Effect Of Propofol On Apoptosis And Cytochrome C Release In Hippocampal Neurons Of Rats

Objectives:To observe the effects of propofol on primary cultured rat hippocampal neuron apoptotic models induced by ischemia-reperfusion injury and the cytosolic cytochrome c release at different time points during the apoptosis, so to explore the mechanism of the cerebral protection of propofol.Methods:1. The embryonic 18 d rat hippocampus was dissected and minced. The cell suspension was harvested by 0.125% trypsin digestion, and incubated in DMEM/F12 medium added with 10% fetal bovine serum, to make the primary culture of rat hippocampal neurons. The growth of the neurons was observed with contrast phase microscope.2. The apoptotic model induced by ischemia reperfusion injury was set up by means of rendering the low-glucose and non-oxygen incubation environment. The medium was changed into low-glucose DMEM/F12 with 10% fetal bovine serum before anaerobic incubation for 6 hours, then replaced with the high-glucose DMEM/F12 added with 10% fetal bovine serum and resumed cultured with oxygen for 24 hours.3. The cells incubated for 7～9 days (5*10⁸cells/L density) were randomly separated into 5 groups. Normal control group (group C), which was cultured with normal methods. Model group (group M), which was treated by hypoxia and ischemia with no propofol given into the medium during the study. Propofol group I, which was medicated with propofol [to the final concentrations of 200μM (group I1) and 20μM (I2), respectively] before the hypoxia intervention. Propofol group II, to which propofol was given [to the final concentrations of 200μM (group II1) and 20μM (II2), respectively] before the re-oxygenation. Propofol group III, to which propofol (final concentration of 200μM) was administered 2 hours after the re-oxygenation.4. The living status of neurons were observed with contrast phase microscope at the following time points: before hypoxia (T₀), end of hypoxic incubation (T₁), two hours after the re-oxygenation incubation (T₂) and 24 hours after the re-oxygenation (T₃). And the concentration of cytochrome C in cytoplasm separation at the above time points.5. The data was statistically analyzed with one -way ANOVA and LSD tests, the size of test, P= 0.05.Results:1. The overview under the contrast phase microscope: Cultured with normal methods for 24 hours, there's less change to the cell of group C. In the group M, some of the neurons got to swollen after the hypoxia incubation. Two hours post re-oxygenation many cells showed morphologic changes of apoptosis such as loss of membrane symmetry, chromatic agglutination, shrinkage, and the apoptotic body appearance. Till the time point T₃, the number of apoptosis cells increased and the active cells almost disappeared. The propofol groups showed better results than the group M at every time point. There was no obvious morphologic change. The numbers of apoptosis were very low, especially in those groups with higher doses of propofol. Among them, the propofol group I₁ took a similar overview with the group C. There's no difference between the propofol groups at the T₃ point.2. The variations of cytosolic cytochrome C:1) Group C: The concentration of cytochrome C for group C had no significant change between T₀, T₁ and T₂; there was a slight increase at the T₃ point but P>0.05 when compared with that of T₀.2) Group M: Compared with that of T₀, the contents of cytochrome C at T₀ and T₂ increased markedly (P<0.05). The later was even higher, (P<0.05). There were significant increases at T₁, T₂ and T₃ when compared to those of the same time points in the group C (P<0.05).3) Propofol groups: Compare with the normal control group (group C), the cytochrome C concentrations in propofol group I₂,II₁,II₂ and III had the same increases at the time point T₁ and T₂, the differences were all significant (P<0.05). While that of the propofol group I₁ was similar to the group C at T₂ (P>0.05). However, there were no difference between the concentrations of the each propofol group and group C at T₃ (P>0.05) .4) Compare with the group M: the concentration of cytochrome C of each of the propofol group had a big step down to at the time point T₁, T₂ and T₃. The LSD test showed significant differences (P<0.05, respectively).5) Amid the propofol groups: The concentrations of cytochrome C had an increase tendency at T₁, T₂ and T₃ when compared with the basal value.6) Among the propofol groups: The values of cytochrome C in group I is lower than those of group II and group III on the time point T₁ and T₂ (P<0.05, respectively), and there was no significant difference between them on T₃ (P>0.05). At T₂ point, the cytochrome C was higher in the group III than that in group II₁ (P<0.05) but at T₃ there was no difference (P>0.05). There was no significant difference observed on both T₂ and T₃ time points when compared with the group II₂ (P>0.05).7) Between the different doses of propofol: The higher dose samples (group I₁ and II₁) showed less cytochrome C value than those of the lower dose groups (group I₂,II₂) on T₂ point (P<0.05). While there were no differences on T₁ and T₃ points (P>0.05).Conclusion:1. There's cytochrome C release from mitochondria to cytoplasm in the early stage of hippocampal neuron apoptosis procedure;2. Earlier, higher dose of propofol can alleviate the cytosolic cytochrome C release during the apoptosis, thus to reduce the injuries from apoptosis on neurons.