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.

The enteric nervous system in patients with calculous and acalculous gallbladder.

BACKGROUND: It is generally thought that gallbladder motility plays a more or less important role in the pathogenesis of gallstones. Some studies have shown that some abnormalities of its intrinsic innervations, but these studies were usually limited to one cell component. AIMS: We investigated the main cell components of gallbladder intrinsic innervation in patients with and without gallstones. METHODS: Archival gallbladder specimens from 39 patients, 27 with gallstones (age range 45-69 yrs) and 12 patients without gallstones (age range 39-71 yrs) were obtained. Full thickness sections were obtained from the gallbladder neck and immunohistochemistry was carried out for enteric neurons (neuron-specific enolase and calretinin), enteric glia (S100) and interstitial cells of Cajal (CD117 and CD34); tryptase staining was also done to distinguish the latter from mast cells. RESULTS: Apart from calretinin-positive neurons, patients with gallstones featured a significant decrease of neurons, enteric glial cells (EGC) and mast cells compared to those without gallstones; interstitial cells of Cajal were extremely few and only found in two patients, one for each group. CONCLUSIONS: The intrinsic innervations of the gallbladder is abnormal in gallstone patients, and this may contribute to gallstone formation in these subjects.

Innervation of Extrahepatic Biliary Tract, With Special Reference to the Direct Bidirectional Neural Connections of the Gall Bladder, Sphincter of Oddi and Duodenum in Suncus murinus, in Whole-Mount Immunohistochemical Study.

Sphincter of Oddi dysfunction is one of the most important symptoms in post-cholecystectomy syndrome. Using either electrical or mechanical stimulation and retrogradely transported neuronal dyes, it has been demonstrated that there are direct neural pathways connecting gall bladder and the sphincter of Oddi in the Australian opossum and the golden hamster. In the present study, we employed whole-mount immunohistochemistry staining to observe and verify that there are two different plexuses of the extrahepatic biliary tract in Suncus murinus. One, named Pathway One, showed a fine, irregular but dense network plexus that ran adhesively and resided on/in the extrahepatic biliary tract wall, and the plexus extended into the intrahepatic area. On the other hand, named Pathway Two, exhibiting simple, thicker and straight neural bundles, ran parallel to the surface of the extrahepatic biliary tract and passed between the gall bladder and duodenum, but did not give off any branches to the liver. Pathway Two was considered to involve direct bidirectional neural connections between the duodenum and the biliary tract system. For the first time, morphologically, we demonstrated direct neural connections between gall bladder and duodenum in S. murinus. Malfunction of the sphincter of Oddi may be caused by injury of the direct neural pathways between gall bladder and duodenum by cholecystectomy. From the viewpoint of preserving the function of the major duodenal papilla and common bile duct, we emphasize the importance of avoiding kocherization of the common bile duct so as to preserve the direct neural connections between gall bladder and sphincter of Oddi.

[NERVOUS REGULATION OF BILIARY TRACT MOTILITY].

Aim: The study of mechanisms of regulation of biliary tract motility by divisions of autonomic nervous system (ANS). Material and methods: Experiments were carried out on rabbits, chinchillas weighing 3.5-4 kg using gentle methods of treatment of experimental animals. Electromotor activity of electromotor (EMA) of the gallbladder and sphincter of Oddi was recorded. Irritation of the nerve produces an electrical pulse duration of 2 ms, the amplitude of 1.5-15 V, frequency of 10 Hz. Results: The mechanism of vagal inhibition of sphincter of Oddi motility and unidirectional stimulatory influence of ANS divisions on the motility of the gallbladder and sphincter of Oddi was studied. It was established that in the mechanism of vagal inhibition of sphincter of Oddi motility involved intramural adrenergic neurons synaptically connected with preganglionic parasympathetic fibers. At the stimulatory effect of vagus on biliary tract motility serotonergic intramural neurons are involved transmitting excitation to serotonin receptors of effector tissue.

Hydrophobic bile salts inhibit gallbladder smooth muscle function via stimulation of GPBAR1 receptors and activation of KATP channels.

Hydrophobic bile salts are thought to contribute to the disruption of gallbladder smooth muscle (GBSM) function that occurs in gallstone disease, but their mechanism of action is unknown. The current study was undertaken to determine how hydrophobic bile salts interact with GBSM, and how they reduce GBSM activity. The effect of hydrophobic bile salts on the activity of GBSM was measured by intracellular recording and calcium imaging using wholemount preparations from guinea pig and mouse gallbladder. RT-PCR and immunohistochemistry were used to evaluate expression of the G protein-coupled bile acid receptor, GPBAR1. Application of tauro-chenodeoxycholate (CDC, 50-100 microm) to in situ GBSM rapidly reduced spontaneous Ca(2+) flashes and action potentials, and caused a membrane hyperpolarization. Immunoreactivity and transcript for GPBAR1 were detected in gallbladder muscularis. The GPBAR1 agonist, tauro-lithocholic acid (LCA, 10 microm) mimicked the effect of CDC on GBSM. The actions of LCA were blocked by the protein kinase A (PKA) inhibitor, KT5720 (0.5-1.0 microm) and the K(ATP) channel blocker, glibenclamide (10 microm). Furthermore, LCA failed to disrupt GBSM activity in Gpbar1(/) mice. The findings of this study indicate that hydrophobic bile salts activate GPBAR1 on GBSM, and this leads to activation of the cyclic AMP-PKA pathway, and ultimately the opening of K(ATP) channels, thus hyperpolarizing the membrane and decreasing GBSM activity. This inhibitory effect of hydrophobic bile salt activation of GPBAR1 could be a contributing factor in the manifestation of gallstone disease.

Serotonergic projection from nucleus raphe pallidus to rostral ventrolateral medulla modulates cardiovascular reflex responses during acupuncture.

We have demonstrated that stimulation of somatic afferents during electroacupuncture (EA) inhibits sympathoexcitatory cardiovascular rostral ventrolateral medulla (rVLM) neurons and reflex responses. Furthermore, EA at P5-P6 acupoints over the median nerve on the forelimb activate serotonin (5-HT)-containing neurons in the nucleus raphe pallidus (NRP). The present study, therefore, examined the role of the NRP and its synaptic input to neurons in the rVLM during the modulatory influence of EA. Since serotonergic neurons in the NRP project to the rVLM, we hypothesized that the NRP facilitates EA inhibition of the cardiovascular sympathoexcitatory reflex response through activation of 5-HT1A receptors in the rVLM. Animals were anesthetized and ventilated, and heart rate and blood pressure were monitored. We then inserted microinjection and recording electrodes in the rVLM and NRP. Application of bradykinin (10 microg/ml) on the gallbladder every 10 min induced consistent excitatory cardiovascular reflex responses. Stimulation with EA at P5-P6 acupoints reduced the increase in blood pressure from 41+/-4 to 22+/-4 mmHg for more than 70 min. Inactivation of NRP with 50 nl of kainic acid (1 mM) reversed the EA-related inhibition of the cardiovascular reflex response. Similarly, blockade of 5-HT1A receptors with the antagonist WAY-100635 (1 mM, 75 nl) microinjected into the rVLM reversed the EA-evoked inhibition. In the absence of EA, NRP microinjection of dl-homocysteic acid (4 nM, 50 nl), to mimic EA, reduced the cardiovascular and rVLM neuronal excitatory reflex response during stimulation of the gallbladder and splanchnic nerve, respectively. Blockade of 5-HT1A receptors in the rVLM reversed the NRP dl-homocysteic acid inhibition of the cardiovascular and neuronal reflex responses. Thus activation of the NRP, through a mechanism involving serotonergic neurons and 5-HT1A receptors in the rVLM during somatic stimulation with EA, attenuates sympathoexcitatory cardiovascular reflexes.

Hypogenesis of intramural vascularity and perivascular plexuses of gallbladder in patients with congenital biliary dilatation.

BACKGROUND: We hypothesized neuronal disorders of the biliary tract as the cause of congenital biliary dilation (CBD). METHODS: Gallbladders were removed from a total of 15 patients who were categorized into two study groups: a CBD group (eight patients) and in a control group (neuroblastoma, acute myelogenous leukemia, wandering gallbladder, Wilms' tumor, glycogen storage disease, familial amyloid polyneuropathy; seven patients). Whole-mount preparations of the gallbladders were made to immunostain the intramural nerves. RESULTS: The intramural vascularity in the gallbladders of the CBD group (5.5 +/- 1.9/cm(2)) was significantly lower than that in the control group (27.6 +/- 14.4/cm(2)). The rate of perivascular plexuses on the surface of intramural vessels was also significantly lower in the CBD group than in the controls (37.7 +/- 18.1 vs. 80.2 +/- 17.4%, respectively). The numbers of ganglion cells per visual field were 38.5 +/- 24.0 and 42.3 +/- 20.6, respectively, in the CBD and control groups; this difference was not statistically significant. CONCLUSIONS: There may be a mechanism in CBD causing contractile failure and dilatation of the biliary tract as a result of decreased intramural blood flow that accompanies the diminished vascularity.

Gerentological studies on the gross and histomorphology of the vesicular gland of gaddi goat (Copra hircus) / Estudios gerontológicos en anatomía e histomorfología de la glándula vesicular de cabra gaddi (Cabra hircus)

The study was conducted on the vesicular glands of 30 entire pure male Gaddi goats from birth to 5yrs and 6 months of age, divided into three equal size groups of 10 animals in each viz; Pre-pubertal (lday old to <18 months of age), Pubertal (18 months to < 5years of age) and Post-pubertal (>5 years) age groups. In new born kid the glands were small, white cord like. It became "?" shaped at 6 months and "S" shaped at 12 months of age. A significant growth in size and weight of gland occurred at 6 month, then up to 12 month and beyond it grew slowly. The secretory acini of the lobules were lined by pseudostratified ciliated columnar epithelium which contained A-tall columnar, B-basal and C-narrow columnar type of cells. The number per unit area and size of the gland increased with age significantly from birth in the prepubertal animals and up to puberty. In post-pubertal animals it did not grow significantly, rather the connective tissue elements were increased in the capsule (adventitia). The intralobular connective tissue however, decreased at the expense of growth of acini at all ages.

Se realizó un estudio de las glándulas vesiculares de 30 cabras Gaddi, macho, totalmente puras desde el nacimiento hasta los 5 años y 6 meses de edad, divididas en tres grupos de 10 animales cada uno, pre-puberal (día 1 de edad a <18 meses de edad), pubertad (18 meses a <5 años de edad) y post-puberal (> 5 años). En el recién nacido las glándulas eran pequeñas, como un cordón blanco. Se presentó en forma de "?" a los 6 meses y de "S" a los 12 meses de edad. Un importante crecimiento en tamaño y peso de la glándula se produjo a los 6 meses, luego de 12 meses y más, creció lentamente. Los acinos secretores de los lóbulos se alinearon como epitelio columnar ciliado pseudoestratificado, los cuales contienen células de tipo A-columnar alto, B-basal y C-columnar estrecho. El número por unidad de área y el tamaño de la glándula aumentó significativamente con la edad desde su nacimiento en los animales prepuberes y hasta la pubertad. En animales post-puberales no creció significativamente, y los elementos del tejido conectivo se incrementaron en la cápsula (adventicia). Sin embargo, el tejido conectivo intralobular, disminuyó a expensas del crecimiento de los acinos, en todas las edades.

Division of the hepatic branch of the anterior vagus nerve in fundoplication: effects on gallbladder function.

BACKGROUND: Cutting the hepatic branch of the anterior vagus nerve makes laparoscopic fundoplication technically easier; however, there is little data about the effect of cutting the nerve on gallbladder function. METHODS: One surgeon (MPä) preserved this nerve until March 2001 when he changed the technique. We investigated patients consecutively operated on before and after this date. A symptom questionnaire was returned by 59 patients, of whom 19 in both groups were successfully further investigated. The follow-up varied from 4 to 9 years postoperatively. The volume of the gallbladder was measured by ultrasonography before and after a fatty test meal. Alkaline phosphatase (ALP), alanine aminotransferase (ALAT), bilirubin, and amylase were determined from plasma. RESULTS: There was no difference in symptoms or use of antireflux medication between the groups. No difference was found in the levels of bilirubin, ALAT, or ALP. A mild elevation in plasma amylase was noted after nerve division (p = 0.041). The gallbladder ejection fraction did not differ between groups, but the fasting gallbladder volume was smaller when the nerve was cut (median 18.1 (range, 6-57.7) ml versus median 23.2 (range, 7.9-66.7) ml; p = 0.049). Both differences in plasma amylase and gallbladder fasting volume were clearer in male patients. CONCLUSIONS: Cutting the hepatic branch of the anterior vagus nerve during fundoplication may reduce the size of gallbladder, but it has no effect on the ejection fraction. No clinical significance has been noted to date. Prospective trials will be necessary to confirm these findings.

[Vegetotropic therapy and hypobaric hypoxic adaptation in gallbladder motor dysmotility].

The study of 67 chronic biliary patients (36 - gallbladder dysfunction, 25 acalculuous cholecystitis, 6 - cholelithiasis) was revealed their functional dysadaptation characterized by gallbladder dysmotility and autonomic dysfunction. Vegetotropic therapy (Propranolol, Metoclopramid, Atropine) according to the autonomic regulation activity has advantages in correction of the gallbladder dysmotility in comparison to the routine pharmacotherapy. Hypobaric hypoxic adaptation results in the increase of gallbladder emptying that opens perspectives for the non-pharmacological correction of the gallbladder hypokinetic dyskinesia.

Adenomyomatous hyperplasia of the gallbladder with perineural invasion: revisited.

We report 9 examples of segmental adenomyomatous hyperplasia of the gallbladder with perineural invasion. Five patients were women and 4 men. Their ages ranged from 49 to 81 years (mean age 64 y). Eight patients had gallbladder calculi. The original pathologic diagnosis of adenocarcinoma was made in 5 patients and of "adenoma malignum" in one. Six patients are disease-free for 2 to 11 years following cholecystectomy, 1 patient died of unrelated causes and 2 were lost to follow-up. Histologically 2 types of adenomyomatous hyperplasia were recognized. The first one characterized by numerous Rokitansky-Aschoff sinuses (RASs) was accompanied by smooth muscle hyperplasia and an expanded subserosal layer containing numerous nerve trunks (6 cases). The second type was characterized by an extensively fibrotic gallbladder wall with numerous RASs but with few or no smooth muscle bundles and an expanded subserosal layer containing abundant nerve-trunks (3 cases). Perineural (7 cases) and intraneural invasion (2 cases) was identified only in the subserosal layer. The lack of p53 reactivity and the very low MIB-1-labeling index provide additional support to the non-neoplastic nature of the lesion. The pseudoinvasive pattern of the RASs, reactive epithelial atypia, and the perineural and intraneural invasion probably contributed to the erroneous diagnosis of adenocarcinoma or "adenoma malignum." The mechanism by which the epithelial structures "invaded" the perineural spaces and the nerves is unclear. We favor the hypothesis that the migration of the benign glandlike structures into the nerves is related to the production of chemotactic factors or signaling substances and the activation of cell receptors.

Role of medullary GABA, opioids, and nociceptin in prolonged inhibition of cardiovascular sympathoexcitatory reflexes during electroacupuncture in cats.

Electroacupuncture (EA) causes prolonged suppression of reflex elevations in blood pressure for 1-2 h in anesthetized preparations. A long-loop pathway involving the arcuate nucleus (ARC), ventrolateral periaqueductal gray, and rostral ventrolateral medulla (rVLM) is involved in sympathoinhibitory cardiovascular EA effects. However, the mechanisms and locations of the prolonged EA inhibition are unknown. We hypothesized that this effect is mediated through a long-loop pathway involving opioid, nociceptin, and gamma-aminobutyric acid (GABA) receptor activation in the rVLM. In anesthetized, ventilated cats application of bradykinin to the gallbladder every 10 min induced consistent reflex increases in blood pressure. Bilateral EA stimulation at the cardiovascular acupoints P5-6 overlying the median nerves reduced the reflex responses for at least 80 min. Bilateral blockade with kynurenic acid in the ARC 60 min after onset of EA inhibition reversed the cardiovascular response, suggesting a role for the ARC in the long-loop pathway during the prolonged inhibitory response. Unilateral microinjection with either an opioid or a GABA(A) antagonist in rVLM 50-60 min after the beginning of the EA response reversed EA inhibition of the cardiovascular excitatory reflex. Gabazine also reversed EA inhibition of cardiovascular premotor sympathetic rVLM neurons. Conversely, microinjection of a nociceptin/orphanin FQ peptide antagonist did not affect the prolonged inhibitory effect. Thus the ARC, an important component in the long-loop pathway in the EA cardiovascular response, is required for prolonged suppression of reflex cardiovascular excitatory responses by EA. Furthermore, in the rVLM, opioids and GABA, but not nociceptin, participate in the long-term EA-related inhibition of sympathoexcitatory cardiovascular responses.

Effects of melatonin on gallbladder neuromuscular function in acute cholecystitis.

Gallbladder stasis is associated to experimental acute cholecystitis. Impaired contractility could be, at least in part, the result of inflammation-induced alterations in the neuromuscular function. This study was designed to determine the changes in gallbladder neurotransmission evoked by acute inflammation and to evaluate the protective and therapeutic effects of melatonin. Experimental acute cholecystitis was induced in guinea pigs by common bile duct ligation for 2 days, and then the neuromuscular function was evaluated using electrical field stimulation (EFS; 5-40 Hz). In a group of animals with the bile duct ligated for 2 days, a deligation of the duct was performed, and after 2 days, the neuromuscular function was studied. The EFS-evoked isometric gallbladder contraction was significantly lower in cholecystitic tissue. In addition, inflammation changed the pharmacological profile of these contractions that were insensitive to tetrodotoxin but sensitive to atropine and omega-conotoxin, indicating that acute cholecystitis affects action potential propagation in the intrinsic nerves. Nitric oxide (NO)-mediated neurotransmission was reduced by inflammation, which also increased the reactivity of sensitive fibers. Melatonin treatment prevented qualitative changes in gallbladder neurotransmission, but it did not improve EFS-induced contractility. The hormone recovered gallbladder neuromuscular function once the biliary obstruction was resolved, even when the treatment was started after the onset of gallbladder inflammation. These findings show for the first time the therapeutic potential of melatonin in the recovery of gallbladder neuromuscular function during acute cholecystitis.

Association between gallstone-evoked pain, inflammation and proliferation of nerves in the gallbladder: a possible explanation for clinical differences.

OBJECTIVE: To investigate whether enhanced neuroproliferation could be involved in the pathogenesis of gallstone pain. MATERIAL AND METHODS: Gallbladders from 117 patients with gallstones and 43 controls were examined. The gallbladder samples were immunostained against the pan-neuronal marker PGP 9.5 and the number of nerves and nerve area per tissue area estimated. RESULTS: More nerves and an increased nerve area per tissue area were found in uncomplicated symptomatic gallstone disease. In comparison, acute cholecystitis displayed a significantly (p=0.01) decreased number of nerves and nerve area per tissue area. In both categories, the gallbladder neck contained more nerves (p=0.06 and 0.04, respectively) and an increased nerve area per tissue area (p=0.034 and 0.008, respectively) than the body. CONCLUSIONS: Uncomplicated disease showed enhanced neuroproliferation, significantly more in the gallbladder neck, whereas significantly fewer nerves were observed in acute cholecystitis. Nerve growth alteration may play a role in uncomplicated gallstone pain but the pathology may be different in inflammation.

Surgical anatomy of innervation of the gallbladder in humans and Suncus murinus with special reference to morphological understanding of gallstone formation after gastrectomy.

AIM: To clarify the innervation of human gallbladder, with special reference to morphological understanding of gallstone formation after gastrectomy. METHODS: The liver, gallbladder and surrounding structures were immersed in a 10 mg/L solution of alizarin red S in ethanol to stain the peripheral nerves in cadavers (n=10). Innervation in the areas was completely dissected under a binocular microscope. Similarly, innervation in the same areas of 10 Suncus murinus (S. murinus) was examined employing whole mount immunohistochemistry. RESULTS: Innervation of the gallbladder occurred predominantly through two routes. One was from the anterior hepatic plexus, the innervation occurred along the cystic arteries and duct. Invariably this route passed through the hepatoduodenal ligament. The other route was from the posterior hepatic plexus, the innervation occurred along the cystic duct ventrally. This route also passed through the hepatoduodenal ligament dorsally. Similar results were obtained in S. murinus. CONCLUSION: The route from the anterior hepatic plexus via the cystic artery and/or duct is crucial for preserving gallbladder innervation. Lymph node dissection specifically in the hepatoduodenal ligament may affect the incidence of gallstones after gastrectomy. Furthermore, the route from the posterior hepatic plexus via the common bile duct and the cystic duct to the gallbladder should not be disregarded. Preservation of the plexus may attenuate the incidence of gallstone formation after gastrectomy.

Gallbladder neuron count in cholelithiasis patients with and without Chagas disease.

Various investigators agree that the incidence of cholelithiasis is greater in patients with Chagas disease. The most plausible explanation for this is based on the parasympathetic denervation that occurs over the whole digestive tract due to Chagas disease. In order to analyze the occurrence of this alteration, gallbladder neuron counts were performed on cholelithiasis patients with and without Chagas disease who were being treated at the Department of Digestive Surgery, Universidade Federal do Triângulo Mineiro, Uberaba, Brazil. In the present study, a notable reduction in the number of neurons in the gallbladder wall was observed in Chagas patients, in comparison with non-Chagas subjects.

Gallbladder neuron count in cholelithiasis patients with and without Chagas disease

Various investigators agree that the incidence of cholelithiasis is greater in patients with Chagas disease. The most plausible explanation for this is based on the parasympathetic denervation that occurs over the whole digestive tract due to Chagas disease. In order to analyze the occurrence of this alteration, gallbladder neuron counts were performed on cholelithiasis patients with and without Chagas disease who were being treated at the Department of Digestive Surgery, Universidade Federal do Triângulo Mineiro, Uberaba, Brazil. In the present study, a notable reduction in the number of neurons in the gallbladder wall was observed in Chagas patients, in comparison with non-Chagas subjects.

Vários autores concordam que a incidência de colelitíase encontra-se elevada nos pacientes portadores de doença de Chagas. A explicação mais plausível para este fato baseia-se na desnervação parassimpática existente em todo o tubo digestivo na doença de Chagas. No intuito de analisar a ocorrência desta alteração, foi realizada contagem neuronal da vesícula biliar de pacientes chagásicos e não chagásicos, portadores de colelitíase, tratados na Disciplina de Cirurgia do Aparelho Digestivo, Universidade Federal do Triângulo Mineiro, Uberaba, Brasil. No presente estudo, observou-se uma redução expressiva do número de neurônios na parede da vesícula biliar dos pacientes chagásicos quando comparado com os não chagásicos.