This study complied with the Declaration of Helsinki and was approved by the Review Board of Ethics Committee of Tongji Hospital. Consent forms were sent to patients and signed by patients or their legal custodians. Full-length resected bowel specimens obtained during pull-through operations for HSCR were collected from ten patients. Three of these patients had a history of preoperative HAEC. Resected tissues included aganglionic and ganglionic segments. Ganglionic segments were taken from the most proximal margin of the resected pull-through specimen while aganglionic segments were taken from the most distal margin of the resected specimen. Control colonic specimens were obtained from the proximal colostomy limb at the time of stoma closure in patients with imperforate anus (n = 10). Tissue specimens were stored in three ways following collection. One portion of each specimen was fixed in formalin at room temperature, for paraffin embedding and immunochemistry. A second portion was snap frozen in a mold containing optimal cutting temperature medium and stored at - 80 °C for immunofluorescence. The remaining specimen was stored at －80 °C for protein or total RNA extraction.
Protein extraction and Western blot
Each protein sample of zebrafish was extracted from 30 embryos. The western blot was then performed as previously described (16). The rabbit anti-NOX5 antibody (Abcam, Cambridge, UK, ab191010) was used at a concentration of 1:1000. The rabbit anti-beta III Tubulin (Tuj1) antibody (Abcam, Cambridge, UK, ab18207). The HRP linked goat anti-rabbit secondary antibody (Abcam, Cambridge, UK) was used at 1:10,000 dilution. Beta actin (dilution 1:1000, Abcam, Cambridge, UK) was used as the loading control. The relative level of protein was determined by the normalized density of each band in the western blot using the ImageJ software.
Sections (4 μm) on silane-coated slides (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) were deparaffinized in xylene and dehydrated in solutions with decreasing concentrations of ethanol. After rehydration and blocking of endogenous peroxidase activity with 3% of hydrogen peroxide for 10 min, heat-induced epitope retrieval was performed for 20min in 0.01M citrate buffer (pH 6.0) in a pressure cooker. Primary antibody for NOX5 was used at 1:100 and incubated for 30 min. After washing and incubation with EnVision™ for 30 min, color products were developed using the Liquid DAB+ as chromogen. The sections were counterstained with hematoxylin before dehydration and coverslipping. Slides processed without primary antibody were prepared as negative controls.
The zebrafish protocols were approved by the Institutional Animal Care and Use Committee at Tongji Hospital. The AB strain zebrafish were maintained according to standard procedures(17). Embryos were raised in E3 medium at 28.5℃ and staged as previously described(18). 0.003% N-phenylthiourea was added to E3 medium to inhibit melanization.
Real-time quantitative PCR
Total RNA was isolated from 0.2, 6, 12, 24, 48, and 72hpf embryos and colon tissues with TRIzol reagent (Life Technologies, Carlsbad, CA). RT-qPCR and data analysis were performed using LightCycler96 (Roche Diagnostics). Relative expression levels were calculated using β-actin as internal reference. The experiments were repeated three times with biological replicates. Zebrafish primers (NOX5 primer: Forward, 5’- ATT CACGGCACT GAAACGGA-3’, Reverse, 5’-GGAGCTCCGCATGATT TACCT A-3’; b-actin primer: Forward, 5’-CGAGCTGTCTTCCCATCCA-3’, Reverse, 5’-TCACC AACGTAGCTG TCTTTCTG-3’). Human primers (Tuj1 primer: Forward, 5’- GGA AGAGGGCGAGATGTACG-3’, Reverse, 5’- GGGTTTAGACACTGCTGGCT-3’; b-actin primer: Forward, 5’- CCTTCCTGGGCATGGAGTC-3’, Reverse, 5’- TGA TCTTCATTGTGCTGGGTG-3’. NOX5 primer: Forward, 5’-CCAGAAAGTGGCTG CTGAGA-3’, Reverse, 5’-AGCTTGGAGAGGTGAGGCTA-3)
Whole-mount in situ hybridization
The 0.2, 6, 12, 48hpf Embryos (n=15 for each phase) were collected and processed for whole-mount ISH as previously described(19). Embryos
A 735-bp fragment of the NOX5 cDNA was amplified using the following primers: Forward: 5’-CGGAGGTCTCTGGATCATGC-3’, Reverse: 5’-ATGTGCAGCCACAA CGTTTC-3’. A T7 promoter was added to the reverse primer. The ISH probe was then generated by in vitro transcription using T7 RNA polymerase.
Microinjection of morpholino antisense oligonucleotides
NOX5 knockdown experiments using MO were carried out as previously described (20). ATG morpholino antisense oligonucleotides targeting NOX5 were designed and synthesized as follows: NOX5-MO 5′-CGGGTGTCATCATCCAGACTCAT-3′, a 5-nucleotide-mismatch morpholino was used as control: 5’- CGGcTGaCtTgATCCAcAC TCAT -3’. Zebrafish embryos were injected with 5ng of the MO at the one cell stage. The knockdown efficiency was validated using western blot.
Whole-mount Immunofluorescent Staining
Whole-mount Immunofluorescent staining with the anti-HuC/D antibody (A-21271, Life Technologies) was performed to examine the enteric neurons along the GI tract. The 5dpf embryos (n=20) were collected and fixed with 4% PFA overnight. The embryos were washed with PBS. After incubation in blocking solution (2% goat serum, 2mg/ml BSA in 1 x PBS) for 1 hour at room temperature, embryos were incubated with the anti-HuC/D antibody (1:500) in blocking solution overnight at 4 ℃. After two washes in PBS for 10 min each time, embryos were incubated in the secondary antibody solution, 1: 1,000 Alexa Fluor rabbit anti-mouse IgG (A11001, Life Technologies) in PBS, for 1 hour at room temperature. Finally, the images were acquired using LSM 800 confocal microscope (Zeiss, Germany). The number of HuC/D-positive cells in the gut was then quantified using ImageJ. All of the experiments were repeated for three times.
Zebrafish intestinal transit assay
The tracer was prepared by mixing 100 mg of egg yolk, 150 μL of yellow-green fluorescent 2.0-um polystyrene microspheres (Invitrogen, Carlsbad, CA, USA) and 50 μL of deionized water as previously described (21). For 7dpf zebrafish larvae (n=65 for Control, n=75 for NOX5-Mo), approximately 2 mg of tracer powder was administered per Petri dish in the morning. After 3, 6, and 9 hours, the larvae were anaesthetized by 0.2%. tricaine (Sigma, St Louis, MO, USA) and imaged using a fluorescent dissecting microscope (Axio Zoom.V16, Zeiss, Germany). For scoring the transit efficiency, the zebrafish intestine was artificially divided into four zones according to anatomical landmarks and the larvae was grouped based on the anterior extent of the tracer.
The embryos were selected by Simple random sampling. Data were analyzed using the GraphPad Prism software package (version 5; GraphPad Software Inc., La Jolla, CA, USA) and are presented as the mean ± standard error of the mean. Differences between two groups were analyzed using an unpaired t-test with Welch's correction. Analysis of variance (ANOVA) was used to compare data of more than two groups. Pearson’s chi-square tests were used to assess the difference between 7 dpf wild-type and NOX5-MO group transit profiles at different time points. The experiments and data analyzing were finished by different researchers. The analyst didn’t know the grouping scheme in advance.