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.

[Perineural invasion in hilar cholangiocarcinoma and distribution of nerve plexuses around porta hepatis].

OBJECTIVE: To explore the incidence of perineural invasion (PNI) in hilar cholangiocarcinoma (HCCA) and summarize the distribution pattern of nerve plexuses around porta hepatis. METHODS: Reported series on PNI in HCCA were systematically reviewed. A clinicopathological study was conducted on sections from 75 HCCA patients to summarize the incidence and modes of PNI. Immunohistochemical stains for CD34 and D2-40 in tumor tissue were performed to clarify the association of PNI with microvessel and lymphoduct. Sections of different decks of hepatoduodenal ligament from 5 autopsy cases were scanned and computerized to display the distribution of nerve plexuses around porta hepatis. RESULTS: The incidence of PNI in HCCA in literature ranged from 59.2% to 100%. In the present study, the overall incidence of PNI was 92.0% (69/75). However, the incidence of PNI showed no remarkable differences among various differentiated groups and Bismuth-Corlette classification groups. Tumor cells could invade microvessels and lymphoduct in HCCA. But no invasion of nerves occurred via microvessels or lymphoduct as demonstrated by immunohistochemistry. Three nerve plexuses in hepatoduodenal ligament and Glisson's sheath were classified and they all surrounded great vessels very closely. CONCLUSION: PNI is generally underreported in HCCA. A surgeon should handle diligently the nerve plexuses around porta hepatis.

Clinical implications of novel aspects of biliary pathophysiology.

Cholangiocytes are the epithelial cells that line the biliary tree; they are the target of chronic diseases termed cholangiopathies, which represent a daily challenge for clinicians, since definitive medical treatments are not available yet. It is generally accepted that the progression of injury in the course of cholangiopathies, and promotion and progression of cholangiocarcinoma are at least in part due to the failure of the cholangiocytes' mechanisms of adaptation to injury. Recently, several studies on the pathophysiology of the biliary epithelium have shed some light on the mechanisms that govern cholangiocyte response to injury. These studies provide novel information to help interpret some of the clinical aspects of cholangiopathies and cholangiocarcinoma; the purpose of this review is thus to describe some of these novel findings, focusing on their significance from a clinical perspective.

Functional anatomy of normal bile ducts.

The biliary tree is a complex network of conduits that begins with the canals of Hering and progressively merges into a system of interlobular, septal, and major ducts which then coalesce to form the extrahepatic bile ducts, which finally deliver bile to the gallbladder and to the intestine. The biliary epithelium shows a morphological heterogeneity that is strictly associated with a variety of functions performed at the different levels of the biliary tree. In addition to funneling bile into the intestine, cholangiocytes (the epithelial cells lining the bile ducts) are actively involved in bile production by performing both absorbitive and secretory functions. More recently, other important biological properties restricted to cholangiocytes lining the smaller bile ducts have been outlined, with regard to their plasticity (i.e., the ability to undergo limited phenotypic changes), reactivity (i.e., the ability to participate in the inflammatory reaction to liver damage), and ability to behave as liver progenitor cells. Functional interactions with other branching systems, such as nerve and vascular structures, are crucial in the modulation of the different cholangiocyte functions.

Adrenergic receptor agonists prevent bile duct injury induced by adrenergic denervation by increased cAMP levels and activation of Akt.

Loss of parasympathetic innervation after vagotomy impairs cholangiocyte proliferation, which is associated with depressed cAMP levels, impaired ductal secretion, and enhanced apoptosis. Agonists that elevate cAMP levels prevent cholangiocyte apoptosis and restore cholangiocyte proliferation and ductal secretion. No information exists regarding the role of adrenergic innervation in the regulation of cholangiocyte function. In the present studies, we investigated the role of adrenergic innervation on cholangiocyte proliferative and secretory responses to bile duct ligation (BDL). Adrenergic denervation by treatment with 6-hydroxydopamine (6-OHDA) during BDL decreased cholangiocyte proliferation and secretin-stimulated ductal secretion with concomitant increased apoptosis, which was associated with depressed cholangiocyte cAMP levels. Chronic administration of forskolin (an adenylyl cyclase activator) or beta(1)- and beta(2)-adrenergic receptor agonists (clenbuterol or dobutamine) prevented the decrease in cholangiocyte cAMP levels, maintained cholangiocyte secretory and proliferative activities, and decreased cholangiocyte apoptosis resulting from adrenergic denervation. This was associated with enhanced phosphorylation of Akt. The protective effects of clenbuterol, dobutamine, and forskolin on 6-OHDA-induced changes in cholangiocyte apoptosis and proliferation were partially blocked by chronic in vivo administration of wortmannin. In conclusion, we propose that adrenergic innervation plays a role in the regulation of biliary mass and cholangiocyte functions during BDL by modulating intracellular cAMP levels.

Role of the neuropeptide, bombesin, in bile secretion.

Since ancient times, bile secretion has been considered vital for maintaining health. One of the main functions of bile secretion is gastric acid neutralization with biliary bicarbonate during a meal or Pavlovian response. Although the liver has many extrinsic and intrinsic nerve innervations, the functional role of these nerves in biliary physiology is poorly understood. To understand the role of neural regulation in bile secretion, our recent studies on the effect of bombesin, a neuropeptide, on bile secretion and its underlying mechanisms will be reviewed. Using isolated perfused rat livers (IPRL) from both normal and 2 week bile duct ligated rats, as well as hepatocyte couplets and isolated bile duct units (IBDU) from normal rat livers, bombesin was shown to stimulate biliary bicarbonate and fluid secretion from bile ducts. Detailed pH studies indicated that bombesin stimulated the activity of Cl-/HCO3- exchanger, which was counterbalanced by a secondary activation of electrogenic Na+/HCO3- symport. Quantitative videomicroscopic studies showed that bombesin-stimulated fluid secretion in IBDU was dependent on Cl- and HCO3- in the media, anion exchanger(s), Cl- and K+ channels, and carbonic anhydrase, but not on the microtubular system. Furthermore, this bombesin response is inhibited by somatostatin but not substance P. Finally, studies of secondary messengers in isolated cholangiocytes and IBDU indicated that bombesin had no effect on intracellular cAMP, cGMP, or Ca++ levels in cholangiocytes. These results provide evidence that neuropeptides such as bombesin can directly stimulate fluid and bicarbonate secretion from cholangiocytes by activating luminal Cl-/HCO3- exchange, but by different mechanisms from those established for secretin. These findings, in turn, suggest that neuropeptides may play an important regulatory role in biliary transport and secretion. Thus, this neuropeptidergic regulation of bile secretion may provide a plausible mechanism for the bicarbonate-rich choleresis seen with meals or Pavlovian response.

Spatiotemporal aspects of sympathetic C-fiber afferent activity in pressor reflex during abdominal ischemia.

Activation of abdominal sympathetic visceral afferents during ischemia elicits excitatory cardiovascular reflexes. The present study examined the time course and discharge patterns of activation of ischemically sensitive sympathetic C-fiber afferents, and then the relationship between summated afferent activity and the pressor reflex induced by prolonged abdominal ischemia was determined. Single-unit activity of abdominal C-fiber afferents was recorded from the right thoracic sympathetic chain of anesthetized cats during 30 min of ischemia. The reflex pressor response to abdominal ischemia was induced by occlusion of celiac and superior mesenteric arteries. Of 68 C-fiber afferents studied, 36 (approximately 53%) were activated during 30 min of ischemia, whereas the activity of the remaining 32 were not altered. Onset latencies of 36 C-fiber afferents activated by ischemia ranged from 1.0 to 17.4 min with an average of 6.1 +/- 0.8 min. The majority of activated afferents manifested a bursting pattern of discharge activity as ischemia was prolonged beyond 10 min. Summated response of activated afferents, but not individual afferent activity, was related closely to the reflex pressor response during 30 min of ischemia. These results suggest that both recruitment of sufficient numbers of C-fiber afferents and adequate discharge frequency of afferents constitute an encoding mechanism for the pressor reflex during abdominal ischemia.

Impact of the site of arterial anastomosis on the risk of biliary complications after liver transplantation.

Biliary complications are an important cause of morbidity after orthotopic liver transplantation (OLT). It has been suggested that the denervation of recipient bile duct and the sphincter of Oddi may contribute to biliary complications. Recipient bile duct and the sphincter of Oddi receive its regulatory innervation through the nerve plexus around the gastroduodenal artery (GDA). These nerves are likely to be severed if recipient hepatic artery is divided at or proximal to the origin of GDA during arterial anastomosis of OLT (proximal arterial anastomosis). This study was carried out to determine the impact of the site of arterial anastomosis on the incidence of biliary complications. A total of 331 transplants by a single surgeon (JACB) were analysed. Patients with Roux-en-Y choledochoduodenostomy were excluded. Biliary complications were noted in 16.9% patients during first three months after transplantation. Incidence of biliary complications in patients with proximal and distal arterial anastomosis was found to be 17.2% and 12.9% respectively (ns). We conclude that the site of arterial anastomosis as an index of sphincter of Oddi denervation does not significantly influence the incidence of biliary complications.

Calcitonin gene-related peptide innervation of the rat hepatobiliary system.

The digestive system is densely innervated by calcitonin gene-related peptide (CGRP)-immunoreactive neurons. The present study investigated a) the distribution and origin of CGRP-immunoreactive fibers in the rat hepatobiliary tract, and b) their relation with substance P/tachykinin (SP/TK) immunoreactivity using immunohistochemical and radioimmunoassay techniques. CGRP-containing fibers form dense networks in the fibromuscular layer of the biliary tree and surrounding the portal vein. Thin, varicose fibers are present at the base of the mucosa of the ducts. In the liver, labeled fibers are restricted to the portal areas and the stromal compartment. Neonatal treatment with capsaicin, a neurotoxin for primary afferent neurons, or celiac/superior mesenteric ganglionectomy depletes CGRP-containing fibers in the biliary tract, and reduces those associated with the portal vein. In contrast, subdiaphragmatic vagotomy does not appreciably modify the density of these fibers. Radioimmunoassay studies show a reduction of CGRP-immunoreactive contents in the biliary tract and portal vein by 84% and 65%, respectively, following capsaicin treatment, and by 80% and 66%, respectively, following ganglionectomy. By contrast, CGRP concentrations in vagotomized animals are comparable to those of controls. Most CGRP-positive fibers appear to contain SP/TK immunoreactivity, as indicated by double-label studies. These results demonstrate that the rat hepatobiliary tract is prominently innervated by CGRP- and CGRP/SP/TK-immunoreactive fibers, which are likely to originate from spinal afferent neurons. The abundance of these fibers and their association with a variety of targets are in line with the involvement of these peptidergic visceral afferents in regulating hepatobiliary activities, including hemodynamic functions of the hepatic vasculature.

An anterograde tracing study of the vagal innervation of rat liver, portal vein and biliary system.

In order to investigate the distribution and structure of the vagal liver innervation, abdominal vagal afferents and efferents were selectively labeled by injecting WGA-HRP or Dil into the nodose ganglia, and DiA into the dorsal motor nucleus, respectively. Vagal afferent fibers produced characteristic terminal-like structures at three locations in the liver hilus: 1. Fine varicose endings preferentially surrounding, but not entering, the numerous peribiliary glands in the larger intra and extrahepatic bile ducts 2. Large, cup-shaped terminals in almost all paraganglia 3. Fine varicose endings in the portal vein adventitia. No fibers and terminals were found in the hepatic parenchyma. While about two thirds of the vagal afferent fibers that originate in the left nodose ganglion, and are contained in the hepatic branch, bypass the liver hilus area on their way to the gastroduodenal artery, a significant number (approx. 10% of the total) of vagal afferents that do innervate the area, originates from the right nodose ganglion, and projects to the periarterial plexus of the common hepatic artery and liver pedicle most likely through the dorsal celiac branch. Varicose vagal efferent fibers were present within the fascicles of the vagal hepatic branch and fine terminal-like structures in a small fraction of the paraganglia. No efferents were found to terminate in the hepatic parenchyma or on the few neurons embedded in nerves or paraganglia. In contrast to the paucity of vagal terminals in the hepatic parenchyma, an abundance of vagal efferent and afferent fibers and terminals with distinctive distribution patterns and structural characteristics was present in esophagus and gastrointestinal tract.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinicopathologic studies on perineural invasion of bile duct carcinoma.

To elucidate the clinical significance of perineural invasion on bile duct cancer, a clinicopathologic study was performed on 70 resected patients with bile duct carcinoma. The overall incidence of perineural invasion in the resected specimen was 81.4%. There seemed to be no correlation between perineural invasion and site, size of the tumor, and lymph node metastasis. A significant correlation was observed, however, between macroscopic type, microscopic type, depth of invasion, and perineural invasion. Perineural invasion index (PNI) was defined as the ratio between the number of nerve fibers invaded by cancer and the total number of nerve fibers with and without cancer invasion. Perineural invasion index was significantly higher at the center compared with the proximal and distal part of the tumor (p less than 0.001). The 5-year survival rate for patients with perineural invasion was significantly lower (p less than 0.05) than that for those without perineural invasion (67% versus 32%).

Distribution of opioidergic, sympathetic and neuropeptide Y-positive nerves in the sphincter of Oddi and biliary tree of the monkey, Macaca fascicularis.

The opioidergic, sympathetic and neuropeptide Y-positive innervation of the sphincter of Oddi (common bile duct sphincter and pancreatic duct sphincter), as well as other segments of the extrahepatic biliary tree was studied in the monkey by use of immunohistochemistry. Methionine-enkephalin-positive nerves were seen to innervate the smooth muscle of all portions of the sphincter of Oddi and also local ganglion cells. No methionine-enkephalin-positive nerves could be detected in the common bile duct, pancreatic duct or gallbladder. Tyrosine hydroxylase-positive nerves occurred between smooth muscle bundles and also ran to local ganglion cells as well as along the common bile duct. Neuropeptide Y-positive nerves were observed within smooth muscle of the sphincter of Oddi (all portions), common bile duct, pancreatic duct and gallbladder. No evidence of any differential innervation of the pancreatic duct and common bile duct sphincters could be detected with these markers.

Regulation of bile duct motility by vagus and sympathetic nerves in the pigeon.

Effects of stimulation of the vagus and sympathetic nerves on bile duct peristalses were studied in pigeons anesthetized with urethane. Vagus stimulation increased the frequency of peristalses. Atropine, hexamethonium and tetrodotoxin abolished this excitatory effect. After atropine, inhibition of peristalses sensitive to tetrodotoxin was produced. Stimulation of sympathetic area in the spinal cord inhibited peristalses. Propranolol converted this effect into an excitatory one, which was abolished by phentolamine. The results suggest that vagal and sympathetic innervations of the bile duct in pigeons are similar to those of the sphincter of Oddi in mammalian species.

Structural and functional biliary tree changes secondary to extrahepatic biliary obstruction.

To assess the potential structural changes of the biliary tree and the liver in patients with extrahepatic biliary obstruction, the resected specimens of 20 patients operated on for benign biliary stricture, were evaluated by means of immunocytochemical, histological and scanning electronmicroscopic studies. Furthermore, liver biopsies were taken for the same purposes. Our results showed that in the dilated segment of the hepatic duct proximal to the stricture, innervation was greatly reduced or completely absent with associated advanced morphological and histological changes and high intrabiliary pressure levels. Similar findings were observed in the liver biopsies, too. These biopsies showed advanced morphological and histological changes associated with reduced innervation. In contrast, the nondilated segment of the hepatic duct, distal to the obstruction, showed normal innervation, normal morphological and histological findings and normal levels of intrabiliary pressure. The present study provides evidence that in cases of extrahepatic biliary obstruction, there is a feature of advanced pathological changes in the biliary tree associated with alterations in innervation. These structural changes are associated with functional changes in both the liver and the biliary tree. These functional changes represent a threat to the patient, in particular if major surgery is required. Increased biliary pressure appears to be a major cause of the development of these changes. Biliary drainage, either surgical or endoscopic, is indicated as the sole alternative, to reduce the intrabiliary pressure and to contribute to a reversal of these structural and functional changes.

Distribution, possible origins and fine structure of neuropeptide Y-containing nerve fibers in the rat liver.

The distribution, possible origins and fine structure of neuropeptide Y (NPY)-containing nerve fibers in the rat liver were investigated by immunohistochemistry, nerve transection and immunoelectron microscopy. Light-microscopic immunohistochemistry showed NPY fibers forming a complex network in and around the walls (tunica adventitia and tunica media) of hepatic vessels. They were also closely associated with interlobular bile ducts. The NPY fibers in the liver were almost completely eliminated by transection of the greater splanchnic nerves just distal to the celiac ganglion. Transection of the greater splanchnic nerves just proximal to the celiac ganglion resulted in a marked decrease in NPY fibers, but a significant number remained intact. Under electron microscopy. NPY terminals without a covering of glial processes were seen not only in proximity to smooth muscle cells within the tunica media of hepatic vessels but also in the subendothelial areas of the tunica intima. Some NPY axon terminals devoid of glial ensheathment were located close to the basal lamina of interlobular bile ducts. Occasionally, single axon terminals with NPY were found in the vicinity of or in contact with hepatic cells. There was a small number of NPY fibers that had lost their glial sheaths while running toward lymphatic capillaries. These findings suggest that hepatic NPY arises from the celiac ganglion and paravertebral sympathetic ganglia, and that it is involved in more complex physiological processes than the previously described neuropeptides in the liver, which are localized exclusively to hepatic vessel walls.

Adrenergic and VIP-ergic relaxatory mechanisms of the feline extrahepatic biliary tree.

Relaxatory mechanisms of the extrahepatic biliary tree were investigated in anesthetized cats allowing separate recordings of the sphincter of Oddi, gallbladder and duodenal wall. Regional intra-arterial administration of vasoactive intestinal peptide (VIP) elicited dose-dependent relaxatory motor responses, which were not influenced by blockade of cholino- or beta-adrenoceptors, but were most probably due to activation of VIP receptors at the smooth muscle membrane. Efferent electrical vagal nerve stimulation unmasked relaxatory motor responses after previous blockade of muscarinic cholinoceptors. The neural transmission did not involve beta-adrenoceptors but was effectively antagonized after additional blockade with hexamethonium. Since both nerve terminals and ganglion cells with VIP-like immunoreactivity were abundant in the feline sphincter of Oddi, VIP is one possible transmitter candidate of the postganglionic inhibitory neurons. Non-selective activation of beta-adrenoceptors by isoprenaline or selective activation of beta 2-adrenoceptors by terbutaline also induced a dose-dependent relaxation of these regions. On a molar basis, relaxation via beta-adrenoceptors was 40-50 times less potent than via VIP. Both types of beta-adrenergic relaxation were antagonized by propranolol. The terbutaline-induced responses were selectively antagonized by beta 2-adrenoceptor blockade. To evaluate the role of beta 1-adrenoceptors, non-selective stimulation with isoprenaline was given; this relaxation was little influenced by blockade of beta 2-adrenoceptors but was completely antagonized by propranolol. In all experiments using beta-adrenoceptor antagonists these drugs each increased the basal tone of the preparation suggesting release of tonic inhibition exerted via beta-adrenoceptors.