COX-2 dependent inflammation increases spinal Fos expression during rodent postoperative ileus

Abstract

Background and aims: Cyclooxygenase 2 (COX-2) and prostaglandins (PGs) participate in the pathogenesis of inflammatory postoperative ileus. We sought to determine whether the emerging neuronal modulator COX-2 plays a significant role in primary afferent activation during postoperative ileus using spinal Fos expression as a marker.

Methods: Rats, and COX-2(+/+) and COX-2(-/-) mice underwent simple intestinal manipulation. The effect of intestinal manipulation on Fos immunoreactivity (IR) in the L(5)-S(1) spinal cord, in situ circumference, and postoperative leucocytic infiltrate of the intestinal muscularis was measured. Postoperative PGE(2) production was measured in peritoneal lavage fluid. The dependence of these parameters on COX-2 was studied in pharmacological (DFU, Merck- Frosst, selective COX-2 inhibitor) and genetic (COX-2(-/-) mice) models.

Results: Postoperative Fos IR increased 3.7-fold in rats and 2.2-fold in mice. Both muscularis leucocytic infiltrate and the circumference of the muscularis increased significantly in rats and COX-2(+/+) mice postoperatively, indicating dilating ileus. Surgical manipulation markedly increased PGE(2) levels in the peritoneal cavity. DFU pretreatment and the genetic absence of COX-2(-/-) prevented dilating ileus, and leucocytic infiltrate was diminished by 40% with DFU and by 54% in COX-2(-/-) mice. DFU reversed postsurgical intra- abdominal PGE(2) levels to normal. Fos IR after intestinal manipulation was attenuated by approximately 50% in DFU treated rats and in COX-2(-/-) mice.

Conclusions: Postoperatively, small bowel manipulation causes a significant and prolonged increase in spinal Fos expression, suggesting prolonged primary afferent activation. COX-2 plays a key role in this response. This activation of primary afferents may subsequently initiate inhibitory motor reflexes to the gut, contributing to postoperative ileus.

Figure 1

Drawing of a section from the L₆ spinal cord in the rat depicting four regions where Fos positive neurones are located. MDH, medial dorsal horn; LDH, lateral dorsal horn; DCM, dorsal commissure; and SPN, sacral parasympathetic nucleus.

Figure 2

Histogram showing the segmental distribution of Fos positive cells (number/section) in a typical rat spinal cord from a control rat and from a rat after intestinal manipulation (IM) (T₁₀–S₂)

Figure 3

Histogram showing the in situ circumference of the intestinal muscularis in (A) control rats with and without DFU, in surgically manipulated (IM) rats with and without DFU, and in rats undergoing laparotomy only, and in (B) COX-2^+/+ and COX-2^−/− control mice and COX-2^+/+ and COX-2^−/− surgically manipulated (IM) mice. Surgical manipulation caused a marked increase in the in situ muscularis circumference indicating dilating ileus (p<0.008). This effect was significantly attenuated in both DFU treated rats and COX-2^−/− mice. n=4–5; **p<0.02.

Figure 4

Histogram of infiltrating leucocytes in muscularis whole mounts from (A) control rats with and without DFU, from surgically manipulated (IM) rats with and without DFU, and from rats undergoing laparotomy only and from (B) COX-2^+/+ and COX-2^−/− control mice and COX-2^+/+ and COX-2^−/− surgically manipulated (IM) mice. Surgical manipulation caused a significant increase in leucocytic infiltrate (p<0.008), which was markedly diminished in both DFU treated rats and COX-2^−/− mice. n=4–5; **p<0.02.

Figure 5

Myeloperoxidase (MPO) staining of jejunal muscularis whole mounts in mice after intestinal manipulation. A low number of MPO positive cells was seen in both COX-2^+/+ and COX-2^−/− controls. Surgical manipulation caused a significant increase in MPO positive cells that was attenuated in COX-2^−/− mice.

Figure 6

Histogram demonstrating a significant increase in prostaglandin E₂ (PGE₂) levels in peritoneal lavage fluid measured 24 hours after intestinal manipulation (IM) using ELISA. This increase was reversed to control levels after administration of DFU. n=6; **p<0.006.

Figure 7

Histogram showing cumulative mean Fos counts in (A) control rats with and without DFU, in surgically manipulated (IM) rats with and without DFU, and in rats undergoing laparotomy only, and in (B) COX-2^+/+ and COX-2^−/− control mice and COX-2^+/+ and COX-2^−/− surgically manipulated (IM) mice. Surgical manipulation caused a significant increase in postoperative Fos expression (p<0.008) which was markedly decreased in both DFU treated rats and COX-2^−/− mice. n=4–5; **p<0.02.

Figure 8

Representative images illustrating distribution of Fos IR in the L₆ spinal cord of a control rat, a surgically manipulated rat, a surgically manipulated rat after DFU treatment, and of a rat that underwent laparotomy only.