| Background and objective:Clinically,acute acalculous cholecystitis (AAC) is absence of specific clinical manifestations and difficult to be diagnosed. It is a kind of serious illness, onset acute and progress fast. The mortality rate of AAC remains at least 30%. After numerous medical profession senior unremitting efforts, the pathogenesis of AAC were explored. The pathogenesis probably includes bile stasis, ischemia-reperfusion injury, activation of endogenous clotting factor and bacterial infection. Impaired smooth muscle contractility is a hallmark of AAC. Common bile duct ligation (CBDL) is recognized as the most credible AAC animal model. CBDL in the guinea pig produces a histologic picture that is nearly identical to that of acute human cholecystitis. Interstitial cells of Cajal (ICC) in the gastrointestinal (GI) tract is considered to be pacemaker cells for slow wave and regulates the rhythmic smooth muscle contraction in the GI tract. Rhythmic Ca2+ transients in ICC is crucial in pace-making. In recent years, interstitial cell of Cajal-like cells were found in the gallbladder, and rhythmic Ca2+ transients were also observed by laser confocal imaging of Ca2+ flashes, which suggested that ICC in the gallbladder could play a role in the generation and propagation of rhythmic electrica activity, and regulate the gallbladder smooth muscle relaxation or contraction. At the earlier stage of AAC, the ultrastructural features of Cajal-like cells were changed in the gallbladder.Objective:The aim of this study was to determine the effects of neutrophils on ICC in the gallbladder using CBDL model in guinea pigs, and to explore the possible mechanism that ICC participated in decreased gallbladder motility during AAC induced by CBDL.Methods:At first, healthy adult guinea pigs were studied in the present research and the model of AAC was structured by CBDL. The color of the bile was observed at 24 h,48h and 72h after CBDL, respectively. The volume of the gallbladder was also measured at each time point. the level of alanine transaminase (ALT) and bilirubin in serum were detected by full automatic biochemical analysis system. Hematoxylin and eosin-stained slides of gallbladder samples were scored for inflammation. The contraction activity of gallbladder muscle strips from guinea pigs before and after CBDL was monitored by RM6240 polygraph. The contractile response to acetylcholine (Ach,10-5mol/l), cholecystokinin octapeptide (CCK-8,10-6mol/l) and potassium chloride (KCl,60mmol/l) was recorded. The ultrastructure changes of gallbladder ICC before and after CBDL was examined by transmission electron microscopy, and the relationship between ICC injury and neutrophils was determined.Then, the guinea pigs of AAC in a reasonable time (CBDL 48h) were selected as a key research object. The animals were sacrificed 48h after CBDL.The color of the bile and the volume of the gallbladder were observed. The level of ALT bilirubin in serum were measured by full automatic biochemical analysis system. Hematoxylin and eosin-stained slides from each gallbladder samples were scored for inflammation, and the myeloperoxidase (MPO) activity was evaluated with MPO test kit. Western Blot detected the expression level of NF-?B. The contractile response of gallbladder muscle strips to Ach, CCK-8 and KCl was registed by tension transducer. The Correlation of cholecystitis score, MPO concentration, NF-kB expression levels and the contractile response of gallbladder muscle strips were analyzed statistically.At last, the gallbladders of guinea pigs were taken in asepsis and blunt dissection was carried out during dissecting gallbladder muscle strips. Gallbladder ICC were obtained by the tissue pieces culture method in vitro and their appearance were observed in upside-down lighr microscope. The cultured gallbladde ICC were stained with the specific tyrosine kinase receptor protein c-kit and identified by immunofluorescence studies. And gallbladder ICC spontaneous electrical activity was recorded, change of calcium-dependent potassium channels(BKca) was observed.Results:The activity of guinea pigs was reduced notablely after CBDL. The color of the bile was light yellow before CBDL, and became dark yellow after CBDL, and turn dark green in the end. In comparison with normal and sham groups at the corresponding time, the volume of the gallbladder, the level of ALT and bilirubin in serum were higher remarkably in CBDL groups (P<0.05). After CBDL, there were white blood cell infiltration, tissue edema, vasodilation and congestion. Inflammation scores in CBDL groups were higher significantly than those in the corresponding normal and sham groups (P<0.05), but the contractile response to Ach, CCK-8, KCl was reduced notablely (P<0.05). The injuries of gallbladder ICC ultrastructure were aggravated with the time extending after CBDL. At CBDL 48h, the cytoplasm of the neutrophils was full of granules, and neutrophils were closely contacted with ICC body and ICC processes.WBC count animals in each group was no significant difference (P> 0.05).Compared with normal and sham groups, Neutrophil percentage of animal in CBDL group was significantly increased (P<0.05),the volume of the gallbladder and the level of ALT,, bilirubin in serum increased significantly (P<0.05) in CBDL group. Inflammation scores and MPO activity in CBDL group were higher remarkably than those in normal and sham groups(P<0.05). After adding Ach, CCK-8, KCl, the contraction ability in CBDL group apparently was less than those of normal and sham groups (P<0.05). After myeloperoxidase activity of gallbladder tissue in each group was detected, the result showed, MPO concentration in CBDL group was (0.657 ± 0.117 u/ g), increased significantly,compared with normal group (0.128 ± 0.026 u/g), sham group (0.135 ± 0.049 u/g) (P<0.05). Compared with normal and sham groups, expression levels of NF-kB gallbladder tissue in CBDL group increased significantly. The Correlation of cholecystitis score, MPO concentration, NF-kB expression levels and the contractile response of gallbladder muscle strips were analyzed negatively..The typical gallbladder ICC had a trilateral or elliptical body with one to two processes by the tissue pieces culture at the third or fourth day. The cultured ICC were growth that form singly or in clumps, and ICC processes were closely contacted with other ICC or gallbladder smooth muscle cells. However, gallbladder ICC did not appear to form a global network. Fluorescent staining with c-kit antibody confirmed that tissue pieces culture was successful in gallbladder muscle layer, and most of cells culture at the third or fourth day were ICC. In the resting state, without any stimulation to the cells, the cells can be recorded to the spontaneous action potential, pacemaker ICC was confirmed. AAC model group and control group maximum potassium current densities were 17.88 ± 2.26 PA·PF-1 and 13.26±2.18 PA· PF-1 (P<0.05).Conclusions:1. AAC animal models can be successfully constructed through CBDL.48h after CBDL, gallbladder ICC ultrastructural damaged, closely contact between neutrophils and damaged soma ICC and ICC projections. This change will inevitably bring about changes in the morphology of functionality.2. Neutrophils of gallbladder tissue may be affect the gallbladder ICC firstly, then involving the gallbladder smooth muscle, which evenutally leading to smooth muscle contraction gallbladder dysfunction. NF-kB signaling pathway plays an important role in the development process of AAC cholecystitis, while its role leading to gallbladder motility disorders could not be ignored.3. After primary cultured tissue block 3-4d, can successfully cultured gallbladder ICC, the cell surface receptor tyrosine kinase c-kit protein positive expression. When using patch-clamp techniques ICC study, target cells by light microscopy to observe cell morphology and cell surface protein expression of c-kit immunofluorescence, confirmed that the ICC; if further method using patch clamp technique was observed that cells spontaneously of the action potential, is more type ICC confirmed pacemaker cells. AAC model in guinea pigs, change of ICC cell calcium-dependent potassium channel (BKca) may lead to the gallbladder smooth muscle contraction disorder. |
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