminal intraduodenal segment. Nerve cell bodies of the gallbladder were found in an inter-connecting network of ganglia that were located in the serosa, muscularis and mucosa. Nerve fibres innervated the muscle, arterioles and the mucosa. Few ganglia were found along the supra sphincteric portion of the common bile duct. Nerve trunks followed the duct and a dense nerve fibre plexus was found in the mucosa. In the sphincter most ganglia were located in two plexuses, the first between the layers of the external sphincter muscle, which was continuous with the external muscle of the duodenum, and the second was associated with the internal sphincter muscle. Nerve fibres were numerous in the sphincter muscle, and were also found in the subepithelial and periglandular plexuses of both the pancreatic and common bile ducts.