Identification and establishment of Sprague-Dawlay rat model with experimental Hirschsprung’s-associated enterocolitis

Abstract

Objective To establish a Sprague-Dawlay (SD) rat model of experimental Hirschsprung’s-associated enterocolitis (HAEC) for providing experimental rationales for elucidating the pathogenesis of HAEC. Methods According to a random number table, a total of 120 SD rats aged 6-8 weeks were divided into control, Hirschsprung’s disease (HSCR) and HAEC groups. Subjects in HAEC group had an intragastric administration of Escherichia coli (E. coli) JM83 (109 CFU/day) for 1 week before modeling. All animal were operated under an anesthesia of 1% phenobarbital sodium. A gauze soaked with 0.1% benzalkonium chloride (BAC) was wrapped around rectal serosa for 45 mins in both HSCR and HAEC groups. And 0.9% saline was wrapped to rectum similarly in control group. The models were examined through gross observations. Myeloperoxidase (MPO) assay, hematoxylin & eosin (HE) staining and the expressions of AchE, glial cell line-derived neurotrophic factor (GDNF) were detected by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) at the end of 1, 3, 5, 7 weeks post-modeling. Colonic wall was harvested for HE staining for identifying successful models in 5 weeks. HE staining, immunofluorescent staining and 16S rRNA were used for detecting the changes of intestinal flora structure at 5 weeks post-modeling. Results After 3-week BAC treatment, abdominal distension and loss of appetite appeared in both groups. Pathological biopsy revealed a narrow segment and marked dilation of proximal segment. Abdominal distension was significantly higher in HAEC group than that in HSCR group after 5-week BAC treatment. Histological examination showed ganglion cells gradually decreased after 5-week BAC treatment and disappeared completely at 7 weeks after BAC treatment. MPO assay showed that the activity in HAEC group (10.4±0.43) was higher than that of HSCR (7.6±0.35) and control (2.8±0.16) groups (P 0.05). The mRNA expression of AchE, GDNF decreased dramatically in both HSCR and HAEC groups after BAC treatment. The mRNA expression of AchE (0.74±0.09, 0.55±0.11 vs. 0.67±0.03, 0.46±0.13) and GDNF (0.47±0.12, 0.40±0.22 vs 0.51±0.06, 0.31±0.09) between HSCR and HAEC groups was lower than those of control group at 5, 7 weeks after BAC treatment (P<0.05). No difference existed in HSCR group compared with HAEC group at 1, 3, 5, 7 weeks after BAC treatment. Colonic mucosa showed typical changes of enterocolitis in HAEC group after modeling at 5 weeks. Twenty-seven bacterial genera were found in rectum, transverse colon and ileum by 16S rRNA gene analysis. Genus Enterobacteriaceae (34.7%, 22.8%, 17.3%) was the most prevalent in HAEC group, following Clostridium (10.4%, 12.4%, 8.7%), Faecium (8.6%, 7.9%, 6.9%) and Fusobacterium (5.6%, 3.8%, 7.6%); genus Lactobacillus (5.7%, 9.4%, 14.5%) was the most prevalent in HSCR group, following Bacteroides (6.3%, 9.4%, 12.6%), Enterobacteriaceae (7.1%, 6.3%, 4.3%) and Clostridium (4.6%, 5.2%, 6.4%). Conclusions A rat experimental model for HAEC is successfully established by applying 0.1% BAC onto rectal serosa plus an intragastric administration of E .coli JM83. This model provides the basis for future studies of exploring the pathophysiology of HAEC. Key words: Models, animal; Rat; Hirschsprung’s disease; Enterocolitis; Intestinal microflora; Benzalkonium chloride