摘要
目的:
通过电针改善慢性应激致抑郁大鼠行为学及大鼠海马星形胶质细胞(AST)损伤的研究,以及电针改善侧脑室注射星形胶质细胞代谢高度选择性的神经毒物L-α-氨基己二酸(L-AAA)造模大鼠行为学及大鼠海马AST损伤的研究,探讨电针在修复星形胶质细胞损伤方面的抗抑郁作用机制。
实验一电针改善慢性应激致抑郁大鼠行为学及大鼠海马AST损伤的研究
方法:
1.动物分组及造模方法
将7-8周龄体重220-250g的成年雌性SD大鼠66只,随机分为4组:正常对照组(n=12)、空白组(n=18)、电针组(n=18)和药物组(n=18);其中正常对照组大鼠不予任何处理,其余三组采用慢性不可预见性温和刺激联合孤养建立抑郁大鼠模型。
2.干预措施
①空白组,造模成功后不予任何处理;②电针组,于造模成功后第二天开始进行电针四关穴治疗,选择G-6805Ⅱ型电针仪,连续波,频率15-30Hz,每次15-20分钟,左右交替进行。治疗第1周每日1次,治疗第2周开始隔日1次,共治疗3周;③药物组,于造模成功后第二天开始给予利鲁唑灌胃治疗,每12小时1次(4mg/kg),连续治疗3周。
3.评价指标
①行为学指标,在造模前以及造模的第7、14、21、28、35天进行体重测量、旷场试验、糖水偏好实验、新环境抑制进食实验,作为检验造模成功与否的重要指标;在治疗第7、14、21天进行上述指标检测,对电针和药物的疗效进行评价。②光镜、电镜、Western-Blot、RT-PCR检测,治疗结束后,运用上述指标对大鼠海马AST的形态、超微结构以及AST标志蛋白GFAP含量以及GFAP mRNA的表达情况进行检测,对各组大鼠AST结构和功能损伤程度进行评价。
4.统计方法
采用PASW statisticsl8.0统计软件对实验数据进行统计分析,实验计量资料若服从正态分布计算x±s,组间比较采用方差分析,不服从正态分布或方差不齐,采用非参数检验;多个不同时点比较采用重复测量方差分析,不满足协方差矩阵或球形检验采用矫正检验(Greenhouse-Gei sser)方法及两两比较,或采用多元方差分析,检验水平α=0.05。
结果:
1.电针对慢性应激致抑郁大鼠行为学的影响
(1)体重
各组体重的基础值无统计学差异(P>0.05),具有可比性。在接受5周慢性应激后,模型大鼠体重增长减缓,与正常对照组比较差异有统计学意义(P<0.05);各造模组大鼠组间比较,差异无统计学意义(P>0.05)。电针四关穴或利鲁唑灌胃治疗3周后,空白组、电针组大鼠体重低于正常对照组,差异有统计学意义(P=0.000,P=0.030);电针组、药物组分别与空白组比较体重升高,差异有统计学意义(P=0.031,P=0.002);电针组与药物组比较,差异无统计学意义(P>0.05)。
(2)旷场实验
①水平运动
各组水平运动的基础值差异无统计学意义(P>0.05),具有可比性。在接受5周慢性应激后,各组造模大鼠的水平运动次数下降,与正常对照组比较差异有统计学意义(P<0.05);各造模组大鼠组间比较差异无统计学意义(P>0.05)。治疗3周后,空白组大鼠水平运动低于正常对照组,两组比较差异有统计学意义P=0.000);与空白组比较电针组和药物组的水平运动次数增多,差异有统计学意义(P=0.000);电针组与药物组比较,差异无统计学意义(P>0.05)。
②垂直运动
各组垂直运动的基础值差异无统计学意义(P>0.05),具有可比性。在接受5周慢性应激后,各组造模大鼠的垂直运动次数下降,与正常对照组比较差异有统计学意义(P<0.05);各造模组大鼠组间比较差异无统计学意义(P>0.05)。治疗3周后,空白组大鼠垂直运动与正常对照组比较差异无统计学意义(P>0.05)。与空白组比较,电针组和药物组垂直运动增多,差异有统计学意义(P=0.011,P=0.008);电针组与药物组、正常对照组之间两两比较,差异无统计学意义(P>0.05)。
(3)糖水偏好实验
各组糖水偏好率的基础值差异无统计学意义(P>0.05),具有可比性。在接受5周慢性应激后,各组造模大鼠的糖水偏好率显著下降,与正常对照组比较差异有统计学意义(P<0.05);各造模组大鼠组间比较差异无统计学意义(P>0.05)。治疗3周后,空白组大鼠糖水偏好率低于正常对照组,两组比较差异有统计学意义(P=0.000);与空白组比较,电针组和药物组糖水偏好率升高,差异有统计学意义(P=0.000);电针组与药物组比较,差异无统计学意义(P>0.05)。
(4)新环境抑制进食实验
各组新环境进食第一口食物花费时间的基础值无统计学差异(P>0.05),具有可比性。在接受5周慢性应激后,各组造模大鼠新环境进食第一口食物花费时间增多,与正常对照组比较差异有统计学意义(P<0.05);各造模组大鼠组间比较差异无统计学意义(P>0.05)。治疗3周后,空白组大鼠新环境进食第一口食物花费时间明显高于正常对照组,两组比较差异有统计学意义(P--O.015);与空白组比较,电针组和药物组新环境进食第一口食物花费时间下降,差异有统计学意义(P=-0.000);电针组与药物组比较,差异无统计学意义(P>0.05)。
2.电针对慢性应激致抑郁大鼠海马AST结构和功能的影响
(1)光镜下观察AST形态和结构
慢性应激致抑郁大鼠海马AST形态变化主要表现为,细胞核的固缩。同时部分脑区(CA1区、CA3区)出现锥体细胞变性、数目减少情况,DG区部分颗粒细胞胞浆空泡化。治疗3周后,电针组,CA1区锥体细胞均恢复正常,星形胶质细胞核固缩情况未见明显好转;CA3区锥体细胞恢复正常,星形胶质细胞核固缩部分恢复;DG区颗粒细胞、星形胶质细胞恢复正常。药物组,CA1区锥体细胞均恢复正常,星形胶质细胞核固缩情况未见明显好转;CA3区锥体细胞恢复正常,星形胶质细胞恢复正常;DG区颗粒细胞恢复正常,星形胶质细胞恢复不明显。
(2)光镜下观察AST数目
在接受5周慢性应激后,造模大鼠海马各区(CA1、CA3、DG区)星形胶质细胞数目均明显减少,与正常对照组比较,差异有统计学意义(P<0.05);治疗3周后,药物组大鼠海马CA1、CA3、DG区星形胶质细胞数目明显增多,与空白组比较,差异有统计学意义(P=0.005,P=-0.019,P=0.020);电针组大鼠CA1区、DG区星形胶质细胞数目增多,与空白组比较,差异有统计学意义(P=0.015,P=-0.035),与药物组比较差异无统计学意义(P>0.05);电针组CA3区星形胶质细胞数目无明显上升,与药物组比较差异有统计学意义(P=0.032),与空白组和正常对照组比较差异无统计学意义(P>0.05)。
(3)电镜下观察AST超微结构
接受5周慢性应激后大鼠海马AST超微结构损伤,主要表现为细胞质中粗面内质网明显扩张和线粒体的嵴疏松变。治疗3周后,电针组和药物组AST胞质中粗面内质网扩张和线粒体嵴疏松变均有所减轻,其他细胞器,核糖体、微丝等也较空白组清晰可见。
(4)电针对慢性应激致抑郁大鼠海马GFAP含量和GFAP mRNA表达的影响
接受5周慢性应激后大鼠海马GFAP含量减少,与正常对照组比较差异有统计学意义(P=-0.004)。治疗3周后,电针组和药物组GFAP含量升高,与空白组比较差异有统计学意义(P=0.007, P=0.001);电针组与药物组比较,差异无统计学意义(P>0.05)。
接受5周慢性应激后大鼠海马GFAP mRNA表达值下降,与正常对照组比较差异有统计学意义(P=-0.001)。治疗3周后,电针组和药物组GFAP mRNA表达值升高,分别与空白组比较,差异有统计学意义(P=-0.029,P=-0.015);电针组与药物组比较,差异无统计学意义(P>0.05)。
实验二电针改善侧脑室注射L-AAA造模大鼠行为学及海马AST损伤的研究
方法:
1.动物分组
将7-8周龄体重220-250g成年雌性SD大鼠78只,随机分为:正常对照组(n=12)、生理盐水组(n=12)、空白组(n=18)、电针组(n=18)和药物组(n=18)。
2.造模方法
①正常对照组大鼠正常饲养,不予任何处理;②空白组、电针组以及药物组侧脑室埋管术后第8天侧脑室注射星形胶质细胞高度选择性神经毒物L-AAA (100ug/ul,lul),注射速率控制在0.25ul/min,注射频次为,前3天每天注射1次,此后分别于造模第6天,第9天,第12天,第15天对大鼠以相同浓度和速率进行侧脑室注射,造模周期为15天,共进行药物注射7次;③生理盐水组,侧脑室埋管术后第8天,于侧脑室注射生理盐水,注射剂量、速率、频次、周期均同侧脑室注射L-AAA造模组。
3.干预措施
①空白组和生理盐水组大鼠造模成功后不予任何处理;②电针组大鼠,于造模结束后第二天开始进行电针四关穴治疗,操作方法和治疗周期同“实验一”。③药物组大鼠,于造模结束后第二天开始进行利鲁唑灌胃治疗,操作方法和治疗周期同“实验一”。
4.评价指标
①行为学指标,在造模前以及造模的第3、6、9、12、15天进行体重测量、旷场试验、糖水偏好实验、新环境抑制进食实验,对造模期间大鼠行为学进行评价;在治疗第7、14、21天检测上述指标,对电针和药物的疗效进行评价。②光镜、电镜、Western-Blot、RT-PCR检测,治疗结束后,运用上述技术对大鼠海马AST的形态、超微结构以及AST标志蛋白GFAP含量以及GFAP mRNA的表达情况进行检测,对各组大鼠AST结构和功能损伤程度进行评价。
5.统计方法
同“实验一”。
结果:
1.电针对侧脑室注射L-AAA造模下大鼠行为学的影响
(1)体重
各组体重的基础值差异无统计学意义(P>0.05),具有可比性。在接受15天L-AAA侧脑室注射后,各组造模大鼠体重增长减缓,与正常对照组和生理盐水组比较,差异有统计学意义(P=0.000);各造模组大鼠组间比较差异无统计学意义(P>0.05),正常对照组与生理盐水组比较差异无统计学意义(P>0.05)。治疗3周后,空白组大鼠体重明显低于正常对照组和生理盐水组,差异均有统计学意义(P=0.000);电针组、药物组分别与空白组比较体重明显升高,差异有统计学意义(P=0.000);电针组与药物组比较,差异无统计学意义(P>0.05)。
(2)旷场实验
①水平运动
各组水平运动的基础值差异无统计学意义(P>0.05),具有可比性。在接受15天L-AAA侧脑室注射后,各组造模大鼠的水平运动次数明显下降,与正常对照组和生理盐水组比较差异有统计学意义(P=0.000),各造模组大鼠组间比较差异无统计学意义(P>0.05),正常对照组与生理盐水组比较差异统计学意义(P>0.05)。治疗3周后,与正常对照组比较,空白组和电针组大鼠水平爬格数目减少,差异均有统计学意义(P=0.000,P=0.013);与空白组比较,电针组和药物组大鼠水平爬格数目增多,差异均有统计学意义(P=0.000);电针组与药物组比较,差异无统计学意义(P>0.05)。
②垂直运动
各组垂直运动的基础值差异无统计学意义(P>0.05),具有可比性。在接受15天L-AAA侧脑室注射后,各组造模大鼠的垂直运动次数明显下降,与正常对照组和生理盐水组比较差异有统计学意义(P=0.000);各造模组大鼠组间比较没有差异无统计学意义(P>0.05),正常对照组与生理盐水组比较差异无统计学意义(P>0.05)。治疗3周后,与正常对照组和生理盐水组比较,空白组大鼠垂直站立次数明显减少,差异有统计学意义(P=0.000,P=0.001);与空白组比较,电针组和药物组大鼠垂直站立次数增多,差异均有统计学意义(P=0.001,P=0.000);正常对照组、电针组、药物组以及生理盐水组组间两两比较,差异无统计学意义(P>0.05)。
(3)糖水偏好实验
各组糖水偏好率的基础值差异无统计学意义(P>0.05),具有可比性。在接受15天L-AAA侧脑室注射后,各组造模大鼠的糖水偏好率明显下降,与正常对照组和生理盐水组比较差异有统计学意义(P=0.000);各造模组大鼠组间比较差异无统计学意义(P>0.05),正常对照组与生理盐水组比较差异无统计学意义(P>0.05)。治疗3周后,空白组大鼠糖水偏好率明显低于正常对照组和生理盐水组,差异有统计学意义(P=0.000),与空白组比较,电针组、药物组大鼠糖水偏好率升高,差异均有统计学意义(P=0.000);正常对照组、电针组、药物组、生理盐水组组间两两比较,差异无统计学意义(P>0.05)。
(4)新环境抑制进食实验
各组新环境进食第一口食物花费时间的基础值差异无统计学意义(P>0.05),具有可比性。在接受15天L-AAA侧脑室注射后,各组造模大鼠的新环境进食第一口食物花费时间明显增多,与正常对照组和生理盐水组比较差异有统计学意义(P=0.000);各造模组大鼠组间比较差异无统计学意义(P>0.05),正常对照组与生理盐水组比较差异无统计学意义(P>0.05)。治疗3周后,与正常对照组和生理盐水组比较,空白组大鼠新环境进食第一口食物花费时间明显增多,差异均有统计学意义(P=0.014,P=0.024);与空白组比较,电针组和药物组大鼠新环境进食第一口食物花费时间明显减少,差异均有统计学意义(P=0.000);正常对照组、生理盐水组、电针组、药物组组间两两比较,差异无统计学意义(P>0.05)。
2.电针对侧脑室注射L-AAA造模大鼠海马AST结构和功能的影响
(1)光镜下观察AST形态结构
经15天侧脑室注射L-AAA造模后,大鼠海马各区星形胶质细胞发生广泛性损害,细胞形态发生变化,主要表现为细胞核的固缩。同时侧脑室注射L-AAA造模还引起CA1区、CA3区部分锥体细胞以及DG区部分颗粒细胞变性、胞浆固缩。侧脑室注射NS后,大鼠海马CA1、CA3区星形胶质细胞未见明显损害,仅DG区少量星形胶质细胞和部分颗粒细胞出现变性、胞浆固缩。治疗3周后,电针组大鼠CA1区、CA3区、DG区星形胶质细胞均得以有效修复,CA1、CA3区锥体细胞修复,DG区颗粒细胞部分修复;药物组大鼠CA1区、CA3区星形胶质细胞和锥体细胞均得以有效修复,DG区星形胶质细胞恢复不明显,颗粒细胞变性部分恢复。
(2)光镜下观察AST数目
接受15天L-AAA侧脑室注射后,在海马CA1区,空白组大鼠AST数目明显减少,与正常对照组比较差异有统计学意义(P=-0.011)。治疗3周后,与空白组比较,电针组与药物组大鼠海马CA1区AST数明显目,差异均有统计学意义(P=0.03,P=0.016);电针组与药物组之间比较,差异无统计学意义(P>0.05)。
在海马CA3区,空白组大鼠AST数目明显减少,与正常对照组和生理盐水组比较差异均有统计学意义(P=0.034, P=0.008)。治疗3周后,电针组与药物组大鼠海马CA3区AST数目显著增多,与空白组比较差异均有统计学意义(P=0.034, P=0.011);电针组与药物组之间比较,差异无统计学意义(P>0.05)。
在海马DG区,空白组大鼠AST数目明显减少,与正常对照组和生理盐水组比较差异均有统计学意义(P=0.007, P=0.017).治疗3周后,电针组与药物组大鼠海马DG区AST数目增多,分别与空白组比较差异均有统计学意义(P=0.022, P=0.007);电针组与药物组之间比较,差异无统计学意义(P>0.05)。
(3)电镜下观察AST超微结构
侧脑室注射L-AAA15天后,空白组大鼠海马星形胶质细胞遭受较为严重的破坏,星形胶质细胞由梭形变为近似椭圆形,胞质减少,胞质内细胞器几乎缺失;生理盐水组大鼠海马AST未见明显损害。
治疗3周后,电针组大鼠AST细胞质内部分细胞器(粗面内质网、核糖体、微丝、线粒体)得以修复,但细胞形状以及胞质稀少情况没有得到很好的改善。药物组大鼠AST超微结构改善情况同电针组相仿。
(4)电针对侧脑室注射L-AAA大鼠海马GFAP含量和GFAP mRNA表达的影响
侧脑室注射L-AAA15天后,与正常对照组和生理豁水组比较,空白组大鼠海马GFAP含量显著减少,差异均有统计学意义(P=0.006, P=0.043);治疗3周后,与空白组比较,电针组和药物组GFAP含量升高,差异均有统计学意义(P=0.011,P=0.009);电针组与药物组比较,差异无统计学意义(P>0.05)。
侧脑室注射L-AAA15天后,与正常对照组和生理盐水组比较,空白组大鼠海马GFAP mRNA表达值显著下降,差异有统计学意义(P=0.000);治疗3周后,与空白组比较,电针组和药物组GFAP mRNA表达值升高,差异有统计学意义(P=0.000);电针组与药物组比较,差异无统计学意义.05)。
结论:
本研究通过上述两个实验相互佐证得出结论:①电针对慢性不可预见性温和刺激联合孤养造模导致的大鼠抑郁样行为及抑郁大鼠海马星形胶质细胞结构和功能的损伤有很好的保护作用;②电针对侧脑室注射L-AAA造模导致的大鼠抑郁样行为及造模大鼠海马星形胶质细胞结构和功能的损伤有很好的保护作用;③电针的上述作用与利鲁唑类似;④实验结果提示,海马星形胶质细胞参与了慢性应激致抑郁以及电针抗抑郁的过程;电针对海马星形胶质细胞的保护作用可能是其发挥抗抑郁的重要机制之
Object ive
Through 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 injury
Methods
1. Animal grouping and modeling methods
66adult 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. Interventions
①After 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. Evaluation
①Behavioral 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 methods
The 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.
Results
1. The influence of electro-acupuncture on the behaviors of depression rats induced by chronic stress
(1) Body Weight Measurement
Baseline 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 Test
①Horizontal motion
Baseline 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 motion
Baseline 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 Test
Baseline 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 Test
Baseline 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 astrocytes
After5weeks 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 astrocytes
After5weeks 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 mRNA
After5weeks 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 injection
Methods
1. Animal grouping and modeling methods
78adult 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 method
①Rats 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. Interventions
①After 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. Evaluation
①Behavioral 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 methods
The same as Part1.
Results
1. The influence of electro-acupuncture on the behaviors of rats modeled by L-AAA lateral cerebral ventricle injection
(1) Body Weight Measurement
Baseline 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 Test
①Horizontal motion
Baseline 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 motion
Baseline 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 Test
Baseline 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 Test
Baseline 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 astrocytes
After15days 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 astrocytes
After15days 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 mRNA
After15days 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).
Conelusions
Through 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.
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