Synthesis of GSNO

GSNO was synthesized by reacting equimolar gluthatione with sodium nitrite in acidic aqueous solutions (chloridric acid 0.5 mol/L, pH 1) as described previously. The compound was diluted in sterile Milli-Q (Merck KGaA, Darmstadt, Germany) water so that the concentrations were 0.01, 0.001, and 0.0001 mmol/L. In each fetus a final volume of 30 μL was applied, thus the final GSNO concentrations were 5, 0.5, and 0.05 μmol/L, respectively.

Animals and groups

This study was submitted to and approved by the Animal Experimentation Ethics Committee of University of Campinas (research project no. 1452-1). Female Sprague Dawley (CEMIB-Unicamp Campinas, São Paolo, Brazil) rats weighing 250-300 g were time mated and the day following mating was considered day 0 of pregnancy after observation of spermatozoids in the vaginal smear (gestation length = 22 days). The animals received chow and water ad libitum, in controlled lighting, temperature, and humidity environment.

The fetuses (n = 60) were divided in 5 groups of 12 fetuses: control (C), gastroschisis (G), gastroschisis + GSNO 5 μmol/L (GNO1), gastroschisis + GSNO 0.5 μmol/L (GNO2), and gastroschisis + GSNO 0.05 μmol/L (GNO3). Besides the above groups, in each pregnant rat operated on, in addition to the gastroschisis group, we had fetuses that were not manipulated (internal control), fetuses that were manipulated with hysterotomy and exteriorized (group sham), as well as internal control fetuses for each NO dose used.

Surgical procedure

Surgery was performed at 18.5 days of gestation. Pregnant rats were anesthetized with ketamine 50 mg/mL (175 mg/kg) (Ketamina Agener; União Química Farmacêutica Nacional S/A, São Paulo, Brazil) associated with xylazine 10 mg/mL (2.5 mg/kg) (Dopaser- Laboratórios Calier S/A, Barcelona, Spain). Their abdominal cavity was opened by median laparotomy in 2 layers. Fetuses were counted from the uterine isthmus and gastroschisis was created on the second fetus in each cornus. Fetuses for the C group were obtained from pregnant rats that were not subjected to surgery.

Gastroschisis was created according to the technique described by Correia-Pinto et al. The lower body of the fetus was exposed and a right paraumbilical incision was performed with exposure of the intestine. In the G group, after creation of gastroschisis, the fetus was carefully placed back in the uterine cavity and the uterus was closed. In the fetuses that received 30 μL of GSNO, the material was applied to the bowel and then the fetus was placed back in the uterine cavity. During the procedure the uterus and the fetus were kept warm by instillation of normal saline solution at 38°C. At the end of the procedure the maternal abdominal wall was closed and supplemental oxygen at 1 L/min was provided during recovery from anesthesia.

Sample harvesting

At 21.5 days of gestation, pregnant rats were anesthetized and underwent cesarean delivery. Fetuses were removed from the uterine cavity, euthanized, and their body weight (BW) was measured. The intestinal tract was resected from the pylorus to the rectum and weighted; intestinal weight (IW) and the IW/BW ratio was calculated. The samples were fixed in 4% paraformaldehyde for morphometric and immunohistochemical analysis or snap frozen at –80°C for Western blotting.

Morphometric and histological analysis

We standardized analysis of the intermediate (jejuno-ileal) segments only. After fixing, the samples were dehydrated in ethanol, diaphanyzed in xylene, and embedded in paraffin. Transversal histological sections of 5 μm were obtained and stained with hematoxylin-eosin for histological analysis. Slides were photographed using a photodocumentation microscope (Eclipse E200 80 i ; Nikon, Tokyo, Japan). A magnification of ×20 was used for measurement of the intestinal diameter and ×200 for measurement of the thickness of intestinal layers, including intestinal wall thickness, mucosa and submucosa, circular muscular, longitudinal muscular, and serosa. Measurements were obtained with the software (Image Pro Plus 6.0; Media Cybernetics Inc, Rockville, MD). All the measurements were performed in radial orientation in the 4 quadrants of 5 sequential nonoverlapping sections for each fetus.

Immunohistochemistry

Histological sections obtained in paraffin were deparaffinized in xylene and rehydrated in ethanol. Then, antigen exposure was performed with sodium borate (0.1 mol/L, pH 7.4) for 1 hour at room temperature. The endogenous peroxidases were eliminated with 10% hydrogen peroxide in phosphate-buffered saline solution (PBS) (0.01 mol/L, pH 7.4) for 30 minutes. Blocking was performed with 1% bovine serum albumin (BSA) in PBS for 1 hour. Sections were incubated with anti-nNOS, anti-iNOS, or anti-eNOS diluted 1:200 in PBS/BSA 3% (sc-648, sc-651, and sc-654, respectively; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. On the second day, sections were washed and incubated with peroxidase conjugated secondary antibody antirabbit raised in goat (sc-2004; Santa Cruz Biotechnology) diluted 1:2000 in PBS/BSA 1% for 1 hour. Then, sections were treated with Vectastain ABC kit (Vector Laboratories, Burlingame, CA) and 3,3 = -diaminobenzidine tetrahydrochloride hydrated (Sigma-Aldrich, St. Louis, MO). As negative control the primary antibody was omitted in one of the slides. Sections were then stained with Harris hematoxylin, dehydrated, and mounted. Slides were analyzed independently and blindly by 2 investigators using a semiquantitative scale ranging from 0-3: grade 0 = no staining; grade 1 = faint staining; grade 2 = moderate staining; and grade 3 = strong staining.

Western blotting

The bowel from 6 animals per group was homogenized in 1 mL of extraction buffer containing: Tris 100 nmol/L (pH 7.4), sodium pyrophosphate 100 nmol/L, sodium fluoride 100 nmol/L, EDTA 10 nmol/L, sodium vanadate 10 nmol/L, and phenylmethylsulfonyl fluoride 2 nmol/L, and aprotinin 0.1 mg/mL, and 1% Triton- X100 at 4°C with a tissue homogenizer (Tecnal, Paulinia, Brazil) operated at maximum speed for 30 seconds. The extracts were centrifuged at 14,000 rpm (9000 g ) at 4°C in a Mikro 200R centrifuge (Hettich, Tuttlingen, Germany) for 30 minutes to remove insoluble material, and the supernatants of these tissues were used for protein quantification using the Bradford method. Then proteins were denatured, run on polyacrylamide gel, and transferred to nitrocellulose membranes. Nonspecific binding sites were blocked with 0.2% BSA in PBS (0.01 mol/L, pH 7.4) for 1 hour. Then, membranes were incubated with anti-nNOS, anti-iNOS, or anti-eNOS diluted 1:200 in PBS/BSA 3% (sc-648, sc-651, and sc-654, respectively; Santa Cruz Biotechnology) overnight at 4°C.

The next day, after washing, the membranes were incubated with peroxidase conjugated secondary antibody antirabbit raised in goat (sc-2004; Santa Cruz Biotechnology) diluted 1:5000 in PBS/BSA 3% for 2 hours. Membranes were then treated with chemoluminescence kit (Pierce, Rockville, IL), exposed to radiographic films (Kodak, Rochester, NY), and developed.

Statistical analysis

Weights and morphometric values were obtained and Western blotting data were analyzed by analysis of variance with Tukey-Kramer posttest. Immunostaining grading was analyzed by Kruskal-Wallis test with Dunn posttest. We used the software GraphPad Prism 5.0 (GraphPad Software Inc, La Jolla, CA). Significance was established for a P value < .05.