Regeneration Of Interstitial Cells Of Cajal And Reconstruction Of The Cellular Networks In Surgical Small Intestines Of Adult Guinea-pigs

Interstitial cells of Cajal (ICCs) were a kind of special interstitial cells, and the cellular networks were distributing within the entire gastrointestinal tract. Recent 20 years, series of researches about ICCs in morphology, ultrastructure, origination and physiology indicated that as pacemaker cells of GI motility, ICCs were responsible for the generation and propagation of slow waves, and also were mediators of inputs from the enteric nervous system to GI smooth muscles. Experiments indicated that slow-waves disappearance and GI dyskinesis were associated with either reduction of ICCs or cellular networks impairment in GI wall. Postoperative GI dysmotilities (eg, transient paralysis of intestine; pseudoileus) following abdominal surgery happened frequently in clinic, and recovered depending on invasive degrees. How surgical manipulation caused a disruption in motility of GI tract was not understood well yet. Recent experiment indicated that ICCs networks broken up and phenotype disappearance might be involved in short-term post-operative dysmotilities. Furthermore some GI dyskinesic diseases (eg, Hirschsprung's disease; slow-transit constipation) have been linked to ICCs reduction and networks impairment. Alrough the underlying mechanisms were not clear yet, they all characterized by reduction of ICCs at different degrees and even totally absense. Supposing ICCs could regenerate after loss, it would provide new ideas for clinic treatment of GI dyskinesis and dysfunction of GI motor after abdominal surgery. Therefore animal models of post-operative dysmotilities in which ICCs networks disrupted were established by using intestinal surgery, morphologic changes of ICCs after surgery were observed by immunohistochemical staining, doule staining, TEM, et al. The animal model enable us not only to observe changes of ICCs after operation, but also to clarify the following questions, including that if ICCs in adult animal could regenerate and restore normal network structure, and if smooth muscle cells and nerves might be involved in this process. Results After careful observation, each anastomotic end could be divided into 3 areas on the longitudinal sections through the anastomotic sites stained with hematoxylin. Smooth muscle layers of cut ends appeared to be thicker than normal (areaⅠ), while adjacent to it, where anastomosis was performed, the muscle layers appeared thinner (areaⅡ), which might caused by suture tension, and area Ⅲwas between oral and aboral ends. 1. Morphologic Changes of ICCs Post-operatively Five and ten days post-operation, the longitudinal sections showed IC-MY and/or IC-DMP disrupted and ICCs partly lost in area Ⅱand area Ⅰby KIT immunohistochemistry, while area Ⅲwas filled with connective tissue where KIT positive cells totally absent. After 30 days of operation, some ICCs-like cell irregularly and scatteredly showed up in the three areas, characterized by 1~2 short and thin processes, oval-shaped nuclei and slim cytoplasm, but these cells were in mess and did not show any sign of normal appearance. Then 50-day post-operatively, distribution of KIT-positive cells and structure of intestinal wall in area Ⅱand Ⅰseem to be in recovery to normal by distinguishable IC-MY and IC-DMP, furthermore processes of them were seen prolonging toward the opposite anastomotic ends. Seventy days after surgery, the anastomotic ends almost restored normal structure, besides KIT-positive cells in area Ⅰincluding IC-DMP and IC-MY increased both in quantity, showing a view of the processes prolonging toward area Ⅲ. Also more ICCs-like cells appeared in area Ⅲwith 2-3 long and thin processes. In 90-day post-operative group, the anastomotic ends had restored to normal structure and ICCs-like cells kept rising in number in area Ⅲ. While 150 days after operation, oral and aboral anastomotic ends interconnected in area Ⅲ, the intestinal wall appeared with normal IC-MY, IC-DMP and smooth muscle layers. As a result, ICCs networks and intestinal structure took about 150 days to restore normal appearance. 2. Identification of Regenerated ICCs Although KIT-positive area strongly increased after surgery, it was not so sure that ICCs owing the ability of regeneration in adult guinea pigs. To detect regenerated cells, BrdU was used, which was a thymidine analogue, capable of being incorporated into DNA in place of thymidine during DNA synthesis. Regenerated ICCs could be distinguished by double staining with BrdU and KIT immunocytochemistry. The control (i.p. injection ofBrdU for 10~30 days, 3mg/other day) and 5 days post-operative group did not show any double staining positive cell, while intestinal epithelial cells revealing BrdU positive. Ten days after surgery, 1~3 double staining cells were seen in each oral or aboral anastomotic end with normal look, while beyond 1 mm from anastomtic sites no double staining cells were observed. The number of regenerated ICCs increased to 6~10 within each anastomotic end 30 days after operation with oval-shaped nuclei with 2~3 slim and long processes, and most of them distributed from 3.0 mm orally or aborally to anastomtic sites. Then at the end of 50 days post-operatively KIT/BrdU double staining cell expanded to 14~18, moreover the positive cells located within 4.5 mm long intestinal wall from anastomtic sites to oral or aboral ends. The regenerated ICCs mainly distributed around DMP (58%~74%). Thinking of BrdU injections might affect the health of guinea-pigs, we canceled injecting after 50 days. 3. Associated Factors with Growth of ICCs As c-kit receptor tyrosine kinase (KIT) and its ligand, stem cell factor (SCF), were required for the development and survival of ICCs. Immunocytochemistry with anti-SCF antibody were used to show the distribution of SCF, and the results revealed that there were positive reaction in smooth muscle tissue and weak positive reaction in nerves respectively. KIT/SCF double staining showed that SCF distributing around ICCs. The changes of smooth muscle cells were examined after surgery by immunohistochemistry staining of anti-α-SMA. The results showed that growth of SMCs were different according to different layers: longitudinal smooth muscle cells began to grow from area Ⅱinto area Ⅲtoward opposite anastomotic ends 30 days after operation and interconnected at 50 days post-operatively, while oral and aboral circular smooth muscle layers did not interconnected with each others until 150 days after operation.