| Background:Alzheimer’s disease is the most common neurodegenerative disease, especially present in the middle aged people. Alzheimer’s disease attack rate was gradually increased along with the proceeding of population aging, grave psychological and financial burdens were induced owing to high lethality and mutilation. The prevention and cure for Alzheimer’ s disease already to be one of the most important topics for the current medicine.Alzheimer’s disease is a neurodegenerative disorder characterized by senile plaque pathology, neurofibrillary tangles, neuronal decrease in cortex and the plaque-like vessels in the neocortex and cerebromeninges. The clinical manifestations of AD are considered to memory impairment or loss, behavioral disturbance and recognition deficit. Now, amyloid hypothesis is the most popular explanation for the pathogenesis of Alzheimer’s disease, which deem Aβ aggregation and deposition plays a crucial role in the pathogenesis of AD disease. Various causes of AD induced dysfunction of Aβ metabolism, such as Aβ overproduction and degradation reduce, Aβ aggregation in the specific region of the brain could trigger various kinds of inflammatory reaction, cascade reaction of neurotoxicity, synapse injury and neuronal death, thus the present of hypomnesis and cognitive dysfunction for AD patients.Although AD was not considered to be a typical inflammatory disease, but findings from experimental and clinical have shown that immune dysfunction was closely related to the pathogenesis of AD disease. The deposition of Aβ form immunogen to activate immunoreactions of body along with the Aβ cleaning had breakdown. The previous study had convinced strong immunoreactions were observed in the brain of the AD patients. Previous data showed humoral immunity and cellular immune dysfunction in AD model animals and AD patients. The effect of AD-related immunotherapy further to convinced immuno-inflammatory mechanism for AD pathogenesis. Active immunity not only can produce specific anti-Aβ antibody to prevent and reduce the formation of amyloid-like plaques, but also reduce clinical symptoms of AD disease, improve cognitive ability in animals. However, a few AD patients had some side effects such as meningoencephalitis after administered with vaccination. The autoreactivity of T cells and infiltration of macrophage was observed in the meninges from the patients by autopsy.Immuno-inflammatory mechanism of the process of AD pathogenesis leads us to consider these problems:Aβ deposition-induced inflammatory mechanism participate in morbility of AD, what is the role of the cellular immune in AD? In particular the T cell has what function in the AD pathology process? Aβ aggregation lead to synapse injury and neuronal death, the existence of T cells whether can affect neuronal apoptosis and neurogenesis in AD pathological process. The effects and its mechanisms of T cells on neuronal apoptosis and neurogenesis in AD pathogenesis were not clear and need to explore in the current field.To investigate the role of T cells in AD pathogenesis, the whole study was designed as follows:Objective:To investigate the effects and mechanisms of T cells on neuronal apoptosis and neurogenesis in Alzheimer’s disease pathogenesis.1. To establish the BALB/c mouse model with Alzheimer’s disease induced by beta-amyloid protein, including BALB/c-wild and BALB/c-nude mouse model. Pathological evaluation was carried out for the new BALB/c mouse model.2. To investigate whether the T cells could affect neuronal apoptosis and neurogenesis in BALB/c mouse model with Alzheimer’s disease.3. To explore the potential mechanisms of T cells on neuronal apoptosis and neurogenesis in the pathogenesis of BALB/c mouse model with Alzheimer’s disease by fluorescent quantitation PCR technology.Methods:1. The animals used in this study were48mice (including BALB/c-WT and BALB/c-nude mice respectively24mice), which are randomly divided into two groups, including the experimental group and the control group. The Aβ1-42oligomerization (4μg/2μl) or same volume saline were bilaterally injected into the CA1region of the hippocampus in mice through microinjection method to mimic main pathological characteristics of AD. The experimental group was divided into two groups:BALB/c-WT+Aβ1-42and BALB/c-nude+Aβ1-42group, twelve mice each group, two groups. The control ones include BALB/c-WT+NS and BALB/c-nude+NS group, twelve mice each group, two groups. All animals were killed with anesthesia, and their brains and Peripheral blood were rapidly removed at7and21days after injection.2. HE staining as routine methods, comparison of inflammatory reactions in injection sites between the experimental groups and the controls using light microscope. To detect the deposition of Aβ1-42in the brain for the experimental groups and the control groups using immunohistochemistry method.3. To detect the expression of Bcl-2and Bax protein in neurons of hippocampus in the experimental groups and the controls by immunohistochemistry method at21days after injection.To evaluate the changes of neuronal apoptosis in each group by TUNEL staining.4. New proliferative neurons were marked by DCX (doublecortin), the positive cells showed newborn neurons. To investigate the changes of neurogenesis in hippocampus for experimental and control groups with DCX marker at7days after injection.5. Using fluorescent quantitative PCR to detect the gene expression of IL-2, IFN-gamma in peripheral blood and IL-1β, TNF-a in brain tissue respectively at7days and21days after injection.6. Average opticals of immuno-reaction product (DCX、Bcl-2、Bax and TUNEL) were detected using Image pro-plus6.0software, and data were carried out by SPSS16.0statistical analysis software, data used for univariate analysis of variance (One-way ANOVA), LSD method multiple comparison in groups. Data from RT-PCR used for repeated measures analysis of variance, and t-test used for two groups at different time point. P<0.05was considered statistically significant.Results:1. The evident neurotoxicity was seen in the region of the hippocampus in mice through HE staining, enlargement of inflammatory injure region in brain in the experimental group when compared to the controls, neuronal degeneration in inflammatory injure region and peripheral region was observed in this study at7day. The existence of A(31-42positive deposition in the injection site was detected by immunohistochemistry method in the experimental group, but no Aβ1-42positive deposition in the control group. 2. Immunohistochemical staining showed:At21day, there were differences for the Bcl-2protein expression in the four groups(F=368.159, P=0.000). Compare with the BALB/c-nude groups, the expression of Bcl-2protein in hippocampal CA3region of the BALB/c-WT groups were significantly increased with highly significant difference (P<0.01). The occurrence of T cell could inhibit neuronal apoptosis by promoting the Bcl-2expression. There were differences for the Bax protein expression in the four groups(Welch F=89.723, P=0.000).Compare with the BALB/c-WT groups, the expression of Bax protein in hippocampal CA3region of the BALB/c-nude groups were significantly increased with highly significant difference (P<0.01). The absences of T cell could promote neuronal apoptosis by promoting the Bax expression. The TUNEL results also further proveed, when the T cell normal hippocampal neuronal apoptosis were inhibited; When the T cell lacks the hippocampal neuronal apoptosis were occurred easily. The result also showed the experimental group (Aβ1-42injection) compare to the control group (NS-injection), the hippocampal neuronal apoptosis were increased obviously. Aβ1-42-injection may damage neuron and lead to apoptosis occurrence in hippocampus.3. Doublecortin (DCX) marker showed the positive cells of newborn neurons mainly existing within the subgranular zone (SGZ) of denate gyrus in hippocampus. At7day, There were differences for the DCX expression in the four groups (F=460.707, P=0.000). Compare with BALB/c-WT mice, the number of DCX positive cells deceased significantly in BALB/c-nude mice. At7day, the experimental Ⅰ group and the controls Ⅰ group compared to the experimental Ⅱ group, the DCX positive cells was significantly increased (P<0.01). Compared with NS-injection group, DCX positive cells was significantly reduced in Aβ-injection group, the experimental Ⅱ group compare to the control Ⅱ group (P<0.01). The result suggests that the presence of T cells could enhance neurogenesis. However, Aβ1-42-injiection can inhibit hippocampal neurogenesis.4. Fluorescent quantitative PCR showed that there were differences for related cytokines expression in each group. In peripheral blood, compared with the Balb/c-Nude group, the IL-2and IFN-y gene expression was significantly increased for Balb/c-WT group at7day and21day (P<.01). With time increasing, the IL-2gene down-regulation in Balb/c-WT group (P<0.01), there were different changes for IL-2gene expression in Balb/c-Nude group. IFN-y gene expression down-regulation in the experimental group (Aβ-injection) and had no differences in the control group (NS-injection). In brain tissue, compared with the Balb/c-Nude group, the IL-1β gene expression was significantly increased for Balb/c-WT group at7day and21day (P<0.01). With time increasing, the IL-1β gene had obviously differences in Balb/c-WT group (P<0.01) and had no differences in Balb/c-Nude group (P>0.05).Compared with the Balb/c-Nude group, the TNF-a gene expression was significantly increased for Balb/c-WT group at7day (P<0.01). At21day, the TNF-a gene expression was significantly increased in the experimental group and had no differences in the control group.Conclusions:1. To establish AD-mimic animal model by hippocampus-intra injection (Aβ1-42and NS) by BALB/c and BALB/c-nude mice, the model could mimic AD-related main pathological characteristics, such as Aβ1-42deposition and inflammatory reaction of neural toxicity.2. T cells were related to the neuronal apoptosis and neurogenesis in hippocampus in a mouse model of Alzheimer’s disease. The present of T cells would inhibit neuronal apoptosis and promote neurogenesis. When the deficiency of T cells would inhibit neurogenesis and promote neuronal apoptosis. Aβ1-42-injiection can inhibit hippocampal neurogenesis and enhance neuronal apoptosis. These results may be concerned with Aβ1-42neurotoxic effect on hippocampal neurons.3. In the AD model mouse, the T cell existence has obvious influence on neuronal apoptosis and neurogenesis in hippocampus, but this kind of influence mechanism is not clear. T cells participate in neuronal apoptosis and also contribute to the maintenance of neurogenesis. There were complex relationship (Cross-talking) between T cell and microglia, which possibly through activates the T cell and the microglia. This effect may be associated with altered expression of cytokines cause-related to the activation of peripheral T cells in peripheral blood and Microglia in central nervous system, such as IL-2, IFN-y, IL-1β and TNF-a.Main creative points:1. We established a new mouse model with Alzheimer’s disease induced by beta-amyloid protein, including BALB/c-wild and BALB/c-nude mouse. The model could mimic AD-related main pathological characteristics.2. In this study, using this mouse model, we demonstrated that T cells participate in the neuronal apoptosis and neurogenesis in hippocampus. The present of T cells would inhibit neuronal apoptosis and promote neurogenesis. Deficiency of T cells would inhibit neurogenesis and promote neuronal apoptosis.3. Further study showed that T cells can affect neuronal apoptosis and neurogenesis in hippocampus might be related to the changes of IL-2and IFN-y gene expression in peripheral blood and IL-1β and TNF-a in brain tissue.4. The research has active significance for revealing the possible contribution of T cells to AD pathogenesis and immunotherapy. |
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