Effects Of Chronic Cholangitis On The Function Of Sphincter Of Oddi

BACKGROUND AND OBJECTSIt is very common that a loose sphincter of Oddi (SO) is encountered during exploration of common bile duct in patients with hepatolithiasis or common bile duct stones. Because of its potential risk to induce reflux cholangitis and result in stones recurrence, loose SO is one of the decisive reasons to adopt cholangiojejunostomy. However, with the long-term complications such as the stenosis and the carcinomatous change of the stoma as well as the high incidence of stones recurrence be recognized, more and more researchers proposed to preserve the structure and the function of SO. This point of view encouraged many researchers to reveal the underlying cause of loose SO accompanied with hepatolithiasis, but completely different results were draw and effects of cholelithiasis on the motility of SO were quiet controversy, it is conceivable that passage of stones through the sphincter can result in loose SO, however, other researchers argued that the passage of stones through SO was not in close relationship to the occurrence of loose SO.Researches on the functional changes of smooth muscle motility in the inflammatory bowel disease (IBD) and acalculous cholecystitis were prevalent in the past few years. Many studies have reported that there is a correlation between the impaired smooth muscle motility and the increased reactive oxygen species (ROS) and /or peroxynitrite, which induce cellular injury via several mechanisms including peroxidation of membrane lipids, and result in impaired calcium homeostasis, abnormal phosphorylation of myosin light chain (MLC) as well as distortion of filamentous actin (F-actin). It is imperative to assume that recurrent cholangitis may be involved in the progress of loose SO, However, few researches focused on the cause and effect of cholangitis and loose SO.Then we assumed that recurrent cholangitis be the most important factor of impaired smooth muscle motility of SO. To identify this hypothesis, the aim of our study was, first of all, to collect clinical data, observe the motility characteristics of loose SO and analyze its related risk factors in hepatolithiasis patients. Then we devised a novel cholangtis model in rabbits and observed the effects of chronic cholangitis on the myoelectric motility of SO in-vivo and in-vitro. Finally, we investigated the effects of chronic cholangitis on the calcium mobilization, phosphorylation of myosin light chain (MLC) as well as the remodeling of filamentous actin (F-actin) in SO smooth muscle cells.MATERIALS AND METHODS1. With the methods of myoelectric and manometry recording via circular electrode (CE) or perfused triplelumen system, we recorded the myoelectric motility and manometry change of SO including the SO basal pressure (SOBP), SO amplitude (SOAP), frequency of SO phasic contraction(SOF), duration of SO contraction, as well as the frequency and amplitude of SO spike burst (SOSB). Then, we analyzed the correlation between loose SO and independent variables involved in the progress of hepatolithiasis including age, gender, course of disease, distribution of stones, size of stones, characteristic of stones, operation frequency, occurrence frequency of cholangitis, and diameter of common bile duct in 293 cases.2. With modified Thomas process, we devised a novel model of myoelectric recording of SO in fully conscious state in rabbits. We recorded the cyclic change of myoelectric motility during fasting, and the myoelectric response to different doses (20 ng·kg-1 or 100ng·kg-1) of cholecystokin (CCK) or feeding.3. To establish the model of chronic cholangitis in rabbits, we introduced three 2-0 silk and sequentially injected E Coli into the bile duct, and then observed the changes of weight, rectal temperature, liver function [alanine aminotransferase (ALT); alkaline phosphatase (ALP);γ-glutamyltransferase (GGT) and direct bilirubin (DB)], as well as inflammation score assessed by pathological changes of SO including inflammatory infiltration and collagen deposition. In in-vivo SO myoelectric motility experiment, the SO myoelectric activity was recorded by cyclic electrode (CE) through the jejunum stump in conscious rabbits or application of CCK. In in-vitro experiment, the SO was completely isolated under operating microscope and the myoelectric activity was recorded by CE in a 10-ml organ bath filled with Krebs solution with or without addition of CCK, KCl, ionomycin or induction of capacitative calcium entry (CCE). All the results from chronic cholangitis rabbits were in comparison with that in control and non-infective subjects.4. To investigate the effects of chronic cholangitis on the calcium mobilization, phosphorylation of MLC as well as the remodeling of F-actin in SO smooth muscle. We isolated the smooth muscle cells from the SO with the modified enzymatic digestion method, then we observed the changes of [Ca2+]i induced by agonists in Fluo-3 AM loaded cells, or the expression of F-actin by FITC-phalloidin loaded smooth muscle cells under laser scanning confocal microscope (LSCM). The phosphorylated MLC was also detected by Western-blot.RESULTS1. 60 cases (20.5%) of loose SO was found in 293 hepatolithiasis patients, in comparison with the intact SO, the SOBP, SOAP, SOF and duration of SOAP recorded by SO manometry were significantly decreased, the frequency and amplitude of SOSBs were also decreased. The recurrent cholangitis and course of disease were in close relationship with loose SO.2. The myoelectric motility was recorded in fully conscious rabbits with this model; sphincter motility exerted a cyclic change because of the cyclical variation of fast waves and almost invariant slow waves. Feeding caused excitation of the sphincter activity as well as the effect of CCK in the dosage of 20 ng·kg-1 or 100 ng·kg-1.3. Chronic cholangitis was successfully established in rabbits. In control (CN) and non-infective (NI) groups, no changes were found in weight, rectal temperature as well as liver function. However, in chronic cholangitis (CC) group, rectal temperature, ALT, ALP, GGT, and DB were significantly elevated at the first 3~4 days. Three months latter, ALP, GGT and inflammation score were significantly higher than that in NI and CN groups, but the ALT and DB was not changed. The cyclic change of myoelectric motility was recorded in 25 rabbits. In comparison with the CN and NI group, the duration of phase 3 was significantly decreased. The frequency and amplitude of SOSBs were lower in CC than that in the CN and NI group in phase 2 and 3, and the myoelectric motility index (MI) was especially lower in phase 3 in CC group. The excitative response of myoelectric motility was decreased in CC group with shortened effective duration, lower amplitude of SOSBs and MI.In-vitro study also showed decreased frequency and amplitude of SOSBs as well as MI in resting state and response to 10 nM CCK in CC group in comparison with NI and CN group. CC group also showed significantly lower MI of elevated myoelectric motility in the presence of 60 mM KCl and induction of CCE. In CC group, agonists (10 nM CCK and 50 nM ionomycin) activated calcium release in calcium-free medium was also fail to induce the significant increase of myoelectric MI as that in CN and NI group.4. With the modified enzymatic digestion method, we successfully isolated and cultivated the smooth muscle cells. Smooth muscle cells of SO isolated from the chronic cholangitis rabbits showed impaired calcium homeostasis including the decrease of 60 mM KCl or CCE-induced calcium influx from extracellular and 10 nM CCK or 10 mM Caffeine-induced calcium release from the calcium pools. In addition, abnormal phosphorylation of MLC and distortion or fragmentation of F-actin was also found in the SO smooth muscle cell in chronic cholangitis rabbits.CONCLUSIONS1. The occurrence rate of loose SO is relatively high in hepatolithiasis patients with decreased SOBP, SOAP, SOF, as well as the frequency and amplitude of SOSB. Course of disease recurrent cholangitis were the independent variable correlated with loose SO.2. The frequency, amplitude and motility index of SO myoelectric activity and of its response to CCK was decreased either in-vivo or in-vitro in chronic cholangitis rabbits.3. Impaired calcium homeostasis and deregulation of MLC phosphorylation as well as remodeling of F-actin might be the key events of impaired motility of SO in chronic cholangitis rabbits.