Leukocyte-derived inducible nitric oxide synthase mediates murine postoperative ileus

Abstract

Objective: To provide evidence that iNOS expression solely in leukocytes plays a role in postoperative ileus.

Summary background data: Intestinal handling initiates a molecular and cellular muscularis inflammation that has been associated with iNOS expression and ileus. The specific cellular source of iNOS is a matter of speculation.

Methods: Chimeric mice were constructed that selectively express the iNOS gene only in their leukocytes or only in their parenchymal cells by lethal radiation and reconstitution with reciprocal bone marrow. Mild intestinal manipulation was used to induce postoperative ileus.

Results: Intestinal manipulation caused a significant leukocyte extravasation into the muscularis of all groups. Postoperative iNOS mRNA expression was evident in iNOS and transplanted iNOS mice with iNOS bone marrow but not in iNOS animals. The loss of the iNOS gene in leukocytes of iNOS mice reduced iNOS mRNA expression by 59%. iNOS-deficient mice and iNOS animals with iNOS leukocytes presented with a significant improvement in postoperative intestinal transit and in vitro smooth muscle contractility, whereas the replacement with iNOS bone marrow in iNOS mice completely reversed this improvement.

Conclusion: These results clearly show that iNOS expressed in leukocytes within the intestinal muscularis plays a major role in mediating smooth muscle dysfunction and subsequently postoperative ileus.

FIGURE 1. MPO staining and F4/80 immunohistochemistry of jejunal muscularis whole mounts demonstrating leukocyte recruitment after intestinal manipulation. A, Occasionally, a few MPO-positive cells could be observed within control whole mounts from iNOS^+/+ mice. B, Infiltrating MPO-positive cells 24 hours after intestinal manipulation (iNOS^−/− mouse with iNOS^+/+ bone marrow). In addition to darkly stained neutrophils, numerous smaller and lighter stained cells, representing monocytes, were observed by light microscopy. F4/80 immunohistochemistry was used to stain monocytes (C and D). C, The near absence of monocytes in whole mounts from control iNOS^+/+ mice. D, A typical whole mount containing numerous F4/80-positive monocytes after intestinal manipulation (iNOS^+/+ mouse). Original magnifications: A–D, ×200.

FIGURE 2. RT-PCR analysis of iNOS mRNA expression 12 hours postoperatively, comparing the genetically different chimeric groups (IM, intestinal manipulation; PC, positive control; NC, negative control). A, Gel bands of muscularis extracts. B, Results of gel band analysis using phosphor imaging. The iNOS signal is absent in control iNOS^+/+ mice and in manipulated iNOS^−/− animals. Levels of iNOS mRNA expression were significantly up-regulated in manipulated iNOS^+/+ mice. The selective deletion of the iNOS gene in leukocytes (iNOS^+/+ BMTX^−/−) resulted in less expression, whereas the addition of the iNOS gene in iNOS^−/− animals (iNOS^−/− BMTX^+/+) caused a distinct postoperative iNOS mRNA up-regulation.

FIGURE 3. Fluorescence micrographs of jejunal muscularis whole mounts from an iNOS^−/− mouse (A) and from an iNOS^−/− BMTX^+/+ mouse (B, both unmanipulated), 1 day after fluorescein-dextran injection. Both panels show an equal distribution of dendritic-appearing resident macrophages within the intestinal muscularis containing fluorescein-labeled dextran. Original magnifications: A, ×150; B, ×400.

FIGURE 4. Fluorescence micrographs of muscularis whole mounts 24 hours after intestinal manipulation, stained with antibodies against iNOS. A, The absence of iNOS immunoreactivity in the muscularis from a manipulated iNOS^−/− animal. B, The occurrence of iNOS-positive infiltrating cells in a manipulated iNOS^−/− animal with iNOS^+/+ bone marrow. C, Postoperative iNOS expression in a muscularis macrophage. D, The concurrent appearance of strong iNOS immunoreactivity in recruited leukocytes and a weaker but noticeable signal in macrophages (c, d: manipulated iNOS^−/− BMTX^+/+ mice). Original magnifications: A, B, ×200; C, ×400; D, ×300.

FIGURE 5. Transit histograms for the distribution of fluorescein-labeled dextran along the gastrointestinal tract 90 minutes after oral administration (IM, intestinal manipulation; stom, stomach; sb, small bowel; col, colon). In control animals, the nonabsorbable marker accumulated in the cecum. Intestinal manipulation in iNOS^+/+ mice caused a significant delay in intestinal transit (A). The loss of the iNOS gene in leukocytes ameliorated the delay caused by manipulation (B). iNOS^−/− mice showed an improvement of postoperative transit similar to the iNOS^+/+ BMTX^−/−, which was completely reversed in iNOS^+/+ bone marrow-transplanted animals (iNOS^−/− BMTX^+/+) (C).

FIGURE 6. Representative traces of spontaneous contractile activity recorded from jejunal circular smooth muscle strips. A, Trace from a control iNOS^+/+ mouse. The last portion of the trace is shown to the right on an expanded time scale, demonstrating characteristic rhythmic and robust contractions. B, Intestinal manipulation results in a marked decrease in spontaneous contractile activity. Traces in C and D demonstrate the amelioration of postoperative in vitro contractility in complete iNOS-deficient mice and iNOS^−/− bone marrow transplanted mice (iNOS^+/+ BMTX^−/−). E, Significant inhibition of spontaneous contractile activity in manipulated iNOS^−/− BMTX^+/+ mice.

FIGURE 7. Bethanechol dose response curves of jejunal circular smooth muscle contractile activity in response to surgical manipulation. A, Significant improvement of contractility in iNOS^+/+ mice with selective loss of the iNOS gene in leukocytes (iNOS^+/+ BMTX^−/−). The improved response to bethanechol in iNOS^−/− animals is completely reversible through an iNOS^+/+ bone marrow replacement (B).