| Background:Depression is a stress-related mental disorder that leads to high morbidity and mortality. According to World Health Organization statistics’data, there are three hundred and fifty million depression patients in worldwide. However, so far the treatment and prognosis of depression is still not optimistic, which is mainly due to the ambiguity of etiology mechanisms in depression. Therefore, in-depth study of the pathogenesis of depression is very important.Stressors exposure is considered to the cause of depression onset and it is considered as a major risk factors which has far-reaching influence on the development of the depression[1,2].Based on the important role of stress on the onset of depression, the stress animal model of depression is now widely applied in studying the etiology of depression and antidepressant pharmacological[3,4,10] However, a part of the individual who subjected to stress stimulation are able to cope with stress and will not develop to depression, which also can be observed in stress stimulus animal models of depression[5-8]. Such phenotype is described as stress resistance, which is particularly important to stress-induced depression study.Limitations of the effects of antidepressants is another major challenge to depression treatment. Selective serotonin reuptake inhibitors (SSRI) are the antidepressant drugs that are widely used in clinical. However, in patients with antidepressant treatment,40%of patients is unable to be clinical remission, shown the resistance to treatment. Also, the phenotype of antidepressants resistance can be easily found in animal stress model of depression. Studying the mechanisms of resistance to drug therapy plays an important role on clarifying the mechanism of antidepressant efficacy. However, so far, relevant research for drug treatment resistance is rarely reported.Due to the requirements of medical ethics and limitations of technique, to in-depth study the mechanism of depression in patients is limited. Therefore building an ideal animal model is the key to further study the mechanism of depression onset and therapeutic, which is the purpse of this study. Chronic unpredictable mild stress model (CUMS) provides a unique platform, which can mimic most of the core symptoms of depression. These " symptoms " can also be reversed by antidepressant[3,4,10]. So the CUMS model provided the feasibility of studing the depression pathogenesis and treatment in one model. In this study, based on CUMS model, we aimed at the establishment of stress sensitivity, stress resistance, fluoxetine treatment response and fluoxetine treatment resistance model, so that we can study the mechanism of depression onset and treatment the from the perspective of stress resistance and drug resistance.Hippocampus is an important area associated with stress stimulation[11,12]. When stressor from both within and outside the brain is inputed into the hippocampus, the neuroplasticity regulation will occur in hippocampus to adapte to the environment. This neuronal plasticity response to stress stimuli is closely related to the mechanisms of depression pathogenesis and antidepressants efficacy. Numerous studies have shown that the hippocampal volume in depression patients is significantly reduced comparing with the normal population’s hippocampal volume[13], And a negative correlation can be find in hippocampus volume decrease between course of the disease[14-16] Further research on hippocampal neurons structural shows the structure changes is presented in hippocampal neurons, such as reducing in cytogenesis,cellular shrinkage, decreased numbers of astroglia, reduced gliogenesis, dendritic atrophy and reduction of dendritic spines [17-19]. In addition, antidepressant treatment will restore the reduction of dendritic spines in the hippocampus of depression[20,2.].The ability of dendritic remodeling induced by antidepressant treatment is the basis for the restoration of behavioral homeostasis by antidepressants[22]. Therefore, abnormal hippocampal neuroplasticity regulation of dendrite and dendritic spines is an important cause of depression onset, and also involved in the mechanisms of antidepressants efficacy[23].Many studies have shown that Eph family proteins is an important protein which regulat dendrites and dendritic spine morphogenesis. Inactivation of Eph receptor in hippocampal neurons will stop dendritic spines development and the number of mushroom-like dendritic spines,the mature dendritic spines will significantly decrease[24]. Also, Eph gene knockout mice present the same dendritic spines dysregulation,the density of immature dendritic spine was significantly reduced[25-27]. In the other hand, Eph family protein also plays an important role in learing and memory regulation[27-29]. In the hippocampal slices from EphB2konkout mice, protein synthesis-dependent long-term potentiation (LTP) was impaired, which is highly related to the mechanisms of learning and memory[30]. In vivo, ephrin-A3-knockout mice shows a significantly reducation in the freezing responses to the contextual[27]. Additionally, research shows infusing EphA antagonist immunoadhesin into hippocampal induces impaired performance in T-maze spontaneous alternation and context-dependent fear condition, while infusing EphA agonist immunoadhesin will result in enhanced performance on these tasks[31]. Moreover, one research combining with high throughput DNA sequencing and microarrays for whole genome scanning shows the localization of Eph are close to some mental disorders related gene locus on same chromosomal, such as schizophrenia and bipolar disorder[32] Due to the biological function of Eph family protein, we can assume that Eph family proteins may also be involved in the neural plasticity regulation of depression pathology. However, research involved Eph family protein in mechanism of depression onset and antidepressants efficacy is rarely reported.Object:Based on the chronic unpredicted mild stress (CUMS) model of depression and selective serotonin Reuptake Inhibitors, we attempt to establish stress sensitivity, stress resistance, fluoxetine treatment response and fluoxetine treatment resistance phenotype from the same CUMS model, so that we can study the mechanism of depression onset and treatment the from the perspective of stress resistance and drug resistance. Meanwhile, we investigated the expression of Eph family protein in stress susceptibility/resilience and drug susceptibility/resistance rats.Method:(1) Rats sucrose intake training and surose intake baseline evaluation: sucrose intake training began at the first5weeks, traning sucrose intake twice a week during the first3weeks and training once a week during in the last2weeks. The mean value of the final four testing was defined as rats sucrose intake baseline value.(2)3weeks of stress continuous exposure:CUMS rats were exposed to various mild stressors for21days. No same stressor was given to any rats for two consecutive days. Control rats would not receive any stress exposure.(3) stress susceptibility or resilience sorting out:measure rats sucrose intake at the end of21days stress exposur. Based on the decrease percentage of sucrose intake, if rats decrease in the sucrose intake>25%, rats were identified as CUMS-susceptible rats(CUMS group); if rats decrease in the sucrose intake<10%, rats were identified as CUMS-resilient rats (CUMS-Res group).(4) stress susceptible rats were stochasticly allocated to receive fluoxetine (CUMS-Flx group)(10mg/kg) or saline (CUMS-V group) administration for28days. CUMS-Flx group and CUMS-V group were received continuous stressor exposure respectively.(5) Sorting CUMS-fluoxetine responders and non-responders:at the end of28days, based on the increase percentage of sucrose intake, if rats increase in the sucrose intake>20%, rats were identified as CUMS-fluoxetine responders rats(CUMS-Flx group); if rats increase in the sucrose intake<10%, rats were identified as CUMS-fluoxetine non-responders rats (CUMS-Flx-Res group).(6) Testing rats body weight and open field spontaneous activity before beginning of chronic stress, end of21days stress exposure and end of28days fluoxetine administration, in order to further assess depression-like behavior.(7) After Anaesthetizing rats, we dissected hippocampus from the whole brain and purify the total protein from the hippocampus. Following by detecting protein’s concentration, western blot were used to measure the expression of EphA4and ephrinA3in each group.Result:(1) after21days of stress exposure, CUMS-susceptible rats (CUMS) and CUMS-resilient rats (CUMS-Res) showed a significantly sucrose consumption difference (P<0.01). Comparing to the control group and CUMS resilience group, the sucrose intake body weight, total distance moved and average velocity in CUMS group were significantly decreased (P<0.01). However, the sucrose intake, body weight, total distance moved and average velocity of CUMS-Res rats had no significant change when comparing to the control rats and CUMS rats (P>0.025).(2) after28days of fluoxetine administration, sucrose consumption showed significant differences among control, CUMS-V group, CUMS-Flx group and CUMS-Flx-Res group (P<0.01). Comparing to CUMS-V group, sucrose consumption in CUMS-Flx group was significantly increased (P<0.01). By fluoxetine administration, the body weight, total distance moved and average velocity in CUMS-Flx group were significantly restored when compared to CUMS-V group (P<0.01). However, CUMS-Flx-Res group showed no response to fluoxetine administration,the sucrose intake, body weight, total distance moved and average velocity in CUMS-Flx-Res group showed no significantly restoration when compared to CUMS-Flx group.(3) Western blot data indicated,in hippocampus the expression of EphA4and ephrinA3protein were changed considerably among control, CUMS group and CUMS-Res group (P<0.01). The expression of EphA4in CUMS group showed significantly decrease(P<0.01), while the expression of ephrinA3showed significantly increase when compared to control group(P<0.01). However, in CUMS-Res group, no change of EphA4and ephrinA3protein expression was found comparing to control group. Moreover, after28days fluoxetine injection, fluoxetine induced increased expression of EphA4protein in CUMS-Flx group, which reach to the expression level of control group; again the expression of ephrinA3protein showed down-regulation by fluoxetine administration comparing to control group. However, fluoxetine administration did not recovery the dysregulation expression of EphA4and ephrinA3in CUMS-Flx-Res group, comparing to the expression of EphA4and ephrinA3protein in CUMS-Flx group (P<0.01).Conclusion:Chronic unpredictable mild stress model can mimic anhedonia-like behavior and also the stress resilient and drug resistance. Stress susceptibility showed remarkablely anhedonia and reducation in body weight and spontaneous activity, aloing with dysregulation of EphA4and ephrinA3protein expression. However, stress resilient rats can cope with the stress exposure and did not show the anhedonia、reducation in body weight and spontaneous activity, also the dysregulation of EphA4and ephrinA3protein expression, which indicated EphA family protein may involved into the mechanisms of stress-induced depression and stress resilience. On the other hand, fluoxetine administration can restore the anhedonia-like behavior, reducation in body weight and spontaneous activity induced by chronic unpredictable mild stress, EphA family protein may also play a role in mechanisms of antidepressant restoration of behavior abnormality. |
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