Ketamina

Surgery was performed at 18.5 days of gestation. Pregnant rats were anesthetized with 50 mg/mL ketamine (Ketamina¯, Pfizer, Brazil) and 10 mg/mL xylazine (Rompum¯, Bayer, Brazil). The abdominal cavity was opened by median laparotomy in 2 layers. Fetuses were counted from the uterine isthmus, and GS was created on the second fetus of each horn according to the technique described by Correia-Pinto et al. (21 (link)). The lower body of the fetus was exposed and a right para-umbilical laparotomy with standardized extension of 5 mm was performed, opening the fetal abdominal cavity with caution not to damage the umbilical vessels and liver. The bowel loops were gently exposed by the delicate compression of the fetal abdomen with cotton swabs. After creating the defect, the fetus was carefully placed back into the uterine cavity, and a previously placed purse string suture closed the uterus.

The fetuses were harvested at 21.5 days of gestation. On harvesting day, pregnant rats were anesthetized with 175 mg/kg ketamine (50 mg/mL, im) (Ketamina^®, Pfizer do Brasil Ltda, Brazil) combined with 2.5 mg/kg xylazine (10 mg/mL) (Rompum^®, Bayer Brasil, Brazil) and the fetuses were collected through a median laparotomy. If necessary, an extra dose (0.1 mL) of ketamine was administered intraperitoneally to the pregnant rat. Before any procedure, each fetus was weighed on an OHAUS model 360 precision scale (Denver Instruments, USA) and processed according to the specific procedures for each group.
Harvesting was performed through the following steps (Figure 1): 1) Two fetuses at a time were removed from the uterus and submitted to hypoxia for 30 min. 2) During this period, one fetus was removed from the uterus and harvested as C group (no manipulation). 3) After hypoxia, the two fetuses were separated, one was harvested as hypoxia group, and the other one was submitted to mechanical ventilation for 30 min. 4) At the end of the 30 min (ventilation), the fetus was harvest as HV group. To form the CV group, at step 1, one of the fetuses was submitted to mechanical ventilation for 30 min, while the other one was submitted to hypoxia.