The Exploration Of The Apoptosis Of Interstitial Cells Of Cajal In Colon Of Hirschsprung’s Disease And Its Allied Disorders

ObjectiveHirschsprung’s Disease(HD) and its allied disorders(HAD), as a result of gastrointestinal motility disorder, are a series of common abnormalities in pediatric surgery, the coordination and cooperation of the intestinal ganglion cells, interstitial cells of Cajal(ICCs) and smooth muscle cells generates and mediates the normal gastrointestinal motility. Some studies have confirmed that ICCs is associated with HD, but the exact mechanism is not yet clear. In this study, by examining the distrubution of ICCs in the colon of HD and HAD, and detecting the expression of caspase-3 、 bcl-2 of ICCs using the immunofluorescence double staining, to find out the level of apoptosis of ICCs and weather the apoptosis of ICCs in the colon plays a role in the pathogenesis of HD and HAD.MethodFrom 2014/2 to 2015/2, 15 cases with HD and 13 cases with HAD were enrolled in this study, all of them were diagnosed by anorectal manometry、barium enema、acetylcholinesterase staining and postoperative pathology. The aganglionic,transitional and dilated gut of HD, and the proximal and distal bowel of HAD were collected in the operation as the experimental group; Another normal colon(10 cases) were also collected as control group. The distrubution of interstitial cells of Cajal(ICCs) in the colon and the expression of caspase-3、bcl-2 of ICCs were identified and counted in double labeled tissue sections, the data was analyzed statistically by SPSS 13.0, and the difference was considered as statistical significance(P<0.05). While the ultrastructural changes in the ICCs were morphologically observed by electron microscopy.1 The distrubution of ICCs in the colonThe distrubution of interstitial cells of Cajal(ICCs) in the colon was counted as /field through the immunofluorescence double staining. Compared to the contral group 17.8± 1.5/field, there was a significant difference among the aganglionic 5.8±1.1/field, transitional 10.0±1.8/field and dilated 13.1±2.1/field gut of HD, and also the distall8.6±1.6/field bowel of HAD(P<0.05), while the distrubution of ICCs in the proximal bowel of HAD was 16.5±2.4/field, there was no significant difference between them(P>0.05). And there was a significant difference among the aganglionic,transitional and dilated gut in HD(P<0.05), also significant difference was found between the proximal and distal bowel in HAD(P<0.05).2 The expression of caspase-3 of ICCs in the colonThe percentage of caspase-3-positive ICCs counted by immunofluorescence double staining in the colon respectively was 6.3± 4.3% in contral group, 77.6±13.8% in aganglionic gut, 53.6±10.2 % in transitional gut, 31.9±8.0% in dilated gut and 23.6±6.8% in proximal bowel, 57.2±12.1% in distal bowel. Compared with the control group, the others were significantly increased(P<0.05), espeacially in the aganglionic gut(P<0.05).3 The expression of bcl-2 of ICCs in the colonThe percentage of bcl-2-positive ICCs counted by immunofluorescence double staining in the colon respectively was 60.3±5.4% in contral group, 18.3±9.3% in aganglionic gut, 31.5±7.3 % in transitional gut, 42.3±7.9% in dilated gut and 48.7±6.5% in proximal bowel, 38.5±6.7% in distal bowel. Compared with the control group, the others were significantly decreased(P<0.05), espeacially in the aganglionic gut(P<0.05).4 The correlation between the distrubution of ICCs and the expression of caspase-3 and bcl-2In HD group, the correlation between the distrubution of ICCs and the expression of caspase-3 was negative(r=-0.872, P<0.01); while it was positive between the ICCs and bcl-2(r=0.676, P<0.01).ResultsIn HAD group, the correlation between the distrubution of ICCs and the expression of caspase-3 was negative(r=-0.915, P<0.01); while it was positive between the ICCs and bcl-2(r=0.575, P<0.01).5 The ultrastructural changes of ICCs in the colonAt the electron microscopy level, the ultrastructural apoptotic changes of ICCs were found in HD and HAD. The nuclear chromatin was condensed and shrinked, the electron density was increased, and the nuclear chromatin was edge set and uneven thickness; but nuclear envelope of ICCs in HD wasn’t possessed, the organelles decreased significantly and cytoplasm was completely empty, the mitochondria, endoplasmic reticulum and lysosomes were swollen, even vacuolar degeneration; while nuclear envelope of ICCs in HAD was still possessed, the organelles decreased slightly and the mitochondria, endoplasmic reticulum and lysosomes were also normal-like.Conclusions1 The distrubution of ICCs in HD and HAD was decreaced while compared to the control group, it means that ICCs may play a role in the the pathogenesis of HD and HAD.2 The expression of caspase-3 in ICCs offers a “negative message” in contrast to the “positive approach” of bcl-2, the correlation between the distrubution of ICCs and the expression of caspase-3 was negative, while it was positive between the ICCs and bcl-2, which points that ICCs which abnormally express caspase-3 and bcl-2 may play a role in the pathogenesis of HD and HAD.3 At the electron microscopy level, the ultrastructural apoptotic changes of ICCs were found, so the apoptosis of ICCs in the colon may be related to the pathogenesis of HD and HAD.