Impact of interstitial cells of Cajal on slow wave and gallbladder contractility in a guinea pig model of acute cholecystitis

Figure 1 Histopathological analysis of a guinea pig model of acute cholecystitis (100 ×). A: Gallbladders in the normal control (NC) group were intact and showed no congestion, edema, or obvious inflammatory cell infiltration. The gallbladders in the acute cholecystitis (AC) groups showed edema, fibroplasia, congestion, mucosal necrosis, considerable inflammatory cell infiltration, and were significantly aggravated over time. In addition, the R-A sinus was present in the lamina propria of the gallbladders in the AC48h group (arrowhead); B: The inflammation score of the NC, AC12h, AC24h, and AC48h groups were 1.20 ± 0.20, 4.00 ± 0.71, 6.80 ± 0.73, and 10.20 ± 0.86, respectively (^aP < 0.05 vs NC groups, ^bP < 0.001 vs NC groups, ^cP < 0.0001 vs NC groups). AC: Acute cholecystitis; NC: Normal control; R-A: Rokitansky-Aschoff; H&E: Hematoxylin and eosin.

Figure 2 Recording of the gallbladder slow wave by electromyogram. A and B: The mean slow wave (SW) frequency in the normal control (NC), methylene blue + light groups were 8.81 ± 0.25 vs 5.71 ± 0.43 (^dP < 0.001 vs NC groups); C and D: The mean SW frequency in the NC, acute cholecystitis 12 h (AC12h), AC24h, and AC48h groups were 10.66 ± 0.51, 7.13 ± 0.20, 6.46 ± 0.16, and 5.75 ± 0.44, respectively (^aP < 0.0001 vs NC groups, ^bP < 0.0001 vs NC groups, ^cP < 0.0001 vs NC groups); E: Illustration of a SW recording of the guinea pig gallbladder. SW: Slow wave; EMG: Electromyogram; NC: Normal control; AC: Acute cholecystitis; MB: Methylene blue.

Figure 3 Effects of cholecystokinin octapeptide-induced contraction of gallbladder muscle strips. A: Destroying interstitial cells of Cajal can induce impairment of gallbladder muscle motility by loading methylene blue with light illumination [0.78 ± 0.08 vs 0.46 ± 0.04, ^aP < 0.01 vs normal control (NC) groups]; B: The cholecystokinin octapeptide-induced effect of the R value in the NC, AC12h, AC24h, and AC48h groups were 0.96 ± 0.21, 0.42 ± 0.09, 0.41 ± 0.03, and 0.20 ± 0.07, respectively (^bP < 0.05 vs NC groups, ^cP < 0.05 vs NC groups, ^dP < 0.01 vs NC groups). CCK-8: Cholecystokinin octapeptide; ICCs: Interstitial cells of Cajal; MB: Methylene blue; NC: Normal control; AC: Acute cholecystitis.

Figure 4 Sections of guinea pig gallbladder were stained with anti-c-Kit and anti-α-SMA antibodies and visualized with 3,3-diaminobenzidine in the normal control and acute cholecystitis groups (200 ×). Interstitial cells of Cajal (ICCs) (arrowheads) were present in the muscular layer of the gallbladder. The number of ICCs was obviously reduced in each acute cholecystitis (AC) group compared with the normal control (NC) group. Notably, there was no significant difference in gallbladder smooth muscle (arrows) morphology and structure between the NC and AC groups. NC: Normal control; AC: Acute cholecystitis; ICCs: Interstitial cells of Cajal; GBSM: Gallbladder smooth muscle.

Figure 5 Transmission electron microscopy results of interstitial cells of Cajal of the guinea pig gallbladder in the normal control and acute cholecystitis groups. A: The normal interstitial cells of Cajal (ICCs) had ovoid or triangular bodies, one to three cytoplasmic processes, large nuclei, abundant mitochondria, endoplasmic reticulum, and caveolae; B: ICCs from the AC12h group presented swollen cell bodies with enlarged mitochondria; C: The impaired ICCs from the AC24h group had more swollen cytoplasm. The distance between ICCs and other cells (mainly smooth muscle cells) was increased; D: The isolated ICCs in loose gallbladder tissue was significant swollen with ruptures in the cytoplasm membrane, and processes diminished. TEM: Transmission electron microscopy; ICCs: Interstitial cells of Cajal; NC: Normal control; AC: Acute cholecystitis; SMCs: Smooth muscle cells; ER: Endoplasmic reticulum. Bar: 10 μm.

Figure 6 Western blot analysis of the normal control and acute cholecystitis groups. A: The protein expression level of c-Kit, cholecystokinin A receptor (CCKAR) and connexin 43 (CX43) in guinea pig acute cholecystitis (AC) model of gallbladders was decreased; B: The mean grayscale values of c-Kit protein levels in the normal control (NC), AC12h, AC24h, and AC48h groups were 0.96 ± 0.12, 0.53 ± 0.09, 0.49 ± 0.09, and 0.37 ± 0.06, respectively (^aP < 0.05 vs NC groups, ^bP < 0.05 vs NC groups, ^cP < 0.01 vs NC groups); C: The mean grayscale values of α-SMA protein levels in the NC, AC12h, AC24h, and AC48h groups were 0.74 ± 0.05, 1.05 ± 0.05, 0.67 ± 0.05, and 0.75 ± 0.05, respectively (^dP < 0.05 vs NC groups); D: The mean grayscale values of CCKAR protein levels in the NC, AC12h, AC24h, and AC48h groups were 1.05 ± 0.11, 0.92 ± 0.12, 0.66 ± 0.10, 0.44 ± 0.12, respectively (^eP < 0.05 vs NC groups); E: The mean grayscale values of CX43 protein levels in the NC, AC12h, AC24h, and AC48h groups were 1.10 ± 0.30, 1.11 ± 0.02, 0.92 ± 0.09, 0.66 ± 0.12, respectively (^fP < 0.01 vs NC groups). NC: Normal control; AC: Acute cholecystitis; CCKAR: Cholecystokinin A receptor; CX43: Connexin 43.