A new animal model of postsurgical bowel inflammation and fibrosis: the effect of commensal microflora

Abstract

Objective: Ileocaecal resection (ICR) is common in Crohn's disease. Inflammation and fibrosis frequently recur at the site of anastomosis or in the small intestine (SI). No animal models of postsurgical inflammation and fibrosis exist. A model of ICR was developed in interleukin 10 (IL10) null and wild-type (WT) mice to test the hypothesis that ICR promotes postsurgical inflammation and fibrosis in the SI or anastomosis of genetically susceptible IL10 null, but not WT or germ-free (GF)-IL10 null mice.

Methods: GF-IL10 null mice were conventionalised (CONV) and 3 weeks later randomised to ICR, transection (T) or no treatment (NoTx). Age-matched conventionally raised (CONV) WT and GF-IL10 null mice received ICR, T or NoTx. Animals were killed 28 days later. Histological scoring, real-time PCR for tumour necrosis factor alpha and collagen, and immunostaining for CD3(+) T cells assessed inflammation and fibrosis.

Results: After ICR, CONV-IL10 null, but not CONV-WT mice, developed significant inflammation and fibrosis in the SI and inflammation in anastomosis compared with NoTx or T controls. Fibrosis occurred in the anastomosis of both CONV-IL10 null and CONV-WT mice following ICR. GF-IL10 null mice developed little or no inflammation or fibrosis in the SI or anastomosis after ICR.

Conclusions: ICR in CONV-IL10 null mice provides a new animal model of postsurgical inflammation and fibrosis in the SI and anastomosis. Absence of inflammation and fibrosis in the SI of CONV-WT and GF-IL10 null mice following ICR indicates that postsurgical small bowel disease occurs only in genetically susceptible IL10 null mice and is bacteria dependent.

Figure 1. Schematic diagram of ileocecal resection (ICR) and tissue harvest

A schematic to indicate the ileocecal resection model where distal ileum, cecum and a small portion of proximal colon are resected, then small intestine and colon joined at the anastomosis. Schematic of tissue harvest shows one cm segment spanning the anastomosis and two one cm segments of small intestine proximal to the anastomosis that were collected for histology (H) or RNA extraction as indicated.

Figure 2. Photographs of dissected small and large bowel from CONV-WT, CONV-IL-10 null or GF-IL-10 null given transection (T) or ICR

Arrows indicate anastomosis. Note the significant small bowel and anastomosis inflammation in CONV-IL-10 null given ICR compared with WT or GF-IL-10 null.

Figure 3. Inflammation in small intestine and anastomosis of IL-10 null, but not WT, after ICR

Histograms show mean ± SEM for inflammation scores in the small intestine and anastomosis of CONV-WT or CONV-IL-10 null mice after 28 days of no treatment (NoTx; WT: n=3; IL-10 null: n= 6), transection (T; WT: n=8; IL-10 null: n=6) or ileocecal resection (ICR; WT: n=10; IL-10 null: n=11). a= p<0.05 compared with all other groups.

Figure 4. Representative photomicrographs of H & E stained sections of IL-10 null or WT small intestine to illustrate inflammation in CONV-IL-10 null mice after ICR

A) Small intestine of CONV-IL-10 null after NoTx, T or ICR and WT after ICR. Magnification is shown at 10x to show inflammation in IL-10 mice following ICR, which is absent in NoTx, T and WT controls. B) High power (20x) of small intestine (SI) or anastomosis (A) of IL-10 null mice after ICR to demonstrate transmural inflammation spanning the mucosa and muscle layers. High power of anastomosis from WT shows little inflammation. C) Immunohistochemical staining for CD3 in small intestine and colon of IL-10 null mice after NoTx or ICR to demonstrate increased infiltration of T-cells into the lamina propria of small intestine after ICR and T cell infiltration in colon. Small intestine of WT after ICR is also shown to demonstrate no increase in T cells.

Figure 5. Serial analyses of inflammation in CONV-IL-10 null mice at 7, 14, and 28 days after ICR

Top: Low power 10x magnification and bottom high power 20x magnification of small intestine of CONV-IL-10 null mice at 7, 14, 28 days after ICR compared with CONV-WT- at 7 days to illustrate that post-surgical small intestine inflammation in CONV-IL-10 null mice is transmural even at early times after ICR and is sustained to 28 days, while WT show no inflammation even at 7 days. Mean inflammation scores after ICR were: CONV-IL-10 null at 7 days 1.8±0.7 n=6; 14 days 2.3±0.7 n=4; and 28 days 2.3±0.2 n=6; WT at 7 days 0.5±0.1 n=8.

Figure 6. Increased expression of TNF α mRNA in small intestine and anastomosis of CONV IL-10 null and not WT following ICR

Histograms show the mean ± SEM for TNF α mRNA abundance in CONV-WT or CONV-IL-10 null small intestine after ICR or T compared with TNFα mRNA in a corresponding region of SI from NoTx controls. Values are expressed as fold change vs. the mean value in WT, NoTx. TNF α mRNA was significantly increased in both the small intestine and anastomosis of IL-10 null mice after ICR when compared with all other groups (a= p ≤ 0.05). N=3 or more animals in each group.

Figure 7. Fibrosis scores in small intestine and anastomosis of CONV-IL-10 null and WT mice

Histograms show mean ± SEM for fibrosis scores in the small intestine and anastomosis of WT and IL-10 null mice, 28 days after NoTx, T, or ICR (N=as in Figure 3). In small intestine ICR caused significant fibrosis in IL-10 null, but not WT. Fibrosis at the anastamosis was not restricted to IL-10 null mice, as WT mice also had significantly higher fibrosis scores than No Tx controls (a= p ≤ 0.05 vs. all other groups; b= p ≤ 0.05 vs. all other SI groups; c= p ≤ 0.05 vs. NoTx).

Figure 8. Representative photomicrographs of Sirius red stained sections of small intestine and anastomosis of CONV IL-10 null and WT mice given NoTx, T or ICR

Photomicrographs of Sirius red stained sections of small intestine (10x magnification) or anastomosis (4x magnification) to demonstrate the increased collagen deposition (stained red) in small intestine of IL-10 null, but not WT after ICR. Fibrosis at the anastomosis is observed in both IL-10 null and WT control mice after ICR, and in a subset of mice given T.

Figure 9. Increased expression of collagen mRNA in small intestine and anastomosis of CONV IL-10 null and not WT following ICR

Histograms show collagen mRNA abundance, expressed as fold change (mean ± SEM), in IL-10 null and WT small intestine and anastomosis compared with small intestine of WT-NoTx controls (N=3 or more per group). Consistent with histological scoring, collagen mRNA abundance in CONV IL-10 null small intestine was significantly higher following ICR than in NoTx, T or WT controls (a=p<0.05). Neither ICR nor T in IL-10 null or WT was associated with significant increases in collagen mRNA at the anastomosis.

Figure 10. No post-surgical inflammation in small intestine and anastomosis of GF IL-10 null mice after ICR

Histograms show mean ± SEM for inflammation in the small intestine and anastomosis of GF- IL-10 null after NoTx (n=6), T (n=6) or ICR (n=8). Inflammation score for CONV-IL-10 null mice after ICR is shown (hashed bars) for comparison. Histological scoring revealed significantly (p=0.009) reduced inflammation in GF-IL-10 null small intestine. Inflammation also tended to be scored lower at the GF-IL-10 null anastomosis.

Figure 11. Representative photomicrographs of H & E stained sections of small intestine and anastomosis of GF IL-10 null mice

A) Photomicrographs of small intestine of GF-IL-10 null mice after NoTx, T or ICR and of anastomosis after T or ICR to demonstrate little or no inflammation in GF-IL-10 null small intestine and anastomosis following ICR (magnification at 10x). B) Immunohistochemical staining for CD3 to demonstrate similar numbers of T-cells infiltrating into GF small intestine following ICR, compared with NoTx.

Figure 12. No significant fibrosis in small intestine or anastomosis of GF IL-10 null mice after ICR

Histograms show mean ± SEM for fibrosis scoring of the SI and anastomosis in GF- IL-10 null after NoTx, T or ICR (N=as for Figure 9). Fibrosis score for CONV-IL-10 null mice after ICR is shown for comparison (hashed bars). Histological scoring revealed significantly lower fibrosis in GF-IL-10 null small intestine and anastomosis (a= p<0.05).

Figure 13. Representative photomicrographs of Sirius red stained sections of small intestine and anastomosis of GF-IL-10 null mice given NoTx, T or ICR

Photomicrographs of Sirius red stained sections of small intestine (10x magnification) or anastomosis (4x magnification) to demonstrate low levels of collagen deposition (red stain) in GF-IL-10 null mice following ICR.