| Background and objective: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 least30%. The pathogenesis of AAC 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+transient 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. Neutrophils are the most important inflammatory cells in early inflammatory response and as a core part in the inflammation. The relationship between neutrophils and ICC injury in the gallbladder is not clear. 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 CBDLMethods: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 at24h,48h and72h after CBDL, respectively. The volume of the gallbladder was also measured at each time point. The level of alanine transaminase (ALT) and glutamic oxalacetic transaminase (AST) 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 RM6240polygraph.The contractile response to acetylcholine (Ach,10-5mol/l), cholecystokinin octapeptide (CCK-8,10-6mol/l) and potassium chloride (KCl,60mmol/1) was recorded. The ultrastructure changes of gallbladder ICC before and after CBDL were 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 (CBDL48h) were selected as a key research object, which neutrophils in the gallbladder wall were closer to ICC injury. The use of anti-polymorphonuclear antibody (anti-PMN) was to reduce the circulating neutrophil counts. Guinea pigs were randomly divided into4groups:normal-2group, sham-2group, CBDL-2group and anti-PMN group, and there were12animals in each group. The color of the bile and the volume of the gallbladder were observed. The level of ALT, AST and 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. The contractile response of gallbladder muscle strips to Ach, CCK-8and KCl was registed by tension transducer and the ultrastructure features of gallbladder ICC were observed.At last, guinea pigs at the age of10-15days were choosed. The gallbladders 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. This research would lay the foundation for the next electro physio logical experiments in gallbladde ICC,Results:The activity of guinea pigs was reduced notable ly 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, AST 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 CBDL48h, the cytoplasm of the neutrophils was full of granules, and neutrophils were closely contacted with ICC body and ICC processes.Compared with normal-2and sham-2groups, the volume of the gallbladder and the level of ALT, AST, bilirubin in serum increased significantly (P<0.05) in CBDL-2and anti-PMN groups, but there was no statistical difference between CBDL-2and anti-PMN groups (P>0.05). Inflammation scores and MPO activity in CBDL-2, anti-PMN group were higher remarkably than those in normal-2and sham-2groups (P<0.05), and they decreased notablely in anti-PMN group compared to CBDL-2group (P<0.05). After adding Ach, CCK-8, KCl, the contraction ability in CBDL-2and anti-PMN groups apparently was less than those of normal-2and sham-2groups (P<0.05), and they were elevated obviously in anti-PMN group compared to CBDL-2group.[Ach:(0.70±0.13vs.0.29±0.03); CCK-8:(0.26±0.06vs.0.12±0.01); KCl:(0.90±0.18vs.0.34±0.08)(P<0.05)]. Transmission electron microscopy confirmed that gallbladder ICC in CBDL-2group were swollen, endoplasmic reticulum dilatation, and cell processes diminished. Compared with CBDL-2group, the ultrastructure of ICC in anti-PMN group shown less swollen, mild dilatation of endoplasmic reticulum, and cell processes increased slightly, and ICC contaction with another ICC or gallbladder smooth muscle was closer.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 cultured at the third or fourth day were ICC.Conclusions:Anti-PMN could decrease the neutrophils infiltration in gallbladder tissues, and reduce inflammation in AAC animal model. Anti-PMN could also alleviate the ultrastructure injury of ICC and elevate the contractile response to Ach, CCK-8, KC1on gallbladder muscle strips. Neutrophils may damage gallbladder ICC firstly, then showed impaired contraction of gallbladder smooth muscle. The method of tissue pieces culture on gallbladder ICC was successful. |
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