The Mechanism On The Repair Of Electro-acupuncture On Hippocampal Astrocytes In Chronic Stress Induced Depression Modle Rats

Object iveThrough the two study, one is the improvement of electro-acupuncture on the behavior and the injured hippocampal astrocytes of depression model rats induced by chronic stress, another is the improvement of electro-acupuncture on the behavior and the injured hippocampal astrocytes of rats with intracerebroventricular injection L-AAA, to discuss the antidepressant mechanism of electric acupuncture on astrocyte injury. Part1:The study of electro-acupuncture to improve chronic stress induced depression rat behaviors and protect the hippocampal astrocytes injuryMethods1. Animal grouping and modeling methods66adult female SD rats(220-250g,7-8weeks) were randomly divided into four groups:normal control group (NC, n=12), depression model group(DM, n=18), electro-acupuncture group(EA, n=18),and riluzole group (RZ, n=18). Normal control group rats would not be any processing, and the rest three groups were exposed to chronic unpredictable mild stress and separation to build depression rat model.2. Interventions①After the successful modeling, rats of DM group would not be any processing;②The day after the successful modeling, rats of EA group accepted Si Guan electro-acupuncture treatment, selecting the G-6805type Ⅱ electric acupuncture apparatus, sparse wave,15-30Hz frequency,15to20minutes at a time, alternately left and right sides. The1st week1times per day, the second and third week treated every other day, for3weeks;③The day after the successful modeling, rats of RZ group accepted riluzole gavage every12 hours (4mg/kg), for3consecutive weeks.3. Evaluation①Behavioral indicators:Body Weight Measurement (BWM), Open-Field Test (OFT), Sucrose Preference Test (SPT) and Novelty Suppressed Feeding Test (NSFT) were carried out before and in the building7th,14th,21th,28th,35th day and treating7th,14th,21th day for detecting group behavior in rats.②Light microscope, electron microscope, Western-Blot and RT-PCR technique were used to observe the morphology and ultrastructure of astrocytes in rat hippocampal and test their GFAP and GFAP mRNA expression after the treatment.4. Statistical methodsThe PASW statisticsl8.0statistical software was used for statistical analysis. If the experimental measurement data follow a normal distribution, calculated x±s and used ANOVA analysis to compare the differences between groups. If the data did not follow a normal distribution or dissatisfied with the homogeneity of variance, non-parametric tests were used. When compare the differences between multiple different measurement points, using ANOVA for repeated measurement. If the data dissatisfied with the sphericity test or Covariance matrix, Using Greenhouse-Geisser method or MANOVA to realize pairwise comparisons, the test level α=0.05.Results1. The influence of electro-acupuncture on the behaviors of depression rats induced by chronic stress(1) Body Weight MeasurementBaseline data of each group in body weight is homogeneous (P>0.05). After5weeks chronic stress, compared with NC group, weight of DM, EA and RZ group dropped significantly (P<0.05). No differences in body weight were observed between DM, EA and RZ group (P>0.05). Over3weeks of treatment, compared with NC group, weight of DM group dropped significantly (P=0.000). Compared with DM group, weight of EA and RZ group increased significantly(P=0.031P=0.002). There is no difference in body weight between EA and RZ group(P>0.05).(2) Open-Field Test①Horizontal motionBaseline data of each group in horizontal motion is homogeneous (P>0.05). After5weeks chronic stress, compared with NC group, horizontal motion of DM, EA and RZ group dropped significantly (P<0.05). No differences in horizontal motion were observed between DM, EA and RZ group(P>0.05). Over3weeks treatment, compared with NC group, horizontal motion of DM group dropped significantly(P=0.000). Compared with DM group, horizontal motion of EA and RZ group increased significantly(P=0.000).There is no difference in horizontal motion between EA and RZ group(P>0.05).②Vertical motionBaseline data of each group in vertical motion is homogeneous (P>0.05). After5weeks chronic stress, compared with NC group, vertical motion of DM, EA and RZ group dropped significantly (P<0.05). No differences in vertical motion were observed between DM, EA and RZ group (P>0.05). Over3weeks treatment, compared with DM group, vertical motion of EA and RZ group increased significantly(P=0.011, P=0.008). There is no differences in vertical motion between EA and RZ group(P>0.05). And no difference was detected in vertical motion between DM and NC group(P>0.05).(3) Sucrose Preference TestBaseline data of each group in sucrose consumption is homogeneous (P>0.05). After5weeks chronic stress, compared with NC group, sucrose consumption of DM, EA and RZ group dropped significantly (P<0.05). No differences in sucrose consumption were observed between DM, EA and RZ group(P>0.05). Over3weeks treatment, compared with NC group, sucrose consumption of DM group dropped significantly (P=0.000). Compared with DM group, sucrose consumption of EA and RZ group increased significantly (P=0.000). There is no differences in sucrose consumption between EA and RZ group(P>0.05).(4) Novelty Suppressed Feeding TestBaseline data of each group in the time of first bite is homogeneous (P>0.05). After5weeks chronic stress, compared with NC group, the time of first bite of DM, EA and RZ group increased significantly (P<0.05). No differences in the time of first bite were observed between DM, EA and RZ group(P>0.05). Over3weeks treatment, compared with NC group, the time of first bite of DM group increased significantly (P=0.015). Compared with DM group, the time of first bite of EA and RZ group dropped significantly (P=0.000). There is no difference in the time of first bite between EA and RZ group(P>0.05).2. The influence of electro-acupuncture on the structure and function injury of hippocampal astrocytes in depression rats induced by chronic stress(1) Electro-acupuncture on the structure of hippocampal astrocytes Over5weeks chronic stress, the morphological changes of hippocampal astrocytes in depression rats mainly exhibited nucleus pyknosis. At the same time, we also found pyramidal cell degeneration and reducing the number in both area CA1and CA3, and granulosa cell cytoplasm vacuoles in area DG.3weeks after treatment, EA group, the area CA1pyramidal cells were returned to normal, astrocytes nucleus pycnosis were not obviously improved; Area CA3pyramidal cells back to normal, astrocytes nucleus pycnosis partial recovery; DG area granulosa cells, astrocytes have returned to normal. RZ group, area CA1pyramidal cells were returned to normal, astrocytes cell nucleus pyknosis situation were not well improved; Area CA3pyramidal cells back to normal, astrocytes had returned to normal; DG area granulosa cells back to normal, astrocytes recovery was not obvious.(2) Electro-acupuncture on the number of hippocampal astrocytesAfter5weeks chronic stress, compared with NC group, number of astrocytes of DM, EA and RZ group dropped significantly (P<0.05).3weeks after treatment, compared with DM group, number of astrocytes of RZ group increased significantly in area CA1.CA3and DG(P=0.005, P=0.019, P=0.020). Compared with DM group, number of astrocytes of EA group increased significantly in area CA1and DG(P=0.015, P=0.035). There is no difference in number of astrocytes between EA and RZ group in area CA1and DG(P>0.05). Compared with RZ group, number of astrocytes of EA group in area CA3decreased significantly (P=0.032). There are no differences in number of astrocytes between EA, DM and NC group in area CA3(P>0.05).(3) Electro-acupuncture on the ultrastructure of hippocampal astrocytesAfter5weeks chronic stress, ultrastructural damage of hippocampus astrocytes in depression rats mainly to the cytoplasm rough endoplasmic reticulum expansion and mitochondrial cristae loose change.3weeks after treatment, The ultrastructure injury of EA group and RZ group astrocytes were both repaired, and other organelles, such as ribosome, microfilament were more clearly visible than DM group(4) Electro-acupuncture on GFAP and GFAP mRNAAfter5weeks chronic stress, compared with NC group, hippocampal GFAP levels of DM, EA and RZ group dropped significantly (P=0.004).3weeks after treatment, compared with DM group, GFAP levels of EA and RZ group increased significantly (P=0.007, P=0.001). There is no difference in GFAP levels between EA and RZ group(P>0.05).After5weeks chronic stress, compared with NC group, hippocampal GFAP mRNA expression value of DM, EA and RZ group dropped significantly (P=0.001).3weeks after treatment, compared with DM group, GFAP mRNA expression value of EA and RZ group increased significantly(P=0.029, P=0.015). There is no difference in GFAP mRNA expression value between EA and RZ group (P>0.05). Part2:The study of electro-acupuncture improve behavior and protect hippocampal astrocytes of rats modeled by L-AAA lateral cerebral ventricle injectionMethods1. Animal grouping and modeling methods78adult female SD rats(220-250g,7-8weeks) were randomly divided into five groups:normal control group (NC, n=12), intracerebroventricular injection of normal saline model group(NS, n=12), intracerebroventricular injection of L-AAA model group (LM, n=18), electro-acupuncture group(EA, n=18),and riluzole group (RZ, n=18).2. Modeling method①Rats of normal control group rats would not be any processing;②Rats of LM, EA and RZ groups would be injected L-AAA, a kind of highly selective neurotoxin of astrocytes, through the lateral ventricle pipe(100ug/uL, luL)8days after surgery. The injection rate was controlled to0.25uL/min. The first3days, injected once a day, then injected in the building6th,9th,12th,15th day respectively. The cycle of intracerebroventricular injection is15days, a total of seven times for injection.③NS group would be injected normal saline through the lateral ventricle pipe. The dose, injection rate, frequency, and cycle were the same as LM group.3. Interventions①After the modeling, rats of LM and NS group would not be any processing;②The day after the modeling, rats of EA group accepted Si Guan electro-acupuncture treatment, the manipulation in details and the treatment frequency and cycle were the same as part1;③The day after the modeling, rats of RZ group accepted riluzole gavage every12hours (4mg/kg), for3consecutive weeks, as the same as part1.4. Evaluation①Behavioral indicators:Body Weight Measurement (BWM), Open-Field Test (OFT), Sucrose Preference Test (SPT) and Novelty Suppressed Feeding Test (NSFT) were carried out before and in the building3th,6th,9th,12th,15th day and treating7th,14th,21th day for detecting group behavior in rats.②Light microscope, electron microscope, Western-Blot and RT-PCR technique were used to observe the morphology and ultrastructure of astrocytes in rat hippocampal and test their GFAP and GFAP mRNA expression after the treatment.5. Statistical methodsThe same as Part1.Results1. The influence of electro-acupuncture on the behaviors of rats modeled by L-AAA lateral cerebral ventricle injection(1) Body Weight MeasurementBaseline data of each group in body weight is homogeneous (P>0.05). After15days lateral cerebral ventricle injection of L-AAA, compared with NC and NS group, weight of LM, EA and RZ group dropped significantly (P=0.000). No differences in body weight were observed between the three groups(P>0.05). No differences in body weight were observed between NC and NS group(P>0.05).3weeks after treatment, compared with NC and NS group, weight of LM group dropped significantly (P=0.000). Compared with LM group, weight of EA and RZ group increased significantly(P=0.000). There is no difference in body weight between EA and RZ group(P>0.05).(2) Open-Field Test①Horizontal motionBaseline data of each group in horizontal motion is homogeneous (P>0.05). After15days lateral cerebral ventricle injection of L-AAA, compared with NC and NS group, horizontal motion of LM, EA and RZ group dropped significantly (P=0.000). No differences in horizontal motion were observed between the three groups (P>0.05). No differences in horizontal motion were observed between NC and NS group(P>0.05).3weeks after treatment, compared with NC and NS group, horizontal motion of LM group dropped significantly (P=0.000, P=0.013). Compared with LM group, horizontal motion of EA and RZ group increased significantly(P=0.000). There is no difference in horizontal motion between EA and RZ group (P>0.05).②Vertical motionBaseline data of each group in vertical motion is homogeneous (P>0.05). After15days lateral cerebral ventricle injection of L-AAA, compared with NC and NS group, vertical motion of LM, EA and RZ group dropped significantly (P=0.000). No differences in vertical motion were observed between the three groups(P>0.05). No differences in vertical motion were observed between NC and NS group(P>0.05).3weeks after treatment, compared with NC and NS group, vertical motion of LM group dropped significantly (P=0.000, P=0.001). Compared with LM group, vertical motion of EA and RZ group increased significantly (P=0.001, P=0.000). There is no difference in vertical motion between NC, NS, EA and RZ group (P>0.05).(3) Sucrose Preference TestBaseline data of each group in sucrose consumption is homogeneous (P>0.05). After15days lateral cerebral ventricle injection of L-AAA, compared with NC and NS group, sucrose consumption of LM, EA and RZ group dropped significantly (P=0.000). No differences in sucrose consumption were observed between the three groups(P>0.05). No differences in sucrose consumption were observed between NC and NS group(P>0.05).3weeks after treatment, compared with NC and NS group, sucrose consumption of LM group dropped significantly (P=0.000). Compared with LM group, sucrose consumption of EA and RZ group increased significantly(P=0.000). There is no difference in sucrose consumption between NC, NS, EA and RZ group(P>0.05).(4) Novelty Suppressed Feeding TestBaseline data of each group in the time of first bite is homogeneous (P>0.05). After15days lateral cerebral ventricle injection of L-AAA, compared with NC and NS group, the time of first bite of LM, EA and RZ group all increased significantly (P=0.000). No differences in the time of first bite were observed between the three groups(P>0.05). No differences in the time of first bite were observed between NC and NS group (P>0.05).3weeks after treatment, compared with NC and NS group, the time of first bite of LM group increased significantly (P=0.014, P=0.024). Compared with LM group, the time of first bite of EA and RZ group dropped significantly(P=0.000). There is no difference in the time of first bite between NC, NS, EA and RZ group(P>0.05).2. The influence of electro-acupuncture on the structure and function of hippocampal astrocytes in L-AAA lateral cerebral ventricle injection model rats(1) Electro-acupuncture on the structure of hippocampal astrocytes Over15days lateral cerebral ventricle injection of L-AAA, rat hippocampal astrocytes were extensively damaged, mainly showed the nucleus pyknosis. At the same time Lateral ventricle injection of L-AAA also caused parts of pyramidal cells in area CA1and CA3and parts of granulosa cell in DG area degeneration, cytoplasm pyknosis. After15days lateral ventricle injection of normal saline, rat hippocampal astrocytes in CA1, CA3area did not show obvious damage, and a small amount of astrocytes and part of the granular cell degeneration, cytoplasm pyknosis were detected in DG area.3weeks after treatment, astrocytes of EA group in CA1, CA3and DG area were effectively repaired, as well as pyramidal cell of CA1and CA3area and part of granulosa cell of DG area were well repaired. Astrocytes of RZ group in CA1and CA3area were effectively repaired, but astrocytes recovery in DG area is not obvious, granular cell degeneration partially restored.(2) Electro-acupuncture on the number of hippocampal astrocytesAfter15days lateral cerebral ventricle injection of L-AAA, compared with NC group, number of astrocytes of LM group dropped significantly in CA1area(P=0.011). There is no difference between LM and NS group in CA1area(P>0.05).3weeks after treatment, compared with LM group, number of astrocytes of EA and RZ group increased significantly in area CA1(P=0.013, P=0.016). There was no difference in number of astrocytes between EA and RZ group in area CA1(P>0.05).After15days lateral cerebral ventricle injection of L-AAA, compared with NC and NS group, number of astrocytes of LM group dropped significantly in CA3area(P=0.034, P-0.008).3weeks after treatment, compared with LM group, number of astrocytes of EA and RZ group increased significantly in area CA3(P=0.034, P=0.011). There was no difference in number of astrocytes between EA and RZ group in area CA3(P>0.05).After15days lateral cerebral ventricle injection of L-AAA, compared with NC and NS group, number of astrocytes of LM group dropped significantly in DG area(P=0.007, P=0.017).3weeks after treatment, compared with LM group, number of astrocytes of EA and RZ group increased significantly in area DG (P=0.022, P=0.007). There was no difference in number of astrocytes between EA and RZ group in area DG(P>0.05).(3) Electro-acupuncture on the ultrastructure of hippocampal astrocytesAfter15days lateral cerebral ventricle injection of L-AAA, the rat hippocampal astrocytes suffered serious damage. Astrocytes were deformed to near oval, Cytoplasm and organelles almost missing; There was no significant damage of astrocytes in NS group.3weeks after treatment, Astrocytes organelles of EA group were partly repaired, including rough endoplasmic reticulum, ribosome, microfilament, mitochondria and et cl. But the deformation and cytoplasmic scarce of astrocytes didn’t get well improvement. Riluzole did the same work as electro-acupuncture in the improvement of astrocytes ultrastructure damaged by L-AAA.(4) Electro-acupuncture on GFAP and GFAP mRNAAfter15days lateral cerebral ventricle injection of L-AAA, compared with NC and NS group, hippocampal GFAP levels of LM group dropped significantly (P=0.006, P=0.043).3weeks after treatment, compared with LM group, GFAP levels of EA and RZ group increased significantly (P=0.011, P=0.009). There is no difference in GFAP levels between EA and RZ group(P>0.05).After15days lateral cerebral ventricle injection of L-AAA, compared with NC and NS group, hippocampal GFAP mRNA expression value of LM group dropped significantly (P<0.05).3weeks after treatment, compared with LM group, GFAP mRNA expression value of EA and RZ group increased significantly (P<0.05). There is no difference in GFAP mRNA expression value between EA and RZ group (P>0.05).ConelusionsThrough the two experiments, we conclude:①Electro-acupuncture can effectively improve the behavior of the depression rats induced by chronic unpredictable mild stress joint solitary raising. And it can also protect the structure and function damage of the hippocampal astrocytes in these model rats.②Electro-acupuncture can effectively improve the depression-like behavior of the rats that were injected by L-AAA through lateral cerebral ventricle. At the same time,to protect the structure and function damage of the hippocampal astrocytes in these model rats.③The curative effect of electro-acupuncture is similar to riluzole.This study suggests that the hippocampal astrocytes involve in the procedure of chronic stress induced depression as well as the procedure of electro-acupuncture antidepressant. The protective effect of electro-acupuncture on the hippocampal astrocytes may be one of the most important mechanisms in antidepressant.