曲古抑素A（TSA）预处理对大鼠局灶性脑缺血再灌注损伤的脑保护作用及机制研究
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摘要
脑缺血再灌注损伤是临床常见的病理生理表现。脑缺血预处理(ischemic preconditioning, IPC)被认为是最强的神经内源性保护现象,但因其是损伤性预适应,所以探讨采用药物诱导预适应替代脑缺血预处理成为必然。
临床缺血性脑血管病中所占比重最大的是发生在大脑中动脉主干分布区的梗死,且常有自发性再通,目前常用的线栓法阻断大脑中动脉复制出的脑缺血再灌注模型(middle cerebral artery occlusion, MCAO)较符合这种病理生理学过程。即使阻断大脑中动脉时间相同,脑梗死范围却不稳定是线栓法复制MCAO模型的缺点。研究采用改良线栓及其他措施来复制稳定性好的MCAO模型,才能为随后的研究奠定可靠的基础。
随着中国步入老龄化社会,缺血性脑血管疾病已成为常见病、多发病。计划生育的实施,人们对独生子女的健康问题更加关注,而神经系统疾病导致的脑功能损伤,是目前亟待解决的儿科难题。虽然对缺血性脑损伤的机制研究甚多,但表观遗传学方面的研究不多。
曲古抑素A (trichostatin A, TSA)作为广谱组蛋白去乙酰化酶抑制剂应用广泛,而其预处理是否对脑缺血再灌注损伤具有保护作用及其作用机制的研究较少,尤其是关于TSA预处理对不同年龄大鼠脑缺血再灌注损伤的保护机制研究尚未见报道。
通过了解TSA预处理对不同年龄大鼠脑缺血再灌注损伤的保护作用,深入研究组蛋白乙酰化/去乙酰化在缺血再灌注过程中脑损伤过程中的作用机制,从多个方面入手对此病理生理进程实施干预,无疑将为积极防治缺血再灌注性脑损伤开拓新的应用前景。
第一部分采用改良线栓法复制稳定的大鼠MCAO模型
1.1研究背景与目的
采用从颈总动脉或颈外动脉置入线栓,阻断大脑中动脉血供一定时间之后再恢复灌注的动物模型,被认为与临床缺血性脑血管病中所占比重最大,且常有自发性再通的大脑中动脉主干分布区脑梗死的病理生理学过程相近似,所以应用较多。进行药物预处理实验时,要求所选动物模型要符合临床实际,所得出的实验结果才有应用价值。所以本实验采用栓塞大脑中动脉复制出的局灶性脑缺血模型(MCAO)进行曲古抑素A预处理的脑保护研究。
线栓法MCAO模型的缺点是即使阻断大脑中动脉时间相同,脑梗死范围却不稳定。一定要复制出稳定性好的MCAO模型,才能为随后的研究奠定可靠的基础。本实验拟采用改良线栓及其它措施,从而复制出稳定性好的MCAO模型。
1.2材料与方法
研究对象：健康清洁SD雄性大鼠随机分为假手术组、缺血再灌注组,每组各6只。
动物模型：改良线栓阻断大鼠大脑中动脉供血90min,再灌注24h制备大鼠MCAO模型。假手术组仅将线栓放置在颈外动脉内。
观测指标：HE染色、Zea-Longa神经功能损害评分、脑梗死体积比。
检测方法：苏木精-伊红(HE)染色法观察手术侧脑组织病理形态改变；缺血再灌注6h、12h、24h时间点使用Zea-Longa评分法进行神经功能损害评分；TTC染色法测定大鼠脑梗死体积比。
数据分析：采用Excel 2007建立数据库并录入数据。神经功能学评分用中位数(范围)表示。采用SPSS17.0进行统计分析,P<0.05表示有统计学差异。
1.3结果
(1)两组大鼠手术侧脑组织HE染色显示不同,缺血再灌注组脑损伤改变明显,假手术组未发生损伤性改变。
(2)两组大鼠Zea-Longa神经功能损害评分于缺血再灌注6h、12h、24h时间点差异有统计学意义(P (3)TTC染色显示缺血再灌注组有明确的梗死灶,对照组则无,差异有统计学意义(P<0.01)。
1.4结论
本实验采用改良线栓阻断MCA法及其它措施复制的MCAO模型,稳定性良好,值得推广应用。
第二部分不同剂量TSA预处理对MCAO大鼠IL-1β、bcl-2的影响
2.1研究背景与目的
脑缺血再灌注损伤是临床常见的病理生理表现。脑缺血预处理被认为是最强的神经内源性保护现象,但由于脑缺血预处理是损伤性预适应,探讨采用药物诱导预适应替代脑缺血预处理成为必然。TSA作为广谱组蛋白去乙酰化酶抑制剂应用广泛,而其预处理是否有减轻脑缺血再灌注损伤作用及其作用机制少见报道。本研究拟探讨不同剂量TSA预处理是否通过对血液和脑脊液中IL-1β含量、抗凋亡蛋白bcl-2等方面来减轻脑缺血再灌注损伤。
2.2材料与方法
研究对象：健康清洁SD雄性大鼠被随机分为缺血再灌注对照组(对照组)、0.1mg/kg TSA预处理组(TSA1组)、0.03mg/kg TSA预处理组(TSA2组)、1%DMSO预处理组(DMSO组),每组又被分为缺血再灌注6h组、12h组、24h组、48h共4个亚组,每亚组6只大鼠。
动物模型：采用前述改良线栓法阻断大脑中动脉供血90min,再灌注6h、12h、24h、48h复制大鼠局灶性脑缺血再灌注模型。TSA1组、TSA2组、DMSO组于缺血前20min分别经尾静脉注入含有相应药物的生理盐水1ml,对照组则注入相同体积的生理盐水。
观测指标：Zea-Longa神经功能损害评分、脑梗死体积比、脑脊液和血液IL-1β含量变化、抗凋亡蛋白bcl-2阳性细胞计数。
检测方法：神经功能损害评分依据Zea-Longa评分法进行；苏木精-伊红(HE)染色法观察缺血侧脑组织病理改变；ELISA法分析法检测脑脊液和血清中IL-1β的含量变化；免疫组化法测定栓塞侧大脑缺血半暗带区bcl-2蛋白阳性细胞数。
数据分析：采用Excel 2007建立数据库并录入数据。采用两因素方差分析处理因素、时间因素及其交互作用对神经功能评分、脑梗死体积比、脑脊液、血液IL-1β和bcl-2阳性细胞计数的影响。如方差分析前提条件未得到满足,采用相应的非参数检验或稳健方法作为替代。选择α=0.05作为统计检验水准,采用SPSS17.0完成统计分析。
2.3结果
(1)与缺血再灌注6h时间点的对照组相比,TSA1组神经功能损害评分降低有统计学意义(P<0.05)外,其余各时间点的各组神经功能损害评分比较无显著差异(P>0.05)。
(2)TSA1组大鼠脑梗死体积比均小于其余3组,差异有统计学意义(P<0.05)；TSA2组及DMSO组脑梗死体积比小于对照组(P<0.05)。
(3) TSA1组血液IL-1β含量低于对照组和DMSO组(P<0.05)；TSA2组与DMSO组无差别(P>0.05),但均低于对照组(P<0.05)
(4) Pearson相关分析显示,脑梗死体积比与血IL-1β、脑脊液IL-1β高度相关,对应的Pearson相关系数分别为0.841和0.618,P<0.05。
(5)再灌注6h时间点,对照组bcl-2计数低于其余3组,DMSO组和小剂量TSA均低于TSA1组(P<0.05)；再灌注12h时间点,对照组、DMSO组和TSA2组之间没有统计学差别(P>0.05),但3组均低于TSA1组(P<0.05)；再灌注24h时间点,对照组bcl-2计数低于其余3组,DMSO组和小剂量TSA均低于TSA1组(P<0.05)；再灌注48h时间点,4组之间bcl-2计数彼此之间均有统计学差别(P<0.05)。
2.4结论
(1)TSA预处理可降低血液和脑脊液中IL-1β的含量,增加抗凋亡蛋白bcl-2阳性细胞数。
(2)TSA预处理可剂量依赖性减少MCAO大鼠缺血再灌注损伤时脑梗死体积比,对MCAO大鼠可能有脑保护作用。
第三部分TSA预处理对MCAO大鼠脑组织中HDAC、乙酰化组蛋白H3、乙酰化组蛋白H4表达水平的影响
3.1研究背景与目的
脑药物预处理是指使用药物刺激机体产生类似机体自身内源性保护物质而呈现脑保护作用,它是在缺血性预处理的基础上发展起来的。组蛋白去乙酰化酶抑制剂(histone deacetylases inhibitors, HDACIs)通过作用于缺血性卒中损伤机制中的多个环节减轻脑组织损伤,增加缺血后神经元可塑性和促进功能恢复。此前研究提示TSA预处理可以减少脑脊液中IL-1β的含量、促进抗凋亡蛋白bcl-2表达,对脑缺血再灌注损伤具有保护作用,但其作用机制尚未明了。本研究拟探讨TSA预处理对MCAO大鼠脑组织中HDAC、乙酰化组蛋白H3 (acetylaced H3, Ac-H3)、乙酰化组蛋白H4 (acetylaced H4, Ac-H4)表达水平的影响。
3.2材料与方法
研究对象：同第二部分。
动物模型：同第一部分。
观测指标：栓塞侧脑组织HDAC活性测定、Ac-H3、Ac-H4表达水平检测。
检测方法：采用比色法检测栓塞侧脑组织HDAC活性；免疫印迹法测定栓塞侧脑组织乙酰化组蛋白H3、H4表达水平。
数据分析：采用Excel 2007建立数据库并录入数据。采用两因素方差分析处理因素、时间因素及其交互作用对HDAC活性、Ac-H3、Ac-H4表达水平的影响。如方差分析前提条件未得到满足,采用相应的非参数检验或稳健方法作为替代。选择α=0.05作为统计检验水准,采用SPSS17.0完成统计分析。
3.3结果
(1)大鼠局灶性脑I/R损伤后HDAC活性随缺血再灌注时间延长呈上升趋势。各组HDAC活性随缺血再灌注时间延长而持续上升。TSAl组大鼠HDAC活性虽有明显上升,但低于对照组和DMSO组,差异有统计学意义(P<0.01)；TSA2组HDAC活性水平与DMSO组没有差别,但低于对照组；DMSO组与对照组没有差别(P>0.05)。
(2)各组Ac-H、Ac-H4表达水平在缺血再灌注6h时间点均呈上升趋势,至缺血再灌注12h时间点达高峰,随后下降。各组在不同处理组和时间点之间乙酰化组蛋白H3表达水平存在差别,TSA1组大鼠Ac-H3表达水平均高于其余3组；TSA2组与DMSO组Ac-H3表达水平均高于对照组,但这两组间没有差别。
(3)各组在不同分组和时间点之间Ac-H4表达水平存在差别,TSA1组大鼠Ac-H4表达水平均高于其余3组；TSA2组Ac-H4表达水平高于对照组,但与DMSO组没有差别；DMSO组与对照组之间没有差别。
(4) HDAC活性改变与Ac-H3、Ac-H4表达水平的Spearman秩相关系数分别为0.400和0.200,没有统计学学意义。但Ac-H3、Ac-H4表达水平的Spearman秩相关系数为0.964,有统计学意义。
(5)Ac-H3表达水平均与脑脊液和血液IL-1β表达水平高度相关,Spearman秩相关系数分别为1.0和0.8；Ac-H4仅与脑脊液IL-1β表达水平相关,秩相关系数为0.8,但与血液IL-1β表达水平的秩相关系数无统计学意义。
(6) Ac-H3、Ac-H4表达水平均与bcl-2表达水平高度相关,Spearman秩相关系数分别为1.0和0.8。
3.4结论
(1)大鼠局灶性脑I/R损伤后脑组织HDAC活性随缺血再灌注时间延长呈上升趋势,TSA对HDAC有抑制作用；
(2)TSA预处理可上调脑组织中Ac-H3、Ac-H4水平,从而对IL-1β、bcl-2产生影响,这可能是TSA预处理的脑保护作用机制之一。
第四部分大剂量TSA预处理对不同年龄组MCAO大鼠IL-1β、bcl-2的影响
4.1研究背景与目的
随着中国步入老龄化社会,缺血性脑血管疾病已成为常见病、多发病。计划生育的实施,人们对独生子女的健康问题更加关注,而神经系统疾病导致的脑功能损伤,是目前亟待解决的儿科难题。虽然对缺血性脑血管疾病的病理生理特点及治疗机制研究甚多,但针对不同年龄大鼠表观遗传学方面的研究不多。此前研究提示TSA预处理对脑缺血再灌注损伤具有保护作用。为深入研究不同年龄大鼠脑缺血再灌注损伤的损伤机制,本研究拟采用大剂量TSA预处理不同年龄组MCAO大鼠,观察其对大鼠血液和脑脊液中IL-1β含量、抗凋亡蛋白bcl-2的影响。
4.2材料与方法
研究对象：SD雄性大鼠分为老年组、青年组、幼年组,均采用0.1mg/kg TSA预处理,每组6只。
动物模型：采用改良线栓法阻断实验大鼠大脑中动脉血供90min,再灌注24h制备大鼠局灶性脑缺血再灌注模型。老年组、青年组、幼年组于缺血前20min分别经尾静脉注入含相应药物的生理盐水共1ml。
观测指标：神经功能损害评分、脑梗死体积比、脑脊液和血液IL-1β、bcl-2阳性细胞计数。
检测方法：Zea-Longa评分法和Chen18分评分法进行神经功能缺陷评分；采用TTC染色法测大鼠脑梗死体积比；苏木精-伊红(HE)染色法观察缺血侧脑组织病理形态改变；采用酶联免疫吸附测定法分析(ELISA)法检测脑脊液和血清中IL-1β的含量变化；免疫组化法测定栓塞侧大脑缺血半暗带区bcl-2蛋白阳性细胞数。
数据分析：采用Excel 2007建立数据库并录入数据。采用两因素方差分析处理因素、时间因素及其交互作用对神经功能评分、脑梗死体积、脑脊液、血液IL-1β和bcl-2计数的影响。如方差分析前提条件未得到满足,采用相应的非参数检验或稳健方法作为替代。选择α=0.05作为统计检验水准,采用SPSS17.0完成统计分析。
4.3结果
(1)不同年龄组大鼠在缺血再灌注6h、12h、24h时间点的神经功能评分的单因素方差分析结果显示,三组间Zea-Longa神经功能损害评分无明显差别(P>0.05)。
(2)三组间缺血再灌注6h、12h时间点的改良神经功能损害程度评分无显著差别(P>0.05),在24h时间点有差别(P<0.05)。3组间两两比较结果显示,老年组与幼年组差别有统计学意义(P<0.05),老年组与青年组、青年组与幼年组之间无差别(P>0.05)。
(3)幼年组大鼠在缺血再灌注24h时间点的平衡总评分明显小于其余两组大鼠(P<0.05)；运动总评分与老年组大鼠相比有差别(P<0.05)；而幼年组大鼠和青年组大鼠的反射总评分小于老年组大鼠,差异有统计学意义(P<0.05)。
(4)不同年龄组大鼠的脑梗死体积比的单因素方差分析结果显示,三组间脑梗死体积比无统计学差别(P>0.05)
(5)单因素方差分析结果显示,不同年龄组大鼠血液和脑脊液中的IL-1β表达水平无差别(P>0.05)
(6)单因素方差分析结果显示,不同年龄组大鼠bcl-2阳性细胞计数3组间存在差别(P<0.05)。3组间两两比较结果显示,幼年组和青年组之间没有统计学差别(P>0.05),但二者bcl-2阳性细胞计数均低于老年组大鼠(P<0.05)。
4.4结论
TSA预处理对脑脊液中IL-1β含量以及抗凋亡蛋白bcl-2表达因年龄而作用不同。大剂量TSA预处理可能对不同年龄组MCAO大鼠具有脑保护作用。
第五部分TSA预处理对不同年龄组MCAO大鼠脑组织中HDAC、Ac-H3、Ac-H4表达水平的影响
5.1研究背景与目的
研究认为缺血耐受指的是特定的时间被出现的特殊刺激激发产生的适应性级联反应,包括基因依赖性反应和非基因依赖性反应,即已存在蛋白的翻译后修饰。预处理是机体器官在基因水平上对缺血反应“重新编程”的过程。组蛋白乙酰化和去乙酰化是目前研究最为深入的蛋白翻译后修饰机制。此前研究提示TSA预处理对脑缺血再灌注损伤具有保护作用。为深入研究不同年龄大鼠脑缺血再灌注损伤的损伤机制,本研究拟针对不同年龄组采用TSA预处理,观察各组大鼠局灶性脑缺血再灌注损伤的脑组织中HDAC、Ac-H3、Ac-H4表达水平的改变,为探索新的治疗方法打下基础。
5.2材料与方法研究对象：同第四部分。动物模型：同第四部分。观测指标：栓塞侧脑组织HDAC活性、Ac-H3、Ac-H4表达水平。
检测方法：采用比色法检测栓塞侧脑组织HDAC活性；免疫印迹法测定栓塞侧脑组织Ac-H3、Ac-H4表达水平。
数据分析：采用Excel 2007建立数据库并录入数据。采用两因素方差分析处理因素、时间因素及其交互作用对HDAC活性、Ac-H3、Ac-H4表达水平的影响。如方差分析前提条件未得到满足,采用相应的非参数检验或稳健方法作为替代。选择α=0.05作为统计检验水准,采用SPSS17.0完成统计分析。
5.3结果
(1)老年组大鼠HDAC活性显著高于幼年组和青年组,差异有统计学意义(P<0.01)；幼年组和青年组HDAC活性水平没有差别(P>0.05)。
(2)不同年龄组之间Ac-H3表达水平于缺血再灌注24h时间点差异有统计学意义(P<0.01)。幼年组和青年组Ac-H3表达水平显著高于老年组,差异有统计学意义(P<0.01)；幼年组和青年组Ac-H3表达水平没有差别(P>0.05)。
(3)不同年龄组大鼠的Ac-H4表达水平单因素方差分析结果显示,三组间Ac-H4于缺血再灌注24h时间点高值,但三组间表达水平无差别。
5.4结论
(1)采用TSA预处理局灶性脑I/R损伤后,不同年龄组大鼠的脑组织HDAC活性改变因年龄而异；
(2)TSA预处理可上调脑组织中H3、Ac-H4表达水平,随年龄不同而有所不同。
PartⅠ:Study on the betterment of suture method to make MCAO model in rats
Background and objective
It was reported that the middle cerebral artery occlusion (MCAO) accounts for the largest proportion of clinic ischemic cerebrovascular diseases and it often has automatic recanalization, which is similar to the pathological symptom of middle cerebral artery occlusion in rats model. Only is the animal experiment applicable for drug pre-conditioning when the selected animal model satisfies clinic need, therefore, this study used MCAO model to learn the functions to the brain tissues when Trichostatin A preconditioned.
The uncertainty of cerebral infarction volume in MCAO rats even if the equal time of middle cerebral artery occlusion is worst defects of this model. To make stable MACO model is the good foundation for later researches. This study used modified suture method to make stable MACO, thus building the base of sequent experiment.
Material and methods
Experimental animals:Healthy clean SD male rats in sham operation group and cerebral ischemia-reperfusion group,6 rats per group.
Animal model:The suture method was used to block the middle cerebral artery for 90 mins, and then a reperfusion for 24 hours was performed in rats. For rats in sham operation group, the suture was merely put in external carotid artery.
Measurement indicators:neurological function disfunction score, cerebral infarction volume, pathological issue change in ischemic rat brain
Testing methods:The hematoxylin-eosin (HE) staining was used to observe pathological changes of ischemic brain issues; Zea-Longa method was used to assessment the neurological disfunction status; triphenyltetrazolium chloride (TTC) staining was used to measure cerebral infarction volume of rats.
Statistical analysis:Excel 2007 was used to build dataset and for data entering. Median and Inter-quartile were used to describe neurological function scores and two-independent sample t test was used to compare the difference in neurological function score between sham group and cerebral ischemia-reperfusion group.'P<0.05'was selected as statistical test level. All of statistical analysis was implemented using the SPSS 17.0.
Results
(1)The result of HE staining between sham group and cerebral ischemia-reperfusion group was different. The damage of the cerebral ischemia-reperfusion group was great and the sham group had no.
(2) Substantial differences in neurological function score between sham group and cerebral ischemia-reperfusion group at I/R6,12 and 24 hours (P<0.05).
(3) TTC staining showed that rats in cerebral ischemia-reperfusion group had clear infarction when compare with the lack of brain infarction in sham control.
Conclusion
The modified suture and other methods are reliable in making MACO model in rats.
PartⅡ:Study of TSA preconditioning on MCAO rats and the relevance to IL-1βand bcl-2
Background and objective
Cerebral ischemia-reperfusion injury is a common clinically pathological symptom. Ischemic preconditioning (IPC) is the most powerful intrinsic nerve protective phenomenon that has been reported currently. Because ischemic preconditioning is an injurious preconditioning, it is necessary to seek for a drug-based preconditioning as a substitute. As a new generation HDACIs, whether does TSA has an cerebral protection effect on cerebral ischemia-reperfusion injury and its mechanism remain unreported. The primary objective of this study aims to examine the cerebral protection impact of TSA preconditioning on MCAO rats and to examine the relationship between cerebral protection of TSA and IL-1βand bcl-2.
Material and methods
Experimental animals:Healthy SD male rats. Four groups were determined as followed:control group,1%DMSO preconditioning group (DMSO group), TSA 0.1mg/kg preconditioning group (TSA 1 group), and TSA 0.03mg/kg preconditioning group (TSA 2 group). Each group included four sub-groups for reperfusion for 6,12,24 and 48 hours, respectively,6 rats per sub-group.
Animal model:The modified suture method was used to block the middle cerebral artery for 90min, then did a reperfusion for 6,12,24 and 48 hours, respectively, thus creating the cerebral ischemia-reperfusion animal model. Four groups were determined as followed: control group, TSA 1 group, TSA 2 group and DMSO group. TSA and DMSO were injected through caudal vein 20 minutes before ischemia implementation. The same volume of normal saline was injected into rats in the control group.
Measurement indicators:neurological function defect score, cerebral infarction volume, cerebrospinal fluid and blood IL-1β, bcl-2 positive cell count.
Testing methods:Zea-Longa method was used to assessment the neurological function status; triphenyltetrazolium chloride (TTC) staining was used to measure cerebral infarction volume ratio of rats; the hematoxylin-eosin (HE) staining was used to observe pathological changes of ischemic brain issues; the enzyme-linked immunosorbent assay (ELISA) was used to test the level of IL-1βin cerebrospinal fluid and blood; and the immunohistochemistry method was used to detect the level of bcl-2 protein.
Statistical analysis:Excel 2007 was used to build dataset and for data entering. Two-way analysis of variance was used to test the main effects of treatment factor, time factor and their interaction on neurological function score, cerebral infarction volume, cerebrospinal fluid and blood IL-1β, and bcl-2 count. Non-parametric test or robust test was used when the assumptions of analysis of variance were violated. The'α=0.05'was selected as statistical test level. All of statistical analysis were implemented using the SPSS 17.0.
Results
(1) The results of analysis of variance showed that there was no significant difference in neurological function scores between four groups (control group, DMSO group, TSA 1 group and TSA 2 group) at all time points (P>0.05) except for that TSA 1 group had lower score than the control group at 6 hours (P<0.05).
(2) Results showed that the cerebral infarction volume ratio of rats in TSA 1 group was less than that of the other three groups (P<0.05); the rats in TSA 2 group and DMSO group had less cerebral infarction volume than those in control group (P<0.05), but did not differ from eAc-H other (P>0.05).
(3) Results displayed that the blood and the cerebrospinal fluid IL-1βof rats in TSA 1 group was lower than that in control and DMSO groups (P<0.05), but show no statistical difference from that in rats of TSA 2 group; The blood IL-1βof rats had no significant difference between TSA 2 group and DMSO group (P>0.05), but both were lower than those for control group (P<0.05).
(4) Pearson correlation showed the cerebral infarction volume highly correlated with blood and cerebrospinal fluid IL-1β(P<0.05), having a correlation coefficient of 0.841 and 0.618, respectively.
(5) Results showed statistical significance between four groups at eAc-H time point. The multi-comparisons revealed that at the 6h, the bcl-2 positive cell count of rats in control group was lower than the other three groups, the rats in DMSO group and TSA 2 group had less bcl-2 positive cell number than those in TSA 1 group (P<0.05); at the 12h, the first three groups had no statistical difference between themselves in bcl-2 positive cell count (P>0.05), while all of them had smaller bcl-2 positive cell count than TSA 1 group (P<0.05); at the 24h, the bcl-2 count of control group was lower than the rest three groups, and DMSO and TSA 2 groups were also less than that of TSA 1 group; and at the 48h, a statistical difference existed between each pair (P<0.05).
Conclusion
(1) TSA preconditioning can reduce cerebral infarction and have a dose-depended effect to lower level of IL-1βin blood and cerebrospinal fluid, and higher sum of anti-apoptosis protein bcl-2 cells.
(2) The protection effect of TSA cerebral ischemia-reperfusion injury is related to the level of IL-1βand anti-apoptosis protein bcl-2, suggesting a cerebral protection of TSA preconditioning to local cerebral ischemia-reperfusion injury in rats.
PartⅢ:Study of TSA preconditioning on MCAO rats and the relevance to HDAC、Ac-H3 and Ac-H4
Backgroud and objective
Cerebral drug pre-conditioning is based on ischemic pre-conditioning and uses drug to stimulate the body to produce endogenous protective secretion, so as to protect brain. Histone deacetylases inhibitors (HDACIs) increase the plasticity of neuron after ischemia and can promote the rehabilitation of function. The previous study reported that TSA preconditioning can decrease the level of IL-1βlevel in the cerebrospinal fluid and raise the expression of apoptosis protein bcl-2, thus yielding protection of cerebral ischemia-reperfusion injury. However, the mechanism of its cerebral protection is unknown. This study was aimed to examine the relationship between TSA pre-conditioning and HDAC, Ac-H3 and Ac-H4 in rats receiving focal cerebral ischemia-reperfusion injury and explore the association between related cerebral protective factors.
Material and methods
Experimental animals:Healthy SD male rats. Four groups were determined as followed:control group, TSA 0.1mg/kg preconditioning group (TSA 1 group), and TSA 0.03mg/kg preconditioning group (TSA 2 group) and 1%DMSO preconditioning group (DMSO group). Each group included four sub-groups for reperfusion for 6,12,24 and 48 hours, respectively,6 rats per sub-group.
Animal model:The modified suture method was used to block the middle cerebral artery for 90min, then did a reperfusion for 6,12,24 and 48 hours, respectively, thus creating the cerebral ischemia-reperfusion animal model. Four groups were determined as followed: control group, TSA 1 group, TSA 2 group and DMSO group. TSA and DMSO were injected through caudal vein 20 minutes before ischemia implementation. The same volume of normal saline was injected into rats in the control group.
Measurement indicators:HDAC level in infracted brain issue, Ac-H3 and Ac-H4 level.
Testing methods:The colorimetry method was used to test the level of HDAC in the infracted brain issue; and the Western blotting was used to test the level of Ac-H3 and Ac-H4 in the infracted brain issue; the enzyme-linked immunosorbent assay (ELISA) was used to test the level of IL-1βin cerebrospinal fluid and blood; and the immunohisto-chemistry method was used to detect the count of bcl-2 protein positive cell.
Statistical analysis:Excel 2007 was used to build dataset and for data entering. Two-way analysis of variance was used to test the main effects of treatment factor, time factor and their interaction on HDAC level, Ac-H3 and Ac-H4. Non-parametric test or robust test was used when the assumptions of analysis of variance were violated. Spearman correlation was used to examine the relationship between related cerebral protective factors. The'α=0.05'was selected as statistical test level. Statistical analysis was implemented using the SPSS 17.0.
Results
(1) After receiving a cerebral ischemia injury, the HDAC level grew over the time of reperfusion in a linear way, with the linear equation HDAC=0.116+0.060×time (P<0.05). Two-way analysis of variance showed that the main effects of treatment factor and time factor were statistically significant (P<0.05), but the effect of interaction of treatment and time was insignificant (P>0.05). Multiple comparisons between four groups revealed:TSA 1 group had lower HDAC level than control group and DMSO group (P<0.05); the HDAC level in TSA 2 group and DMSO group were lower than control group (P<0.05); and there no statistical difference between other groups (P>0.05).
(2) For Ac-H3 and Ac-H4, two-way analysis of variance showed that that the main effects of treatment factor and time factor were statistically significant (P<0.05), but the effect of interaction of treatment and time was insignificant (P>0.05). Both Ac-H3 and Ac-H4 increased from 6 hours and reAc-Hed the peak at 12 hours, then decreased gradually at all of four groups.
Multiple comparisons for Ac-H3 showed that the level of Ac-H3 in TSA 1 group was statistically higher than the other three groups (P<0.05), and TSA 2 group and DMSO group had higher Ac-H3 level than control group (P<0.05). There no statistical difference between other groups (P>0.05).
(3)Multiple comparisons for Ac-H4 showed that the level of Ac-H4 in TSA 1 group was statistically higher than the other three groups (P<0.05), and TSA 2 group had higher Ac-H4 level than control group (P<0.05). There no statistical difference between other groups (P>0.05).
(4)The bcl-2 positive cell count significantly correlated with HDAC, Ac-H4, with the Spearman rank correlation coefficient of -0.775 and 0.346 (P<0.01). The correlation coefficient between Ac-H3 and Ac-H4 was 0.800 (P<0.01). The rest of correlation were not statistically significant (P>0.05).
(5) Ac-H3 had a higher correlation with cerebrospinal fluid IL-1βand blood IL-1β(rs=1.0 and 0.8, P<0.01); the cerebrospinal fluid IL-1βhighly correlated with Ac-H4 (rs=0.8, P<0.01); and the correlation coefficient between cerebrospinal fluid IL-1βand blood IL-1βwas 0.8 (P<0.01). The rest of correlation were not statistically significant (F>0.05).
Conclusion
(1) The HDAC level rise over the time of reperfusion after the rats receive cerebral ischemia injury. TSA can reduce the expression of HDAC.
(2) TSA pre-conditioning can raise the level of histone Ac-H3 and Ac-H4 in cerebral issue. Histone Ac-H3 positively associate with cerebrospinal fluid IL-1βand blood IL-1β; and cerebrospinal fluid IL-1βhas a high and positive correlation with Ac-H4.
PartⅣ:Study of Association between age and TSA preconditioning and related factors (IL-1β, bcl-2)
Backgroud and objective
As China step into the aging society, ischemic cerebral vasnacular diseases have become more and more frequent than before. The implementation of family planning policy has attracted more and more attention to the health of singe child for eAc-H family. The cerebral function injury from neurological system diseases is one of important and unresolved pediatric research problem. Few studies involve the epigenetics of ischemic cerebral vanascular diseases although there are lots of researches exploring the pathological characteristics and treatment. It has been reported that TSA preconditioning can decrease the level of IL-1βlevel in the cerebrospinal fluid and raise the expression of apoptosis protein bcl-2, thus yielding protection of cerebral ischemia-reperfusion injury. However, the mechanism of its cerebral protection at different ages of rats keeps unreported. To deepen the mechanism of cerebral ischemia-reperfusion injury at rats of different ages, this study aimed to examine the relationship between age and cerebral protection of TSA preconditioning and related factors (IL-1β, bcl-2).
Material and methods
Experimental animals:18 healthy and clean SD male rats that received TSA preconditioning (0.1mg/kg) were divided into three groups based on their age:young group, adult group and old group.
Animal model:The modified suture method was used to block the middle cerebral artery for 1.5 hours, and then a 24-hour reperfusion was done, thus creating the cerebral ischemia-reperfusion animal model. TSA was injected through caudal vein 20 minutes before ischemia implementation.
Measurement indicators:neurological function score, cerebral infarction volume, cerebrospinal fluid and blood IL-1β, bcl-2 positive cell count.
Testing methods:Zea-Longa method was used to assessment the neurological function status; triphenyltetrazolium chloride (TTC) staining was used to measure cerebral infarction volume ratio of rats; the hematoxylin-eosin (HE) staining was used to observe pathological changes of ischemic brain issues; the enzyme-linked immunosorbent assay (ELISA) was used to test the level of IL-1βin cerebrospinal fluid and blood; and the immunohistochemistry method was used to detect the count of bcl-2 protein positive cell.
Statistical analysis:Excel 2007 was used to build dataset and for data entering. One-way analysis of variance was used to compare the difference in neurological function score, cerebral infarction volume ratio, cerebrospinal fluid and blood IL-1β, and bcl-2 positive cell count between three age groups. Non-parametric test or robust test was used when the assumptions of analysis of variance were violated. The 'α=0.05'was selected as statistical test level. Statistical analysis was performed using the SPSS 17.0.
Results
(1) One-way analysis of variance revealed that there was no statistical difference in Zea-Longa neurological function score between three age groups at 6h,12h and 24h (P>0.05).
(2)No significant difference in modified neurological function score were observed between three age groups at 6h and at 12h (P>0.05); but there the differences at 24h were statistically significant between three age groups. The mult-comparion between three age group at 24h showed, the scores of old group were different from those of young group (P<0.05), and there no significant difference between other groups (P>0.05).
(3)The total balance scores of young group were obviously lower than those of the other age groups (P<0.05); the total sport scores of young group differed from those of old group (P<0.05); The total reflection scores of young group and adult group were less than those of old group (P<0.05); the total sensation scores of three age groups were not significantly different from each other (P>0.05).
(4) One-way analysis of variance showed that no statistical difference was found in cerebral infarction volume between three age groups (P>0.05).
(5) One-way analysis of variance showed that rats of three age group showed no significant difference in cerebrospinal fluid and blood IL-1βlevel (P>0.05).
(6) One-way analysis of variance showed that the bcl-2 count differed between three age groups (P<0.05). Multi-comparisons between three groups showed that the old group had higher bcl-2 count than the other two groups (P<0.05), but no significant difference was detected between young group and adult group (P>0.05). Conclusion
Age is related to the cerebral protection of TSA pre-conditioning. The different function of the level of cerebrospinal fluid IL-1βand the bcl-2 count of the three groups changed with TSA preconditioning.
PartⅤ:Study of Association between age and TSA preconditioning and related factors (HDAC, Ac-H3 and Ac-H4)
Background and objective
Previous study reported that the occurrence of ischemic resistance is the adoptive chain of reaction, including genetic dependent reaction and non-genetic dependent reaction, the existed decoration of protein translation. The pre-conditioning is the re-programming process of organs to ischemia reaction at genetic level. Histone acetylation and deacetylation is the the most explored decoration mechanism after protein translation. Prior studies suggested that TSA pre-conditioning can produce protection to cerebral ischemia-reperfusion injury. To deepen the mechanism of cerebral ischemia-reperfusion injury in rats of different age, this study aimed to explore the relationship between age and HDAC, Ac-H3 and Ac-H4 in rats those receive TSA preconditioning, so as to provide base for the exploration of new treatment method.
Material and methods
Experimental animals:18 healthy and clean SD male rats that received TSA preconditioning (0.1mg/kg) were divided into three groups based on their age:young group, adult group and old group.
Animal model:The modified suture method was used to block the middle cerebral artery for 1.5 hours, and then a 24-hour reperfusion was done, thus creating the cerebral ischemia-reperfusion animal model. TSA was injected through caudal vein 20 minutes before ischemia implementation.
Measurement indicators:HDAC level in infracted brain issue, acetylaced H3 and H4 level.
Testing methods:The colorimetry method was used to test the level of HDAC in the infracted brain issue; and the Western blotting was used to test the level of acetylaced H3 and H4 in the infracted brain issue.
Statistical analysis:Excel 2007 was used to build dataset and for data entering. One-way analysis of variance was used to compare the difference in HDAC, Ac-H3 and Ac-H4 between three age groups. Non-parametric test or robust test was used when the assumptions of analysis of variance were violated. The'α=0.05'was selected as statistical test level. Statistical analysis was performed using the SPSS 17.0.
Results
(1) The HDAC level showed significant differences between three age groups (P<0.05). The old group had higher HDAC level than young and adult groups (P<0.05); there was no significant difference in HDAC level between the young group and the adult group (P<0.05).
(2) Rats of three groups showed statistical difference in Ac-H3 level (P<0.05). The old group had lower Ac-H3 level than the other two groups (P<0.05), but no significant difference was detected between the young group and the adult group (P>0.05). There was significant differences in Ac-H4 level between three age groups (P>0.05).
(3) Rats of three groups showed no statistical difference in Ac-H4 level (P<0.05). The old group had higher H4 level than the other two groups, but no significant difference was detected of the three groups (P>0.05).
Conclusion
(1) Age has an effect on the expression of HDAC in rats that receive TSA pre-conditioning.
(2) Age affects the expression of Ac-H3 in rats receiving TSA pre-conditioning but has no effect on the expression of Ac-H4.
临床缺血性脑血管病中所占比重最大的是发生在大脑中动脉主干分布区的梗死,且常有自发性再通,目前常用的线栓法阻断大脑中动脉复制出的脑缺血再灌注模型(middle cerebral artery occlusion, MCAO)较符合这种病理生理学过程。即使阻断大脑中动脉时间相同,脑梗死范围却不稳定是线栓法复制MCAO模型的缺点。研究采用改良线栓及其他措施来复制稳定性好的MCAO模型,才能为随后的研究奠定可靠的基础。
随着中国步入老龄化社会,缺血性脑血管疾病已成为常见病、多发病。计划生育的实施,人们对独生子女的健康问题更加关注,而神经系统疾病导致的脑功能损伤,是目前亟待解决的儿科难题。虽然对缺血性脑损伤的机制研究甚多,但表观遗传学方面的研究不多。
曲古抑素A (trichostatin A, TSA)作为广谱组蛋白去乙酰化酶抑制剂应用广泛,而其预处理是否对脑缺血再灌注损伤具有保护作用及其作用机制的研究较少,尤其是关于TSA预处理对不同年龄大鼠脑缺血再灌注损伤的保护机制研究尚未见报道。
通过了解TSA预处理对不同年龄大鼠脑缺血再灌注损伤的保护作用,深入研究组蛋白乙酰化/去乙酰化在缺血再灌注过程中脑损伤过程中的作用机制,从多个方面入手对此病理生理进程实施干预,无疑将为积极防治缺血再灌注性脑损伤开拓新的应用前景。
第一部分采用改良线栓法复制稳定的大鼠MCAO模型
1.1研究背景与目的
采用从颈总动脉或颈外动脉置入线栓,阻断大脑中动脉血供一定时间之后再恢复灌注的动物模型,被认为与临床缺血性脑血管病中所占比重最大,且常有自发性再通的大脑中动脉主干分布区脑梗死的病理生理学过程相近似,所以应用较多。进行药物预处理实验时,要求所选动物模型要符合临床实际,所得出的实验结果才有应用价值。所以本实验采用栓塞大脑中动脉复制出的局灶性脑缺血模型(MCAO)进行曲古抑素A预处理的脑保护研究。
线栓法MCAO模型的缺点是即使阻断大脑中动脉时间相同,脑梗死范围却不稳定。一定要复制出稳定性好的MCAO模型,才能为随后的研究奠定可靠的基础。本实验拟采用改良线栓及其它措施,从而复制出稳定性好的MCAO模型。
1.2材料与方法
研究对象：健康清洁SD雄性大鼠随机分为假手术组、缺血再灌注组,每组各6只。
动物模型：改良线栓阻断大鼠大脑中动脉供血90min,再灌注24h制备大鼠MCAO模型。假手术组仅将线栓放置在颈外动脉内。
观测指标：HE染色、Zea-Longa神经功能损害评分、脑梗死体积比。
检测方法：苏木精-伊红(HE)染色法观察手术侧脑组织病理形态改变；缺血再灌注6h、12h、24h时间点使用Zea-Longa评分法进行神经功能损害评分；TTC染色法测定大鼠脑梗死体积比。
数据分析：采用Excel 2007建立数据库并录入数据。神经功能学评分用中位数(范围)表示。采用SPSS17.0进行统计分析,P<0.05表示有统计学差异。
1.3结果
(1)两组大鼠手术侧脑组织HE染色显示不同,缺血再灌注组脑损伤改变明显,假手术组未发生损伤性改变。
(2)两组大鼠Zea-Longa神经功能损害评分于缺血再灌注6h、12h、24h时间点差异有统计学意义(P
1.4结论
本实验采用改良线栓阻断MCA法及其它措施复制的MCAO模型,稳定性良好,值得推广应用。
第二部分不同剂量TSA预处理对MCAO大鼠IL-1β、bcl-2的影响
2.1研究背景与目的
脑缺血再灌注损伤是临床常见的病理生理表现。脑缺血预处理被认为是最强的神经内源性保护现象,但由于脑缺血预处理是损伤性预适应,探讨采用药物诱导预适应替代脑缺血预处理成为必然。TSA作为广谱组蛋白去乙酰化酶抑制剂应用广泛,而其预处理是否有减轻脑缺血再灌注损伤作用及其作用机制少见报道。本研究拟探讨不同剂量TSA预处理是否通过对血液和脑脊液中IL-1β含量、抗凋亡蛋白bcl-2等方面来减轻脑缺血再灌注损伤。
2.2材料与方法
研究对象：健康清洁SD雄性大鼠被随机分为缺血再灌注对照组(对照组)、0.1mg/kg TSA预处理组(TSA1组)、0.03mg/kg TSA预处理组(TSA2组)、1%DMSO预处理组(DMSO组),每组又被分为缺血再灌注6h组、12h组、24h组、48h共4个亚组,每亚组6只大鼠。
动物模型：采用前述改良线栓法阻断大脑中动脉供血90min,再灌注6h、12h、24h、48h复制大鼠局灶性脑缺血再灌注模型。TSA1组、TSA2组、DMSO组于缺血前20min分别经尾静脉注入含有相应药物的生理盐水1ml,对照组则注入相同体积的生理盐水。
观测指标：Zea-Longa神经功能损害评分、脑梗死体积比、脑脊液和血液IL-1β含量变化、抗凋亡蛋白bcl-2阳性细胞计数。
检测方法：神经功能损害评分依据Zea-Longa评分法进行；苏木精-伊红(HE)染色法观察缺血侧脑组织病理改变；ELISA法分析法检测脑脊液和血清中IL-1β的含量变化；免疫组化法测定栓塞侧大脑缺血半暗带区bcl-2蛋白阳性细胞数。
数据分析：采用Excel 2007建立数据库并录入数据。采用两因素方差分析处理因素、时间因素及其交互作用对神经功能评分、脑梗死体积比、脑脊液、血液IL-1β和bcl-2阳性细胞计数的影响。如方差分析前提条件未得到满足,采用相应的非参数检验或稳健方法作为替代。选择α=0.05作为统计检验水准,采用SPSS17.0完成统计分析。
2.3结果
(1)与缺血再灌注6h时间点的对照组相比,TSA1组神经功能损害评分降低有统计学意义(P<0.05)外,其余各时间点的各组神经功能损害评分比较无显著差异(P>0.05)。
(2)TSA1组大鼠脑梗死体积比均小于其余3组,差异有统计学意义(P<0.05)；TSA2组及DMSO组脑梗死体积比小于对照组(P<0.05)。
(3) TSA1组血液IL-1β含量低于对照组和DMSO组(P<0.05)；TSA2组与DMSO组无差别(P>0.05),但均低于对照组(P<0.05)
(4) Pearson相关分析显示,脑梗死体积比与血IL-1β、脑脊液IL-1β高度相关,对应的Pearson相关系数分别为0.841和0.618,P<0.05。
(5)再灌注6h时间点,对照组bcl-2计数低于其余3组,DMSO组和小剂量TSA均低于TSA1组(P<0.05)；再灌注12h时间点,对照组、DMSO组和TSA2组之间没有统计学差别(P>0.05),但3组均低于TSA1组(P<0.05)；再灌注24h时间点,对照组bcl-2计数低于其余3组,DMSO组和小剂量TSA均低于TSA1组(P<0.05)；再灌注48h时间点,4组之间bcl-2计数彼此之间均有统计学差别(P<0.05)。
2.4结论
(1)TSA预处理可降低血液和脑脊液中IL-1β的含量,增加抗凋亡蛋白bcl-2阳性细胞数。
(2)TSA预处理可剂量依赖性减少MCAO大鼠缺血再灌注损伤时脑梗死体积比,对MCAO大鼠可能有脑保护作用。
第三部分TSA预处理对MCAO大鼠脑组织中HDAC、乙酰化组蛋白H3、乙酰化组蛋白H4表达水平的影响
3.1研究背景与目的
脑药物预处理是指使用药物刺激机体产生类似机体自身内源性保护物质而呈现脑保护作用,它是在缺血性预处理的基础上发展起来的。组蛋白去乙酰化酶抑制剂(histone deacetylases inhibitors, HDACIs)通过作用于缺血性卒中损伤机制中的多个环节减轻脑组织损伤,增加缺血后神经元可塑性和促进功能恢复。此前研究提示TSA预处理可以减少脑脊液中IL-1β的含量、促进抗凋亡蛋白bcl-2表达,对脑缺血再灌注损伤具有保护作用,但其作用机制尚未明了。本研究拟探讨TSA预处理对MCAO大鼠脑组织中HDAC、乙酰化组蛋白H3 (acetylaced H3, Ac-H3)、乙酰化组蛋白H4 (acetylaced H4, Ac-H4)表达水平的影响。
3.2材料与方法
研究对象：同第二部分。
动物模型：同第一部分。
观测指标：栓塞侧脑组织HDAC活性测定、Ac-H3、Ac-H4表达水平检测。
检测方法：采用比色法检测栓塞侧脑组织HDAC活性；免疫印迹法测定栓塞侧脑组织乙酰化组蛋白H3、H4表达水平。
数据分析：采用Excel 2007建立数据库并录入数据。采用两因素方差分析处理因素、时间因素及其交互作用对HDAC活性、Ac-H3、Ac-H4表达水平的影响。如方差分析前提条件未得到满足,采用相应的非参数检验或稳健方法作为替代。选择α=0.05作为统计检验水准,采用SPSS17.0完成统计分析。
3.3结果
(1)大鼠局灶性脑I/R损伤后HDAC活性随缺血再灌注时间延长呈上升趋势。各组HDAC活性随缺血再灌注时间延长而持续上升。TSAl组大鼠HDAC活性虽有明显上升,但低于对照组和DMSO组,差异有统计学意义(P<0.01)；TSA2组HDAC活性水平与DMSO组没有差别,但低于对照组；DMSO组与对照组没有差别(P>0.05)。
(2)各组Ac-H、Ac-H4表达水平在缺血再灌注6h时间点均呈上升趋势,至缺血再灌注12h时间点达高峰,随后下降。各组在不同处理组和时间点之间乙酰化组蛋白H3表达水平存在差别,TSA1组大鼠Ac-H3表达水平均高于其余3组；TSA2组与DMSO组Ac-H3表达水平均高于对照组,但这两组间没有差别。
(3)各组在不同分组和时间点之间Ac-H4表达水平存在差别,TSA1组大鼠Ac-H4表达水平均高于其余3组；TSA2组Ac-H4表达水平高于对照组,但与DMSO组没有差别；DMSO组与对照组之间没有差别。
(4) HDAC活性改变与Ac-H3、Ac-H4表达水平的Spearman秩相关系数分别为0.400和0.200,没有统计学学意义。但Ac-H3、Ac-H4表达水平的Spearman秩相关系数为0.964,有统计学意义。
(5)Ac-H3表达水平均与脑脊液和血液IL-1β表达水平高度相关,Spearman秩相关系数分别为1.0和0.8；Ac-H4仅与脑脊液IL-1β表达水平相关,秩相关系数为0.8,但与血液IL-1β表达水平的秩相关系数无统计学意义。
(6) Ac-H3、Ac-H4表达水平均与bcl-2表达水平高度相关,Spearman秩相关系数分别为1.0和0.8。
3.4结论
(1)大鼠局灶性脑I/R损伤后脑组织HDAC活性随缺血再灌注时间延长呈上升趋势,TSA对HDAC有抑制作用；
(2)TSA预处理可上调脑组织中Ac-H3、Ac-H4水平,从而对IL-1β、bcl-2产生影响,这可能是TSA预处理的脑保护作用机制之一。
第四部分大剂量TSA预处理对不同年龄组MCAO大鼠IL-1β、bcl-2的影响
4.1研究背景与目的
随着中国步入老龄化社会,缺血性脑血管疾病已成为常见病、多发病。计划生育的实施,人们对独生子女的健康问题更加关注,而神经系统疾病导致的脑功能损伤,是目前亟待解决的儿科难题。虽然对缺血性脑血管疾病的病理生理特点及治疗机制研究甚多,但针对不同年龄大鼠表观遗传学方面的研究不多。此前研究提示TSA预处理对脑缺血再灌注损伤具有保护作用。为深入研究不同年龄大鼠脑缺血再灌注损伤的损伤机制,本研究拟采用大剂量TSA预处理不同年龄组MCAO大鼠,观察其对大鼠血液和脑脊液中IL-1β含量、抗凋亡蛋白bcl-2的影响。
4.2材料与方法
研究对象：SD雄性大鼠分为老年组、青年组、幼年组,均采用0.1mg/kg TSA预处理,每组6只。
动物模型：采用改良线栓法阻断实验大鼠大脑中动脉血供90min,再灌注24h制备大鼠局灶性脑缺血再灌注模型。老年组、青年组、幼年组于缺血前20min分别经尾静脉注入含相应药物的生理盐水共1ml。
观测指标：神经功能损害评分、脑梗死体积比、脑脊液和血液IL-1β、bcl-2阳性细胞计数。
检测方法：Zea-Longa评分法和Chen18分评分法进行神经功能缺陷评分；采用TTC染色法测大鼠脑梗死体积比；苏木精-伊红(HE)染色法观察缺血侧脑组织病理形态改变；采用酶联免疫吸附测定法分析(ELISA)法检测脑脊液和血清中IL-1β的含量变化；免疫组化法测定栓塞侧大脑缺血半暗带区bcl-2蛋白阳性细胞数。
数据分析：采用Excel 2007建立数据库并录入数据。采用两因素方差分析处理因素、时间因素及其交互作用对神经功能评分、脑梗死体积、脑脊液、血液IL-1β和bcl-2计数的影响。如方差分析前提条件未得到满足,采用相应的非参数检验或稳健方法作为替代。选择α=0.05作为统计检验水准,采用SPSS17.0完成统计分析。
4.3结果
(1)不同年龄组大鼠在缺血再灌注6h、12h、24h时间点的神经功能评分的单因素方差分析结果显示,三组间Zea-Longa神经功能损害评分无明显差别(P>0.05)。
(2)三组间缺血再灌注6h、12h时间点的改良神经功能损害程度评分无显著差别(P>0.05),在24h时间点有差别(P<0.05)。3组间两两比较结果显示,老年组与幼年组差别有统计学意义(P<0.05),老年组与青年组、青年组与幼年组之间无差别(P>0.05)。
(3)幼年组大鼠在缺血再灌注24h时间点的平衡总评分明显小于其余两组大鼠(P<0.05)；运动总评分与老年组大鼠相比有差别(P<0.05)；而幼年组大鼠和青年组大鼠的反射总评分小于老年组大鼠,差异有统计学意义(P<0.05)。
(4)不同年龄组大鼠的脑梗死体积比的单因素方差分析结果显示,三组间脑梗死体积比无统计学差别(P>0.05)
(5)单因素方差分析结果显示,不同年龄组大鼠血液和脑脊液中的IL-1β表达水平无差别(P>0.05)
(6)单因素方差分析结果显示,不同年龄组大鼠bcl-2阳性细胞计数3组间存在差别(P<0.05)。3组间两两比较结果显示,幼年组和青年组之间没有统计学差别(P>0.05),但二者bcl-2阳性细胞计数均低于老年组大鼠(P<0.05)。
4.4结论
TSA预处理对脑脊液中IL-1β含量以及抗凋亡蛋白bcl-2表达因年龄而作用不同。大剂量TSA预处理可能对不同年龄组MCAO大鼠具有脑保护作用。
第五部分TSA预处理对不同年龄组MCAO大鼠脑组织中HDAC、Ac-H3、Ac-H4表达水平的影响
5.1研究背景与目的
研究认为缺血耐受指的是特定的时间被出现的特殊刺激激发产生的适应性级联反应,包括基因依赖性反应和非基因依赖性反应,即已存在蛋白的翻译后修饰。预处理是机体器官在基因水平上对缺血反应“重新编程”的过程。组蛋白乙酰化和去乙酰化是目前研究最为深入的蛋白翻译后修饰机制。此前研究提示TSA预处理对脑缺血再灌注损伤具有保护作用。为深入研究不同年龄大鼠脑缺血再灌注损伤的损伤机制,本研究拟针对不同年龄组采用TSA预处理,观察各组大鼠局灶性脑缺血再灌注损伤的脑组织中HDAC、Ac-H3、Ac-H4表达水平的改变,为探索新的治疗方法打下基础。
5.2材料与方法研究对象：同第四部分。动物模型：同第四部分。观测指标：栓塞侧脑组织HDAC活性、Ac-H3、Ac-H4表达水平。
检测方法：采用比色法检测栓塞侧脑组织HDAC活性；免疫印迹法测定栓塞侧脑组织Ac-H3、Ac-H4表达水平。
数据分析：采用Excel 2007建立数据库并录入数据。采用两因素方差分析处理因素、时间因素及其交互作用对HDAC活性、Ac-H3、Ac-H4表达水平的影响。如方差分析前提条件未得到满足,采用相应的非参数检验或稳健方法作为替代。选择α=0.05作为统计检验水准,采用SPSS17.0完成统计分析。
5.3结果
(1)老年组大鼠HDAC活性显著高于幼年组和青年组,差异有统计学意义(P<0.01)；幼年组和青年组HDAC活性水平没有差别(P>0.05)。
(2)不同年龄组之间Ac-H3表达水平于缺血再灌注24h时间点差异有统计学意义(P<0.01)。幼年组和青年组Ac-H3表达水平显著高于老年组,差异有统计学意义(P<0.01)；幼年组和青年组Ac-H3表达水平没有差别(P>0.05)。
(3)不同年龄组大鼠的Ac-H4表达水平单因素方差分析结果显示,三组间Ac-H4于缺血再灌注24h时间点高值,但三组间表达水平无差别。
5.4结论
(1)采用TSA预处理局灶性脑I/R损伤后,不同年龄组大鼠的脑组织HDAC活性改变因年龄而异；
(2)TSA预处理可上调脑组织中H3、Ac-H4表达水平,随年龄不同而有所不同。
PartⅠ:Study on the betterment of suture method to make MCAO model in rats
Background and objective
It was reported that the middle cerebral artery occlusion (MCAO) accounts for the largest proportion of clinic ischemic cerebrovascular diseases and it often has automatic recanalization, which is similar to the pathological symptom of middle cerebral artery occlusion in rats model. Only is the animal experiment applicable for drug pre-conditioning when the selected animal model satisfies clinic need, therefore, this study used MCAO model to learn the functions to the brain tissues when Trichostatin A preconditioned.
The uncertainty of cerebral infarction volume in MCAO rats even if the equal time of middle cerebral artery occlusion is worst defects of this model. To make stable MACO model is the good foundation for later researches. This study used modified suture method to make stable MACO, thus building the base of sequent experiment.
Material and methods
Experimental animals:Healthy clean SD male rats in sham operation group and cerebral ischemia-reperfusion group,6 rats per group.
Animal model:The suture method was used to block the middle cerebral artery for 90 mins, and then a reperfusion for 24 hours was performed in rats. For rats in sham operation group, the suture was merely put in external carotid artery.
Measurement indicators:neurological function disfunction score, cerebral infarction volume, pathological issue change in ischemic rat brain
Testing methods:The hematoxylin-eosin (HE) staining was used to observe pathological changes of ischemic brain issues; Zea-Longa method was used to assessment the neurological disfunction status; triphenyltetrazolium chloride (TTC) staining was used to measure cerebral infarction volume of rats.
Statistical analysis:Excel 2007 was used to build dataset and for data entering. Median and Inter-quartile were used to describe neurological function scores and two-independent sample t test was used to compare the difference in neurological function score between sham group and cerebral ischemia-reperfusion group.'P<0.05'was selected as statistical test level. All of statistical analysis was implemented using the SPSS 17.0.
Results
(1)The result of HE staining between sham group and cerebral ischemia-reperfusion group was different. The damage of the cerebral ischemia-reperfusion group was great and the sham group had no.
(2) Substantial differences in neurological function score between sham group and cerebral ischemia-reperfusion group at I/R6,12 and 24 hours (P<0.05).
(3) TTC staining showed that rats in cerebral ischemia-reperfusion group had clear infarction when compare with the lack of brain infarction in sham control.
Conclusion
The modified suture and other methods are reliable in making MACO model in rats.
PartⅡ:Study of TSA preconditioning on MCAO rats and the relevance to IL-1βand bcl-2
Background and objective
Cerebral ischemia-reperfusion injury is a common clinically pathological symptom. Ischemic preconditioning (IPC) is the most powerful intrinsic nerve protective phenomenon that has been reported currently. Because ischemic preconditioning is an injurious preconditioning, it is necessary to seek for a drug-based preconditioning as a substitute. As a new generation HDACIs, whether does TSA has an cerebral protection effect on cerebral ischemia-reperfusion injury and its mechanism remain unreported. The primary objective of this study aims to examine the cerebral protection impact of TSA preconditioning on MCAO rats and to examine the relationship between cerebral protection of TSA and IL-1βand bcl-2.
Material and methods
Experimental animals:Healthy SD male rats. Four groups were determined as followed:control group,1%DMSO preconditioning group (DMSO group), TSA 0.1mg/kg preconditioning group (TSA 1 group), and TSA 0.03mg/kg preconditioning group (TSA 2 group). Each group included four sub-groups for reperfusion for 6,12,24 and 48 hours, respectively,6 rats per sub-group.
Animal model:The modified suture method was used to block the middle cerebral artery for 90min, then did a reperfusion for 6,12,24 and 48 hours, respectively, thus creating the cerebral ischemia-reperfusion animal model. Four groups were determined as followed: control group, TSA 1 group, TSA 2 group and DMSO group. TSA and DMSO were injected through caudal vein 20 minutes before ischemia implementation. The same volume of normal saline was injected into rats in the control group.
Measurement indicators:neurological function defect score, cerebral infarction volume, cerebrospinal fluid and blood IL-1β, bcl-2 positive cell count.
Testing methods:Zea-Longa method was used to assessment the neurological function status; triphenyltetrazolium chloride (TTC) staining was used to measure cerebral infarction volume ratio of rats; the hematoxylin-eosin (HE) staining was used to observe pathological changes of ischemic brain issues; the enzyme-linked immunosorbent assay (ELISA) was used to test the level of IL-1βin cerebrospinal fluid and blood; and the immunohistochemistry method was used to detect the level of bcl-2 protein.
Statistical analysis:Excel 2007 was used to build dataset and for data entering. Two-way analysis of variance was used to test the main effects of treatment factor, time factor and their interaction on neurological function score, cerebral infarction volume, cerebrospinal fluid and blood IL-1β, and bcl-2 count. Non-parametric test or robust test was used when the assumptions of analysis of variance were violated. The'α=0.05'was selected as statistical test level. All of statistical analysis were implemented using the SPSS 17.0.
Results
(1) The results of analysis of variance showed that there was no significant difference in neurological function scores between four groups (control group, DMSO group, TSA 1 group and TSA 2 group) at all time points (P>0.05) except for that TSA 1 group had lower score than the control group at 6 hours (P<0.05).
(2) Results showed that the cerebral infarction volume ratio of rats in TSA 1 group was less than that of the other three groups (P<0.05); the rats in TSA 2 group and DMSO group had less cerebral infarction volume than those in control group (P<0.05), but did not differ from eAc-H other (P>0.05).
(3) Results displayed that the blood and the cerebrospinal fluid IL-1βof rats in TSA 1 group was lower than that in control and DMSO groups (P<0.05), but show no statistical difference from that in rats of TSA 2 group; The blood IL-1βof rats had no significant difference between TSA 2 group and DMSO group (P>0.05), but both were lower than those for control group (P<0.05).
(4) Pearson correlation showed the cerebral infarction volume highly correlated with blood and cerebrospinal fluid IL-1β(P<0.05), having a correlation coefficient of 0.841 and 0.618, respectively.
(5) Results showed statistical significance between four groups at eAc-H time point. The multi-comparisons revealed that at the 6h, the bcl-2 positive cell count of rats in control group was lower than the other three groups, the rats in DMSO group and TSA 2 group had less bcl-2 positive cell number than those in TSA 1 group (P<0.05); at the 12h, the first three groups had no statistical difference between themselves in bcl-2 positive cell count (P>0.05), while all of them had smaller bcl-2 positive cell count than TSA 1 group (P<0.05); at the 24h, the bcl-2 count of control group was lower than the rest three groups, and DMSO and TSA 2 groups were also less than that of TSA 1 group; and at the 48h, a statistical difference existed between each pair (P<0.05).
Conclusion
(1) TSA preconditioning can reduce cerebral infarction and have a dose-depended effect to lower level of IL-1βin blood and cerebrospinal fluid, and higher sum of anti-apoptosis protein bcl-2 cells.
(2) The protection effect of TSA cerebral ischemia-reperfusion injury is related to the level of IL-1βand anti-apoptosis protein bcl-2, suggesting a cerebral protection of TSA preconditioning to local cerebral ischemia-reperfusion injury in rats.
PartⅢ:Study of TSA preconditioning on MCAO rats and the relevance to HDAC、Ac-H3 and Ac-H4
Backgroud and objective
Cerebral drug pre-conditioning is based on ischemic pre-conditioning and uses drug to stimulate the body to produce endogenous protective secretion, so as to protect brain. Histone deacetylases inhibitors (HDACIs) increase the plasticity of neuron after ischemia and can promote the rehabilitation of function. The previous study reported that TSA preconditioning can decrease the level of IL-1βlevel in the cerebrospinal fluid and raise the expression of apoptosis protein bcl-2, thus yielding protection of cerebral ischemia-reperfusion injury. However, the mechanism of its cerebral protection is unknown. This study was aimed to examine the relationship between TSA pre-conditioning and HDAC, Ac-H3 and Ac-H4 in rats receiving focal cerebral ischemia-reperfusion injury and explore the association between related cerebral protective factors.
Material and methods
Experimental animals:Healthy SD male rats. Four groups were determined as followed:control group, TSA 0.1mg/kg preconditioning group (TSA 1 group), and TSA 0.03mg/kg preconditioning group (TSA 2 group) and 1%DMSO preconditioning group (DMSO group). Each group included four sub-groups for reperfusion for 6,12,24 and 48 hours, respectively,6 rats per sub-group.
Animal model:The modified suture method was used to block the middle cerebral artery for 90min, then did a reperfusion for 6,12,24 and 48 hours, respectively, thus creating the cerebral ischemia-reperfusion animal model. Four groups were determined as followed: control group, TSA 1 group, TSA 2 group and DMSO group. TSA and DMSO were injected through caudal vein 20 minutes before ischemia implementation. The same volume of normal saline was injected into rats in the control group.
Measurement indicators:HDAC level in infracted brain issue, Ac-H3 and Ac-H4 level.
Testing methods:The colorimetry method was used to test the level of HDAC in the infracted brain issue; and the Western blotting was used to test the level of Ac-H3 and Ac-H4 in the infracted brain issue; the enzyme-linked immunosorbent assay (ELISA) was used to test the level of IL-1βin cerebrospinal fluid and blood; and the immunohisto-chemistry method was used to detect the count of bcl-2 protein positive cell.
Statistical analysis:Excel 2007 was used to build dataset and for data entering. Two-way analysis of variance was used to test the main effects of treatment factor, time factor and their interaction on HDAC level, Ac-H3 and Ac-H4. Non-parametric test or robust test was used when the assumptions of analysis of variance were violated. Spearman correlation was used to examine the relationship between related cerebral protective factors. The'α=0.05'was selected as statistical test level. Statistical analysis was implemented using the SPSS 17.0.
Results
(1) After receiving a cerebral ischemia injury, the HDAC level grew over the time of reperfusion in a linear way, with the linear equation HDAC=0.116+0.060×time (P<0.05). Two-way analysis of variance showed that the main effects of treatment factor and time factor were statistically significant (P<0.05), but the effect of interaction of treatment and time was insignificant (P>0.05). Multiple comparisons between four groups revealed:TSA 1 group had lower HDAC level than control group and DMSO group (P<0.05); the HDAC level in TSA 2 group and DMSO group were lower than control group (P<0.05); and there no statistical difference between other groups (P>0.05).
(2) For Ac-H3 and Ac-H4, two-way analysis of variance showed that that the main effects of treatment factor and time factor were statistically significant (P<0.05), but the effect of interaction of treatment and time was insignificant (P>0.05). Both Ac-H3 and Ac-H4 increased from 6 hours and reAc-Hed the peak at 12 hours, then decreased gradually at all of four groups.
Multiple comparisons for Ac-H3 showed that the level of Ac-H3 in TSA 1 group was statistically higher than the other three groups (P<0.05), and TSA 2 group and DMSO group had higher Ac-H3 level than control group (P<0.05). There no statistical difference between other groups (P>0.05).
(3)Multiple comparisons for Ac-H4 showed that the level of Ac-H4 in TSA 1 group was statistically higher than the other three groups (P<0.05), and TSA 2 group had higher Ac-H4 level than control group (P<0.05). There no statistical difference between other groups (P>0.05).
(4)The bcl-2 positive cell count significantly correlated with HDAC, Ac-H4, with the Spearman rank correlation coefficient of -0.775 and 0.346 (P<0.01). The correlation coefficient between Ac-H3 and Ac-H4 was 0.800 (P<0.01). The rest of correlation were not statistically significant (P>0.05).
(5) Ac-H3 had a higher correlation with cerebrospinal fluid IL-1βand blood IL-1β(rs=1.0 and 0.8, P<0.01); the cerebrospinal fluid IL-1βhighly correlated with Ac-H4 (rs=0.8, P<0.01); and the correlation coefficient between cerebrospinal fluid IL-1βand blood IL-1βwas 0.8 (P<0.01). The rest of correlation were not statistically significant (F>0.05).
Conclusion
(1) The HDAC level rise over the time of reperfusion after the rats receive cerebral ischemia injury. TSA can reduce the expression of HDAC.
(2) TSA pre-conditioning can raise the level of histone Ac-H3 and Ac-H4 in cerebral issue. Histone Ac-H3 positively associate with cerebrospinal fluid IL-1βand blood IL-1β; and cerebrospinal fluid IL-1βhas a high and positive correlation with Ac-H4.
PartⅣ:Study of Association between age and TSA preconditioning and related factors (IL-1β, bcl-2)
Backgroud and objective
As China step into the aging society, ischemic cerebral vasnacular diseases have become more and more frequent than before. The implementation of family planning policy has attracted more and more attention to the health of singe child for eAc-H family. The cerebral function injury from neurological system diseases is one of important and unresolved pediatric research problem. Few studies involve the epigenetics of ischemic cerebral vanascular diseases although there are lots of researches exploring the pathological characteristics and treatment. It has been reported that TSA preconditioning can decrease the level of IL-1βlevel in the cerebrospinal fluid and raise the expression of apoptosis protein bcl-2, thus yielding protection of cerebral ischemia-reperfusion injury. However, the mechanism of its cerebral protection at different ages of rats keeps unreported. To deepen the mechanism of cerebral ischemia-reperfusion injury at rats of different ages, this study aimed to examine the relationship between age and cerebral protection of TSA preconditioning and related factors (IL-1β, bcl-2).
Material and methods
Experimental animals:18 healthy and clean SD male rats that received TSA preconditioning (0.1mg/kg) were divided into three groups based on their age:young group, adult group and old group.
Animal model:The modified suture method was used to block the middle cerebral artery for 1.5 hours, and then a 24-hour reperfusion was done, thus creating the cerebral ischemia-reperfusion animal model. TSA was injected through caudal vein 20 minutes before ischemia implementation.
Measurement indicators:neurological function score, cerebral infarction volume, cerebrospinal fluid and blood IL-1β, bcl-2 positive cell count.
Testing methods:Zea-Longa method was used to assessment the neurological function status; triphenyltetrazolium chloride (TTC) staining was used to measure cerebral infarction volume ratio of rats; the hematoxylin-eosin (HE) staining was used to observe pathological changes of ischemic brain issues; the enzyme-linked immunosorbent assay (ELISA) was used to test the level of IL-1βin cerebrospinal fluid and blood; and the immunohistochemistry method was used to detect the count of bcl-2 protein positive cell.
Statistical analysis:Excel 2007 was used to build dataset and for data entering. One-way analysis of variance was used to compare the difference in neurological function score, cerebral infarction volume ratio, cerebrospinal fluid and blood IL-1β, and bcl-2 positive cell count between three age groups. Non-parametric test or robust test was used when the assumptions of analysis of variance were violated. The 'α=0.05'was selected as statistical test level. Statistical analysis was performed using the SPSS 17.0.
Results
(1) One-way analysis of variance revealed that there was no statistical difference in Zea-Longa neurological function score between three age groups at 6h,12h and 24h (P>0.05).
(2)No significant difference in modified neurological function score were observed between three age groups at 6h and at 12h (P>0.05); but there the differences at 24h were statistically significant between three age groups. The mult-comparion between three age group at 24h showed, the scores of old group were different from those of young group (P<0.05), and there no significant difference between other groups (P>0.05).
(3)The total balance scores of young group were obviously lower than those of the other age groups (P<0.05); the total sport scores of young group differed from those of old group (P<0.05); The total reflection scores of young group and adult group were less than those of old group (P<0.05); the total sensation scores of three age groups were not significantly different from each other (P>0.05).
(4) One-way analysis of variance showed that no statistical difference was found in cerebral infarction volume between three age groups (P>0.05).
(5) One-way analysis of variance showed that rats of three age group showed no significant difference in cerebrospinal fluid and blood IL-1βlevel (P>0.05).
(6) One-way analysis of variance showed that the bcl-2 count differed between three age groups (P<0.05). Multi-comparisons between three groups showed that the old group had higher bcl-2 count than the other two groups (P<0.05), but no significant difference was detected between young group and adult group (P>0.05). Conclusion
Age is related to the cerebral protection of TSA pre-conditioning. The different function of the level of cerebrospinal fluid IL-1βand the bcl-2 count of the three groups changed with TSA preconditioning.
PartⅤ:Study of Association between age and TSA preconditioning and related factors (HDAC, Ac-H3 and Ac-H4)
Background and objective
Previous study reported that the occurrence of ischemic resistance is the adoptive chain of reaction, including genetic dependent reaction and non-genetic dependent reaction, the existed decoration of protein translation. The pre-conditioning is the re-programming process of organs to ischemia reaction at genetic level. Histone acetylation and deacetylation is the the most explored decoration mechanism after protein translation. Prior studies suggested that TSA pre-conditioning can produce protection to cerebral ischemia-reperfusion injury. To deepen the mechanism of cerebral ischemia-reperfusion injury in rats of different age, this study aimed to explore the relationship between age and HDAC, Ac-H3 and Ac-H4 in rats those receive TSA preconditioning, so as to provide base for the exploration of new treatment method.
Material and methods
Experimental animals:18 healthy and clean SD male rats that received TSA preconditioning (0.1mg/kg) were divided into three groups based on their age:young group, adult group and old group.
Animal model:The modified suture method was used to block the middle cerebral artery for 1.5 hours, and then a 24-hour reperfusion was done, thus creating the cerebral ischemia-reperfusion animal model. TSA was injected through caudal vein 20 minutes before ischemia implementation.
Measurement indicators:HDAC level in infracted brain issue, acetylaced H3 and H4 level.
Testing methods:The colorimetry method was used to test the level of HDAC in the infracted brain issue; and the Western blotting was used to test the level of acetylaced H3 and H4 in the infracted brain issue.
Statistical analysis:Excel 2007 was used to build dataset and for data entering. One-way analysis of variance was used to compare the difference in HDAC, Ac-H3 and Ac-H4 between three age groups. Non-parametric test or robust test was used when the assumptions of analysis of variance were violated. The'α=0.05'was selected as statistical test level. Statistical analysis was performed using the SPSS 17.0.
Results
(1) The HDAC level showed significant differences between three age groups (P<0.05). The old group had higher HDAC level than young and adult groups (P<0.05); there was no significant difference in HDAC level between the young group and the adult group (P<0.05).
(2) Rats of three groups showed statistical difference in Ac-H3 level (P<0.05). The old group had lower Ac-H3 level than the other two groups (P<0.05), but no significant difference was detected between the young group and the adult group (P>0.05). There was significant differences in Ac-H4 level between three age groups (P>0.05).
(3) Rats of three groups showed no statistical difference in Ac-H4 level (P<0.05). The old group had higher H4 level than the other two groups, but no significant difference was detected of the three groups (P>0.05).
Conclusion
(1) Age has an effect on the expression of HDAC in rats that receive TSA pre-conditioning.
(2) Age affects the expression of Ac-H3 in rats receiving TSA pre-conditioning but has no effect on the expression of Ac-H4.
引文
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