| The mental and emotional problems of left-behind chrldrens are easier to be noticed and observed, while cognitive deficits are often easily ignored. Nowadays, increased experimental and clinical findings have shown that left-behind children, which is similar to children exposed to long-term undesirable stress events, such as ignores, discrimination, diatribe and maltreatment, has problems in cognitive functions, including spatial memory, working memory, judgment and decision-making ability. At early developmental, chronic stress can damage body’s memory capacity to adul thood. In China, the problem of left-behind children become getting more serious, according to the report of the sixth census data, which has shown that the number of rural left-behind children is more than 60 million, among this population, preschool left-behind children (0-5 years old) accounted for 38.37%, and there is rising trend. A chronic lack of this population even without parental care and guardianship were long living in full harmful stimulating environment, and some of them already have mental defects and emotional problems and the mental development also lagged behind the normal growth of the same age children in their family.In our country, a directional thinking has been recognized by many academics and experts, and they hold the opinion that left-behind children means separation from parents, therefore the animal models should be choosed the mother-child separation. However, in foreign countries, this thinking is inaccurate, the animal model of mother-child separation can not mimic the separation of human society between parents and young children, and vast majority of research results in the field in abroad has shown that mother-child separation after 30 minutes to 3 hours (currently the longest time for mother-child separation) is beneficial to the young rather than harmful. One of the possible reasons is that the dam will give more caress stimulation after mother-child separation more than 30 minutes to 3 hours.In this study, we adopt a well-designed and investigated early-life stress animal model to substitute mother-child separation animal model, and then, to research the effect of chronic stress on dendritic spines and excitatory synaptic of hippocampal pyramidal neurons by applying the morphological, molecμl ar biology techniques and behavioral tests.Objectives1. To observe the changes of body weight, weight of global cerebral, thymus and bilateral adrenal at P9, P21, and P120 mice that experienced 1 week chronic stress started at P2.2. To observe the changes of dam-pups behavior including the time of lactation, the frequency of dams leaving nest, the time of pups leaving nest. The behaviors will be measured at P2-P9 when the pups are experiencing the early-life stress,1 h per day.3. To analyze the dendrites and dendritic spines of hippocampal pyramidal neurons at ages of P9, P21, and P120 by using Golgi staining method.4. To detect the capacity of cognition and memory, and analyze the psychological and emotional characters of ES mice and compared with their controls at16 weeks of age via object recognition and object location tasks, sucrose preference test and open field test.5. To investigate the expression of synaptic function-associated proteins including GluRl and PSD-95 using immunocytochemistry when the mice are 4 months old.Methods1. To establish early-life stress animal models:C57BL/6J mice were used in current study and grouped into ES and control groups. Rearing environment was changed started from P2 to P9, and last 1 week.2. After establishing early-life stress animal models, we record the body weight of animals at P9/P21/P120 three time points. During the day P2-9 when pups are experiencing early-life stressthe time of lactation, the frequency of dams leaving nest, and the time of pups leaving nest were observed for 1 hour per day. In the morning (0900) of P9/P21/P120, some of ES/Ctl mice are sacrificed by cervical dislocation after weighing, then to collect their blood and isolate their global cerebral, thymus and bilateral adrenal. Moreover, weighing some of 4 months old ES/Ctl mice, perfusion fixation via their left ventricle (aortic arch) successively with physiological saline and 4% paraformaldehyde for immunochemistry.3. Using ELISA CORT kit to detect plasma levels of corticosterone from the supernatant stored.4. At 16th week, to complet behavioral experiments in sequence, including object recognition test, object location test, sucrose preference test and open field test. Some of 4 months old ES/Ctl male mice are used to Golgi staining.5. Some of 4 months old ES/Ctl mice are used to immunohistochemical, and then to analyse the GluR1 /PSD-95 expression and distribution on dendritic spines and excitatory synaptic of hippocampal pyramidal neurons.Results1. During P2-9, within an hour, the frequency of dams leaving the nest and the time of pups leaving the nest show significantly difference, the ES groups are higher than the control group (P<0.05), while the time of dam-pups lactation show no significant difference (P>0.1)2. From P2 to P9, pups’ body weight gain show that the ES mice significantly lower than the control group (P<0.05). At P9 and P21, the thymus body weight ratio of ES pups significantly lower than the control group (P<0.05),while the ES pups’ bilateral adrenal body weight ratio show no significant difference (P>0.1)3. At P9, the corticosterone level of pups show that the ES pups significantly higher than the control group (P<0.05)4. Golgi staining show that the number of mature dendritic spines is down-regulated, especially, the proportion of mature dendritic spines is significantly reduced.5. Object recognition and object location tests show that the discrimination index (DI) of ES group is significantly lower than the control group (P<0.05) after familiarization 1h (Short-term memory test), while the control group DI show significantly higher the ES mice (P<0.05) after familiarization 24h (Long-term memory test). Sucrose preference tests shows that sugar preference scores of ES mice is significantly lower the control group (P<0.05). The result of open field tests show that ES mice is more likely to be anxiety, the scores that reflect the psychology and emotion of ES mice significantly lower than the control group (P<0.05)6. At P120, immunohistochemic for 4 old months mice show that the expression of synaptic function-associated proteins-PSD95 and GLUR1, is down-regulated in the area of hippocampal pyramidal cells.ConclusionWhen the young body in poor stress for a long period, HPA-axis and the pathway of hippocampal edge loop are activated, stress regμl atory factor increased in hippocampus negative regμl ator synapses in dendrites and dendritic spines, then influence establishment and improvement of normal nerve fibers pathway, eventually, leading to cognitive impairment. |
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