Innervation of the proximal human biliary tree.

The autonomic nervous system plays a role in a variety of liver regenerative and metabolic functions, including modulating bile secretion and cholangiocyte and hepatobiliary progenitors of the canals of Hering. However, the nature and location of nerves which link to the proximal biliary tree have remained uncertain. We investigate the anatomic relationship of nerves to the proximal biliary tree including the putative stem/progenitor cell niche of the canal of Hering. Using double immunostaining (fluorescence, histochemistry) to highlight markers of cholangiocytes (biliary-type keratins), nerves (S100, neurofilament protein, PGP9.5, tyrosine hydroxylase), and stellate cells (CRBP-1), we examined sections from normal adult livers from autopsy or surgical resections. There is extensive contact between nerves and interlobular bile ducts, bile ductules, and canals of Hering (CoH). In multiple serial sections from 4 normal livers, biliary-nerve contacts were seen in all of these structures and were more common in the interlobular bile ducts (78/137; 57%) than in the ductules and CoH (95/294; 33%) (p < 0.001). Contacts appear to consist of nerves in juxtaposition to the biliary basement membrane, though crossing through basement membrane to interface directly with cholangiocytes is also present. These nerves are positive for tyrosine hydroxylase and are, thus, predominately adrenergic. Electron microscopy confirms nerves closely approximating ductules. Nerve fiber-hepatic stellate cell juxtaposition is observed but without stellate cell approximation to cholangiocytes. We present novel findings of biliary innervation, perhaps mediated in part, by direct cholangiocyte-nerve interactions. The implications of these findings are protean for studies of neuromodulation of biliary physiology and hepatic stem/progenitor cells.

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus.

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the biliary tract in Suncus murinus (S. murinus ). First, the specimen was dissected from S. murinus and fixed in 4% paraformaldehyde (PFA). Second, an enzymatic treatment and potential endogenous peroxidase inactivation were performed. The specimen was then exposed to the primary antibody, anti-neurofilament protein antibody, for 3-6 days. It was then incubated with the secondary antibody conjugated with horseradish peroxidase. The color reaction was revealed by reacting the specimen with a 3,3'-diaminobenzidine (DAB) substrate. This method can be applied to analyze the innervation of all visceral organs. Furthermore, this protocol can also be adapted to test other neuronal antibodies, but optimization of the antibodies should be done first. This method was originally introduced by Kuratani and Tanaka1,2,3.

Updates on Precancerous Lesions of the Biliary Tract: Biliary Precancerous Lesion.

Precursor lesions of invasive adenocarcinoma of the bile duct (cholangiocarcinoma) have been increasingly recognized during the past decade because of the results of multiple studies on the carcinogenesis of cholangiocarcinoma, technologic advancements in diagnostic imaging modalities, and an increase in the volume of elective procedures. The two main precursor lesions of cholangiocarcinoma that have evolved are biliary intraepithelial neoplasia and intraductal papillary neoplasm of the bile duct. These lesions demonstrate histomorphologic similarities to pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasm of the pancreas, respectively, whereas mechanisms of carcinogenesis and risk of progressive disease appear distinct. An enhanced understanding of the clinical presentation and pathologic features of precursor lesions of the biliary tract and use of the correct terminology will facilitate efficient communication between surgeons, oncologists, and pathologists and improve quality of patient care.

Alpha-synuclein immunohistochemistry of gastrointestinal and biliary surgical specimens for diagnosis of Lewy body disease.

In Lewy body disease, Lewy pathology (LP: the accumulation of α-synuclein in neuronal perikarya and processes as Lewy bodies and Lewy neurites and dots, respectively) is observed in the central and peripheral nervous systems. Previous autopsy or biopsy studies of individuals with Lewy body diseases (LBDs) indicated that LP could be observed in the peripheral nerves of the gastrointestinal (GI) systems. The aim of this study is to clarify whether examination of GI and biliary surgical specimens would be useful for diagnosing LBD. We analyzed eight patients diagnosed clinically with LBD and with medical histories of GI or biliary surgery at our hospital. LP was identified by using α-synuclein immunohistochemistry in GI and biliary surgical specimens obtained before, at or after the clinical onset of LBD. LP was frequently observed in Auerbach's plexus, Meissner's plexus and the subserosal nerve fascicles within the GI and biliary surgical specimens. LP was observed in the specimens obtained 7 years before the onset of LBD. Our approach does not require any invasive procedures for patients. The immunohistochemical analysis of anti- α-synuclein antibody to archival GI or biliary surgical specimens from patients with clinically suspected LBD may contribute to clinical diagnosis of LBD.

Hypothermic oxygenated machine perfusion prevents arteriolonecrosis of the peribiliary plexus in pig livers donated after circulatory death.

BACKGROUND: Livers derived from donation after circulatory death (DCD) are increasingly accepted for transplantation. However, DCD livers suffer additional donor warm ischemia, leading to biliary injury and more biliary complications after transplantation. It is unknown whether oxygenated machine perfusion results in better preservation of biliary epithelium and the peribiliary vasculature. We compared oxygenated hypothermic machine perfusion (HMP) with static cold storage (SCS) in a porcine DCD model. METHODS: After 30 min of cardiac arrest, livers were perfused in situ with HTK solution (4°C) and preserved for 4 h by either SCS (n = 9) or oxygenated HMP (10°C; n = 9), using pressure-controlled arterial and portal venous perfusion. To simulate transplantation, livers were reperfused ex vivo at 37°C with oxygenated autologous blood. Bile duct injury and function were determined by biochemical and molecular markers, and a systematic histological scoring system. RESULTS: After reperfusion, arterial flow was higher in the HMP group, compared to SCS (251±28 vs 166±28 mL/min, respectively, after 1 hour of reperfusion; p = 0.003). Release of hepatocellular enzymes was significantly higher in the SCS group. Markers of biliary epithelial injury (biliary LDH, gamma-GT) and function (biliary pH and bicarbonate, and biliary transporter expression) were similar in the two groups. However, histology of bile ducts revealed significantly less arteriolonecrosis of the peribiliary vascular plexus in HMP preserved livers (>50% arteriolonecrosis was observed in 7 bile ducts of the SCS preserved livers versus only 1 bile duct of the HMP preserved livers; p = 0.024). CONCLUSIONS: Oxygenated HMP prevents arteriolonecrosis of the peribiliary vascular plexus of the bile ducts of DCD pig livers and results in higher arterial flow after reperfusion. Together this may contribute to better perfusion of the bile ducts, providing a potential advantage in the post-ischemic recovery of bile ducts.

[Biliary dysfunctions and their prevention].

THE AIM OF REVIEW: To present the up-to-date methods of prophylaxis of biliary system pathology. ORIGINAL POSITION: The number of patients with biliary tract pathology is constantly growing up. Participation of psychovegetative state in the development of gallbladder dysfunction was confirmed. CONCLUSION: Revealing and treatment of gallbladder dysfunction, treatment of disorders of psychovegetative disorders system in patients with dysfunction of gallbladder is the necessity for successful prophylaxis of chronic cholecystitis, cholelithiasis, pancreatitis.

Expression of NCAM in activated portal fibroblasts during regeneration of the rat liver after partial hepatectomy.

In the portal tract of the regenerating liver after partial hepatectomy, vascular and bile ductular remodeling takes place in response to the portal hyperdynamic state and parenchymal hyperplasia. In order to reveal phenotypical changes in the portal fibroblasts, we immunohistochemically investigated neural cell adhesion molecules (NCAM) and alpha smooth muscle actin (alphaSMA) expression and the ultrastructural changes in them during liver regeneration. In the control rat liver, portal fibroblasts were negative for both NCAM and alphaSMA. They became positive for both markers two days after partial hepatectomy, increased in staining intensity, reached a maximum at three to four days, then decreased, being still clearly positive at 14 days. Under an electron microscope, portal fibroblasts from the regenerating liver had larger amounts of cytoplasm and rough endoplasmic reticulum than those from the control liver; thus they might be activated. Additionally, periportal hepatic stellate cells in the regenerating liver were activated with alphaSMA, but without NCAM. The present study has demonstrated that portal fibroblasts express NCAM and alphaSMA in the regenerating liver after partial hepatectomy via transformation into myofibroblasts following reconstruction of the portal tracts.

Prediction of lymph node metastasis and perineural invasion of biliary tract cancer by selected features from cDNA array data.

OBJECTIVE: To better understand the nature of the malignancy of biliary tract carcinoma and evaluate the feasibility of its prediction by gene expression profiles. METHODS AND RESULTS: We explored the gene expression profiles characteristic of progression and invasiveness in the cDNA array data obtained from 37 biliary tract carcinomas (15 bile duct, 11 gallbladder, 11 of ampulla of Vater). We pre-selected 51 and 100 genes for the presence versus absence of lymph node metastasis and perineural invasion on the basis of statistical difference. To search optimized sets of genes for prediction, we applied a sequential forward feature selection, minimizing leave-one-out error rates on a k-nearest neighbor classifier. We could predict lymph node metastasis and perineural invasion with an accuracy of 94 and 100%, respectively. When the 6-stage IA cancers without perineural invasion were precluded, a marked difference in gene expression (147 gene), discriminable with 100% accuracy, was noted between positive versus negative perineural invasion, suggesting that the acquisition of invasive character is rather a later molecular pathological event in biliary tract cancer. CONCLUSION: The present method provides a powerful means of classifying biliary tract carcinomas. We also suggest that perineural invasion is an important target of array databased pattern classification, which may predict patient outcomes and facilitate the determination of the extent of surgery to minimize the risk of recurrence.

On the problem of innervation of the biliary tracts.

We studied the possibility of co-directed stimulatory effects of the sympathetic and parasympathetic systems on the motility of gallbladder, Oddi sphincter, and adjacent ascending portion of the duodenum. It is established that serotoninergic intramural neurons transmitting excitatory influences to(5)HT(1,2)-serotonin receptors of the effector tissues participate in this stimulatory effect.

[Serotonergic systems of the biliary tract, stomach and duodenum in the norm and at the experimental stomach ulcer]. / Serotoninérgicheskie sistemy biliarnogo trakta, zheludka i dvenadtsatiperstnoi kishki v norme i pri éksperimental'noi iazve zheludka

Stimulation of the sympathetic trunk against the background of the vagus nerve irritation was shown to cause the enhancement of the electromotor activity of the gallbladder, Oddi's sphincter, stomach and duodenum blocked by ganglionary and peripheral serotonin blockers. A lateral-medial gradient of the serotonergic innervation of the biliary tract was revealed. The gastro- and duodenoprotective action of the 5-HT 1,2-serotonin blocker was established at the experimental stomach ulcer.

[An investigation of the mode of local spreading of biliary tract carcinoma to microvascular and neural structure].

To understand the spreading mode of extrahepatic bile duct carcinoma and gallbladder carcinoma, 170 specimens (86 of which had gallbladder carcinoma while the remaining 84 had extrahepatic bile duct carcinoma) were histo-pathologically studied. In addition, experiments utilizing a canine model were also concurrently conducted in order to gain a greater understanding of relationship between micrometastasis of extrahepatic bile duct carcinoma and microstructures of lymphatics, veins and nerves of biliary tract. The results were as follows: 1) Invasion of the local lymphatics, veins and nerves was found to increase as the grade of tumor penetration into the wall of the biliary tract increased. In addition, it was observed in cases of extrahepatic bile duct carcinoma, a high rate of lymphatic involvement when tumor invasion involved the fibrous muscle layer. However, in cases of gallbladder carcinoma there was no lymphatic involvement even though it also had tumor invasion of the proper muscular layer. 2) In the case of gallbladder carcinoma, correlation of the micrometastasis to lymphatics, veins and nerves was seen, but in cases of extrahepatic bile duct carcinoma, no relationship to micrometastasis of veins and nerves was observed. 3) Anatomically, the study noted rich networks of lymphatics and nerves in the hepatoduodenal ligament. Thus, the rate of micrometastasis to these lymphatics and nerves was more extensive than the observed positive rate of the micrometastasis to veins. 4) Although no direct relationship between nerves and lymphatics was found, it was noted that perineural space was extended in the cases with lymph stasis. There was a network of blood-vessels of nerves and the pericholedocal vascular plexus in the hepatoduodenal ligament. Perineural invasion of carcinoma cells account for the anatomical distribution the surrounding lymphatics and veins. In conclusion, the localization and distribution of micrometastasis from extrahepatic bile duct carcinoma to the involved lymphatics, veins and nerves structures seemed to correlate to the microanatomy of the biliary.

Nitric oxide synthase-containing nerve fibres and neurones in the gall bladder and biliary pathways of the guinea-pig.

We investigated the distribution pattern of nitric oxide (NO) synthesizing nerve cell bodies and axons in the biliary system of the guinea-pig using immunohistochemistry for nitric oxide synthase (NOS). Nerve fibres staining for NOS were found to contact non-vascular smooth myocytes and to course beneath the epithelium. No perivascular NOS fibres were observed. The innervation density varied in different parts of the biliary tree. The lower portion of the common bile duct was more richly innervated than the remaining parts of the duct system. NOS-containing neurones encompassed a subpopulation of intramural ganglion cells. Sympathetic neurones in the coeliac ganglion were not stained. It is suggested that intrinsic NOergic neurones are involved in inhibitory motor control of the biliary musculature, including the sphincter of Oddi.

Muscarinic receptors in vagal routes to the biliary system in dogs.

Although muscarinic receptor subtypes in the biliary system have been characterized, those in the vagal routes have not. This study was performed to characterize the muscarinic receptors in the vagal routes to the biliary system. The effects of four types of muscarinic antagonists, pirenzepine, AF-DX 116, p-F-HHSiD, and atropine, on excitatory responses in the gallbladder and sphincter of Oddi induced by electrical stimulation of the dorsal motor nucleus of the vagus (DMV) were studied in cervical cord-transected anesthetized dogs. Atropine 10 micrograms/kg and over and p-F-HHSiD 50 micrograms/kg and over significantly decreased the excitatory responses in both effectors, but it required a high dose of AF-DX 116 (1-2 mg/kg) to reduce those responses. Pirenzepine (50-300 micrograms/kg) significantly decreased the excitatory response in the gallbladder, but not that in the sphincter of Oddi. A high dose of atropine (0.5 mg/kg) abolished the gallbladder response, whereas a slight excitatory response remained in the sphincter. With the exception of pirenzepine, there was no significant difference in the effects of the three muscarinic blockers on the gallbladder response and sphincter response after reduction of the mean value of the atropine resistant responses from those of the sphincter responses after the muscarinic blockers. These results indicate that the excitatory vagal routes to the gallbladder include at least M1 receptors and those to the sphincter of Oddi may include M3 receptors.

Perineural invasion of carcinoma of the pancreas and biliary tract.

Carcinoma of the pancreatobiliary system often produces perineural invasion extending to extrabiliary and extrapancreatic sites. A surgical technique has been developed to manage this invasion and has been used since 1974. Serial sections were prepared from 90 resected specimens and examined for perineural involvement of intramural and extramural biliary plexuses as well as the pancreatic nerve plexus. Perineural invasion was seen in 34 of 40 patients with carcinoma of the common bile duct, in ten of 14 with cancer of the gallbladder and in four of 15 with carcinoma of the papilla of Vater. Invasion extended to the extramural biliary or pancreatic nerve plexuses in 24 of 40 patients with carcinoma of the bile duct. Involvement of the intrapancreatic nerves was seen in all 21 patients with carcinoma of the pancreatic head and that of the pancreatic nerve plexus in 17 of 21. Perineural invasion was often found at the most progressive margin of the tumour. The survival rate of patients with perineural invasion was low compared with that of those without such invasion, although survival of patients with perineural invasion tended to be longer after extensive resection. The perineural space should be regarded as an important route for the spread of pancreatic and biliary carcinoma.

Effects of selective vagal stimulation on the gallbladder and sphincter of Oddi and peripheral vagal routes mediating bile evacuative responses induced by hypothalamic stimulation.

Peripheral routes of the vagus nerves to the biliary system were studied in anesthetized dogs using various selective vagal stimulation. Efferent stimulation of the gastric, hepatic, or celiac vagal branch as well as the cervical or thoracic vagal trunk induced gallbladder and Oddi's sphincter contractions, but those induced by hepatic vagal stimulation were rather small. The contraction responses in the gallbladder and sphincter of Oddi induced by thoracic vagal stimulation were greatly reduced after an external ligation around the pyloric sphincter. After administration of sympathetic blockers and atropine, vagally-induced gallbladder contractions were completely abolished and slight relaxation was seen in some animals. On the other hand, relaxation or transient relaxation followed by enhanced contractions was elicited in the sphincter of Oddi by vagal stimulation after atropine and sympathetic blockers. The relaxation response in the gallbladder after atropine and sympathetic blockers was abolished and that in the sphincter of Oddi was greatly reduced after the ligation around the pyloric sphincter. Stimulation of a ventral part of the anterior hypothalamic area induced gallbladder contraction and simultaneous relaxation of the sphincter of Oddi. These responses were completely abolished by the ligation around the pyloric sphincter in six dogs, while a slight relaxation response in the sphincter of Oddi remained in two dogs. These results suggest that the vagal fibers passing across the pyloric sphincter region are important for regulation of canine biliary motility and that extragastric vagal routes play a minor role in the nervous control of canine bile evacuation.

Neuroendocrine innervation of the hepatic vessels in the rat and in man.

An extensive array of nerve fibers ramify around the afferent blood vessels of the liver and the extrahepatic and intrahepatic biliary pathways, and are thought to be involved in regulation of blood flow. Although the role of sympathetic innervation is established, little is known about the location or role of regulatory peptidergic innervation in the liver. We examined the anatomic distribution of a wide variety of regulatory peptides and several neural antigens by in situ immunohistochemistry in the rat and in man. A rich peptidergic plexus of nerve fibers and ganglion cells was observed around the arterial vessels in both species, with intense immunoreactivity for neuron-specific enolase, neurofilaments, neuropeptide Y, substance P, and vasoactive intestinal polypeptide. S-100 protein immunoreactivity was seen principally in large nerve bundles, suggesting that the majority of nerves in this area were unmyelinated. In contrast, the portal vessels revealed very little peptidergic innervation. No staining was observed with antibodies directed against insulin, glucagon, gastrin, serotonin, met-enkephalin-Arg-Gly-Leu, cholecystokinin, or growth hormone. These findings indicate the presence of a rich, although selective, peptidergic plexus surrounding afferent hepatic blood vessels. This plexus may play an important role in regulation of hepatic blood flow.

Effect of vagotomy on biliary-tract motor activity in the opossum.

In this study, we evaluated the effect of truncal vagotomy in the opossum on changes in gallbladder and sphincter-of-Oddi (SO) contractile activity that occur normally during fasting and after a meal. In six animals, bipolar electrodes were implanted on the SO, gastric antrum, duodenum, and jejunum. A catheter secured in the gallbladder fundus was used to monitor gallbladder volume. After control studies, truncal vagotomy and pyloroplasty were done in each animal. Before vagotomy, the SO exhibited cyclic changes in spike-burst rate, from 2 to 5/min, that were synchronized with corresponding phases of the duodenal MMC cycle. The gallbladder exhibited partial emptying during the second half of the duodenal MMC cycle with refilling during the first half of the next cycle. By two weeks after vagotomy, the MMC-related changes in SO and gallbladder contractile activity were normal. In contrast, vagotomy retarded the increase in SO spike-burst rate and delayed as well as diminished the pronounced gallbladder emptying that normally occurs after a meal. The same effects were observed for changes in SO and gallbladder activity induced by intraduodenal infusion of Isocal. After vagotomy, the SO and gallbladder exhibited exaggerated responses to intravenous infusion of CCK-OP or motilin. We conclude that truncal vagotomy in the opossum does not affect the fasting pattern of SO and gallbladder contractile activity, but it significantly alters the normal postprandial pattern of enhanced SO spike-burst rate and pronounced gallbladder emptying. The precise mechanism(s) whereby vagotomy produces these changes remains to be determined.

Cyclic motor activity; migrating motor complex: 1985.

Most of the gastrointestinal tract and the biliary tract have a cyclic motor activity. The electric counterpart of this motor activity is called cyclic myoelectric activity. A typical motor cycle in the LES, stomach, and small intestine is composed of a quiescent state, followed by progressively increasing amplitude and frequency of contractions culminating in a state of maximal contractile activity. The colonic motor cycle has only the quiescent and the contractile states. In the small intestine, these motor complexes migrate in an aborad direction, and in the colon in both orad and aborad directions. The mechanisms of initiation and migration of these complexes are best understood in the small intestine. Both the initiation and migration of these complexes seem to be controlled by enteric neural mechanisms. The functions of the enteric mechanisms may be modulated by the central nervous system and by circulating endogenous substances. The mechanisms of initiation of these complexes are not completely understood in the rest of the gastrointestinal tract and in the biliary tract. The physiologic function of these motor complexes that occur only after several hours of fast in the upper gastrointestinal tract of nonruminants may be to clean the digestive tract of residual food, secretions, and cellular debris. This function is aided by a coordinated secretion of enzymes, acid, and bicarbonate. In ruminants, phase III activity is associated with the distal propulsion of ingested food. The function of colonic motor complexes that are not coordinated with the cyclic motor activities of the rest of the gastrointestinal tract may be only to move contents back and forth for optimal absorption.

Chagas' disease: a model of denervation in the study of digestive tract motility.

The enteric nervous system (ENS) is an important neural network that integrates, modulates and coordinates various motor functions of the digestive tract. Chagas' disease, to a variable extent and in a nonsystematic way, causes destruction of ENS neurons throughout the digestive tract. The studies carried out on the pathophysiology of the digestive form of this disease have provided, as a corollary, a better understanding of the participation of the ENS in the regulation of the normal motor activities of the digestive tract and have led to the conclusion that the disease is a true model of intrinsic denervation of the human digestive tract. Among the motor alterations caused by impairment of the digestive tract in Chagas' disease are: absence of peristalsis in the esophagus, achalasia of the cardia, loss of the accommodation reflex in the proximal stomach, gastric emptying disorders, changes in the migrating motor complex (MMC), disappearance of postprandial motor phenomena in the colon and rectum, lack of relaxation of the internal sphincter of the anus, and anomalous responses of the gall bladder to cholekinetic stimuli.