| Stress is a non-specific systemic body reactions caused by a change in the external environment. The body reactions include neuropsychiatric, endocrine system, immune system et al. Among the crowd suffered from stress, the military is a special group. The occupational characteristics of military means they are often faced with high intensity stress. Combat stress is a stress reaction which often appeared in battlefiled, containing acute stress and chronic stress. Acute combat stress appeared immediately or within hours when the military entered into the sudden, unexpected, horror, intense, dangerous, brutal environment or a few hours after the fight. The performance is physiological over-arousal even life threatened, which last a few minutes or hours. When the military suffer combat stress reaction, they will show poor memory, impaired concentration, diminished mental alertness, visual and verbal ability, aimless movement, decreased ability to judge, difficult to perform even simple tasks, decreased military operations ability, even post-traumatic stress disorder (PTSD). Chronic combat stress is a reaction when the military spent a long time on the battlefiled. The performance is low activity, out of the group, depression, alcohol and substance abuse, violation of discipline and so on. Combat stress finally not only seriously affected the effectiveness of troops, but also is one of the main causes of war wounded troops downsizing. For the militry, the duration of the acute combat stress is the key period when they execute the task of fight, rescue, maintenance of stability and the parade. And in the morden with high technology, cognition plays the increasing weight in battle. So, if there are methods to prevent the cognitive impairment caused by acute stress in the battlefield, the combat effectiveness will be greatly improved. The stress response and tolerance have much individual differences. Due to the current screening of high stress sensitive people is still a lack of specific biological indicators, so assessment scale is still a clinical diagnosis with the shortcomings that subjectivity, lower repetition, accuracy and specificity. Furthermore, the main interventions for acute combat stress are psychological counseling, psychotherapy, which belongs to post-intervention, lacking of pre-intervention and medicine for prevention and treatment. In this study, for cognitive impairment caused by acute combat stress, on one hand is to find biomarkers for screening anti-stress people, on the other hand is to find that the drug for prevention.To achieve the both aims above, first of all, in this research, a model of cognitive impairment induced by acute multimodal stress was established by simulating the sudden acute complex stress factors in battlefield. Then the learning and memory ability and concentration of corticosterone in plasma of the model were investigated. Secondly, dendrite and synapse structure of the model was observed employing Golgi staining and transmission electron microscope. Thirdly, the mRNA expression of 90 genes associated with stress and cognition was detected by PCR array. Correlation between differentially expressed genes and dendritic morphology alteration was performed using two-tailed Pearson analysis, which also applied for differentially expressed genes between different regions among hippocampus, cerebral cortex and peripheral blood. Then bioinformatics were applied to analyze the differentially expressed genes and found the marker molecules related with cognitive impairment induced by acute multimodal stress. Finally, the prevention of LW active fraction combination (LW-AFC) to cognitive impairment induced by acute multimodal stress was detected and found LW-AFC could improve the cognitive impairment by regulating some differentially expressed rmolecules in cerebral cortex. The results as follows:Part one:Mechanism of cognitive impairment induced by acute multimodal stress1. Preparation of animal model exposed to acute multimodal stressTo simulate the gusty acutely complex stress factors in the life, the BALB/c mice were used for preparation of an animal model under combat stress which contains 5-hr jostling, noise, bright light, food and water deprivation, restraint, oscillation collide with each other, companion uncomfortable state. Acoording to the different parameters of mild (67,200 or 600Lux),jostling (77,130 or 220rpm) and noise (83,100 or 120dB), the stress were devided into three different degrees. Then the ability of learning and memory and the concentration of corticosterone in plasma were investigated under the three conditions. The results are as follows:1.1 The ability of learning and memory of the animal model exposed to acute multimodal stress1.1.1 Passive avoidance responseThe passive avoidance response of BALB/c mice was investigated by step-down test after three different-degree stress followed by mood recovery period 90min after stress. The results show that the percentage of successful mice and latency was degree-dependently decreased after stress, in which the latency of severe stress group was significantly decreased compared with the control group (P<0.05). Moreover, the number of errors was elevated degree-dependently by stress, in which the number of errors of severe stress group was significantly increased compared with the control group. These data indicated that acute multimodal stress induced a degree-dependent decrease of latency and percentage of successful mice while increase of the number of errors in the step-down test. This indicated that acute multimodal stress degree-dependently impaired the passive avoidance response of mice.1.1.2 Object recognition memoryObject recognition memory of BALB/c mice was detected by the object recognition test. The results show that there was a significant longer exploration time of new object than that of old object in control group and mild stress group, exactly there was obvious difference of exploration time between new object and old object. This indicated that the animals still have the characteristics of being interested in the novel objects. While there was no difference in exploration time of the old and new object between medium stress group and severe stress group, it indicated that stress damaged the instinct of being interested in the novel objects. These results indicated that medium and severe acute multimodal stress resulted in object recognition memory impairment.1.2 The corticosterone level in plasma of the animal model exposed to acute multimodal stressTo detect the change of corticosterone level in plasma of tested mice under different-degree acute multimodal stress, we obtained the plasma from the blood of animals in the test after the start of three different-degree stresses 30,60,90 and 120 min and 4,8 and 16min after the end of stress. And then the concentration of corticosterone was detected by ELISA. The results show that corticosterone concentrations in three different-degree stress groups were detected significantly higher than control group during the stress, but there was no significant difference between the three different-degree stress groups. The results suggest that stress can increase the corticosterone concentration in plasma, but there are no significant relationship between the increased rate of corticosterone concentration and the degree of stress.2. The effects of acute multimodal stress on dendritic and synaptic morphologyAt 30 min after BALB/c mice exposed to acute multimodal stress, each group was sacrificed by decapitation, and their brains were removed. The hemi-brains of mice in each group were fixed for Golgi staining for observeing dendritic morphology and transmission electron microscope for observeing synaptic morphology. Then the other hemi-brains of all the mice were dissected out the cerebral cortex and hippocampus for PCR array.The quantitative analysis of whole slide images showed that there was no difference between control and stressed mice for the number of neuron in the hemi-brain, hippocampus and cerebral cortex. Otherwise hours-long multimodal stress deteriorated dendritic and synaptic morphology in hippocampus and cerebral cortex.2.1 Acute multimodal stress damaged the dendritic morphologyThe dendritic length, number of dendritic branch and density of dendritic spine were measured quantitatively in cerebral cortex and sub-region of cortex including of visual cortex, auditory cortex, and olfaction cortex of control and stressed mice. Results showed that severe hours-long multimodal stress decreased the density of dendritic spine in cerebral cortex. The medium and severe hours-long multimodal stress reduced the dendritic length in visual cortex comparing with control group. The severe hours-long multimodal stress decreased the density of dendritic spine in auditory cortex comparing with control group, mild or medium stress group. The mild hours-long multimodal stress increased the number of dendritic branch in auditory cortex.As well, the dendritic length, number of dendritic branch and density of dendritic spine were measured quantitatively in hippocampus and sub-region of hippocampus including of dentate gyrus, CA1, CA2 and CA3 region of control and stressed mice. Results showed that hours-long multimodal stress decreased the density of dendritic spine in hippocampus and CA1 area of hippocampus comparing with control group. The mild hours-long multimodal stress reduced the dendritic length in CA1 area of hippocampus. The severe hours-long multimodal stress decreased the number of dendritic branch in CA3 area of hippocampus compared with control group.2.2 Acute multimodal stress damaged the synaptic morphologyIn order to investigate hours-long multimodal stress effect on synaptic morphology, the synapse structure was observed in the hippocampus of control and stressed mice employing transmission electron microscopy. Results showed that hours-long multimodal stress reduced the number of synaptic vesicle docking on postsynaptic membrane, lessened the curvature of synapse, decreased thickness of postsynaptic dense material, induced synaptic cleft shorten.3. The influence on the mRNA expression of central and peripheralWe detected the expression of 90 genes associated with stress and cognition by PCR array in cerebral cortex, hippocamopus and peripheral blood. The results as follows:3.1 The mRNA expression of hippocampusIn hippocampus, among 90 genes, mild stress induced the expression of 12 genes ACHE, NEFH, BACE1 and so on increased, while KCNA4, GRIA2, GRIA3, PSEN2 and ROCK1 decreased. Medium stress induced the expression of 20 genes ACHE, APBB1, UBQLN1、FGF2 and so on increased, while KCNA4, GRIA3 and PSEN1 decreased. Severe stress induce the expression of gene SNCA、NTRK3、KCNA4 and NOTCH3 decreased. There were seven styles of the change of expression of the mRNA along with different degree of stress, while the expression of the mRNA of KCNA4, NOTCH3, NTRK3 and SNCA degree-dependently decrease after acute multimodal stress.3.2 The mRNA expression of cerebral cortexIn cerebral cortex, among 90 genes, mild stress induced the expression of 50 genes APBB1, CLSTN1, KCNA4 and so on decreased, while KCNA1 increased. Medium stress induced the expression of 26genes GSN, ITM2C, APBB1 and so on decreased, while KCNA1 increase. Severe stress induced the expression of gene APBB1, CLSTN1, KCNA4, NOTCH3, PLAU, RPS6KA1, SYP and TGFB1 decreased, while KCNA1 increased. There were five styles of the change of expression of the mRNA along with different degree of stress.3.3 The mRNA expression of peripheral bloodIn peripheral blood, among 90 genes, mild stress induced the expression of gene BACE1, CRYAB, FGF2, GRIA3, MAPT, LRP1 and RPS6KA1 decreased, while FOS increased. Medium stress induced the expression of gene BACE1, CRYAB, FGF2, GRIA3 and MAPT decrease, while AGER, APP, PSEN1, S100A11 and FOS increased. Severe stress induced the expression of gene BACE1, CRYAB, FGF2, GRIA3 and MAPT decrease, while FOS increased. There were five styles of the change of expression of the mRNA along with different degree of stress. The mRNA expression of BACE1 was downregulation gradually along with different degree of stress, while FOS was upregulation gradually along with different degree of stress. This result indicated that the alteration degree of expression of BACE1 and FOS is closely related with the stress degree and may realted with the degree of cognitive impairment. So the molecules BACE1 and FOS have the potential as the indicator to the degree of cognitive impairment induced by different degree stress.3.4 The correlation analysis between differentially expressed genes and dendritic morphologyIn order to find abnormally expressed genes related with the impaired dendrite structure in hippocampus and cerebral cortex of stressed mice, Pearson correlation analysis was employed to perform analysis of correlation between the expression of genes APBB1, CLSTN1, KCNA4, NOTCH3, PLAU, RPS6KA1, SYP, TGFB1 and KCNA1 and dendritic morphology, including the dendritic length, number of dendritic branch and density of dendritic spine. The results showed that in cerebral cortex, the expression of gene CLSTN1, PLAU and NOTCH3 has negative correlation with the number of dendritic branch in auditory cortex of combat stressed mice, while the expression of gene TGFB1 has positive correlation with the density of dendritic spine in visual cortex.Furthermore in hippocampus expression of gene NOTCH3 has positive correlation with the number of dendritic branch in CA3 region. This indicated that CLSTN1, PLAU, NOTCH3 and TGFB1 may be the most important molecules being response to combat stress.3.5 The correlation analysis of differentially expressed genes among different regionsIn addintion, we found in our study that it was positively correlated of the gene expression alteration of BACE1 in the cortex and in peripheral blood. The results suggest that the mRNA expression of BACE1 detected in peripheral blood may be as an indicator of the expression of BACEl in brain.4. The molecular network involving dendritic morphology damage induced by acute multimodal stressIn order to identifythe distinct molecular phenotype being response to hours-long multimodal stress,CLSTN1, PLAU, NOTCH3 and TGFB1 related with dendritic morphology were used as seed molecular to construct the distinct PPI network being response to stress. These results showed that the network inferred from PPI contained 59 nodes and 136 edges with network diameter 6. In the PPI network, the degree of TGFBl. PLAU, NOTCH3 and CLSTN1 were respectively 28,12,9 and 6, at the core of the network. According to the results of experiment and network analysis, TGFB1, PLAU, NOTCH3 and CLSTN1 may be the key molecules asssociated with dendritic structural damage induced by combat stress.Furthermore, we made functional analysis about moleculesin the PPI networksapplying for GO functional annotation and KEGG pathway enrichment respectively based on DAVID platforms. The results of GO functional annotation show that transmembrane receptor protein serine/threonine kinase signaling pathway, TGF-beta receptor signaling pathway and notch signaling pathway were lie the top three.Then results of KEGG pathway enrichment show notch signaling pathway, complement and coagulation cascades and TGF-beta signaling pathway lie the top three.In combination with the results of GO functional annotation and KEGG pathway enrichment,we found that notch signaling pathway and TGF-beta signaling pathway are most closely related with dendritic structural damage induced by combat stress. So it indicated that acute multimodal stress induced dendritic structural damage then eventually leaded to cognitive dysfunction most likely by Notch and TGF-beta signaling pathway.Part two:Research on the prevention of LW-AFC to acute multimodal stress induced cognitive dysfunction1. Improvement of LW-AFC on the impairment of passive avoidance response ability induced by acute multimodal stressThe passive avoidance response of BALB/c mice was investigated by step-down test. The results show that the percentage of successful mice was in stress was obviously lower than control group. But the positive drug group and LW-AFC goup (three doses) all can relieve the decrease of the percentage of successful mice induced by stress, and in wich the medium dose and high dose had the best effects. The latency of stress group decreased significantly compared with control group, while the positive drug group and LW-AFC goup (three dose) all can relieve the decrease of the latency induced by stress, and in wich the medium dose and high dose had the best effectsMoreover, the number of errors was elevated significatly in stress group which was compared with control group and the medium dose and high dose had the best effects. These data indicated that acute multimodal stress induced a decrease of latency and percentage of successful mice while increase of the number of errors in the step-down test. This indicated that acute multimodal stress impaired the passive avoidance response of mice. LW-AFC dose-dependently increase the latency and percentage of successful mice while decrease of the number of errors.It indicated that LW-AFC administration in advance can relieve the impairment of passive avoidance response induced by acute multimodal stress.2. Regulation of LW-AFC to the differentially expressed genes induced by acute multimodal stressWe detected the expression of 90 genes associated with stress and cognition with PCR array in cerebral cortex after step-down test. The results showed that among 90 genes, the expression of gene NEFH, NEFM, NSF, PLAU, PRKCA, STAT3, STUB1, APBB1, NTRK3, GRIA2 and NOTCH3 decreased in the cerebral cortex after acute multimodal stressed mice comparing with control group. LW-AFC has the tendency to upregulate NEFH, PLAU, STAT3 and STUB1.3. Regulation of LW-AFC to the differentially expressed genes induced by acute multimodal stressIn order to identify the distinct molecular network regulated by LW-AFC in cerebral cortex to improve the cognitive impairment induced by acute multimodal stress, we found four differentially expressed genes NEFH, PLAU, STAT3 and STUB1 which were regulated by LW-AFC.Then the four genes were used as seed molecular to construct the distinct PPI network. These results showed that the network inferred from PPI contained 9 nodes and 10 edges with network diameter 4. The molecules in the PPI network were MAPK1, STAT3, AR, STUB1, NEFH, PLAT, PLAU, MET and HGF. So,it indicated LW-AFC may improve the cognitive impairment induced by Combat stress by regulating the molecular network consist of MAPK1, STAT3, AR, STUB1, NEFH, PLAT, PLAU, MET and HGF.Furthermore, we made functional analysis about moleculesin the PPI networksapplying for GO functional annotation and KEGG pathway enrichment respectively based on DAVID platforms. The results of GO functional annotation show that enzyme linked receptor protein signaling pathway lie the top location.Then results of KEGG pathway enrichment show pathways in cancer lie the top location. So it indicated that LW-AFC may improve the cognitive impairment induced by acute multimodal stress by regulating enzyme linked receptor protein signaling pathway and pathways in cancer.Through the studies above, the preliminary conclusions as follows:1. Different degree of acute multimodal stress can degree-dependently damage the passive avoidance response, object recognition memory, dendritic and synaptic morphology in brain, which may result from the key molucules, PLAU, NOTCH3, TGFB1 and the key pathway, Notch and TGF-beta signaling pathway.2. The altration degree of expression of BACE1 and FOS in peripheral blood is closely related with the stress degree and may realted with the degree of cognitive impairment. So the molecules, BACE1 and FOS, have the potential as the biomarker to indicate the degree of cognitive impairment induced by different degree of stress.3. LW-AFC has a protective effect on the congnitive impairmentinduced by combat stress and the mechnism may regulated the the molecular network consist of MAPK1, STAT3, AR, STUB1, NEFH, PLAT, PLAU, MET and HGF, Which may have the function of enzyme linked receptor protein signaling pathway and pathways in cancer. |
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