Effects Of Propofol On Apoptosis Of The Developping Hippocampal Neurons And The Role Of Rho GTPases In Rats

Part Ⅰ The viability and apoptosis of propofol on the developpinghippocampal nuronseObjective: To evaluate the effects of propofol with different concentrations and time on the viability of developping hippocampal neurons in culture by MTT assay, to observe the morphology of neurons by inverted phase contrast microscope. To assess the effects of propofol with different concentrations on the apoptosis of the developping primary hippocampal neurons cultivated for 24 h by Annexin Ⅴ-FITC kit and Hoechst33258 immunofluorescence staining.Methods: Cell cultures were prepared from newborn Sprague-Dawley rats for 4 days,then the cultures were exposed to propofol with different concentrations(0μg/ml, 1μg/ml, 10μg/ml, 20μg/ml, 40μg/ml, 60μg/ml, 80μg/ml) or cultivated for 6h, 12 h, 24 h and 48 h with propofol(40μg/ml), respectively. The primary culture medium was adjusted with 0.04%DMSO in each group(we had previously proved that a 0.04% DMSO vehicle alone did not affect survival and viability of neurons). The primary culture medium with 0μg/ml propofol was designed as control group. Then the OD value of each group were detected. Cell viability rate was determined from the formula as Rate=(control group-blank group)/(experimental group-blank group). The roughly morphology of the neuron was observed by inverted phase contrast microscope. According to the results of MTT, the immature hippocampal neurons were exposed to propofol with different concentrations(20μg/ml, 40μg/ml, 60μg/ml, 80μg/ml) for 24 h, the samples of each group was prepared, and then the apoptosis rate were analyzed by flow cytometer with AnnexinⅤ-FITC kit. Apoptotic morphology was detected by Hoechst33258 immunofluorescence staining to verify the result of FCM in each group.Results: 1 The viability of neurons was evaluated by MTT assay. Propofol decreased hippocampal neuron viability with dose and time dependent. First, the cultures exposed to increasing concentrations of propofol, high concentrations of propofol(≥20μg/ml) led to a significant impact(P<0.01) on cell viability compared with control group. But low concentrations of propofol(1μg/ml, 10μg/ml) did not affect cell survival. Exposure of cultures to propofol(60μg/ml) up to 6h, the hippocampal neuron survival remained unaltered, but treated up to 12 h or longer time(24h, 48h), neurons in culture exhibited a distinct loss of viability(P<0.01). 2 The morphology of the hippocampal cell was observed in inverted phase contrast microscope. On the 4th day of the culturing process, the cells were well-rounded, and the neurites were elongated and enlarged significantly and the network generated initially. It showed that the morphology of the neuron in cultures changed significantly when the concentration of agent increased. High concentration of the propofol(60μg/ml) led to a massive loss of neurons, and the residual cells were distinctly atrophied, the network fracture, leaved granuliform pieces. 3 The flow cytometer assay with AnnexinⅤ-FITC kit showed that with the increase of propofol concentration, the cell apoptosis increased accordingly in a dose-dependant manner. We found that significant apoptosis occurred after cells were treated with propofol at 40μg/ml, 60μg/ml and 80μg/ml(P<0.05, P<0.01, P<0.01). No significant difference was observed in cell apoptosis between 20μg/ml propofol-treated and control group. 4 Hoechst 33258 staining results were consistent with the flow cytometer assay and MTT, propofol induced hippocampal neurons apoptosis, the rate of apoptosis was increased with dose dependent.Conclusions: Propofol induced injuries in developing hippocampal neurons accordingly in a dose- and time-dependant manner. Low concentration of propofol could affected the neuron survival, high concentration led to massive apoptosis in developing hippocampal neurons,and the apoptosis increased with the agent concentration rising. And high concentration of propofol affected the neuron morphogenesis. Part Ⅱ The role of Rho GTPases in the propofol neurotoxicity on thedeveloping hippocampal neuronsObjective: To detect the effect of propofol on the neural cellular expression levels of Rho A, ROCK1, Rac1 and PAK1 by western blot and investigate the effect of Rho/ROCK inhibitor-Y27632 on the propofol neurotoxicity in vitro.Methods: The hippocampal neurons cultivated for 4 days were divided into four groups, each group was treated with different concentrations of propofol(0μg/ml、20μg/ml、40μg/ml、80μg /ml), The primary culture medium was adjusted with 0.04% DMSO every group, the group of 0μg/ml was the vehicle group. To examined the 12 h post-treated expression of Rho A, ROCK1, Rac1 and PAK1 by western blot every group; Another part, the hippocampal neurons cultivated for 4 days were divided into four groups, the vehicle group(0.04% DMSO, C), 60μg/ml propofol group(P), 60μg/ml propofol plus 10μM Y27632(P+Y) and 10μM Y27632 group(Y), cultures were exposed to agent for 24 h, then examine the viability of the hippocampal neurons by MTT assay, apoptosis of the cells was assessed using two different assays, flow cytometer assay with AnnexinⅤ-FITC kit and cleaved-caspase-3 by western blot.Results: 1 Western blot was performed to assess the expression level of Rho A, ROCK1, Rac1 and PAK1. Western blot analysis detected Rho A expression increased significantly with the concentration of propofol increasing(20μg/ml, 40μg/ml, 80μg/ml) compared with the vehicle group(P<0.05, P<0.01, P<0.01), expression of ROCK1 increased significantly with the concentration of propofol increasing(20μg/ml、40μg/ml、80μg /ml) compared with the vehicle group(P<0.05, P<0.01, P<0.01), too. The expression level of Rac1 decreased with the concentration of propofol increasing, but difference was not statistically significant versus the vehicle group. Compare with the vehicle group, PAK1 expression level decreased significantly with the concentration of propofol increasing(P<0.01). 2 The MTT assay was performed to assess Y27632 influencing on the cell viability impaired by propofol, the results suggest that, the cell viability decreased significantly(P<0.01) suffer from propofol(P group), but no statistical difference in Y group compared with C group. Co-treated with Y27632 and propofol for 24 h, the cell viability increased significantly versus P group(P<0.01). 3 The flow cytometer assay was carried out to determine the amount of apoptosis that occurs after the different treatments. There was only a few neurons death in the C and Y group, and no statistical difference between two groups. The ratio of apoptotic neurons increased significantly after exposure to 60μg/ml propofol( P group) for 24 h compared with the C group(P<0.01). This increase was prevented by the addition of Y27632. 4 Western blot was carried out to assess the expression level of cleaved-caspase-3 to estimate the neuron apoptosis after different treatments. Western blot analysis detected significantly increased cleaved-caspase-3 expression in neurons of the propofol group compared with those in the control group(P<0.01). Cleaved-caspase-3 expression was uniform in neurons of the Y27632 group versus the control group. The effect of propofol treatment was inhibited by Y27632co-treatment(P<0.01). These results were consistent with the flow cytometer assay results.Conclusions: Propofol could promote the Rho A and ROCK1 expression but reduce the PAK1 expression. The neuron damage exposure to propofol could be prevented by inhibiting the Rho/ROCK pathway. The Rho kinase activation might be one mechanism of the propofol-induced apoptosis. Part Ⅲ The effects of propofol on apoptosis in developing hippocampalneurons and persistent learning/memory functionsObjective: Propofol was administered to 7-day-old rats as a single dose for 7 days. To investigate the effects of propofol on the apoptosis of hippocampal neurons and long-term learning/memory functions.Methods: The 78 healthy 7-day-old Sprague-Dawley(SD) rats were randomly divided into 3 groups. Propofol group(P group): the rats were peritoneally injected with propofol(50mg/kg) once a day at regular 24-h intervals for 7 days; Solvent control group(S group): the rats were peritoneally injection with the same amount of lipid emulsion once a day at regular 24 h intervals for 7 days; Blank control group(C group): the rats were peritoneally injection with the same amount of saline once a day at regular 24-h intervals for 7 days. Throughout the anesthesia procedure, the rats were kept in a neonatal incubator to maintain their body temperature and provided with low-flow oxygen to reduce potential stressors. Until they could successfully perform the righting reflex, all rats were separated from their mothers less than 300 min. The blood glucose level and the Sp O2 were monitored, randomly selected rats(n=5) from each group; Nissl staining was performed to observed histopathological changes, randomly selected rats(n = 3) from each group; The TUNEL staining was performed to observe the apoptotic cells(per 0.01 mm2) in the hippocampal CA1, CA3 regions and the dentate gyrus, randomly selected rats(n=5) from each group;The activity of caspase-3 was detected using a commercial kit, randomly selected rats(n=3) from each group; At two months of age(60days), the learning and memory abilities were tested using the Morris water maze in the rest rats.Results: 1 The blood glucose and the Sp O2 level did not differ significantly among three groups(P>0.05). 2 Histopathological changes in the hippocampal CA1, CA3 regions and the dentate gyrus region were observed with a light microscope after Nissl staining. The number of neurons in the hippocampus(CA1) of pups repeatedly exposed to propofol(P group) significantly fewer than those in the saline treated group(C group), the shape of neurons were defected,and the amount of Nissl body was limited in the neurons of CA1. There is no significantly difference between C and S group in CA1 region. And there is no significantly difference in CA3 and dentate gyrus among all groups. 3 TUNEL staining was performed to detect the apoptosis of hippocampal neurons at regions CA1, CA3 and the dentate gyrus. The results showed that the number of apoptotic cells per mm2 at CA1,CA3 and the dentate gyrus had a significant difference in P group compared with C group(P<0.01). There is no significantly difference in all regions between C and S group. 4 The activity of caspase-3 assay showed that the caspase-3 activity was significantly increased in P group versus C group(P<0.01). The caspase-3 activity was no statistical difference between C and S group. These results were consistent with TUNEL assay.5 Morris water maze test showed that the latency for rats to find the platform was no statistical differences among all groups in the first three days. Further day analysis showed that the rats in group P took longer time to find the hidden platform on the fourth and fifth training days compared to the controls(P<0.01).There was no statistical differences of the latency between group C and S in the training period. In the probe trial, the times of crossing the platform of group P was significantly fewer than the controls(P<0.01), and the crossing times of C and P group were no statistical differences.Conclusions: The propofol administered to 7-day-old rats as a single dose for 7 days, will induce hippocampal neuron injury, widespread neuroapoptosis in the neonatal brain and can cause persistent learning deficits as the animal matures. Part Ⅳ The mechanism study of hydroxylfasudil against propofol–induced apoptosis in developing hippocampal neuronsObjective: To investigate whether the administration of hydroxylfasudil could exert neuroprotective effect against propofol–induced apoptosis in developing hippocampal neurons and to explore the role of p38 MAPK pathway, which will provide the experimental evidence for clinical neuroprotection against propofol –induced apoptosis.Methods: The 84 healthy 7-day-old Sprague-Dawley(SD) rats were randomly divided into 4 groups. Propofol group(P group): the rats were peritoneally injected with propofol(50mg/kg) once a day at regular 24-h intervals for 7 days; Propofol +Hydroxylfasudil(PF group): the rats were peritoneally injected with propofol(50mg/kg) and Hydroxylfasudil(10mg/kg); Hydroxyl Fasudil(F group): the rats were peritoneally injected with Hydroxylfasudil(10mg/kg); Blank control group(C group): the rats were peritoneally injected with the same amount of saline once a day for 7 days. Throughout the anesthesia procedure, the rats were kept in a neonatal incubator to maintain their body temperature and provided with low-flow oxygen to reduce potential stressors, until they could successfully perform the righting reflex. All rats were separated from their mothers less than 300 min. The blood glucose level and the Sp O2 were monitored, randomly selected rats(n=3) from each group; The TUNEL staining was performed to observe the apoptotic cells(per 0.01 mm2) in the hippocampal CA1 regions, randomly selected rats(n=5) from each group. The protein levels of p38 MAPK, p-p38 MAPK, Bcl-2 and Cleaved-caspase-3 were measured by western blot analysis, randomly selected rats(n=3) from each group; At two months of age(60days old), the learning and memory abilities were tested using the Morris water maze in the rest rats.Results: 1 TUNEL staining results showed that the number of apoptotic cells per mm2 at CA1 had a significant difference in P group compared with C group(P<0.01). There is no significantly difference in CA1 regions between C and F group. Co-treated with hydroxylfasudil and propofol(PF group), the apoptotic cells decreased significantly versus P group(P<0.01). 2 Western blot analysis detected p38 MAPK expression of group F was lower than the controls(P<0.05). There is no statistical difference of p38 MAPK expression among other groups; The p-p38 MAPK expression of group P was significantly higher(P<0.01) and group F was lower(P<0.05) than the controls. And Co-treated with hydroxylfasudil and propofol(PF group), the p-p38 MAPK expression was significantly lower than the group P(P<0.01); The Bcl-2 expression of group P was significantly lower(P<0.01) and group F was higher(P<0.05) than the controls, Bcl-2 expression of group PF was significantly higher than group P(P<0.01); The activatied-caspase-3 expression of group P was significantly higher than the controls(P<0.01), but the expression of group F was no statistical difference with the group C. Activatied-caspase-3 expression of group PF was significantly lower than group P(P<0.01).These results were be consistent with the TUNEL assay. 3 Morris water maze test showed that the latency for rats to find the platform was no statistical differences among all groups in the first three days. Further day analysis showed that the rats in group P took longer time to find the hidden platform on the fourth and fifth training days compared to the controls(P<0.01).This result was consistent with the previous research in the Part 3 of this study. The latency for rats of the group PF to find the platform was shorter than group P on the fourth and fifth training days(P<0.05). There was no statistical difference of the latency between group C and F in the training period. In the probe trial, the times of crossing the platform of group P was significantly fewer than the controls(P<0.01), and the crossing times of C and F group were no statistical differences, Co-treated with hydroxylfasudil and propofol(PF group), the crossing times was more than the group P P<0.05).Conclusions: Hydroxylfasudil had the neuroprotective effect against the propofol induced-on apoptosis in developing hippocampal neurons. And the protective effects of fasudil might be mediated by the inhibition of p38 MAPK pathway.