MOLECULAR MEDICINE REPORTS 16: 4797-4802, 2017 The impact of HuD protein on the intestinal nervous system in the terminal rectum of animal models of congenital anorectal malformation MENG KONG1,2*, YURUI WU1* and YUANMEI LIU2* 1Department of Pediatric Surgery, Qilu Children's Hospital of Shandong University (Jinan Children's Hospital), Jinan, Shandong 250022; 2Department of Pediatric Surgery, Affiliated Hospital of Zunyi Medical College, Zunyi, Guizhou 563003, .R.P China Received January 20, 2016; Accepted January 19, 2017 DOI: 10.3892/mmr.2017.7204 Abstract. Patients with congenital anorectal malformation study may provide a useful theoretical reference for the treat- (ARM) often present with different degrees of defecation ment of postoperative defecation dysfunction in patients with dysfunction severity following corrective operations. Therefore, ARM. studies on how to improve the postoperative defecation func- tion of patients with ARM are of clinical importance. The Introduction present study investigated the expression of the HuD protein in the terminal rectum of ARM embryonic rats and explored the Congenital anorectal malformation (ARM) is one of the effect of HuD expression on the development of the intestinal more common digestive tract abnormalities in children nervous system. Pregnant Sprague Dawley rats were random- with an incidence of ~1/1,500-1/5,000 (1). With advances in ized into a control or ARM (induced by ethylene thiourea) surgical techniques, the ARM recovery rate has improved. group. The terminal rectums of the embryonic rats were However, certain patients continue to suffer from defecation obtained during pregnancy (20 days). The histological changes dysfunctions, including fecal soiling and constipation, with an of the terminal rectum were observed using hematoxylin incidence of ~20-30% due to the multifactorial pathogenesis and eosin staining. The expression of the HuD protein was mechanisms and complex pathological changes associated assessed by immunohistochemistry and western blot analysis. with ARM. However, morphological abnormalities can be In the control group, the histological structure of the terminal corrected following surgery (2), which greatly influence the rectum was well‑defined and a large number of submucosal quality of life of patients (3). Previously, these dysfunctions and intermuscular neurons with a rich cytoplasm and strong were thought to be associated with a postoperative megacolon neuritis were observed. In the ARM group, the histological or megarectum or postoperative rectal weakness. It is suggested layers were ill‑defined and the number of neurons was small. that maldevelopment of features, including the perianal muscle Immunohistochemistry and western blot analysis demon- groups and self-enteric nervous system (self-ENS), causes a strated that the concentration of the HuD protein in the ARM change in enteric dynamics, which is closely associated with group was significantly lower compared with the control group the development of postoperative defecation dysfunction in (312.90±53.40:456.40±57.13; 0.24±0.05:0.45±0.06, P<0.05). patients with ARM (3). Patients with ARM present with no HuD was abnormally expressed in the terminal rectum of the anus however additionally with noticeable enteric nervous ARM embryonic rats and may be involved in the development maldevelopment, which is particularly associated with the and maturation of the enteric nervous system. The present maldevelopment of the ENS in the terminal rectum (4). Originating from neural crest cells, the ENS is a complex, self-created somatic nervous system. Subsequent to entering the intestinal tract and phases of gradual migration, prolif- eration and agglomeration, it develops into gangliocytes and Correspondence to: Professor Yuanmei Liu, Department of colloid cells (5). During this process, the disturbance of any Pediatric Surgery, Affiliated Hospital of Zunyi Medical College, step can affect the development of gangliocytes, resulting in 201 Dalian Road, Zunyi, Guizhou 563003, P.R. China E-mail: [email protected] ENS maldevelopment. The ENS forms different layers of nerve plexuses in the digestive tract, including the myenteric nervous *Contributed equally plexus (MP) and submucosal nervous plexus (SP). The MP is located between the circular muscle and longitudinal muscle Key words: malformation, intermuscular neuron, immuno- layers, while the SP lies between the stratum mucosum and the histochemistry, protein circular layer. The MP and SP are responsible for processing the signals from the enteric cavity and intestinal wall and regulating the contraction, secretion and blood supply of the 4798 KONG et al: HuD IN ENS intestinal tract. Therefore, they are of great significance for the administered physiological saline at the same dose. At day 20, formation of the defecation reflex (6). Maldevelopment of the 10% chloral hydrate (NanJing SenBeiJia Biotechnology Co., ENS in the terminal rectum reaches up to 60% in cases of anal Ltd., Nanjing, China) was given at 0.4 ml/100 g to the animals atresia (7). Kong et al (8) identified that there are no notice- and then the intrauterine embryonic rats were removed. able intestinal glands distributed throughout the rectal cecum; Abnormalities were judged by an experienced chief physician however, proliferated squamous epithelial tissue and thickened using a blinding method. A section of the terminal rectum muscular layers are present in ARM embryonic rats and the was stored at 80˚C and the remaining specimen fixed in 4% number of gangliocytes in the terminal rectum of ARM rats formaldehyde for 12‑36 h. Routine dehydration and paraffin were significantly decreased compared with that in the normal wax embedding were then performed. group. The ENS is a critical system for regulating the functions All the procedures were approved by the Institute of of the intestinal tract. Defecation function is closely associated Animal Ethics of the Affiliated Hospital of Zunyi Medical with the developmental condition of the ENS in the terminal College (Zunyi, China). rectum (4). Therefore, recovery of the normal function of the ENS in the terminal rectum has become a primary focus in the Hematoxylin and eosin (HE) staining. Paraffin wax‑embedded treatment of ARM in recent years. pelvic perineal tissue and terminal rectum tissue were seri- HuD is a member of the highly conserved, neuron‑specific ally sectioned at 4 mm thickness. Routine HE staining was RNA-binding Hu family, which is homologous to the ELAV then performed. The structures of the terminal rectums from protein in Drosophila. Following transcription, HuD regulates the two groups were observed under an optical microscope. the expression of neuron-specific genes and serves impor- A total of 10 sections were then obtained from each group tant roles in the growth, development and differentiation of and 10 fields were randomly selected in each section to neurons (9-11). It is a necessary protein for the formation and calculate the average number of intermuscular and submu- regeneration of nervous processes (12-15). HuD demonstrates cosal neurons. abnormal expression in diseased intestinal canals of patients with a congenital megacolon, which indicates that HuD has a Immunohistochemistry. A total of 10 paraffin-embedded close association with the development of the ENS. The intes- sections were randomly selected from each group. They tinal canal in a congenital megacolon and the terminal rectum were stored at 60˚C for 2 h and then brought to room of ARM children demonstrate gangliocyte maldevelop- temperature. Deparaffinization was performed with xylene. ment (16). ARM tends to be accompanied by a megacolon (17). The specimens were then blocked in 3% H2O2 at room How HuD is expressed in the terminal rectum of the ARM temperature for 15-30 min. A citrate antigen-retrieval embryonic rat model and whether its expression influences solution (pH 6.0) was used for room-temperature cooling the development of ENS in the terminal rectum of the model of the microwave-processed specimens (heated to boiling remains to be elucidated. for 6 min at a power level of 100 W). The specimens were Based on the aforementioned studies, an ARM animal incubated with a rabbit anti-mouse HuD antibody (1:200; cat. model was established in the present study using ethylene- no., 300065; CapitalBio, Beijing, China) at 4˚C overnight and thiourea (ETU) (18). The expression of HuD in the terminal then washed with PBS. A goat anti-rabbit secondary antibody rectum of the embryonic rats with ARM was assessed by (cat. no. 400087; OriGene Technologies, Inc., Beijing, China) immunohistochemistry and western blotting. Its role in the was applied for 20 min at room temperature. Hematoxylin development of the nervous system in the intestinal wall was counterstaining, dehydration, mounting and 3,3'-diami- then explored. nobenzidine coloration were then performed. Cells with cytoplasm and nuclei stained yellow-brown were considered Materials and methods HuD-positive. The expression of HuD was analyzed with a Leica QWin relative optic density analytical system (Thermo ARM model establishment and grouping. A total of 40 healthy, Fisher Scientific, Inc., Waltham, MA, USA) and Image‑pro nulliparous Sprague Dawley rats aged 6-8 weeks (comprising plus version 6.0 image analysis system (Media Cybernetics, 30 females and 10 males, specific pathogen‑free grade) and Inc., Rockville, MD, USA) and an average integral optical weighing 200-250 g were supplied by the animal center of density (IOD) was calculated. the Third Military Medical University (Chongqing, China). The animals were caged at a female-male ratio of 3:1 for Western blot analysis. A total of 10 normal specimens were mating. At 8:00 a.m. on the second day, a vaginal smear test randomly selected from each group. The total protein was was performed. The smears were evaluated using dark field extracted according to the protocol provided with the bicin- microscopy (x100 magnification). An observation of sperm choninic acid (BCA) protein concentration assay kit (P0009; was noted as pregnancy day 0. Each pregnant rat was weighed Beyotime Institute of Biotechnology, Nantong, China). Protein at day 0 and 10.