Intrahepatic bile ducts guide establishment of the intrahepatic nerve network in developing and regenerating mouse liver.

Epithelial organs consist of multiple tissue structures, such as epithelial sheets, blood vessels and nerves, which are spatially organized to achieve optimal physiological functions. The hepatic nervous system has been implicated in physiological functions and regeneration of the liver. However, the processes of development and reconstruction of the intrahepatic nerve network and its underlying mechanisms remain unknown. Here, we demonstrate that neural class III ß-tubulin (TUBB3)+ nerve fibers are not distributed in intrahepatic tissue at embryonic day 17.5; instead, they gradually extend along the periportal tissue, including intrahepatic bile ducts (IHBDs), after birth. Nerve growth factor (Ngf) expression increased in biliary epithelial cells (BECs) and mesenchymal cells next to BECs before nerve fiber extension, and Ngf was upregulated by hairy enhancer of slit 1 (Hes family bHLH transcription factor 1; Hes1). Ectopic NGF expression in mature hepatocytes induced nerve fiber extension into the parenchymal region, from where these fibers are normally excluded. Furthermore, after BECs were damaged by the administration of 4,4-diaminodiphenylmethane, the nerve network appeared shrunken; however, it was reconstructed after IHBD regeneration, which depended on the NGF signal. These results suggest that IHBDs guide the extension of nerve fibers by secreting NGF during nerve fiber development and regeneration.

Proliferating cholangiocytes: a neuroendocrine compartment in the diseased liver.

In the last 15 years, the intrahepatic biliary tree has become the object of extensive studies, which highlighted the extraordinary biologic properties of cholangiocytes involved in bile formation, proliferation, injury repair, fibrosis, angiogenesis, and regulation of blood flow. Proliferation is a "typical" property of cholangiocytes and is key as a mechanism of repair responsible for maintaining the integrity of the biliary tree. Cholangiocyte proliferation occurs virtually in all pathologic conditions of liver injury where it is associated with inflammation, regeneration, and repair, thus conditioning the evolution of liver damage. Interestingly, proliferating cholangiocytes acquire the phenotype of neuroendocrine cells, and secrete different cytokines, growth factors, neuropeptides, and hormones, which represent potential mechanisms for cross talk with other liver cells. Many studies suggest the generation of a neuroendocrine compartment in the injured liver, mostly constituted by cells with cholangiocyte features, which functionally conditions the progression of liver disease. These insights on cholangiocyte pathophysiology will provide new potential strategies for the management of chronic liver diseases. The purpose of this review is to summarize the recent findings on the mechanisms regulating cholangiocyte proliferation and the significance of the neuroendocrine regulation of cholangiocyte biology.

Heterogeneity of the intrahepatic biliary epithelium.

The objectives of this review are to outline the recent findings related to the morphological heterogeneity of the biliary epithelium and the heterogeneous pathophysiological responses of different sized bile ducts to liver gastrointestinal hormones and peptides and liver injury/toxins with changes in apoptotic, proliferative and secretory activities. The knowledge of biliary function is rapidly increasing because of the recognition that biliary epithelial cells (cholangiocytes) are the targets of human cholangiopathies, which are characterized by proliferation/damage of bile ducts within a small range of sizes. The unique anatomy, morphology, innervation and vascularization of the biliary epithelium are consistent with function of cholangiocytes within different regions of the biliary tree. The in vivo models [e.g., bile duct ligation (BDL), partial hepatectomy, feeding of bile acids, carbon tetrachloride (CCl4) or alpha-naphthylisothiocyanate (ANIT)] and the in vivo experimental tools [e.g., freshly isolated small and large cholangiocytes or intrahepatic bile duct units (IBDU) and primary cultures of small and large murine cholangiocytes] have allowed us to demonstrate the morphological and functional heterogeneity of the intrahepatic biliary epithelium. These models demonstrated the differential secretory activities and the heterogeneous apoptotic and proliferative responses of different sized ducts. Similar to animal models of cholangiocyte proliferation/injury restricted to specific sized ducts, in human liver diseases bile duct damage predominates specific sized bile ducts. Future studies related to the functional heterogeneity of the intrahepatic biliary epithelium may disclose new pathophysiological treatments for patients with cholangiopathies.

S-100-positive nerve fibers in hepatocellular carcinoma and intrahepatic cholangiocarcinoma: an immunohistochemical study.

The aim of the present study was to determine whether or not liver carcinomas are innervated, since there have been no previous reports on the distribution of nerve fibers in human hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). We investigated nerve fibers by immunohistochemical and morphometric methods in 63 cases of HCC and 28 cases of ICC. An antibody to S-100 protein was used to visualize nerve fibers. In HCC, S-100-positive nerve fibers were absent in the tumoral region, including the sinusoids, and tumoral fibrous septa, while in the capsule of HCC some S100-positive nerve fibers (density: 0.08 +/- 0.03/mm2) were present in contact with vasculatures. In ICC, a few nerve fibers were noted in the tumoral stroma (density: 0.02 +/- 0.01/mm2). No nerve fibers were seen in the neovasculized vessels (tumor vessels) in both HCC and ICC. In invasive regions of HCC and ICC, there were S-100-positive nerve fibers in pre-existing residual portal tracts (density: HCC, 0.11 +/- 0.03/mm2; ICC, 0.13 +/- 0.04/mm2). In non-tumor regions of HCC and ICC, there were many S100-positive nerve fibers (density: 0.41 +/- 0.13/mm2) in portal tracts and, to a much lesser degree, in the sinusoids. These results suggest that tumor cells and vasculatures in HCC and ICC are rarely influenced by nerve fibers.

Bombesin stimulates bicarbonate secretion from rat cholangiocytes: implications for neural regulation of bile secretion.

BACKGROUND & AIMS: Bombesin is a neuropeptide with many biological functions and is known to stimulate bile secretion. The aim of this study was to determine the role of bombesin in bile secretion and its site of action. METHODS: The effects of bombesin on bile secretion were examined using isolated perfused rat livers, hepatocyte couplets, and isolated bile duct units (IBDU) from rat liver. RESULTS: Bombesin (100 nmol/L) increased bile pH, bicarbonate concentration, and output in isolated perfused rat livers from both normal and 2-week bile duct-ligated rats, although bile flow increased only in the latter model. Bombesin (10-100 nmol/L) also had no effect on canalicular bile secretion in isolated hepatocyte couplets. However, bombesin produced a dose-dependent increase in secretion in IBDU, which was inhibited almost completely by a specific bombesin receptor inhibitor, [Tyr4, D-Phe12]-bombesin (1 micromol/L). This bombesin (10 nmol/L)-stimulated secretion in IBDU was accompanied by an increase in luminal pH and was dependent on bicarbonate and chloride in the medium. Somatostatin but not substance P inhibited the bombesin response. CONCLUSIONS: Neuropeptides such as bombesin can directly stimulate fluid and bicarbonate secretion at the level of cholangiocytes, suggesting that neuropeptides play an important regulatory role in biliary transport and secretion.

Distribution of neuronal nitric oxide synthase in the rat liver.

We studied the distribution of neuronal nitric oxide synthase (nNOS) in the rat liver with a specific polyclonal antibody by using immunocytochemical procedures in the light microscopic level. Immunoreactive varicose nerve fibers were found forming a dense plexus around the interlobular hepatic artery and the interlobular bile duct in the hepatic hilus, and in the hepatic artery ramifications of the portal triads. The density of nNOS positive nerve fibers decreases with successive portal ramifications, and some non-immune positive nerve fibers were found in the distal portions of the arterial vessels. The presence of the nNOS positive nerve fibers suggests that the possible main functional role could be related with the regulation of hepatic blood circulation and hepatobiliary activities.

Perineural tumor invasion and its relation with the lymphogenous spread in human and experimental carcinoma of bile duct. A computer-aided 3-D reconstruction study.

The pathogenesis of perineural tumor invasion was studied by computer aided 3-D reconstruction of bile duct wall from two patients submitted to surgery for hepatohilar carcinoma, in order to analyze how and via what route carcinoma reaches the perineural spaces. Rabbits with VX2 carcinoma implanted in the wall of the common bile duct were also examined. It was found that carcinomas growing along the perineural spaces had abundant connections with the tumors growing outside the nerve, especially those lurking in the lymphatics. In an additional analysis on the wall tissues of bile duct from 35 patients operated for carcinoma, the degree of invasion into perineural spaces proved to correlate with that into lymphatics much higher than with venular invasion. Thus it is likely that tumors reach distant nerves mainly via lymphatics, i.e., forming satellite lymphogenous foci around nerves and then, as a second step, breaking into the perineural spaces.

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%).

Innervation of intrahepatic bile ducts and peribiliary glands in normal human livers, extrahepatic biliary obstruction and hepatolithiasis. An immunohistochemical study.

Innervation of the intrahepatic biliary tree was examined in human normal livers, extrahepatic biliary obstruction and hepatolithiasis. Nerve fibers were immunohistochemically identified on formalin-fixed and paraffin-embedded sections by antibodies to S-100 protein (S-100) and neuron specific enolase (NSE). S-100 and NSE-immunoreactive nerve fibers were present in the walls of intrahepatic large, medium-sized and septal bile ducts as well as in peribiliary glands. Some nerve fibers were in close contact with epithelia of bile ducts and peribiliary glands. Serial section observations showed that the nerve fibers arising from nerve bundles approached, and came to lie in close contact with epithelia of the bile ducts and peribiliary glands. Nerve fibers were sparse around the interlobular bile ducts and bile ductules. These immunoreactive nerve fibers of the intrahepatic biliary tree were rather sparse in normal livers, intermediate in extrahepatic biliary obstruction and dense in hepatolithiasis. These findings suggest that intrahepatic bile ducts and peribiliary glands are innervated and biliary functions are regulated in part by these nerve fibers. Increased nerve fibers may have altered effects on biliary functions in hepatolithiasis.