| ObjectiveOur previous experimental studies have shown, AST glial f ibrillary acidic protein (glial fibrillary acidic protein, GFAP) expression change has an important role in the pathogenesis of depression, EA can fix chronic stress-induced depression in rats hippocampal astrocytes morphological damage. In this study, Chronic mild stress through acupuncture (chronic unpredictable mild stress CUMS)-induced rat model of depression and astrocytes via glutamate transporter antagonist-induced depression of glutamate in rat astrocytes intake of two experiments to verify the impact of acupuncture on astrocyte glutamate function. This study differs from previous studies of acupuncture are mostly confined to the antidepressant antidepressant mechanism of acupuncture research focus of this new field of research ideas traditional signal transduction pathway is.Experiments1Experimental study of an acupuncture school and CUMS rats induced astrocyte glutamate function of behavioral models of depression in ratsMethodsThe9-10week old male SD rats weighing250-300g of56, were randomly divided into four groups:control group (n=11), control group (n=15), acupuncture group (n=15) and Drug group (n=15); among the control group of rats received no treatment, and the remaining three groups of chronic unpredictable mild stimulus to support the establishment of a joint isolated rat model of depression. Control group, no treatment was successful modeling. Acupuncture group, on the second day after the successful model started off four acupuncture points (gu, Taichong) treatment. First week of treatment a day, beginning the second week of treatment every other day, were treated for5weeks. Drug group, on the second day after the successful model given riluzole treatment administered every24hours1(4mg/kg),5weeks of continuous treatment. Evaluation rat rat behavior and body mass index. Important indicator before the first7,14,21,28,35day modeling and modeling and treatment were body mass measurements, sucrose preference test,50g of food intake as a test of the modeling and treatment success. The use of transmission electron microscopy, immunohistochemistry, Western-Blot, RT-PCR technique to detect indicators observed four groups of rats hippocampus and prefrontal cortex in each group structure and morphology of astrocytes, glutamine synthetase (GS), the prefrontal cortex and changes in protein and gene expression in the hippocampus star keratinocytes high affinity excitatory amino acid transporter (EAAT1, EAAT2).Using SPSS20.0statistical software for the experimental data for statistical analysis, experimental measurement data if the subject is too distribution, the results are expressed as mean±standard deviation(x±s), differences between each group and compared using ANOVA (One-ANOVA) least significant difference method (LSD), with p<0.05was considered statistically significant. Groups were compared using analysis of variance, is not normal or heterogeneity of variance, using non-parametric tests; several different time points were compared using repeated measures analysis of variance, covariance matrix or dissatisfied with the Football Association correction of spherical test using test (Greenhouse-Geisser) method and pairwise comparisons, or the use of multivariate analysis of variance test level a=0.05.Results1. An acupuncture induced behavioral depression rats learned to CUMSBy measuring the body mass, sucrose preference test,50g intake experiments to prove chronic mild stress stimulation modeling, modeling of the indicators in each group of rats decreased symptoms of depression, the control group and the control group was statistically significant, the difference was not statistically significant.After five weeks to complete the treatment and control group indexes increased depressive symptoms improved, statistically significant difference compared with the control group; compared with the control group, the acupuncture group and the drug group indexes rising, depression symptoms improved, the difference was statistically significant; acupuncture group compared with the medication group, the difference was not statistically significant. Showed that acupuncture on chronic stress-induced depression model rats learned behavior has a significant impact.2. TEM observation of acupuncture on CUMS-induced depression in rats astrocyte structure and morphologyAfter the depression modeling, control, acupuncture and drug group AST ultrastructural damage in rat hippocampus, mainly within the cytoplasm of dilated rough endoplasmic reticulum and mitochondria cristae loose change nuclear condensation. Hippocampus (CA1region-CA3region) pyramidal cell degeneration occurs, reducing the number of cytoplasmic actin, microtubules significantly reduced density of mitochondria and vacuoles case, DG granular cytoplasmic vacuoles to some of edema or until the cells dense like changes, changes occur early necrosis and apoptosis. Prefrontal cortex occur within the cytoplasm dilated rough endoplasmic reticulum and mitochondria crest loose change, nuclear condensation, actin, microtubules significantly reduced density of mitochondria and vacuoles situation. Blank rat hippocampus and prefrontal cortex without significant ultrastructural damage. After five weeks to complete treatment, acupuncture group, hippocampus (CA1region-CA3region) pyramidal cells were normal, increasing the number of actin, microtubules increased density of mitochondria and vacuoles situation has improved, astrocytes pyknotic nuclei recover part quality; DG to some granulosa cells, astrocytes back to normal. Prefrontal cortical granule cell cytoplasmic vacuoles until edema, dense like change the situation returns, astrocyte morphology gradually restored. Drug group, hippocampus (CA1region-CA3region) pyramidal cells were normal astrocytes karyopyknosis no significant improvement in the situation; DG District granulosa cells return to normal astrocytes recovery is not obvious. Prefrontal cortex cytoplasm rough endoplasmic reticulum situation improves, becomes dense mitochondria, nucleus recovery, actin, microtubules began to increase, mitochondrial density and vacuolization conditions improve. Acupuncture group and the drug group considerable effect, and the control group there was no significant difference between the control group and there are significant differences.3. Immunohistochemical observation of acupuncture on CUMS-induced depression in rats GS, shape EAAT1, EAAT2and the number of positive cells. After five weeks to complete treatment, acupuncture and drug hippocampal damage area GS, EAAT1-positive cells at different time points after injury CUMS caused damage to the surrounding cortical area stained darker conditions have recovered a large number of vascular proliferation conditions improve, CA1, CA3, DG area can be seen fewer GS, EAAT1positive cells are small vacuolization, lightly stained, no projections, protrusions or short, can also be seen a small cell in the body, dyed darker, longer projections GS, EAAT1-positive cells, and reduce the number of positive cells. Acupuncture group and drug group prefrontal cortex damaged area GS, EAAT2-positive cells in the cortex of the visible damage to the surrounding EAAT2antagonists soma less clear-cut, dark olive edge, center lightly stained, projections obvious GS, EAAT2-positive cells, and concentrated around the damaged area, reducing the number of positive cells. There was no significant difference compared to recovery after treatment and control group, and the control group were significantly different. The number of positive cells in the control group and the control group was significantly reduced compared to the number at each time point after injury, no significant change. There were significant differences between the two.4. Western blot technique to observe the impact of acupuncture-induced rat model of depression protein transporter of astrocytes CUMSIn this study, the hippocampus GS Western Blot technique to detect rat EAAT1protein expression levels detected in rat prefrontal cortex GS, expression of EAAT2protein showed GS depression after modeling the hippocampus of rats, EAAT1protein content was significantly reduced in the prefrontal cortex GS, EAAT2protein content was significantly reduced, there was significant difference compared with the control group, with the control group, the difference was not statistically significant. Tip rat hippocampus and prefrontal cortex depression AST function or structure affected, leading to its GS, EAAT1, EAAT2protein synthesis levels decline. After five weeks of treatment is completed, GS rat hippocampus, EAAT1protein comparison with the control group showed no significant difference, rat prefrontal cortex GS, EAAT2protein content with the control group showed no statistical significance, with the control group, the difference was statistically significant. And two, the difference was not statistically significant, the effect is quite. 5. RT-PCR technique to observe the impact of acupuncture-induced gene expression in rat model of depression transporter of astrocytes CUMSIn this study, RT-PCR technique to detect GS rats, mRNA expression of EAAT1, EAAT2, the results show that depression in rat hippocampus after modeling GS mRNA, EAAT1mRNA, the prefrontal cortex GS mRNA, EAAT2mRNA expression levels decreased, suggesting that GS, EAAT1, EAAT2protein replication is reduced. After the result of chronic unpredictable mild irritation, hippocampus GS mRNA, EAAT1mRNA and prefrontal cortex GS mRNA, EAAT2mRNA result of reduced protein content consistent. There are differences with the control group was statistically significant, similar to the gene expression level in the control group, the difference was not statistically significant. After acupuncture or riluzole administered intervention GS mRNA in rat hippocampus, EAAT1mRNA, the prefrontal cortex GS mRNA, EAAT2mRNA expression levels increased comparing with the control group showed no significant difference, and two by considerable effect, statistically significant difference compared with the control groupTwo experimental studies of acupuncture on EAAT1, EAAT2glutamate antagonist induced learn and function of astrocytes in a rat model of depressive behaviorMethodsThe7-9weeks of age weighing270-290g75adult male SD.rats were randomly divided into:control group (n=5). EAAT1group (n=30rats), which EAAT1control group (n=10), EAAT1acupuncture group (n=10), EAAT1drug group (n=10). EAAT2group (n=30rats), which EAAT2in the control group (n=10), EAAT2acupuncture group (n=10), EAAT2drug group (n=10). Blank normal feeding rats, no treatment. EAAT1buried in the lateral ventricle after the first eight days of intracerebroventricular injection of astrocyte transporter EAAT1antagonist (DL-threo-β-benzyloxyaspartate, TBOA)(100ug/ul, lul), injection rate control in0.25ul/min, the frequency of injections, one week every two days before the injection of1total of3times, then every other day for rats at the same concentration and rate of intracerebroventricular injections of three weeks, a period of four weeks modeling of conduct drug injection15times; EAAT2in rat prefrontal cortex buried eight days after the first injection of the prefrontal cortex astrocytes transporter EAAT2antagonists (dihydrokainic acid, DHK),(100ug/ul,1ul), injection rate, dose, frequency, period EAAT1group were injected with the lateral ventricles.EAAT1EAAT2control group and the control group after successful modeling without any treatment. EAAT1and EAAT2acupuncture rats, at the end of the second day after the model started off four acupuncture points (hegu, Taichong) treatment, the specific methods of operation and the treatment cycle with the experiment." EAAT1and EAAT2drugs rats, at the end of the second day after the model started riluzole treatment administered, the specific methods of operation and the treatment cycle with the "experiment." Evaluation of the behavior of rats indexes.Before modeling, and modeling the first day were7,14,21,28weight measurement, sucrose preference test, open field test test modeling as an important indicator of success; at14,21days after therapy detection of these indicators, the efficacy of acupuncture and drug evaluation. After the acupuncture and medication, the use of light microscopy, transmission electron microscopy, Western-Blot, RT-PCR detection of four technical indicators observed in hippocampus and prefrontal cortex structure and morphology of astrocytes, glutamine synthetase (GS), changes in the prefrontal cortex and hippocampus star keratinocytes high affinity excitatory amino acid transporter (EAAT1, EAAT2) protein and gene expression Statistical methods with the experimental oneResults1. Acupuncture on injection EAAT1, EAAT2antagonists induced rat model of depressionBy measuring the body mass, sucrose preference test, open-field experiments to prove intracerebroventricular injection EAAT1antagonists, the prefrontal cortex after injection of EAAT2antagonists modeling, control group and the control group was statistically significant, each modeling the difference between the two groups of rats were no statistically significant. Three weeks after the end of treatment, the control group indexes rose, with the blank group, the difference was statistically significant; compared with the control group, the acupuncture group and the drug group difference was statistically significant; acupuncture group compared with the medication group, the difference was not statistically significant; acupuncture group and the drug group and the control group, the difference was not statistically significant. Showed that acupuncture on injection EAAT1, EAAT2antagonist -induced rat model of depression behavior has a significant impact on learning.2. Light microscope acupuncture on injection EAAT1, EAAT2antagonists morphology and structure induced depression model rat astrocytesIn an interview five weeks after the injection of the lateral ventricles and the prefrontal cortex, modeling rat hippocampus (CA1, CA3, DG District) star significantly reduce the number of glial cells, astrocytes number prefrontal cortex were significantly reduced, and the blank group, the difference was statistically significant, compared with the control group, the difference was not statistically significant; after3weeks of treatment, the drug hippocampal CA1, CA3, DG zone astrocytes the number of cells increased, the number of astrocytes in the prefrontal cortex were significantly increased compared with the control group, the difference was statistically significant, compared with the control group,-the difference was not statistically significance; acupuncture rats hippocampal CA1, CA3region, increase in the number of astrocytes in DG, the number of astrocytes in the prefrontal cortex were significantly increased compared with the control group, the difference was statistically significance, with the control group and the drug group, the difference was not statistically significant; acupuncture group prefrontal cortex has a small number of astrocytes increased drug group and the control group the difference was not statistically significant, with the control group, the difference was statistically significant.3. TEM observation on acupuncture injection EAAT1, EAAT2antagonists induced rat model of depression astrocytes structure and functionDepression after modeling the hippocampus of rats AST ultrastructural damage, mainly within the cytoplasm of dilated rough endoplasmic reticulum and mitochondria cristae loose change, nuclear condensation. Hippocampal CA1region, CA3pyramidal cell degeneration area appears to reduce the number of cytoplasmic actin, microtubules significantly reduced density of mitochondria and vacuoles case, DG to some granular cytoplasmic vacuoles until cell edema or dense like changes, changes occur early necrosis and apoptosis. Prefrontal cortex occur within the cytoplasm dilated rough endoplasmic reticulum and mitochondria crest loose change, nuclear condensation, actin, microtubules significantly reduced density of mitochondria and vacuoles situation. Comparison with the control group, there were significant differences. Compared with the control group, no significant difference. After3weeks of treatment, acupuncture group, CA1region, CA3pyramidal cells were normal, increasing the number of actin, microtubules increased density of mitochondria and vacuoles situation has improved, astrocytes part karyopyknosis recovery; DG to some granulosa cells, astrocytes back to normal. Prefrontal cortical granule cell cytoplasmic vacuoles until edema, dense like change the situation returns, astrocyte morphology gradually restored. Drug group, CA1region, CA3pyramidal cells were normal astrocytes karyopyknosis no significant improvement in the situation; DG District granulosa cells return to normal astrocytes recovery is not obvious. Prefrontal cortex cytoplasm rough endoplasmic reticulum situation improves, becomes dense mitochondria, nucleus recovery, actin, microtubules began to increase, mitochondrial density and vacuolization conditions improve. And blank group, no significant differences. And the control group, there were significant differences. Acupuncture group and the drug group is quite effective.4. Western blot technique of acupuncture injection EAAT1, EAAT2antagonists induced astrocyte transporter protein expression in rat model of depressionAfter modeling antagonist depression, depression in rat hippocampal GS, EAAT1protein content was significantly reduced in the prefrontal cortex GS, significantly reducing the EAAT2protein content, there was significant difference compared with the control group, and control There was no significant difference between groups, prompted depression rat hippocampus and prefrontal cortex AST function or structure affected, leading to its GS, EAAT1, EAAT2protein synthesis levels decline. Acupuncture and riluzole administered intervention GS hippocampus of rats, EAAT1protein content increased, comparing with the control group showed no significant difference, with the control group, the difference was statistically significant, rat prefrontal cortex GS, EAAT2protein content increased, comparing with the control group showed no significant difference, with the control group, the difference was statistically significant. And two, the difference was not statistically significant, and two similar role.5. RT-PCR technique acupuncture for injection EAAT1, EAAT2antagonists induced astrocyte transporter gene expression in rat model of depressionIn this study, RT-PCR technique to detect the hippocampus and prefrontal cortex of rats GS, mRNA expression of EAAT1, EAAT2, the results showed that the hippocampus GS mRNA, EAAT1mRNA, GS mRNA in the prefrontal cortex and the lateral ventricle of depression after the prefrontal cortex of rats modeling, EAAT2mRNA expression decreased, suggesting that GS, EAAT1, EAAT2protein replication decrease was statistically significant difference compared with the control group, with the control group, the difference was not statistically significant. The results suggest that the injection of EAAT1, EAAT2antagonists modeling, the hippocampus GS mRNA, EAAT1mRNA and prefrontal cortex GS mRNA, EAAT2mRNA protein content decreased. After acupuncture or riluzole administered intervention GS mRNA in rat hippocampus, EAAT1mRNA, the prefrontal cortex GS mRNA, EAAT2mRNA expression level rise, with the control group the difference was not statistically significant, and the control group the difference was statistically significant, and two similar role.GonclusionThe research evidence to prove to each other through these two experiments: Acupuncture for chronic unpredictable mild stimulus to support joint modeling of solitary rat hippocampal astrocytes depression structure and function of the damage caused by a very good regulatory role; depressive-like behavior in rats acupuncture on intracerebroventricular injection EAAT1, EAAT2antagonists prefrontal cortex injection modeling and modeling lead in rat hippocampus, prefrontal cortex damage to the structure and function of astrocytes have a good regulation; these effects similar to acupuncture with riluzole; experimental results suggest that acupuncture regulation of hippocampal astrocytes function may be one of the important mechanisms play an antidepressant. |
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