| Objective: To evaluate the effect of the hyperbaric oxygenation therapy on the apoptosis of the hippocampal neurons, and the cognitive function of the newborn rats suffering from bilirubin encephalopathy.Methods: One hundred and forty-five five-day-old SD rats were randomly divided into three groups: control group(C); bilirubin encephalopathy group(B);hyperbaric oxygenation treated group(HBO). Normal sodium or equivalent bilirubin solution (10mg/mL) was injected into rats'Cerebellomedullary cistern,the injected dose was 10μL per gram weight. Observing the shapes of the nerve cells by Hematoxylin-Eosin (HE) Stain and Nissle stain, the expression of Cytochrome C and the Caspase-3 in hippocampus neurons were detected by immunohistochemistry and immunofluorescence respectively, at the time of 6 hours (h), 1 day (d), 3 d, 5 d and 7 d after intra-cisternal injection. Neuronal apoptosis was checked by TUNEL method. The cognitive function was tested by Morris experiment, when the three group rats were twenty eight-day-old, it was at 23 days after intra-cisternal injection.Results: Rats of both B group and HBO group appeared abnormal behaviors in different degree, but the food take, activity and survival rates of rats in HBO treated group (80%) were better than those of B group (60%).In light microscope: the apoptosis and nicrosis of neurocytes in B group were more visible than the control group, The neurons'arrangement and blue-stain of endochylema in HBO group were better than those in B group. Detected the expression of Cytochrome C by immunohistochemistry: the tendency of expression in both HBO and B group were similar, it was very visible at the time of 6 h after the injection: HBO (0.199±0.002),B (0.216±0.012),and it reached the peak at the time of 1 d after injection: HBO (0.232±0.011),B(0.263±0.029), afterward it began to fall-off ,but the expressions of the Cytochrome C in HBO group were lower than those in B group and higher than C group at various time point (P <0.05). Detected the expression of Caspase-3 by immunoflourescence: the intensity in HBO group (0.385±0.004) had no difference to the C group (0.385±0.004)at the time of 6 h after injection(P>0.05),B group (0.425±0.004) was visible at the same time point; afterward the tendency of expression in both HBO and B group were similar, they expressed maximum at the time of 1 d time point, HBO (0.448± 0.003),B (0.463±0.003), afterward they began to fall-off , The HBO group (0.4±0.001) had no difference to the B group (0.404±0.002) (P>0.05). The TUNEL positive cell rate: The TUNEL positive cell rate in HBO group (27.2±1.79)% was less than that in B group (37.6±6.58)% (P <0.05). In Morris experiment: the rats of HBO group were easier to find the safe platform than those of B group(P <0.05), for example : HBO group need (46.05±9.41) seconds to find the safe platform,but the B group only need (59.29±1.54) seconds in the 4th day ; in the 7th day the HBO group need (30.87±4.05) seconds and the B group need (56.35±3.65) seconds, and also, the frequency of crossing the safe platform were eight times to the B group (P <0.05) in Space pilot test.Conclusions: By reducing the expressions of Cytochrome C and Caspase-3, hyperbaric oxygenation therapy could inhibit bilirubin induced hippocampal neurons'apoptosis partly. In result, it improved cognitive function of the rats suffering from bilirubin encephalopathy.
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