PMID: 29325538 PMCID: PMC5765622 DOI: 10.1186/s12871-018-0471-2
Methods
This study was approved by the Ethics Committee of Affiliated Shengjing Hospital of China Medical University, and specific pathogen free SD pregnant rats weighing 380–420 g were purchased from the Experimental Animal Center of Affiliated Shengjing Hospital of China Medical University. Animals were housed at 22–24 °C, 40–60% humidity with a 12-h light /dark cycle and had free access to food and water. Rats at the gestational age of 21 days (E21) were used in subsequent experiments. According to the isoflurane dose, rats were divided into 3 groups: the Iso1 group (1.3% isoflurane), the Iso2 group (2.0% isoflurane) and the control group (0% isoflurane; O2).

In the absence of anesthesia, intratracheal intubation was difficult in the control group. Thus, all the rats retained spontaneous breathing and did not receive intratracheal intubation. Inhalation of isoflurane at a high concentration may inhibit respiration and cause hypoxia. Thus, in our pilot study, pregnant rats at the gestational age of 20 days (E20) were anesthetized intraperitoneally with pentobarbital sodium and catheter indwelling was done in the right carotid artery; rats were then allowed to recover at room temperature. At E21, rats were placed in a box filled with prefilled gas according to the following groups: 50% O2 was administered in the control group; 1.3% isoflurane was administered in the Iso1 group (50% oxygen, balanced with nitrogen); 2.0% isoflurane was administered in the Iso2 group (50% oxygen, balanced with nitrogen). All rats were retained spontaneous breathing and exposed in the box for 3 h (the concentrations of isoflurane and oxygen were monitored). The mean arterial blood pressure was continuously monitored via a catheter in the carotid artery, and arterial gas analysis was performed hourly. The results showed that inhalation of isoflurane at 1.3% or 2.0% had no influence on the arterial gas and mean arterial blood pressure. Rats used in pilot study will not be used for formal study.

In this study, a total of 48 rats at E21 were randomly assigned into 3 groups and exposed to isoflurane at the predesigned concentration for 3 h. Animals were allowed to recover at room temperature and housed until they delivered. The number of fetuses was recorded, and healthy male neonatal rats were used in the experiments. At day 28 after birth (P28), the male offsprings were randomly assigned into two groups: one for Morris water maze (MWM) test to evaluate memory and learning and the other one were housed until day 90 after birth (P90) to receive the same MWM test.

MWM test used a round swimming pool sized 150 cm in diameter and 60 cm in height with a platform sized 10 cm in diameter in the maze. The removable platform was 1.5 cm lower than the water surface. The visual cues (a variety of figures) on the maze’s inner wall remained unchanged during the study. Training and examination were performed in the water at 20 °C. After each examination, rats were dried under a lamp and returned to the cages.

Place navigation test was performed for consecutive 5 days. In brief, platform were placed in a quadrant (the 4th quadrant in this study). At predesigned time point, rats were placed in a random quadrant (once for each quadrant). If the rat found the platform within 90s, it was allowed to stay on the platform for 15 s and then placed out of the pool. The spatial navigation test was performed on the 6th day to evaluate memory. In brief, the platform was removed, rats were placed in a random quadrant and the swimming trajectory was recorded within 90s. In the test, the proportion of swimming distance in the platform quadrant to the total swimming distance and the times of crossing the platform were calculated. The swimming distance in the platform quadrant reflects spatial localization and the times of crossing the platform reflects the accuracy of spatial memory. Before training, the platform was visible above the water surface, which may exclude rats with visual defects that were unable to find the platform. In addition, rats with poor performance in the test, such as those could not find the hidden platform and swam along the wall, were also excluded from this study.

Two hours after the spatial navigation test, rats were intraperitoneally anesthetized with pentobarbital sodium. Half of each group of the rats were used to collect brain and followed by the separation of hippocampus. The hippocampus was weighed and lysed for total protein extraction. Samples were then stored at −80 °C for later use. Western blotting was performed to detect the protein expression of CREB and p-CREB in the hippocampus. The half of the rats were transcardially perfused with 4% paraformaldehyde and the brain was collected and fixed in 4% paraformaldehyde. Immunohistochemistry was performed to detect CREB and p-CREB expression. (Fig. 1).
The neonatal rats were randomly assigned into different groups to reduce variation. We normalized CREB and p-CREB protein expression in control group as 1. CREB and p-CREB expression in the Iso1 and Iso2 group was compared with the controls. All data are expressed as mean ± standard deviation. Statistical analyses were performed by using SPSS software (version 21.0; IBM, Corp., Armonk, NY, USA). One-Way ANOVA was used to compare the means between groups. A value of P < 0.05 indicated significance.