不同时机针刺介入对TBI模型大鼠脑神经元的保护作用及机理研究
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摘要
目的
1.使用eCCI仪器,研究制备颅脑损伤(traumatic brain injury,TBI)大鼠动物模型,为针刺治疗TBI实验研究提供操作可控、损伤程度稳定、重复性好、死亡率低的模型制备方法。
2.利用该方法制备SD大鼠TBI模型,研究不同时间点针刺介入治疗TBI的效果,揭示针刺干预对TBI动物神经元的保护作用及部分机理,为临床针刺治疗TBI的最佳介入时间提供实验依据、丰富针刺治疗TBI的科学内涵。
方法
1.选用65只SD大鼠,随机分为空白对照组、模型1组、模型2组、模型3组和假手术组,使用eCCI选定不同打击参数(模型1组速度3m/s、深度3mm；模型2组速度4m/s、深度3mm；模型3组速度5m/s、深度3mm)制备不同损伤程度的TBI模型,通过以下几方面评测模型的制备效果：
(1)动物神经功能、平衡能力和行走能力评价。
(2)HE染色光镜下观察脑组织形态学改变情况。
(3)电镜观察脑组织超微结构的变化。
(4) ELISA法检测动物血清中S100B、NSE含量的动态变化。
(5)免疫组化法检测脑组织NGF、BDNF表达情况。
(6)免疫组化法检测脑组织Bax、Bcl-2表达情况。
2.选用70只SD大鼠,随机分为空白对照组、针刺1组、针刺2组、针刺3组和模型对照组,使用eCCI仪器,选用打击参数为速度4m/s、深度3mm制备TBI动物模型。取百会、人中、内关(左侧)和足三里(左侧),分别在脑损伤后第1日、第3日、第5日开始针刺治疗,各组治疗持续10次,等待结束点,在第15日取材,观察、检测相关指标：
(1)光镜下HE染色观察脑组织病理改变情况。
(2)免疫组化法、Western-blot法检查脑组织中NGF、BDNF蛋白的定位、含量及荧光定量PCR法检测NGFmRNA、BDNF mRNA的表达变化。
(3)免疫组化法、Western-blot法检查脑组织中Bcl-2、Bax蛋白的定位、含量及荧光定量PCR法检测Bcl-2mRNA、Bax mRNA的表达变化。
结果
1.TBI模型大鼠制备研究
(1)SD大鼠颅脑受到不同速度的外力打击后,模型1组、模型2组、模型3组出血量均随打击强度增加而增多,呼吸减慢,死亡动物数随之上升。于打击后第1日,对其神经功能、平衡能力、行走能力进行评价,结果显示神经功能、平衡能力、行走能力均随打击速度的增加而下降,且各组间有显著性差异(P<0.01)；于第3日进行评估,各组神经功能、平衡能力、行走能力均较第1日明显改善,但各组之间仍存在显著性差异(P<0.01)。
(2)采用HE染色光镜下观察脑组织形态学变化,空白组和假手术组脑组织皮层细胞分布清晰致密、排列均匀、胞体饱满、胞核呈蓝色、轮廓清楚、神经元染色正常；模型各组受伤皮质可见不同程度红细胞溢出、大量炎性细胞浸润、间质水肿、组织疏松、血管周围间隙增宽,且随打击强度增加而明显加重。
(3)电镜观察脑组超微结构变化,正常组和假手术组神经元整体结构完整、清晰,线粒体、核糖体、粗面内质网等细胞器含量丰富,线粒体具有清晰可见的内外膜结构、嵴排列整齐；而模型各组神经元细胞出现核固缩、溶解,胞浆变少,细胞器含量减少,线粒体、内质网肿胀等,且随打击力度增加而加重。
(4)ELISA法动态检测血清中S100B、NSE含量的变化,模型各组动物当损伤发生后第1日、第2日、第3日均呈上升趋势,同空白组和假手术组比较均有显著性差异(P<0.01)；模型1组、模型2组、模型3组之间表现为随打击力度的增加S100B、NSE含量显著性增高,且各组之间具有显著性差异(P<0.01)。
(5)免疫组化法检测脑组织中NGF、BDNF、Bax、Bcl-2蛋白表达情况。阳性表达物主要分布在神经细胞内,以小颗粒为主、部分呈现为聚集分布。空白组和假手术组大鼠损伤脑组织大脑皮质、海马及小脑神经元胞浆中NGF、BDNF、 Bax、Bcl-2均有弱阳性反应。模型各组损伤后脑组织损伤区大脑皮质、海马、小脑神经元胞浆中NGF、BDNF、Bax、Bcl-2呈强阳性反应,表达明显增多,且随损伤打击速度的增加而增多,各组之间具有统计学意义(P<0.01)。
2.不同时机针刺介入治疗TBI对脑组织NGF、BDNF、Bax和Bcl-2调控的研究。
(1)HE染色脑组织形态学变化。空白组脑神经细胞密集,可清楚地看到细胞的大体形态；模型组皮层神经细胞较少、神经元皱缩、胞质深染、核不清晰,有坏死神经细胞；针刺各组胶质细胞增多、凋亡小体减少、坏死细胞明显减少,神经元形态接近正常,坏死面积缩小,血液循环明显改善。
(2)采用免疫组化检测脑组织中NGF、BDNF、Bax、Bcl-2蛋白表达定位及含量,又结合Western-blot定量检测其蛋白含量变化,针刺可上调脑组织损伤皮质中NGF、BDNF、Bcl-2蛋白的含量、下调脑组织损伤皮质中Bax蛋白的含量；同时,荧光定量PCR检测也发现,针刺可上调脑组织损伤皮质中NGF、BDNF、Bcl-2相应基因mRNA含量、下调脑组织损伤皮质中Bax基因1mRNA含量,且针刺1组优于针刺2组、针刺3组,组间比较具有统计学意义(P<0.01)。
结论
1.利用eCCI仪器制备TBI模型大鼠能够通过打击速度、打击深度参数的设置而精确控制模型的损伤程度,具有易操作、打击强度可控制、重复性好、死亡率可控等优点,能够根据研究需要制备各种理想损伤程度的动物模型。
2.TBI后针刺介入治疗的时间点应尽可能早,更有利于发挥针刺上调脑组织神经元的神经生长因子(NGF、BDNF)及相应mRNA表达、调控凋亡因子(Bax、 Bcl-2)及相应mRNA的表达,达到保护脑组织神经元的作用。
Objective:
1. To study on preparation of TBI rats model by eCCI, and provide a model preparation method of easy operation, the degree of damaged controlled, good repeatability and low mortality for the TBI experimental study.
2. To prepare TBI model of SD rat by the method, and study the effects of acupuncture treatment of TBI at different periods. To reveal protective effects and mechanism of the acupuncture to the injured nerve cells of TBI brain, provide experimental basis of acupuncture treatment of acute phase TBI at the best time, and to add the scientific contents of acupuncture treatment of TBI.
Methods:
1.65SD rats, which were randomly divided into blank control group, model group1, model group2, model group3and sham-operation group, were produced as the animal model of brain injury in different degree by eCCI from selected different fighting parameters. The speed was3m/s and the depth was3mm in model group1. The speed was4m/s and the depth was3mm in model group2. The speed was5m/s and the depth was3mm in model group3. Evaluated the model quality through the following:
(1) Evaluation of animal behavior, nerve function, balance ability and walking ability.
(2) To observe the morphological changes of brain tissue by HE under light microscope.
(3) To observe the ultrastructure changes of brain tissue by electron microscopy.
(4)To detect the dynamic changes of S100B and NSE were by ELIASA in animal serum.
(5) To detect the contents of NGF and BDNF of brain tissue by immunohistochemical method.
(6)To detect the contents of BAX and Bcl-2of brain tissue by immunohistochemical method.
2.70SD rats were randomly divided into blank control group, acupuncture group1, acupuncture group2, acupuncture group3and model control group. The TBI animal models were prepared with the eCCI of the speed of4m/s and the depth of3mm. Than rats were treated by acupuncturing Baihui(DU20), Renzhong(DU26), the left Neiguan((PC6) and the left Zusanli(ST36)) after the brain injured at1day,3days,5days. The treatments of each group were repeated10times. The materials were collected at the15th day and to observe and examine the related index signs:
(1) To observe the pathological changes of brain tissue by HE staining light microscope.
(2)By immunohistochemical and Western-blot to examine the localization and content of NGF and BDNF in the brain tissue protein, to detect the mRNA changes of NGF and BDNF by Fluorescence metered PCR method.
(3) By immunohistochemical and Western-blot to examine the localization and content of Bcl-2and Bax in the brain tissue protein, and detect the mRNA changes of Bcl-2and Bax by Fluorescence metered PCR method.
Results:
1. Study on preparation of TBI models of rats
(1) After the brains of SD rats had been damaged by different speeds of outside force, the amount of bleeding with the stroke strength increasing was on the increase, the breath slowly, the dead animals increasing in model group1,2and3. On the1st day after hit, the nerve function, balance and walking ability were evaluated. The results showed that nerve function, balance and walking ability were decreased with the increase of hit, and there were significant differences in groups(P<0.01). On the third day, each group of nerve function, balance and walk ability were obviously improved than the first day, and there were still differences between the groups after evaluation (P<0.01).
(2) Observed by HE under light microscope, the cells of brain tissues were distributed clear and fine,Arranged well, chubbiness, and had clear contour, blue nucleus. The colour of nerve cells been stained was normal in the blank group and sham operation group. In model groups the injured cortex showed that they bled in different degrees. There were infiltration of a large number of inflammatory cells, edema of interstitial brain, wide perivascular space, and they obviously increased with the increase of impact strength. By electron microscopic in normal group and sham operation group, the structure of brain cells was complete and clear. Mitochondria, ribosomes and rough endoplasmic reticulum were rich.The mitochondria had clear inner and outer membrane structure and neat crest. In the model groups, the brain cells showed that there were pyknosis of the nucleus, the nucleus dissolved, and little cytoplasm. The content of organelles was reduced. The mitochondria and endoplasmic reticulum were swelling with the increase of the hit.
(3) The content of S100B and NSE in serum was increased on the first day, second day, third day after animal damaged in model group by ELISA. In the model group and sham operation group, there were significantly different (P<0.01).In model group1,2and3, The content of S100B and NSE was increased significantly with the increase of hit (P<0.01).
(4) The NGF, BDNF, Bax and Bcl-2protein were detected by immunohistochemistry in brain tissue. The positive expressions were mainly distributed in nerve cells, mainly as small particles and partly as aggregated distribution. The NGF, BDNF, Bax and Bcl-2in the brain tissue of cerebral cortex, hippocampus and cerebellum cytoplasm of neurons showed weak positive reaction in control group and sham operation group. The NGF, BDNF, Bax and Bcl-2of damaged brain in model group showed positive reaction and the expression was markedly increased with the increase of hit (P<0.01).
2. Study of time of acupuncture to regulate NGF, BDNF, Bax and Bcl-2of brain tissue
(1) Morphological changes of brain tissue by HE. The nerve cells in blank group were dense and the cell morphology can be clearly seen. In model group, the cortical neuron were, little and shrinkage, the cytoplasm deeply colored, illegibility of nuclear, and some died. In acupuncture group, glial cells increased. Apoptotic body and necrotic cells were less obviously reduced. The neurons were close to normal, the area of necrosis reduced, the blood circulation improved.
(2) The expression and content of NGF, BDNF, Bax, Bcl-2were detected by immunohistochemistry and Western-blot in brain tissue. Acupuncture can up-regulate NGF, BDNF, Bcl-2, and down-regulation Bax. And by PCR, acupuncture can increase the mRNA of NGF, BDNF, Bcl-2, and decrease the mRNA of Bcl-2. After TBI occurred, while acupuncture treatment was used as soon as possible, the acupuncture was more obvious on the regulation effect. And there was significant differences among the three groups (P<0.01).
Conclusion:
1. By eCCI TBI rat model can be prepared with regulation of hit speed and depth. The damage of model was related to the hit speed. The method of TBI model preparation has the advantages of easy operation, good control ability, repeatability, low mortality rate, and can according to the need be used to make various animal model for the study.
2. Acupuncture treated TBI as early as possible.The results of this study showed that after1day of TBI occurred if acupuncture was used immediately, the curative effect was better than3days and5days latter.
1.使用eCCI仪器,研究制备颅脑损伤(traumatic brain injury,TBI)大鼠动物模型,为针刺治疗TBI实验研究提供操作可控、损伤程度稳定、重复性好、死亡率低的模型制备方法。
2.利用该方法制备SD大鼠TBI模型,研究不同时间点针刺介入治疗TBI的效果,揭示针刺干预对TBI动物神经元的保护作用及部分机理,为临床针刺治疗TBI的最佳介入时间提供实验依据、丰富针刺治疗TBI的科学内涵。
方法
1.选用65只SD大鼠,随机分为空白对照组、模型1组、模型2组、模型3组和假手术组,使用eCCI选定不同打击参数(模型1组速度3m/s、深度3mm；模型2组速度4m/s、深度3mm；模型3组速度5m/s、深度3mm)制备不同损伤程度的TBI模型,通过以下几方面评测模型的制备效果：
(1)动物神经功能、平衡能力和行走能力评价。
(2)HE染色光镜下观察脑组织形态学改变情况。
(3)电镜观察脑组织超微结构的变化。
(4) ELISA法检测动物血清中S100B、NSE含量的动态变化。
(5)免疫组化法检测脑组织NGF、BDNF表达情况。
(6)免疫组化法检测脑组织Bax、Bcl-2表达情况。
2.选用70只SD大鼠,随机分为空白对照组、针刺1组、针刺2组、针刺3组和模型对照组,使用eCCI仪器,选用打击参数为速度4m/s、深度3mm制备TBI动物模型。取百会、人中、内关(左侧)和足三里(左侧),分别在脑损伤后第1日、第3日、第5日开始针刺治疗,各组治疗持续10次,等待结束点,在第15日取材,观察、检测相关指标：
(1)光镜下HE染色观察脑组织病理改变情况。
(2)免疫组化法、Western-blot法检查脑组织中NGF、BDNF蛋白的定位、含量及荧光定量PCR法检测NGFmRNA、BDNF mRNA的表达变化。
(3)免疫组化法、Western-blot法检查脑组织中Bcl-2、Bax蛋白的定位、含量及荧光定量PCR法检测Bcl-2mRNA、Bax mRNA的表达变化。
结果
1.TBI模型大鼠制备研究
(1)SD大鼠颅脑受到不同速度的外力打击后,模型1组、模型2组、模型3组出血量均随打击强度增加而增多,呼吸减慢,死亡动物数随之上升。于打击后第1日,对其神经功能、平衡能力、行走能力进行评价,结果显示神经功能、平衡能力、行走能力均随打击速度的增加而下降,且各组间有显著性差异(P<0.01)；于第3日进行评估,各组神经功能、平衡能力、行走能力均较第1日明显改善,但各组之间仍存在显著性差异(P<0.01)。
(2)采用HE染色光镜下观察脑组织形态学变化,空白组和假手术组脑组织皮层细胞分布清晰致密、排列均匀、胞体饱满、胞核呈蓝色、轮廓清楚、神经元染色正常；模型各组受伤皮质可见不同程度红细胞溢出、大量炎性细胞浸润、间质水肿、组织疏松、血管周围间隙增宽,且随打击强度增加而明显加重。
(3)电镜观察脑组超微结构变化,正常组和假手术组神经元整体结构完整、清晰,线粒体、核糖体、粗面内质网等细胞器含量丰富,线粒体具有清晰可见的内外膜结构、嵴排列整齐；而模型各组神经元细胞出现核固缩、溶解,胞浆变少,细胞器含量减少,线粒体、内质网肿胀等,且随打击力度增加而加重。
(4)ELISA法动态检测血清中S100B、NSE含量的变化,模型各组动物当损伤发生后第1日、第2日、第3日均呈上升趋势,同空白组和假手术组比较均有显著性差异(P<0.01)；模型1组、模型2组、模型3组之间表现为随打击力度的增加S100B、NSE含量显著性增高,且各组之间具有显著性差异(P<0.01)。
(5)免疫组化法检测脑组织中NGF、BDNF、Bax、Bcl-2蛋白表达情况。阳性表达物主要分布在神经细胞内,以小颗粒为主、部分呈现为聚集分布。空白组和假手术组大鼠损伤脑组织大脑皮质、海马及小脑神经元胞浆中NGF、BDNF、 Bax、Bcl-2均有弱阳性反应。模型各组损伤后脑组织损伤区大脑皮质、海马、小脑神经元胞浆中NGF、BDNF、Bax、Bcl-2呈强阳性反应,表达明显增多,且随损伤打击速度的增加而增多,各组之间具有统计学意义(P<0.01)。
2.不同时机针刺介入治疗TBI对脑组织NGF、BDNF、Bax和Bcl-2调控的研究。
(1)HE染色脑组织形态学变化。空白组脑神经细胞密集,可清楚地看到细胞的大体形态；模型组皮层神经细胞较少、神经元皱缩、胞质深染、核不清晰,有坏死神经细胞；针刺各组胶质细胞增多、凋亡小体减少、坏死细胞明显减少,神经元形态接近正常,坏死面积缩小,血液循环明显改善。
(2)采用免疫组化检测脑组织中NGF、BDNF、Bax、Bcl-2蛋白表达定位及含量,又结合Western-blot定量检测其蛋白含量变化,针刺可上调脑组织损伤皮质中NGF、BDNF、Bcl-2蛋白的含量、下调脑组织损伤皮质中Bax蛋白的含量；同时,荧光定量PCR检测也发现,针刺可上调脑组织损伤皮质中NGF、BDNF、Bcl-2相应基因mRNA含量、下调脑组织损伤皮质中Bax基因1mRNA含量,且针刺1组优于针刺2组、针刺3组,组间比较具有统计学意义(P<0.01)。
结论
1.利用eCCI仪器制备TBI模型大鼠能够通过打击速度、打击深度参数的设置而精确控制模型的损伤程度,具有易操作、打击强度可控制、重复性好、死亡率可控等优点,能够根据研究需要制备各种理想损伤程度的动物模型。
2.TBI后针刺介入治疗的时间点应尽可能早,更有利于发挥针刺上调脑组织神经元的神经生长因子(NGF、BDNF)及相应mRNA表达、调控凋亡因子(Bax、 Bcl-2)及相应mRNA的表达,达到保护脑组织神经元的作用。
Objective:
1. To study on preparation of TBI rats model by eCCI, and provide a model preparation method of easy operation, the degree of damaged controlled, good repeatability and low mortality for the TBI experimental study.
2. To prepare TBI model of SD rat by the method, and study the effects of acupuncture treatment of TBI at different periods. To reveal protective effects and mechanism of the acupuncture to the injured nerve cells of TBI brain, provide experimental basis of acupuncture treatment of acute phase TBI at the best time, and to add the scientific contents of acupuncture treatment of TBI.
Methods:
1.65SD rats, which were randomly divided into blank control group, model group1, model group2, model group3and sham-operation group, were produced as the animal model of brain injury in different degree by eCCI from selected different fighting parameters. The speed was3m/s and the depth was3mm in model group1. The speed was4m/s and the depth was3mm in model group2. The speed was5m/s and the depth was3mm in model group3. Evaluated the model quality through the following:
(1) Evaluation of animal behavior, nerve function, balance ability and walking ability.
(2) To observe the morphological changes of brain tissue by HE under light microscope.
(3) To observe the ultrastructure changes of brain tissue by electron microscopy.
(4)To detect the dynamic changes of S100B and NSE were by ELIASA in animal serum.
(5) To detect the contents of NGF and BDNF of brain tissue by immunohistochemical method.
(6)To detect the contents of BAX and Bcl-2of brain tissue by immunohistochemical method.
2.70SD rats were randomly divided into blank control group, acupuncture group1, acupuncture group2, acupuncture group3and model control group. The TBI animal models were prepared with the eCCI of the speed of4m/s and the depth of3mm. Than rats were treated by acupuncturing Baihui(DU20), Renzhong(DU26), the left Neiguan((PC6) and the left Zusanli(ST36)) after the brain injured at1day,3days,5days. The treatments of each group were repeated10times. The materials were collected at the15th day and to observe and examine the related index signs:
(1) To observe the pathological changes of brain tissue by HE staining light microscope.
(2)By immunohistochemical and Western-blot to examine the localization and content of NGF and BDNF in the brain tissue protein, to detect the mRNA changes of NGF and BDNF by Fluorescence metered PCR method.
(3) By immunohistochemical and Western-blot to examine the localization and content of Bcl-2and Bax in the brain tissue protein, and detect the mRNA changes of Bcl-2and Bax by Fluorescence metered PCR method.
Results:
1. Study on preparation of TBI models of rats
(1) After the brains of SD rats had been damaged by different speeds of outside force, the amount of bleeding with the stroke strength increasing was on the increase, the breath slowly, the dead animals increasing in model group1,2and3. On the1st day after hit, the nerve function, balance and walking ability were evaluated. The results showed that nerve function, balance and walking ability were decreased with the increase of hit, and there were significant differences in groups(P<0.01). On the third day, each group of nerve function, balance and walk ability were obviously improved than the first day, and there were still differences between the groups after evaluation (P<0.01).
(2) Observed by HE under light microscope, the cells of brain tissues were distributed clear and fine,Arranged well, chubbiness, and had clear contour, blue nucleus. The colour of nerve cells been stained was normal in the blank group and sham operation group. In model groups the injured cortex showed that they bled in different degrees. There were infiltration of a large number of inflammatory cells, edema of interstitial brain, wide perivascular space, and they obviously increased with the increase of impact strength. By electron microscopic in normal group and sham operation group, the structure of brain cells was complete and clear. Mitochondria, ribosomes and rough endoplasmic reticulum were rich.The mitochondria had clear inner and outer membrane structure and neat crest. In the model groups, the brain cells showed that there were pyknosis of the nucleus, the nucleus dissolved, and little cytoplasm. The content of organelles was reduced. The mitochondria and endoplasmic reticulum were swelling with the increase of the hit.
(3) The content of S100B and NSE in serum was increased on the first day, second day, third day after animal damaged in model group by ELISA. In the model group and sham operation group, there were significantly different (P<0.01).In model group1,2and3, The content of S100B and NSE was increased significantly with the increase of hit (P<0.01).
(4) The NGF, BDNF, Bax and Bcl-2protein were detected by immunohistochemistry in brain tissue. The positive expressions were mainly distributed in nerve cells, mainly as small particles and partly as aggregated distribution. The NGF, BDNF, Bax and Bcl-2in the brain tissue of cerebral cortex, hippocampus and cerebellum cytoplasm of neurons showed weak positive reaction in control group and sham operation group. The NGF, BDNF, Bax and Bcl-2of damaged brain in model group showed positive reaction and the expression was markedly increased with the increase of hit (P<0.01).
2. Study of time of acupuncture to regulate NGF, BDNF, Bax and Bcl-2of brain tissue
(1) Morphological changes of brain tissue by HE. The nerve cells in blank group were dense and the cell morphology can be clearly seen. In model group, the cortical neuron were, little and shrinkage, the cytoplasm deeply colored, illegibility of nuclear, and some died. In acupuncture group, glial cells increased. Apoptotic body and necrotic cells were less obviously reduced. The neurons were close to normal, the area of necrosis reduced, the blood circulation improved.
(2) The expression and content of NGF, BDNF, Bax, Bcl-2were detected by immunohistochemistry and Western-blot in brain tissue. Acupuncture can up-regulate NGF, BDNF, Bcl-2, and down-regulation Bax. And by PCR, acupuncture can increase the mRNA of NGF, BDNF, Bcl-2, and decrease the mRNA of Bcl-2. After TBI occurred, while acupuncture treatment was used as soon as possible, the acupuncture was more obvious on the regulation effect. And there was significant differences among the three groups (P<0.01).
Conclusion:
1. By eCCI TBI rat model can be prepared with regulation of hit speed and depth. The damage of model was related to the hit speed. The method of TBI model preparation has the advantages of easy operation, good control ability, repeatability, low mortality rate, and can according to the need be used to make various animal model for the study.
2. Acupuncture treated TBI as early as possible.The results of this study showed that after1day of TBI occurred if acupuncture was used immediately, the curative effect was better than3days and5days latter.
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