| Stress is a sum of nonspecific response for the body to cope with various endogenous or exogenous stimuli.Moderate stress factors can make human body to adjust self psychological and physiological state reasonably,is beneficial for the improvement of their adaptive ability and activity efficiency.But excessive stress or long-term chronic stress could affect the body's psychosomatic health and quality of life.In the face of the same or similar stressors,different individuals showed different adaptive capacity.This is associated with the physical condition,physical quality,individual experience and cognitive evaluation,is the so-called of the individual diversity and subjectivity to stress response.It have been proved that in the process of aging,the aging brain also are more likely to suffer from degenerative disease in nervous system.Meanwhile,the changes of body's internal environment also affect the brain's response to stress.Then whether the changes of brain structure and function result from brain aging can make the body's response to stress more strongly? Whether the cognitive decline caused by stress is affected by the brain aging? What are the mechanisms? Although a lot of research have explored the effect of stress on brain function,the relationship between chronic stress,aging and cognitive function and it's related mechanisms still needs to be further explored.Unpredictable Chronic Mild Stress(UCMS)is a recognized and commonly used model to study the mechanism of stress.It use a variety of psychological and/or environmental stressors to simulate the stress condition which similar to human environment.Weeks of chronic stress can make the rodents showed various pathologic manifestation related to stress,including changes in emotion,neuroendocrine,neurochemistry and behavior,etc.SAMP8 mice,an ideal model to study aging,is characterized by cognitivedecline(rapid aging)and appears similar neuropathological changes to alzheimer's disease in early stage.SAMR1 mice is resistant to early senescence and are usually set as controls.Morris water maze and Novel object recognition test are two classic experiment method to examine cognitive function of rodents,evaluating the spatial and episodic memory changes in cognitive function related to hippocampus respectively.Synaptic plasticity gradually changed after birth,is relatively stable in adult stage and slowly degenerate in the phase of old age.Hippocampal synaptic plasticity plays an important role in the process of learning and memory formation,and development of neurodegenerative disease as well.The structure and function changes in the hippocampal synapse can lead to the change of cognitive function.Thus,hippocampal synaptic plasticity can be regarded as neurobiological basis of brain aging related cognitive dysfunction.Fluoxetine,a kind of selective serotonin reuptake inhibitors,is mainly used for the treatment of depression.Fluoxetine can prevents neural toxicity,effectively promotes the differentiation of neurons,enchances synaptic plasticity,possesses neuroprotective effect,ameliorates the cognitive dysfunction.A previous study in our research group showed that fluoxetine can improves cognitive function and depressive behavior in adult SD rats,while no research focus on the effect of fluoxetine on cognition in chronic stress challenged aged SAMP8 mice.In this study,we used SAMP8 and SAMR1 mice to observe the influence of UCMS on neuropathology,learning and memory ability and structural synaptic plasticity.We further explored the therapeutical effect of SSRI drugs fluoxetine on stress-induced cognitive dysfunction in SAMP8 and SAMR1 mice,and investigated it's potential mechanisms.Part one The effect of chronic stress on the cognitive function and thePart one The effect of chronic stress on the cognitive function and the ultrastructure of neuron in SAMP8 miceObjective: To observe the effect of chronic stress on the cognitive function and the ultrastructure of neuron in SAMR1 and SAMP8 mice,and explore whether there is difference in stress tolerance of brain between two kinds of mice.Methods:1 Male 6-month-old SAMP8 and SAMR1 mice were randomly divided into 4 groups as follows:1)R1,SAMR1 mice that were not exposed to chronic mild stress;2)UCMS+R1,UCMS-challenged SAMR1 mice;3)P8,SAMP8 mice that were not exposed to chronic mild stress;4)UCMS+P8,UCMS-challenged SAMP8 mice.Mice were exposed to various randomly scheduled,low-intensity social and environmental stressors 2-3 times a day for4 weeks.2 Weighed the mice at onset and end of the UCMS experiment to reflect the general condition of mice.3 After 4 weeks of UCMS,the cognitive function among different groups was tested by NOR and MWM.4 TEM was used to observe the neurons and synaptic ultrastructure in hippocampus CA1 area among different groups after behavioral tests.Results:1 Before the experiment,there was no difference on the body weight between R1 and UCMS+R1 group(P>0.05),so does P8 and UCMS+P8 group(P>0.05).At the end of the experiment,there was no difference on the body weight between R1 and UCMS+R1 group(P>0.05);compared with the P8 mice,the weight of UCMS+P8 mice was decreased(P<0.05).The results show that chronic stress reduces the weight of SAMP8 mice,but has no obvious effect on the body weight of SAMR1 mice.2 NOR: The total time of the mice touching the two objects was no significant difference among different groups during the NOR test at 1h or 24h(P>0.05).Compared with R1 group,the cognitive index was decreased in P8 group at either 1 h or 24 h(P<0.05),compared with P8 group,the cognitive index was decreased in UCMS+P8 group at either 1 h or 24 h(P<0.05);There was no difference on the cognitive index between R1 and UCMS+R1 group(P>0.05).3 To determine the effect of chronic stress on cognition in the SAMR1 and SAMP8 mice,we evaluated hippocampal-dependent spatial learning and memory ability by MWM experiment.For the navigation test: There was no difference on the escape latency between R1 and UCMS+R1 group(P>0.05);compared to the R1 group,the escape latency was significantly increased in the P8 group(P<0.05);However,the chronic mild stress challenge further increased this latency(P<0.05,UCMS+P8 versus P8 group).For the probe test,after the removal of the platform,the number of crossings in the platform area was significantly decreased in the P8 group compared to the R1 group(P<0.05);However,the chronic mild stress challenge further decreased the number of platform crossings(P<0.05,UCMS+P8 versus P8).No statistically significant difference was found in the platform crossings between the R1 and UCMS+R1 groups.These data suggest that cognitive functions decreased with brain aging,and that chronic stress aggravated the cognitive deficit in the SAMP8 mice.The stressors in our experiment had no effect on the cognition of the SAMR1 mice.4 TEM was used to observe the neurons and synaptic ultrastructure in hippocampus CA1 area among different groups.R1 group: Hippocampal neuron membrane can be seen clearly,cytoplasmic matrix and ribosome distributed evenly,structure of mitochondrial membrane and mitochondrial crista were intact,rough endoplasmic reticulum rendered laminar or tubulovesicular form,golgi complexes consists of single layer(single layer form four to eight parallel arranged flat sac,and the lumen was formed),nucleoplasm distributed evenly;Number of synapses were abundant,the structure of presynaptic membranes,postsynaptic membranes and synaptic clefts were clear,synaptic links were obvious,number of synaptic vesicles were abundant.UCMS+R1 group: there were no significant difference between R1 and UCMS+R1 group.SAMP8 group: Hippocampal neuron membrane fairly intact,cytoplasmic matrix and ribosome still distributed evenly,mitochondrial crista and membrane mixed partially,rough endoplasmic reticulum swelled mildly,membrane structure of some golgi complexes were vague,structure of the nucleus and nuclear membrane fairly intact,edema can be seen in cytoplasm and nucleus;Synaptic structure less clear,presynaptic terminal swelling can be seen occasionally.UCMS+P8group: Very poor state of hippocampal neurons,cell membranes were vague,edema can be seen in cytoplasm,the cytoplasm appeared uneven matrix,the number of various organelles were reduced(such as mitochondria,endoplasmic reticulum,golgi complexes),mitochondrial crista and membrane mixed seriously,rough endoplasmic reticulum degranulation,chromatin edge moved and nuclear membranes were indistinct;Presynaptic membranes were swelled and destroyed,postsynaptic membranes thickened,synaptic clefts unclear.Part two The effect of chronic stress on the synaptic plasticity associated markers in SAMP8 miceObjective: To observe the protein and m RNA expression of synaptic plasticity associated markers in hippocampus of SAMR1 and SAMP8 mice,then investigate the molecular biological mechanisms of chronic stress,clear the reason of the lower stress tolerance in SAMP8 mice.Methods:1 The animal grouping and model establishment were similar to part one.2 The protein expression of PSD95,SYN,BDNF and Trk B in hippocampus among different groups were detected by western blotting analysis.3 The m RNA expression of PSD95,SYN,BDNF and Trk B in hippocampus among different groups were detected by RT-PCR method.Results:1 PSD95 and SYN expression in the P8 group were significantly decreased compared to the R1 group(P<0.05).PSD95 and SYN expression in the UCMS+P8 group was further decreased compared to the P8 group(P<0.05).No statistically significant difference was found in the protein expression between the R1 and UCMS+R1 groups.The RT-PCR results were similar to the results for protein expression.PSD95 and SYN m RNAs were high in the R1 groupcompared with the P8 group(P<0.05).The mRNA expression in the UCMS+P8 group was significantly lower compared to that of the P8 group(P<0.05).No statistically significant difference was found in the m RNA expression between the R1 and UCMS+R1 groups.These results suggest that chronic stress downregulates the protein and m RNA expression of PSD95 and SYN.2 BDNF and Trk B expression in the P8 group were significantly decreased compared to the R1 group(P<0.05).BDNF and Trk B expression in the UCMS+P8 group was further decreased compared to the P8 group(P<0.05).The RT-PCR results were similar to the results for protein expression.BDNF and Trk B m RNAs were high in the R1 groupcompared with the P8 group(P<0.05).The m RNA expression in the UCMS+P8 group was significantly lower compared to that of the P8 group(P<0.05).No statistically significant difference was found in the protein and m RNA expression between the R1 and UCMS+R1 groups.These results suggest that chronic stress downregulates the protein and m RNA expression of BDNF and Trk B.Part three The effect of fluoxetine on the cognitive function in chronic stress-challenged SAMP8 mice and exploration of its mechanismsObjective: To study the effect of fluoxetine(SSRI)on the cognitive function in chronic stress-challenged SAMP8 mice via UCMS model and explore its potential mechanisms.Methods:1 Male SAMP8 mice(6-month-old)were randomly divided into 3 groups:P8 group(SAMP8 mice with no chronic mild stress),UCMS group(UCMS-challenged SAMP8 mice),and Flu group(UCMS-challenged SAMP8 mice receiving fluoxetine).We chose male SAMR1 mice(6-month-old)as the normal control(R1 group).2 Unpredictable Chronic Mild Stress(UCMS)and experimental drugs Mice were exposed to various randomly scheduled,low-intensity social and environmental stressors 2-3 times a day for 6 weeks.The stressors were same as part one.Fluoxetine was prepared with saline(Na Cl 0.9%)to a final concentration of 2mg/ml,and injected intraperitoneally in a concentration of 20mg/kg(10ml/kg).When exposure to chronic stress for two weeks,mice in Flu group were administrated with fluoxetine for 4 consecutive weeks.Mice in other groups were injected intraperitoneally with equal doses of saline.3 Weighed the mice at onset and end of the UCMS experiment to reflect the general condition of mice.4 After finishing fluoxetine treatment,the depressive-like behavior was tested by SPT,the cognitive function among different groups was tested by MWM.5 TEM was used to observe the neurons and synaptic ultrastructure in hippocampus CA1 area among different groups after behavioral tests.6 The protein expression of PSD95,SYN,BDNF,Trk B,Bcl-2 and Bax in hippocampus among different groups were detected by western blotting analysis.7 The m RNA expression of PSD95,SYN,BDNF and Trk B in hippocampus among different groups were detected by RT-PCR method.Results:1 Before the experiment,there was no difference on the body weight between P8 and UCMS+P8 group(P>0.05).At the end of the experiment,compared with the P8 mice,the weight of UCMS mice was decreased(P<0.05);Compared with the UCMS mice,the weight of UCMS mice was increased(P<0.05).The results show that chronic stress reduces the weight of SAMP8 mice,while fluoxetine treatment increases the body weight of UCMS-challenged SAMP8 mice.2 SPT: Compared with the P8 mice,the sucrose preference value of UCMS mice was decreased(P<0.05);Compared with the UCMS mice,the sucrose preference value of UCMS mice was significantly increased(P<0.05).The results indicate that UCMS induces the depressive-like behavior of SAMP8 mice,while fluoxetine treatment ameliorates the behavior.3 To determine the effect of fluoxetine on cognition in male SAMP8 mice after chronic stress administration,we evaluated hippocampal-dependent spatial learning and memory ability by a MWM experiment.Compared to the R1 group,the escape latency was significantly increased in the P8 group(P<0.05).However,the chronic mild stress challenge further increased this latency(P<0.05).The escape latency was significantly decreased after fluoxetine treatment(P<0.05,Flu versus UCMS group).For the probe test,after the removal of the platform,the number of crossings in the platform area was significantly decreased in the P8 group compared to the R1 group(P<0.05),the chronic mild stress challenge further decreased the number of platform crossings(P<0.05).However,the number of platform crossings was significantly increased after fluoxetine treatment(P<0.05,Flu versus UCMS group).These data suggest that chronic stress aggravated the cognitive deficit in the SAMP8 mice,while fluoxetine treatment reversed the cognitive deficit.4 Fluoxetine treatment improved the damaged neuronal and synaptic ultrastructure of hippocampus CA1 area in chronic stress-challenged SAMP8 mice.5 Compared to the R1 group,PSD95 and SYN expression(protein and m RNA)in the P8 group were significantly decreased(P<0.05).Compared to the P8 group,PSD95 and SYN expression in the UCMS group was further decreased(P<0.05).Compared to the UCMS group,PSD95 and SYN expression in the Flu group was increased(P<0.05).The results indicate that chronic stress downregulated the protein and m RNA expression of PSD95 and SYN in the SAMP8 mice,while fluoxetine treatment reversed the changes.6 Compared to the R1 group,BDNF and Trk B expression(protein and m RNA)in the P8 group were significantly decreased(P<0.05).Compared to the P8 group,BDNF and Trk B expression in the UCMS group was further decreased(P<0.05).Compared to the UCMS group,BDNF and Trk B expression in the Flu group was increased(P<0.05).The results indicate that chronic stress downregulated the protein and m RNA expression of BDNF and Trk B in the SAMP8 mice,while fluoxetine treatment reversed the changes.7 Compared to the R1 group,the protein level of Bcl-2 in the P8 group was decreased(P<0.05);Compared to the P8 group,the protein level of Bcl-2in the UCMS group was further decreased(P<0.05);Compared to the UCMS group,the protein level of Bcl-2 in the Flu group was increased(P<0.05).Compared to the R1 group,the protein level of Bax in the P8 group was increased(P<0.05);Compared to the P8 group,the protein level of Bax in the UCMS group was further increased(P<0.05);Compared to the UCMS group,the protein level of Bax in the Flu group was decreased(P<0.05).The results indicate that UCMS decreased the protein expression of Bcl-2 and increased the protein expression of Bax in the SAMP8 mice,while fluoxetine treatment reversed the changes caused by chronic stress.Conclusions:1 Compared to the SAMR1 mice,the cognitive ability was declined in SAMP8 mice,accompanied by the damaged neuronal and synaptic ultrastructure of hippocampus CA1 area.The results of behavioral tests and TEM showed that four consecutive weeks UCMS have no effect on the cognition,neuronal and synaptic ultrastructure in SAMR1 mice,but aggravated the cognitive deficit,lead to neuronal and synaptic ultrastructure seriously damaged in the SAMP8 mice.The effect of CUMS on the cognitive function and neuropathology were different in two kinds of mice,6-month old SAMR1 mice showed almost normal modulation to chronic stress,while6-month old SAMP8 mice exhibited decreased stress toleration.This indicate that aging is accompanied by decreased stress toleration,and more sensitive to the damage caused by the stress.2 Compared to the 6-month old SAMR1 mice,the expression of hippocampal synaptic plasticity associated markers and BDNF-Trk B were further decreased in the age-matched SAMP8 mice after exposure to UCMS.In the influence of endogenous factors(aging)and exogenous factors(UCMS),SAMP8 mice showed decreased stress toleration,the impairments of hippocampal synaptic plasticity and BDNF-Trk B pathway might be the neurobiological mechanism of chronic stress-induced cognitive decline in SAMP8 mice.The present study improves our understanding of chronic stress-induced cognitive impairment in the elderly,and maybe offers more theoretical basis for research on the effective treatment of cognitive impairment or dementia.3 Fluoxetine treatment ameliorated the depressive-like behavior and cognitive dysfunction in chronic stress-challenged SAMP8 mice,improved the damaged neuronal and synaptic ultrastructure of hippocampus.Fluoxetine exerts its neuroprotective effect through multiple-pass.Fluoxetine treatment increased the expression of hippocampal synaptic plasticity associated markers in chronic stress-challenged SAMP8 mice,the improvement of hippocampal structural synaptic plasticity and inhibition of apoptosis pathway might be a potential mechanism of fluoxetine-related modulation of cognition in chronic stress-challenged SAMP8 mice.The activition of BDNF-Trk B signaling pathway might play a crucial part in the fluoxetine-related modulation of synaptic plasticity and inhibition of apoptosis pathway.These studies provides more evidence for the treatment of cognitive dysfunction after chronic stress. |
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