1. Volume 125, Issue 4, Pages 1137-1147 (October 2003)
Postoperative ileus is maintained by intestinal immune infiltrates that activate inhibitory neural pathways in mice1  
Wouter J De Jonge, René M Van Den Wijngaard, Frans O The, Merel-Linde Ter Beek, Roel J Bennink, Guido N.J Tytgat, Ruud M Buijs, Pieter H Reitsma, Sander J Van Deventer, Guy E Boeckxstaens 
Gastroenterology 
Volume 125, Issue 4, Pages (October 2003)
DOI: /S (03)

2. Figure 1
Gastric emptying is delayed after abdominal surgery. (A ) A representative series of planar scintigraphic scans of mice that underwent laparotomy (L) or intestinal manipulation (IM) is shown. The position of the stomach is indicated (st) with a dotted circle. From these scans, gastric emptying could be repetitively assessed for each mouse individually by determining the amount of radioactivity present in the gastric region compared with the total abdominal region. Note the difference in radioactivity in the intestinal region between L and IM mice (arrows) at t = 80 minutes. (B) Half-emptying time (t 1 2; open symbols) and gastric retention after 64 minutes (Ret64; filled symbols) as a function of time after L (squares) or IM (circles). Intestinal manipulation, performed at t = 0 hours, resulted in a significant (P < 0.05) increase in t 1 2 and Ret64 compared with laparotomy at t = 6, 12, and 24 hours after surgery. Similar results were obtained with use of a caloric, solid test meal; t 1 2 was significantly increased after intestinal manipulation compared with mice that underwent L only (C ). ∗Significant difference from L with 1-way analysis of variance, followed by Dunnett’s multiple comparison test. Data represent mean ± SEM of 8–15 mice.
Gastroenterology  , DOI: ( /S (03) )

3. Figure 2
Ileal myeloperoxidase (MPO) activity was selectively increased at 12, 24, and 48 hours after surgery with intestinal manipulation (IM). MPO activity was determined in whole homogenates of ileum isolated 6, 12, 24, and 48 hours after surgery as indicated. MPO activity was significantly increased 12, 24, and 48 hours after laparotomy with IM (gray bars) compared with laparotomy only (L; white bars). ∗Significant difference from L for each time point with a Student t test (P < 0.05). Data represent mean ± SEM of 6–8 mice.
Gastroenterology  , DOI: ( /S (03) )

4. Figure 3
Intestinal manipulation results in an increase in MPO activity measured in ileal muscularis. MPO activity was measured in homogenates of ileal muscularis tissue isolated 24 hours after surgery. Laparotomy (L) with intestinal manipulation (IM) was associated with significantly increased MPO activity in ileal muscularis tissue compared with L alone. Treatment with ICAM-1- and LFA-1-blocking antibodies before IM prevented the increase in MPO activity (IM+ab). Treatment with hexamethonium did not affect the increased MPO activity found 24 hours after IM (IM+hex). ∗Significant difference from L with 1-way analysis of variance (P < 0.05) followed by Dunnett’s multiple comparison test. Data represent mean ± SEM of 5–8 mice.
Gastroenterology  , DOI: ( /S (03) )

5. Figure 4
Focal leukocyte infiltrates after intestinal manipulation in the ileal muscularis tissue. (A and B) Transverse sections of the ileal intestinal muscularis 24 hours after laparotomy (A ) and intestinal manipulation (B) were stained with mouse-specific monoclonal rat antibodies against LFA-1 (CD11a). Note the presence of LFA-1+ leukocytes in the ileal muscularis after (B) intestinal manipulation (arrows), but not after (A ) laparotomy. Sections were counterstained with hematoxylin. MPO activity-containing leukocytes were visualized in whole mounts of ileal muscularis tissue (C-F) isolated 24 hours after surgery. Intestinal manipulation (D), but not laparotomy (C ), was associated with a focal influx of MPO-containing leukocytes. Preoperative treatment of the mice with monoclonal rat-blocking antibodies against ICAM-1 (CD54), combined with rat monoclonal antibodies against LFA-1, prevented leukocyte influx (E ). Postoperative treatment with hexamethonium did not affect the presence of MPO-staining cells 24 hours after laparotomy with intestinal manipulation (F ). (G and H ) Transverse sections of gastric antrum stained with monoclonal antibody against LFA-1. Note the lack of LFA-1+ cells in the antral muscularis after laparotomy (G), as well as laparotomy with intestinal manipulation (H ). Sections were counterstained with hematoxylin. Bar is 75 μm (A, B, G, and H ) or 0.6 mm (C, D, E, and F ).
Gastroenterology  , DOI: ( /S (03) )

6. Figure 5
Gastroparesis after intestinal manipulation (IM) is prevented by blocking leukocyte infiltration or neural blockade by hexamethonium or guanethidine treatment. Gastric emptying, determined by scintigraphic imaging of the abdomen after oral administration of a semiliquid noncaloric meal at 6 and 24 hours (A ) after IM, was compared with laparotomy alone (L). Values in (A ) are given as relative gastric content compared with the total abdominal region. Corresponding t 1 2 (B) with semiliquid noncaloric (gray bars) and caloric solid (white bars) test meals were significantly (P < 0.05) increased at 6 and 24 hours after IM, compared with L. Preoperative treatment with anti-ICAM-1 and anti-LFA-1 antibodies (IM+MAb) normalized the t 1 2 of semiliquid and solid test meals (B) at 24 hours after surgery. Postoperative injections of hexamethonium (IM+hex) or guanethidine (IM+gua) normalized t 1 2 at 6 and 24 hours (B). Values are averages ± SEM of 8–12 mice per treatment group. Significant differences (P < 0.05), determined by 1-way analysis of variance with treatment groups as variants, are indicated.
Gastroenterology  , DOI: ( /S (03) )

7. Figure 6
Expression of c-fos in the spinal cord 24 hours after intestinal manipulation. (A ) c-fos-labeled nuclei in the left and right hemispheres of the lumbar dorsal horn of mice 24 hours after control laparotomy (L), intestinal manipulation (IM), or IM with pretreatment with neutralizing antibodies against ICAM-1 and LFA-1 (IM + MAb). Images are representative of 3 mice examined in each group. The number of nuclei labeled per section was significantly increased after IM (B) compared with control. Pretreatment of mice with neutralizing antibodies against ICAM-1 and LFA-1 prevented increased c-fos expression after intestinal manipulation. Significant differences (P < 0.05), determined by 1-way analysis of variance with treatment group as variants, are indicated. Values are averages ± SEM of 3 mice per treatment group.
Gastroenterology  , DOI: ( /S (03) )

8. Figure 7
Postoperative hexamethonium treatment accelerates postoperative gastric emptying, but not intestinal transit. Transit was measured as a percentage distribution of the nonabsorbable 99mTc-Albures (albumin microcolloid) over the gastrointestinal tract after oral intake of a caloric solid test meal. Stomach and 6 equal segments of small bowel, cecum, and colon were isolated 80 minutes after oral ingestion of the caloric test meal (baked egg yolk), and radioactivity was counted in each segment. In mice that underwent intestinal manipulation (IM) and received vehicle (saline) (dark gray bars), the distribution of radioactivity indicates a delayed gastric emptying and an impaired small-intestinal transit time compared with control mice that underwent only laparotomy (L; black bars). The geometric center (GC) was significantly lower (P < 0.05; 1-way analysis of variance) in mice that received IM + saline. Postoperative treatment with hexamethonium prevented the surgery-induced delay in gastric emptying (IM + hexamethonium; light gray bars), but not intestinal transit. Consequently, the geometric center was not different from that in mice that underwent IM + saline. The impaired intestinal transit after manipulation is highlighted by a higher percentage of radioactivity found in intestinal fragments 1 and 2 in manipulated intestine compared with L and by the lower percentage of radioactivity in fragments 5 and 6 (indicated by the dotted boxes). Numbers shown are averages ± SEM of 8 mice per group.
Gastroenterology  , DOI: ( /S (03) )

9. Figure 8
Ileal circular muscle carbachol dose-response curves 24 hours after laparotomy or intestinal manipulation. (A ) Intestinal manipulation (open squares) significantly suppresses contraction to higher doses of carbachol compared with laparotomy (open circles). (B) The addition of hexamethonium (3 × 10−5 mol/L) to the organ bath did not reverse the impaired contractility of mice that underwent intestinal manipulation (filled squares). Values are mean ± SEM of 6 mice. Contractions are expressed in grams of contraction per gram of tissue per square millimeter. ∗Significant differences (P < 0.05) after unpaired Student t tests.
Gastroenterology  , DOI: ( /S (03) )

10. Figure 9
In vitro gastric contractility of mice that underwent intestinal manipulation was not altered. Lack of effect of intestinal manipulation on in vitro contractility of longitudinal muscle strips of gastric fundus (A ) and antrum (B) or circular muscle strips of the antrum (C ) on different receptor agonists and electric field stimulation is shown. Dose-response curves after electrical pulse stimulation (left), carbachol (middle), or prostaglandin F2α (right) are shown. There was no difference in the neuromotor responses of mice that underwent laparotomy (filled symbols) or intestinal manipulation (open symbols). Contractions are expressed in grams of contraction per gram of tissue per square millimeter. Values shown are means ± SEM (n = 6−7). No significant differences (P < 0.05) were found after 1-way analysis of variance followed by a Dunnett’s multiple comparison test.
Gastroenterology  , DOI: ( /S (03) )