Volume 127, Issue 2, Pages 535-545 (August 2004) Mast cell degranulation during abdominal surgery initiates postoperative ileus in mice  Wouter J. de Jonge, Frans O. The, Dennis van der Coelen, Roelof J. Bennink, Pieter H. Reitsma, Sander J. van Deventer, René M. Van den Wijngaard, Guy E. Boeckxstaens  Gastroenterology  Volume 127, Issue 2, Pages 535-545 (August 2004) DOI: 10.1053/j.gastro.2004.04.017

Figure 1 Delayed gastric emptying after intestinal manipulation is prevented by pretreatment with mast cell stabilizers ketotifen or doxantrazole. (A) Gastric emptying curves determined by scintigraphic imaging of the abdomen after oral administration of solid caloric meal at 24 hours after intestinal manipulation (IM + vehicle; solid squares), or IM after pretreatment with ketotifen (open squares), compared with laparotomy (L + vehicle; gray circles). Values shown are percentages of gastric content compared with the total abdominal region. (B) Treatment with doxantrazole prevents gastroparesis 24 hours following bowel surgery (IM + doxantrazole), compared with vehicle treatment (IM + vehicle). Lower panel: corresponding deduced half-emptying time (t1/2) as well as the retention after 60 minutes. Ret60min is significantly increased after IM, irrespective of vehicle used, compared with L. Pretreatment with either ketotifen or doxantrazole restores both t1/2 to normal. Note that Ret60min is restored to normal only following ketotifen treatment. Treatment with ketotifen or doxantrazole did not alter basal emptying after L. Values are averages ± SEM of 5–10 mice per treatment group. Asterisks indicate significant differences with respective L + vehicle group at P < 0.05. Gastroenterology 2004 127, 535-545DOI: (10.1053/j.gastro.2004.04.017)

Figure 2 The increase in ileal myeloperoxidase (MPO) activity after surgery with intestinal manipulation is prevented by ketotifen or doxantrazole pretreatment. MPO activity was determined in whole homogenates of ileum, isolated 24 hours after surgery. The MPO activity after IM in mice pretreated with ketotifen vehicle is increased, but no increase is seen after pretreatment with mast cell stabilizer ketotifen (solid bars). Similarly, doxantrazole pretreatment prevented postoperative increase in MPO activity, compared with its respective vehicle treated control (grey bars). Treatment with ketotifen or doxantrazole had no effect on basal MPO activity in control animals. Asterisks indicate significant differences in ketotifen or doxantrazole treatment group using a 1-way ANOVA (P < 0.05), followed by Dunnett’s multiple comparison test. Data represent means ± SEM of 5–8 mice. Gastroenterology 2004 127, 535-545DOI: (10.1053/j.gastro.2004.04.017)

Figure 3 The appearance of leukocyte infiltrates in ileal muscularis after intestinal manipulation is prevented by ketotifen or doxantrazole pretreatment. (A—D) Whole mount preparations of ileal intestinal muscularis tissue 24 hours after L (A), IM with ketotifen vehicle (B), IM with ketotifen pretreatment (C), and IM with doxantrazole pretreatment (D) are stained for MPO positive leukocytes. IM with either ketotifen or doxantrazole (not shown) vehicle pretreatment induced a massive influx of MPO-positive leukocytes to the ileal muscularis, compared with L (A and B). Pretreatment with ketotifen (C) or doxantrazole (D) prevented this influx of inflammatory cells. Preparations shown are representative for 5–8 mice per treatment group. Bar is 0.6 mm. (E) Shows that the significant increase in the number of MPO positive leukocytes per mm2 of muscularis tissue after IM with (ketotifen) vehicle pretreatment was prevented by ketotifen or doxantrazole pretreatment. Asterisk indicates significant difference using a 1-way ANOVA (P < 0.05), followed by Dunnett’s multiple comparison test. Values shown are the mean cell counts ± SEM of muscularis prepared from 5–8 mice. Gastroenterology 2004 127, 535-545DOI: (10.1053/j.gastro.2004.04.017)

Figure 4 Intestinal exposure to C48/80 delays gastric emptying. A Gastric emptying curves, determined by scintigraphic imaging of the abdomen after oral administration of solid caloric meal at 24 hours after exposure to C48/80 (solid circles), exposure to C48/80 after pretreatment with ketotifen (open circles), and L alone (squares). Exposure to C48/80 results in a delay in gastric emptying, which can be prevented by ketotifen pretreatment. Values are given as percentage of gastric content compared with the total abdominal region. Corresponding half-emptying time (t1/2) as well as the retention after 60 minutes. Ret60min is significantly increased after C48/80 exposure, compared with vehicle (saline) (B). Pretreatment with ketotifen restores both t1/2 as well as Ret60min back to normal. Values are means ± SEM of 8–12 mice per treatment group. Asterisks indicate significant differences at P < 0.05. Gastroenterology 2004 127, 535-545DOI: (10.1053/j.gastro.2004.04.017)

Figure 5 Mast cell degranulation results in infiltration of leukocytes in ileal muscularis. (A and B) Whole mount preparations of ileal intestinal muscularis tissue 24 hours after exposure to vehicle (0.9% NaCl) (A) or C48/80 (B) are stained for MPO-positive leukocytes. Extensive inflammatory infiltrates were observed after exposure to C48/80 but not saline. Preparations shown are representative for 6–8 mice per treatment group. Bar is 0.6 mm. Panel C shows that the number of MPO-positive leukocytes was significantly increased after incubation with C48/80, compared with incubation with vehicle. Asterisk indicates significant difference (P < 0.05). Values shown are the means ± SEM of 6–8 mice. Gastroenterology 2004 127, 535-545DOI: (10.1053/j.gastro.2004.04.017)

Figure 6 Mast cell degranulation elicits an increase in ileal MPO activity that can be prevented by ketotifen pretreatment. MPO activity was determined in whole homogenates of ileum isolated 24 hours after exposure to C48/80, or vehicle only (saline). The MPO activity after exposure to C48/80 is significantly increased, whereas exposure to saline did not affect MPO activity, irrespective of ketotifen pretreatment. Note that the increase in MPO activity elicited by C48/80 exposure was prevented by ketotifen pretreatment. Asterisk indicates significant difference using a 1-way ANOVA (P < 0.05), followed by Dunnett’s multiple comparison test. Data represent means ± SEM of 6–8 mice. Gastroenterology 2004 127, 535-545DOI: (10.1053/j.gastro.2004.04.017)

Figure 7 Reconstituted mast cells in Kit/Kitv mutant mice have a normal phenotypic appearance. (A and C) Mast cells were absent in Kit/Kitv small intestinal muscularis and Peyer’s patch (LM, longitudinal muscle layer; CM, circular muscle layer; PP, Peyer’s patch), as well as in peritoneal fluid (E). Sections of small intestinal muscularis (B and D) and peritoneal fluid (F) of Kit/Kitv mice reconstituted with cultured bone marrow-derived Kit/WT wild-type mast cells. The number of mast cells recovered in reconstituted mice is similar to that in wild-type mice, and they have a normal histology and granule content (arrows). Giemsa staining. Sections are representative of 5 mice examined in each group. Bar is 75 μm. Gastroenterology 2004 127, 535-545DOI: (10.1053/j.gastro.2004.04.017)

Figure 8 Intestinal inflammation after intestinal manipulation depends on the presence of mast cells. MPO activity was determined in whole homogenates of ileum isolated 24 hours after L or IM. A significant increase in MPO activity and inflammation was observed after IM in wild-type mice but not in mast cell deficient Kit/Kitv mutants (solid bars). Reconstitution of Kit/Kitv mutant mice with cultured Kit/WT mast cells restored the granulocyte infiltration after intestinal manipulation to wild-type levels. Asterisks indicate significant differences (P < 0.05). Data represent means ± SEM of 5 mice. Gastroenterology 2004 127, 535-545DOI: (10.1053/j.gastro.2004.04.017)

Figure 9 Granulocyte infiltration into the intestinal muscularis after intestinal manipulation in mast cell deficient- and mast cell-reconstituted mice. (A—D) Whole mount preparations of ileal intestinal muscularis tissue 24 hours after IM stained for MPO-positive leukocytes. Extensive inflammatory infiltrates were observed after IM in Kit/WT mice (A), but the number was drastically reduced in Kit/Kitv mutant mice (B). Reconstitution of Kit/Kitv mutant mice with Kit/WT mast cells restored the inflammatory response to IM (D), whereas reconstitution with PBS did not (C). Preparations shown are the representative for 5 mice per treatment group. Bar is 0.6 mm. The number of MPO-positive leukocytes was significantly decreased after IM in Kit/Kitv mutant mice, compared with Kit/WT wild-type, whereas mast cell reconstitution restored the inflammatory response to IM. Asterisk indicates significant difference (P < 0.05). Values shown are the means ± SEM of 5 mice. Gastroenterology 2004 127, 535-545DOI: (10.1053/j.gastro.2004.04.017)