All animal experiments were carried out according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (publication no. 85-23, revised 1985). The experiments were approved by the Institutional Animal Care and Use Committee of the Nanjing Medical University (no: 15030254). Sprague-Dawley (SD) dams with pups were bred in our colony in a temperature-controlled (22-23°C) room on a 12 h light/dark cycle (lights on at 8:00 AM) with free access to food and water. Fourty postnatal day 7 (PND-7) SD male rat pups (11-14 g) were used in our experiment and were randomly assigned to ketamine-treated and sham-treated groups. The grouping method was performed by using the methods described in our previous study . In the anesthesia group, ketamine was diluted in 0.9% normal saline, and PND-7 rats were intraperitoneally administered with 40 mg/kg doses of ketamine in four injections at 1 h intervals (40 mg/kg×4 injections). The ketamine injection program was explored through the preliminary experiment. In the sham-treated group, rats received an equal volume of 0.9% normal saline. Temperature probes were used to facilitate the control of temperature at 36.5±1°C by using computer-controlled heater/cooler plates that were integrated into the floor of the chamber. Between each injection, animals were returned to their individual chambers to help in maintaining body temperature and to reduce stress. We found that the movement and the righting reflex were disappeared in all animals after 40 mg/kg ketamine injection, and animals were completely unresponsive during the 1 h intervals between injections. These findings suggested that four injections of 40 mg/kg ketamine with 1 h intervals could exert the satisfactory anesthesia effect and all animals could survive after the anesthesia.
Morris Water Maze Test (MWM)
The apparatus and behavioral procedures of the MWM test have been previously described . Behavioral testing was conducted in a circular, black painted pool (180 cm diameter, 50 cm deep). The water temperature was maintained at 25±1℃. An invisible platform (10 cm diameter) was submerged 1 cm below the water surface and was placed in the quadrant III, which was determined by using four starting locations (defined as Ⅰ, Ⅱ, Ⅲ and Ⅳ) . There was a 90 degree angular offset between each pair of starting locations. During five consecutive training days, the experiments were conducted in a dimly lit and quiet laboratory setting, we placed a lamp in a corner of the laboratory and kept the same light level used for both training and testing period, all animals could detect extra maze visual cues and learn how to locate the platform. The rats were trained four times per day, with the different starting position being randomized for each rat. When the rat found the platform, it was allowed to stay on the platform for 30 s. If a rat did not find the platform within 120 s, the rat would be gently guided to the location and allowed to stay on the platform for 30 s, and the latency time in finding the hidden platform was recorded as 120 s. The average escape latency from the 4 trials was represented as the daily result for each of the rats. For a given rat, the starting position was different across all five days of training. Following the completion of the training, spatial memory was assessed in the probe tests, in which the hidden platform was removed. The animals were placed in the quadrant opposite to the quadrant that had contained the platform and allowed to swim freely for 120 s. The paths of each of the animals were tracked by using a computerized video system. The number of times that the entire body of a rat crossed the previous platform area were recorded. The total swim distance and the average speed of each animal during the probe test were also need to be analyzed. After every trial, each rat was placed on a heater plate for 1 to 2 min until they were dry, after which they were returned to their chambers. The data were analyzed by using software for the MWM (Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University, Xuzhou, China.).
Experiment 1 evaluated the survival rate of developmentally generated granule neurons in the hippocampal DG during adult stage. The PND-7 rats received three consecutive BrdU (5-bromo-2-deoxyuridine; Sigma) injections intraperitoneally, at a dosage of 100 mg/kg, on PND-7, 8 and 9 after administered with normal saline or ketamine, then two groups of rats were weaned at PND-35, after which they were housed in cages with free access to food and water for up to 2 months (5 animals per group) (Fig. 3A). Then, all animals were deeply anesthetized with 40 mg/kg ketamine at 2 months old and transcardially perfused with 0.9% normal saline, followed by a transfusion with 4% paraformaldehyde. To visualize the dividing neurons in the early postnatal DG, the NeuN+/BrdU+ cells in the hippocampal DG were examined by using double-immunofluorescence staining (5 tissue sections per group).
Experiment 2 evaluated the integration rate of developmentally generated granule neurons into the hippocampus-dependent memory networks in the DG (Fig. 1). The PND-7 rats received three consecutive BrdU injections intraperitoneally on PND-7, 8 and 9 after administered with normal saline or ketamine, then two groups of rats were weaned at PND-35, after which they were housed in cages with free access to food and water for up to 3 months old (6 animals per group). Hippocampus-dependent memory was assessed following the training period in the MWM task. Then, all animals were deeply anesthetized with 40 mg/kg ketamine and transcardially perfused with 0.9% normal saline, followed by a transfusion with 4% paraformaldehyde. The previous study had suggested that the expression of c-Fos was regulated by the neural activity that occurs as an animal performs the hidden platform version of the water maze. The c-Fos expression in NeuN+/BrdU+ cells was examined by triple-immunofluorescence staining. This approach was used to estimate whether developmentally generated granule neurons had been functionally integrated into hippocampal memory networks during adult stage. In this experiment, two groups of animals were sacrificed immediately after the completion of the MWM testing. The integration rate of developmentally generated granule neurons into the hippocampal memory networks was estimated by calculating the proportion of c-Fos+/NeuN+/BrdU+ cells in the hippocampal DG (5 tissue sections per group).
Tissue Preparation and Immunofluorescence
The brains were postfixed in 4% paraformaldehyde and the coronal sections of the brains were cut consecutively at a thickness of 30 μm, at the point in which the hippocampus was initially exposed, the fifteenth section was taken and stored in PBS. The position of the hippocampus coronal sections selected in our study was approximately 2.80-2.85 mm posterior to the bregma for the 2 months old rats and approximately 2.90-2.95 mm posterior to the bregma for the 3 months old rats [15,16].
For the NeuN/BrdU double-immunofluorescence staining, the BrdU antigen was exposed by incubating the sections in 2-normal hydrochloric acid for 30 min at 37°C, then the sections were washed by PBS. The blocking of nonspecific epitopes with 10% donkey serum in PBS (which contained 0.3% Triton-X) for 2 h at room temperature preceded an overnight incubation at 4°C with the primary antibodies against NeuN (Mouse anti-NeuN monoclonal antibody; 1:200; Millipore, Massachusetts, USA) and BrdU (Rabbit anti-BrdU monoclonal antibody; 1:500; Abcam, San Francisco, USA). On the next day, the sections were incubated with the appropriate secondary fluorescent antibodies (Invitrogen Carlsbad, USA) for 2 h at room temperature.
For the Fos/NeuN/BrdU triple labeling, identical procedures were performed by using a primary rabbit anti-c-Fos polyclonal antibody (1:200; Abcam), a mouse anti-NeuN antibody (1:200; Millipore) and a rat anti-BrdU monoclonal antibody (1:500; Abcam). On the next day, the sections were incubated with the appropriate secondary fluorescent antibodies (Invitrogen) for 2 h at room temperature.
The single-plane images of the stained sections were taken by using a laser scanning confocal microscope (Fluoview 1000, Olympus, Japan), and a skilled pathologist, who was blinded to the study conditions, examined the labeled sections and portrayed the scale of hippocampal DG in the brain slice in the fluorescence image. The numbers of double-positive or triple-positive cells in the hippocampal DG were manually quantified by using Image-Pro Plus software (Media Cybernetics Inc., Bethesda, USA).
Brain Tissue Harvest and Western Blot Analysis
In order to observe whether neonatal ketamine exposure induces neural apoptosis in the hippocampal DG at 2 months old and 3 months old, levels of caspase-3 expression were measured at these time points. Rats in the sham group and ketamine group were deeply anesthetized with ketamine and decapitated at 2 months old (3 animals per group) or 3 months old (3 animals per group). The hippocampal DG tissue was dissected carefully with an stereo microscope (Leica EZ4HD). The harvested hippocampal DG tissues were homogenized on ice using lysate buffer plus protease inhibitors. The lysates were centrifuged at 14,000 rpm for 15 min at 4°C and were resolved by 12% polyacrylamide gel electrophoresis, and the target proteins were transferred to nitrocellulose membranes. The blots were incubated with blocking buffer for 2 h at room temperature and then incubated for 24 h at 4°C with the primary antibodies rabbit anti-caspase-3 antibody (1:1000 dilution; Cell Signaling Technology) and β-tubulin (1:10000 dilution; Abcam). The membranes were then incubated with the appropriate secondary alkaline phosphatase-conjugated antibody (Abcam, dilution factors included Tris-Hcl, NaCl, tween20) for 1 h at room temperature. The band intensity was quantified using Image J software. We quantified the Western blots in two steps. First, we used β-tubulin levels to normalize (e.g., determining the ratio of caspase-3 to β-tubulin amount) protein levels. Second, we presented changes in ratio levels in rats undergoing ketamine anesthesia as a percentage of those in the control group. One hundred percent of ratio level changes refer to control group for the purpose of comparison with experimental conditions.
The statistical analysis was conducted by using SPSS 13.0 (SPSS Inc., Chicago, USA), and the graphs were created by using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, USA). The data were analyzed by using the Mann-Whitney U test. The interaction between the time and group factors, which was determined by using a two-way ANOVA, was used to analyze the differences in escape latency between the rats in the control group and the rats that were treated with ketamine in the MWM. The data are presented as the mean±SD, and P<0.05 was considered to be statistically significant.