| Objective: To investigate AChE, SYN and Jmjd6 (Jumonji C domain containing gene 6) expression in the hippocampal CA3 neurons of juvenile rats for investigating the effects of intrauterine hypoxia on hippocampal CA3 neurons, as well as the mechanisms of learning-memory in juvenile rats. Method: Healthy pregnant Sprague Dawley rats (n=14) were randomly separated into control (n=6) and hypoxia (n=8) groups. The hypoxia group pregnant rats were placed in a three-gas incubator (O2: 130 mL/L, CO2: 0.3 mL/L-0.4 mL/L, Temperature: 25℃, Relative humidity: 75%-80%) starting from day 14 of pregnancy for 2 h/d for 5 consecutive days (gestational 14-18th days) to establish a fetal rat intrauterine hypoxia model. Control group lacked the hypoxic conditions. After birth, two neonatal rats were selected randomly from each litter and fed for 30 days (in order to balance the neonatal rats number among litters, the remaining neonatal rats were killed). The 30-day-old juvenile rats were tested by Morris water maze for 11 days, searching time (T1) in the target quadrant in probe trail of water maze experiment and searching time (T2) in the target quadrant in reversal probe trail of water maze experiment were recorded. And then these juvenile rats were perfused and fixed with formaldehyde (40 g/L), after that brain tissues were removed, dehydrated, paraffin-embedded and sectioned serially for hippocampal coronal sections (thickness: 5μm) to proceed with immunohistochemistry detection. In section immunohistochemistry detection, a monoclonal anti-AChE antibody (1:200 dilution), a monoclonal anti-SYN antibody (1:150 dilution) and a polyclonal anti-Jmjd6 antibody (1:80 dilution) were used. Specific steps of immunohistochemistry detection in this experiment were as follows:①Conventional dewaxing in water.②Preconditioned the sections according to the special requirements of the primary antibodies.③Incubated for 10 min in 3% H2O2 deionized water to block the endocardial peroxide enzyme, washed with PBS, 2 min×3 times.④Added the primary antibodies, 4℃overnight in refrigerator, washed with PBS, 2 min×3 times.⑤Incubated with secondary antibodies according to the origin of the primary antibodies for 30 min in 37℃, washed with PBS, 2 min×3 times.⑥Stained with DAB for 5-10 minutes in the dark (time was controlled under the microscope).⑦Washed fully with distilled water.⑧Then the sections were routinely dehydrated, made transparent, and sealed. Slices of hippocampal CA3 neurons were acquired with a Motic DigiLab II microscope DMBA200 (photomicrographs were taken at 400×magnification) and IPP6.0 software, IOD (integral optical density) values of the positive cells were recorded. Data were analyzed by means of independent sample T-test. Results: In the Morris water maze, the searching time of the probe trail of hypoxia group (T1 = 18.47±4.41 s) exhibited asignificantly (P < 0.05) shorter than the searching time of the probe trail of control group (T1 = 24.38±4.89 s), and the searching time of the reversal probe trail of hypoxia group (T2 = 15.38±6.10 s) also exhibited a significantly (P < 0.05) shorter than the searching time of the reversal probe trail of control group (T2 = 22.04±5.57 s). IOD values of AChE, SYN and Jmjd6 positive cells of hypoxia group were compared with control group, respectively. Mean of IOD values of AChE positive cells of hypoxia group (12.09±1.93) exhibited a significantly (P < 0.05) higher than control group (10.36±1.42); mean of IOD values of SYN positive cells of hypoxia group (12.51±2.65) exhibited a significantly (P < 0.05) lower than control group (17.63±2.53); mean of IOD values of Jmjd6 positive cells of hypoxia group (7.99±1.75) exhibited a significantly (P < 0.05) higher than control group (6.24±0.52).Conclusion: Intrauterine hypoxia in a certain period of time (2 h/d, for 5 consecutive days) and a certain degree of oxygen concentration (130 mL/L) could negatively affect learning and memory in juvenile rats. The mechanisms may be ascribed to the increased quantity of AChE (deduced quantity of ACh relatively), deduced quantity and reduced expression of SYN and the weakened function of Jmjd6, which induced by hypoxia in juvenile rats'hippocampal CA3 neurons.
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