[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.

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%).

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.

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.

[Structural basis of peripheral innervation mechanisms in the walls of the biliary tract]. / Strukturnye osnovy perifericheskikh innervatsionnykh mekhani zmov v stenkakh zhelchevyvodiashchikh putei

Several types of giant neurons exist in the extrahepatic bile ducts in the dog. The intrinsic innervation of the ducts wall has the characteristics as follows: 1) within the nerve, fascicless axons and nerve terminals come into a different relation to the glial cytoplasm; 2) two categories of the nerve terminals are identified by their glial accompaniment--"closed" and "open" types; 3) separation of the nerve terminals containing synaptic vesicles from the several effector elements is usually about several thousand A and with no synapses; typical synaptic complexes were identified between nerve terminals and neurons, although not as commonly as would have been expected considering the large amount of nerve terminals surrounding some of the neurons; 4) within the interstitial space the terminals are often of the "open" type. The morphological data suggest that the peripheral vegetative fibers may form transitional provisional "functional" nerve endings.

An acetylcholinesterase method for in toto staining of peripheral nerves.

UNLABELLED: Stomach, small intestine, uterus, urinary bladder, vagina, mesentery, mesometrium and joint capsule of rats, gall bladder, cystic duct and bile duct of dogs and uteri of young children are stained in toto. PROCEDURE: Tissue is perfused with saline containing hyaluronidase, then pinned on a flat layer of Paraplast and fixed for 24 hr in cold sucrose formol solution. Stomach, urinary bladder and gall bladder are also fixed in toto. Rinse for 2 days in cold 0.22 M sucrose in a sodium cacodylate buffer pH 7.2. Incubate in medium consisting of 60 mM acetate-buffer pH 5.0 or pH 5.6 (for human material only), 2 mM acetylthiocholine iodide, 15 mM Na citrate, 3 mM Cu sulphate, 0.5 mM K3Fe(CN)6, 5 times 10-4 M iso-OMPA, 1% Triton X 100 at 37C. Rinse in doubly distilled water. Dehydrate in glycerine/water mixtures of increasing glycerine content. Store in glycerine or delaminate under dissecting microscope. Delaminated specimens are mounted on gelatinized object glasses, cleared in xylene and coverslipped with Malinol. Specimens stored in glycerine can be studied microscopically. Stained specimens can also be embedded in Paraplast and sections can be studied after counterstaining.