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.

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.

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%).