| Objective:Through establishing the Chronic unpredictable mild stress rats model during pregnancy (Chronic unpredictable mild stress, CUMS), observe systematically the state of maternal reproductive function of pregnancy rat during stress, the ability of learning, memory ability and the influence of the mood of offspring, discuss the possible mechanism of injury starting from the hippocampus neurotransmitter and plasticity and probe into the intervention mechanism for learning and memory ability damage after intervening measure of a rich environment. To understand and predict the stress was harm for person, start the next step of genetic epidemiological studies and provide a scientific basis for intervention. Methods:Twenty female Wistar rats were divided into control group and stress group, the model was established by using chronic unpredictable mild stress stimulation combined with lonely raising for twenty-one days (mating at the beginning of the third day).1) Weight and behavior changes of model rats and the offspring were observed, plasma levels of cortisol were measured by radio-immune assay, behavior determined by open-field test, fluid consumption was from sucrose preference test, and analysed the basic situation of the reproductive ability. The ability of learning and memory was measured using Morris water maze and Y maze test, the mood of offspring was measured using liquid consume experiment, open-field test and the tail suspension experiment, at the same time, analysis of the changes.2) The brain morphology observed by using HE staining and electron microscopy in offspring, the central neurotransmitter content and activity of Hippocampus was measured by ELISA method, hippocampus neurotrophic factors BDNF and IGF-Ⅱ, nuclear transcription factor NF-κB, Arc mRNAs and protein expression were respectively mearsured by using real-time fluorescence quantitative-PCR and Western Blotting and analyze its relevance.3) Given respectively rich environment intervention to model and control children, then conducted behavior, mind, the neurotransmitter levels and synaptic related protein expression. Rusults:1) The repeated measurement analysis of variance showed that chronic stress had a significant impact on the circulatory corticosterone level and the weight (P<0.05), weight increase rate of model group rats was lower than the control group, the corticosterone level of model group rats had drastically changed along with stress time. In addition to the waste grain number, the behavior indicators of model group rats had statistically significant with the control group (all P<0.05), the change of mother’s behavior index had relationship with stress duration, there was no interaction relationship between time and stress factors (all P>0.05). The water consumption of model and the control group had slight difference (P>0.05), and sugar water consumption, total liquid consumption and1%sucrose preference had statistical differences between two groups (all P<0.05), it showed that stress affected fluid consumption index of rats during pregnancy, time factor was statistically significant (all P<0.05). The comparison of each point in time:total liquid, sugar water, water consumption, sugar percentage preference didn’t have difference between a baseline of normal control and model group (P>0.05), After exposure to stress for1day, sucrose preference and total liquid consumption underwent a significant decline among rats in the CUMS and depression model groups (P<0.05), following subjection to stress for14and21days, the depression model rats manifested a substantially reduced sucrose-intake compared with control rats (P<0.05). There was statistical significance of the number of offspring and days of pregnancy between two groups (P<0.05), the number of offspring and days of pregnancy in model was less than the control group.2) At postnatal day28and42, the weight of model offspring were lower than control (P<0.05), the plasma corticosterone was higher (P<0.05). The results of morris water maze task:the escape latency of model offspring compared with control difference was statistically significant, there were significant differences in terms of the escape latency between two groups at different time points (P<0.05), there was no interaction relationship between time and stress factors (P>0.05). The escape latency time of model offspring was leader than control and had a tendency to shorten (P<0.05), number of cross-platform had statistical differences between the two offspring groups (P<0.05). The results of Y maze experiment:the required number, correct response remembering to keep tests training of model offspring was statistically significant compared with control group rat difference (P<0.05). The results of behavioral experiments:besides cleaning, the index of behavioral experiments of the model offspring had statistical differences with control (P<0.05). The results of liquid consumption:there were statistical differences in the consumption of sugar water and1%sucrose preference percentage between two offspring groups (P<0.05), we didn’t find statistical differences in respect of water consumption and total liquid consumption (P>0.05). The results of tail suspension test:the rest time and the struggle number of model offspring rat were statistical differences with control group (P<0.05).3) Hippocampal structure form and ultrastructure of model offspring changed. The ACh content, ChAT and AChE activity of hippocampal cholinergic neurotransmitter of model and contrast group offspring had statistical differences (P<0.05), the content of ACh and activity of ChAT in model offspring declined, AChE activity increased. Hippocampal monoamine neurotransmitter NE, DA and5-HT were statistical differences between model offspring and contrast (P<0.05), all they were declined. Hippocampal Glu and GABA were statistical differences between model offspring and contrast (P<0.05), excitatory neurotransmitter Glu reduced and inhibiting neurotransmitter NO content increased. NO content of hippocampal had statistical differences between model offspring and contrast, NO content decreased.4) The results of Real-time fluorescent quantitative PCR:the differences existed statistical significance of hippocampus BDNFmRNA, IGF-Ⅱ mRNA and NF-κB mRNA between model offspring and contrast (P<0.05), BDNFmRNA, IGF-Ⅱ mRNA and NF-κB mRNA expression of model offspring declined, the Arc mRNA statistical significance difference wasn’t found (P>0.05). The detection of western blotting:the differences existed statistical significance of hippocampus BDNF、IGF-Ⅱ、Arc and NF-κB proteins between model offspring and contrast (All P<0.05), compared with the control offspring, the BDNF、IGF-Ⅱ、Arc and NF-κB proteins of hippocampus decreased.5) Correlation analysis:the escape latent period and in the water maze training times of offspring existed positive correlation with maternal corticosterone (P<0.05), however, negatively correlated to maternal horizontal movement, vertical movement and1%sucrose preference (P<0.05). Cross-platform times in water maze and correct response rate of Y maze in offspring were negatively correlated to maternal corticosterone (P<0.05), however, positively correlated to maternal horizontal movement, vertical movement and1%sucrose preference (P<0.05). the escape latent period and in the water maze training times of offspring were positively correlated to plasma corticosterone and GABA in the hippocampus of offspring, but negatively correlated to ACh、ChAT、NE、DA、5-HT and NO in the hippocampus. Cross-platform times in water maze and correct response rate of Y maze in offspring were negatively correlated to plasma corticosterone and GABA in the hippocampus of offspring (P<0.05), at the same time, positively correlated to ACh、ChAT、NE、DA、5-HT and NO in the hippocampus. The relationship between cross-platform times in water maze and training times of Y maze and IGF-Ⅱ protein expression was not found, other indicators were found they had some correlation, what shows respectively that cross-platform times in water maze and correct response rate of Y maze in offspring were positively correlated to proteins related-synaptic (P<0.05), the escape latent period and in the water maze training times of offspring were negatively correlated to proteins related-synaptic (P<0.05).6) The influence of rich environment on the offspring weight:the weight of offspring in model and model&rich environment was lower than control offspring and control&rich environment, after the rich environment:weight of four groups had statistical differences (P<0.05). Impact rich environment on plasma corticosterone levels of offspring:plasma corticosterone of four groups had statistical differences (P<0.05), the statistical significance differences existed model offspring and other groups (P<0.05), the plasma corticosterone of control&rich environment offspring group was minimum in the four groups, the model offspring group was the highest.7) The ability of learning and memory after rich environment:four groups had differences statistically significant in the escape latency in morris water maze and control offspring (P<0.05), the escape latency period of two rich environment group growed, number of cross-platform had statistical differences between the four groups (P<0.05), cross-platform of control&rich environment offspring group was at most, model offspring group was the least. In the number of learning and memory in the Y maze test was statistically significant difference in four groups (P<0.05), learning and memory ability was improved after accepted rich environment. Horizontal movement and vertical movement existed statistically significant difference in four groups of comparison (P<0.05), horizontal and vertical movement ability improved after rich environment. Sugar in water consumption, water consumption and1%sucrose preference percentage comparison difference had statistical significance in four offspring groups (P<0.05), sugar water consumption increased after rich environment, compared four groups on the static time and the number of struggling was statistically significant difference (P<0.05), the static time of model offspring group was longest, however, struggling time was least, and as long as the rich environmental model rat group, the rest time would be shorten, struggling number increased.8) Rich environment affected hippocampus neurotransmitter of offspring:the hippocampus cholinergic neurotransmitter ACh content, the activity of ChAT and AChE, single amine neurotransmitter NE, DA and5-HT content, amino acid neurotransmitter Glu and GABA levels in four offspring groups were statistically significant difference (P<0.05), it hinted that rich environment had influence on these. Hippocampus NO content in four groups had statistical differences (P<0.05), NO content rised after rich environment intervention.9) The impacted of rich environment on protein expression:hippocampus BDNF mRNA, IGF-Ⅱ mRNA and NF-κBmRNA and protein expression in four offspring groups had statistical significance differences (P<0.05), hippocampus BDNF mRNA, IGF-Ⅱ mRNA and NF-κBmRNA and protein expression reduced after rich environment. Conclusion:1) Prenatal stress can make the maternal behavior, activity habit and the ability of fertility alter.2) Chronic stress during pregnancy to offspring will access to high stress state, at the same time, the ability of learning memory and work space decreased, moods of them may appear similar phenomenon of anxiety and depression.3) The decrease in the ability of learning and memory after chronic stress during pregnancy affecting offspring was associated with elevated plasma corticosterone in maternal and child.4) The decrease in the ability of learning and memory after chronic stress during pregnancy affecting offspring was related with the changes of hippocampus central neurotransmitter and decreased of BDNF, IGF-Ⅱ, Arc and NF-κB protein expression.5) Rich environment intervention can improve the ability of learning and memory of offspring, increase the activity of them, enhance exploration and the ability to adapt to the new environment, boost the feeling of pleasure.6) Improving the ability of learning and memory and changed mood of offspring during pregnancy of chronic stress after rich environment are caused by decreased plasma corticosterone, increased central neurotransmitter secretion and boosted Brain-derived neurotrophic factor, The insulin-like growth factor receptor-Ⅱ, Nucleartranscri-ption factor-κB, Synaptic plasticity related proteins-Arc in hippocampus. |
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