Establishment Of Hirschsprung’s Disease Rat Model And Study On The SCF/c-kit Signaling Pathway

Hirschsprung’s disease （aganglionic megacolon, HD or HSCR） is characterised bythe absence of the myenteric and submucosal plexuses. This congenital and developmentaldisorder causes functional intestinal obstruction with an incidence of1/5000in new-bornbabies. It is a common disease in pediatric surgery and is the second leading cause of thegastrointestinal malformations. Interstitial cells of Cajal （ICC）, a kind of special interstitialcells distributed widely in the entire digestive tract, is considered as the pacemaker cellsof spontaneous rhythmic electrical activity of smooth muscle. Emerging evidences showthat there is a close relationship between colonic motility dysfunction and the abnormaldistribution and functions of ICC. The signaling pathway initiated via binding of stem cellfactor （SCF） to c-kit receptor is essential for development and maintenance of interstitialcells of Cajal and were considered to be involved in the pathogenesis of HD. Therefore, futher achievement of in-depth knowledge on this field would certainly herald a syntheticunderstanding of the very complex pathogenesis of HD. In the present study, weestablished the different types of neonatal HD rat models and then explore the associationbetween SCF/c-kit pathway and HD deficit. The available data should provide theoreticalbasis for HD etiological research.ObjectiveWe aimed to establish several different kinds of Hirschsprung’s disease neonatal ratmodels and then to study the expression level of SCF/c-kit pathway in the models andafter intervence in vitro experiment, thus providing the experimental basis for future studyon potential involvement of SCF/c-kit signaling in ICC morphology and function changes.MethodsExperiment1: Establishment and identification of several different kinds of HDneonatal rat modelFour litters neonatal SD rats of6to8days old were randomly divided into fourgroups （three experimental groups and one control group）. After different lengths ofmicroinjection catheter were carefully placed through the anus, we slowly injecteddifferent doses and concentrations of benzalkonium chloride （benzalkonium chloride,BAC） to establish three types of Hirschsprung animal models. After treatment with BAC,animals were sacrificed and examined through general observation at postoperative2,4,6and8weeks. After dehydration, the samples including the whole colon and rectum wereembedded in paraffin and further processed into5-μm-thick sections forhematoxylin-eosin （H&E） staining. Parallel immunohistochemical analysis of protein geneproduct9.5（PGP9.5） were carried out to further confirm the animal models. Experiment2: Evaluation of expression of SCF/c-kit signaling in rat HD modelsPresence of SCF/c-kit along the colon tissue was studied using immunofluorescentstaining. The expression level of SCF/c-kit in narrow segment of colon tissue was alsoassessed at the transcriptional level and the translational level by quantitative Real-TimePCR （RT-qPCR） and western-blotting, respectively.Experiment3: Primary culture of ICC from HD model rats and effect ofactivation/inhibition of SCF/c-kit pathway on ICCICC were isolated from HD model and normal control rats and were cultured asdescribed previously. In detail, the colon tissues were dissected, minced and the cells wereseeded onto six-well plates precoated with poly-L-lysine at the density of110⁶/well.The medium of cell culture was changed overall after24h and then replaced with freshmedium every2days onwards. The cells were divided into four groups. The cells ofnormal control group and HD group received no treatment except changing the mediumwhen it was needed. The medium of HD+S group was replaced with new containing SCF（100ng/ml） after24h of seeding and then cells were cultured for nine days with constantpresence of SCF. At the7^thday, Glivec was added into the medium at the finalconcentration of10μM and the cells were cultured for another48h （HD+S+G group）.Morphological alteration of ICC was assessed by immunofluorescent staining. Cellsuspensions were sorted by flow cytometry and the expression changes of SCF or c-kitwere detected by RT-qPCR and western blotting at the transcriptional level and thetranslational level, respectively.Results1. Some rats began to exhibit symptoms of abdominal distention, reduced bowelmovements and smaller fecal particles four weeks after treatment with BAC. Afterdissection, we found apparent stenosis of the bowel in HD group when compared tothe control group. The results of immunohistochemical staining also showed that ganglion cells disappeared gradually after BAC treatment, which were not observed inthe control group.2. When compared to the control group, the expression levels of c-kit and SCF reducedsignificantly in the colon of HD model rats as reflected by western-blot, qT-PCR andimmunofluorescence analyses.3. There was apparent morphological difference in the primary cultured ICC amongdifferent experimental groups. Compared with the control, the axon length of ICC inHD group was shorter. After being cultured in the medium containing SCF （100ng/ml）for nine days, the ICC axons were significantly elongated. The effect of SCF could beblocked by Glivec.4. In the primary cultured ICC, the expression of c-kit and SCF of HD group weredecreased remarkably compared to the control（P<0.05）. Treatment of the cells withSCF could reverse the down-regulation of c-kit and SCF expression （P<0.05）. Thiseffect of SCF could be blocked by Glivec.ConclusionWe successfully established three different kinds of megacolon neonatal rat modelsby injecting different concentrations and doses of benzalkonium chloride through anus.This model was stable and could be easily repeated. It laid a reliable foundation forin-depth study of ICC function in the pathogenesis of HD. By studing the expressionprofile of c-kit and SCF in HD model rats and in primary cultured ICC, we found thatchanges of SCF/c-kit signaling pathway was indeed related to the alterations of HD. Oursystematic work should pave the way for future mechanism study.