低氧预适应刺激脑海马区内源性神经干细胞的增殖

背景:缺血/缺氧/低氧等刺激均能导致内源性神经干细胞的增殖和分化,起到脑组织修复作用,但低氧预适应能否影响内源性神经干细胞的增殖尚不清楚。目的:探讨低氧预适应对小鼠脑海马区内源性神经干细胞增殖的影响。方法:清洁级Balb/c近交系小鼠随机分为3组,低氧对照组小鼠放入广口瓶内,立即用橡皮塞封紧,以动物出现第1次喘呼吸为低氧耐受极限的标志,完成低氧暴露1次;低氧预适应组小鼠按此法重复操作4次;正常对照组小鼠不进行低氧暴露。通过免疫荧光和激光共聚焦显微镜等技术,测定海马BrdU阳性细胞数和荧光强度。结果与结论:与低氧对照组比较,低氧预适应组小鼠耐受时间显著延长(P0.01)。正常对照组海马区BrdU标记的内源性神经干细胞荧光强度微弱,海马区可见少量BrdU阳性细胞;低氧对照组、低氧预适应组荧光强度及BrdU阳性细胞数均明显增加(P0.01),且低氧预适应组增加幅度大于低氧对照组(P0.01)。证实在低氧预适应过程中,海马区内源性神经干细胞明显增殖,可能参与低氧预适应脑保护机制。     

天门冬氨酸盐及氯氨酮对反复缺氧小鼠海马中NOS表达的影响(英文)

兴奋性氨基酸、ＮＭ ＤＡ 受体和一氧化氮合酶／一氧化氮系统参与了缺氧性脑损伤的形成,急性重复缺氧则能通过缺氧预适应显著提高小鼠对缺氧的耐受性。本研究旨在观察外源离子型ＮＭ ＤＡ 受体激动剂天门冬氨酸和抑制剂氯氨酮对小鼠缺氧预适应的形成以及形成过程中ＮＯＳ在小鼠海马脑区表达的影响。取昆明小鼠随机分为天冬氨酸、氯氨酸和正常对照三组,分别腹腔注射天门冬氨酸（３ ｇ／ｋｇ）、氯氨酮（１００ ｍ ｇ／ｋｇ）和生理盐水（３ ｍ ｌ／ｋｇ）;分别造成各组动物急性重复缺氧１ 次和４ 次同时测定各组缺氧耐受时间,并采用ＮＡＤＰＨ ｄ 组织化学技术观察各组动物脑内ＮＯＳ的表达。结果表明：天冬氨酸和氯氨酮分别显著地缩短和延长了小鼠的标准耐受时间。缺氧１ 次后,各组动物脑内ＮＯＳ的表达增强,而４ 次缺氧后,由于缺氧预适应的形成ＮＯＳ的表达未继续增加而保持不变。三组动物在缺氧４ 次后脑内ＮＯＳ的表达依次为天门冬氨酸组＞ 正常对照组＞ 氯氨酮组。上述结果提示：ＮＯＳ的抑制可能有益于缺氧耐受,并可能是预适应的机制之一。离子型ＮＭＤＡ 受体的激活和抑制分别不利于和有利于缺氧预适应的形成,这也可能是因为受体的激活和抑制分别促进和抑制了ＮＯＳ在海马的表达。     

缺氧预适应小鼠中一氧化氮合酶与缺氧诱导因子-1的表达

目的　探讨缺氧预适应小鼠海马组织中内皮型一氧化氮合酶 (eNOS)和缺氧诱导因子 1α的表达。　方法　HeLa细胞分为 :不缺氧 (H0 组 )、缺氧 1h(H1 组 )和缺氧 4h组 (H4组 ) ;小鼠分为 3组 :不缺氧 (M0 组 )、急性缺氧 1次 (M1 组 )和重复缺氧 4次组 (M4组 ) ,SDS PAGE和Western印迹法检测HIF 1α和eNOS的表达。　结果　H0 组HeLa细胞中未见HIF 1α ,H1 组出现少量HIF 1α ,H4组HIF 1α有所增加。M0 组小鼠海马中几乎检测不到HIF 1α ,可见eNOS ;M1 组可检测到较少的HIF 1α,eNOS无明显变化 ;M4组HIF 1α和eNOS水平均增高。　结论　慢性缺氧HeLa细胞中出现HIF 1α ,可作为检测HIF 1α的阳性对照 ;随着小鼠缺氧次数的增加 ,HIF 1α和eNOS的水平均升高 ,提示它们可能参与预适应的保护作用     

DNA甲基转移酶1、3A和3B在低氧预适应小鼠大脑中的表达简

 目的 研究DNA甲基转移酶（DNMT）1、3A和3B在低氧预适应小鼠大脑中的变化,探讨低氧预适应形成过程中DNMTs的变化与脑保护作用的关系。方法 小鼠随机分成常氧组（H0）、低氧1次组（H1）或4次（H4）后取大脑皮质组织,应用实时定量PCR 技术、免疫印迹技术和DNA甲基转移酶活性检测DNMT1、DNMT3A和DNMT3B mRNA和蛋白质及DNMT活性。结果H1组和H4组DNMT3A和DNMT3B的mRNA和蛋白质水平较H0组显著降低（p<0.05）,H4组DNMT活性较H0组降低（p<0.05）。结论DNMT3A和3B水平及DNMT活力的变化可能与低氧预适应小鼠获得脑保护的关系密切。     

急性重复低氧预适应小鼠海马组织DNA甲基转移酶表达的变化

目的:检测小鼠脑海马组织中三种DNA甲基转移酶在急性重复缺氧条件下的变化,探讨缺氧预适应过程中DNA甲基转移酶的变化在低氧神经保护中的作用。方法:昆明小鼠随机分成正常对照组(常氧组,H0)、低氧1次组(H1)或低氧4次(H4)组,实验后取海马组织,应用Real-time PCR技术、Western bolt技术和DNA甲基转移酶活性实验对三种DNA甲基转移酶的变化进行研究。结果:DNA甲基转移酶1的mRNA和蛋白质水平在三组之间无明显变化;DNA甲基转移酶3A和3B mRNA和蛋白质水平在三组之间有变化,DNA甲基转移酶3A和3B的mRNA在H4组中表达较H0组降低,差异有统计学意义(P<0.01),DNA甲基转移酶3A和3B的蛋白质变化与mRNA变化相似,H4组与H0组相比有所降低(P<0.05);DNA甲基转移酶活性在H4组较H0组降低。结论:DNA甲基转移酶的变化可能参与缺氧预适应小鼠获得脑保护。     

低氧预适应小鼠脑组织内P38 MAPK磷酸化水平增高

目的探讨P38丝裂原激活蛋白激酶(mitogen-activated protein kinase,P38 MAPK)在小鼠脑低氧预适应形成过程中的作用。方法用成年雄性BALB/c小鼠制备小鼠整体低氧预适应模型,小鼠随机分为正常对照(H0)、早期(H1～H4)和延迟性(H5～H6)低氧预适应等7组;应用Westernbolt并结合GelDoc凝胶成像系统,定量检测小鼠脑组织内P38MAPK磷酸化水平和蛋白表达量。结果与H0组小鼠相比,H2~H6组的海马和皮层及H3～H6组的下丘脑中P38MAPK磷酸化水平明显增高(P<0·05,每组n=6);H1~H6组的皮层、海马和下丘脑中P38MAPK蛋白表达量无明显变化。结论P38MAPK磷酸化激活而非蛋白表达量的变化可能参与了小鼠脑低氧预适应的发生、发展过程。     

通心络对缺氧预适应小鼠脑组织细胞凋亡因子表达的影响

目的:观察缺氧预适应小鼠脑组织细胞凋亡因子表达的变化以及通心络的干预作用。方法:将小鼠随机分为预适应组(对照组)与通心络组2组,通心络组按1.52g生药·kg-1·d-1,灌胃给药,5d后进行实验。2组动物分别进行0次(H0)、1次(H1)、3次(H3)、5次(H5)重复缺氧暴露,小鼠置于低氧密闭罐中,通过小鼠呼吸消耗罐内氧造成罐内低氧,以小鼠出现喘呼吸为低氧耐受极限,然后将小鼠转到另一低氧密闭罐中,依此类推,连续进行5次低氧。观察它们的缺氧耐受时间,以及脑组织缺氧诱导因子1α(HIF-1α)、B细胞白血病/淋巴瘤相关抗原相关X蛋白(Bax)、B细胞白血病/淋巴瘤相关抗原2(Bcl-2)蛋白的表达。结果:随着缺氧次数的增加,对照组与通心络组缺氧耐受时间均依次增强,HIF-1α、Bcl-2蛋白表达逐渐增强(P0.01或P0.05);Bax蛋白在1次缺氧时明显增加,后逐渐降低(P0.01或P0.05)。与对照组比较,通心络组各次缺氧时间增长,HIF-1α、Bcl-2蛋白表达增强,Bax蛋白表达下降(P0.01或P0.05)。结论:缺氧预适应具有强大的自适应能力,HIF-1α、Bcl-2蛋白表达增强和Bax表达下降可能是缺氧预适应小鼠缺氧自适应的机制之一。通心络能够明显增强机体的自适应能力,提高小鼠对急性缺氧的耐受性。     

低氧预适应小鼠脑内ERK1/2磷酸化水平和蛋白表达量的改变

目的：初步探讨细胞外信号调节激酶（Extraeellular signal-regulated kinases，ERK1／2）在脑低氧预适应发生发展过程中的作用。方法：按已建小鼠整体低氧预适应模型，将BALB／C小鼠（18-22g，雌雄不限）随机分为正常对照（H0）、早期（H1-H4）和延迟性（U5-H6）低氧预适应等7组（每组至少6只动物）。应用SDS-PAGE和Western blot等生化技术，并结合Gel Doc凝胶成像系统，半定量检测小鼠脑组织内ERK1／2的磷酸化水平和蛋白表达量。结果：①早期低氧预适应形成过程中，随低氧暴露次数的增加（H1-H4），小鼠海马和皮层组织内ERK1／2磷酸化水平显著降低（P〈0．05，n=6），而ERK1／2蛋白表达量并无显著变化；②延迟性低氧预适应中（H5-H6），小鼠大脑皮层和海马组织内ERK1／2的蛋白表达量显著降低（P〈0．05，n=6）。结论：ERK1／2的活性降低（磷酸化水平降低），以及ERK1／2蛋白表达量下调可能分别参与了脑早期低氧预适应和晚期延迟性低氧预适应的发生发展过程。     

低氧预适应小鼠脑组织内CaMKⅡ磷酸化水平增高

目的探讨钙调蛋白依赖性蛋白激酶(CaMKⅡ)在小鼠脑低氧预适应(HPC)发生发展中的作用。方法成年雄性BALB/c小鼠随机分为正常对照(H0)、早期(H1～H4)和延迟性(H5～H6)低氧预适应等共计7组,制备小鼠HPC模型;用Western blot并结合GelDoc凝胶成像系统,检测小鼠脑组织内CaMKⅡ磷酸化水平和蛋白表达量;用免疫组化检测小鼠脑皮层和海马CaMKⅡ磷酸化水平。结果与H0组小鼠相比,H3～H5组海马和皮层的CaMKⅡ磷酸化水平明显升高(P<0.05);而H1～H6组的皮层和海马的CaMKⅡ蛋白表达量无明显变化;H3、H6组皮层和海马p-CaMKⅡ阳性细胞数目增多和灰度增强。结论CaMKⅡ磷酸化水平的升高可能参与了小鼠脑HPC的发生发展过程。     

反复低氧对小鼠脑内神经肽Y免疫反应活性的影响

目的:观察反复低氧对小鼠脑内神经肽Y(NPY)免疫反应活性的影响。方法:昆明小鼠40只随机分为对照组、低氧1次(H₁)组、低氧2次(H₂)组、低氧3次(H₃)组和低氧4次(H₄)组。对低氧各组动物分别行不同次数的反复低氧。各组动物脑内NPY的免疫反应活性采用放射免疫测定法测定。结果:小鼠对低氧的标准耐受时间随反复低氧次数的增加而显著增加。经1次低氧和2次低氧后小鼠脑内NPY的免疫反应活性明显提高,分别为正常对照组的145.5%±3.2%和147.3%±2.5%(P＜0.01);而低氧3次后则降到正常对照水平。结论:反复低氧通过预适应能显著提高小鼠对低氧的耐受性,并可诱导小鼠脑内NPY免疫反应活性在预适应形成的早期一过性地上升。     

在缺氧预处置中钙离子的作用(英文)

本文用 Fura/AM荧光法和钙离子选择电极方法分别测定了急性重复缺氧小鼠脑细胞内和全脑匀浆液中钙离子浓度。结果表明 ,与正常对照组相比 ,所有缺氧组的脑细胞内和全脑匀浆液中钙离子含量明显高于对照组 (P<0 .0 5 ) ,但是随着缺氧次数的增加 ,却未见明显的钙离子浓度的升高。这些结果提示 ,在急性重复缺氧这种缺氧预处置中 ,钙离子增加的向下回调可能与其自身具有的神经性保护功能有关     

Effect of Hypoxic Preconditioning on Free Radical Processes in Tissues of Rats with Different Resistance to Hypoxia

We studied the effect of single hypoxic preconditioning exposure (hypobaric hypoxia, 5000 m, 60 min) on free radical processes, glutathione system, and antioxidant defense enzymes in tissues of rats with different resistance to acute hypoxia. The intensity of free radical processes was shown to increase or decrease on day 1 after hypoxic preconditioning. These changes were tissue-specific and opposite in animals with genetically determined differences in the resistance to hypoxia. Hypoxic preconditioning contributes to the immediate resistance. The effect was more pronounced in low resistant animals, who did not exhibit signs of oxidative stress in tissues during the early posthypoxic period. By contrast, hypoxic preconditioning was followed by activation of free radical processes in tissues of highly resistant animals. These rats were characterized by low ability for the development of immediate resistance. Activation of free radical processes in the early period of adaptation (first hours after hypoxic preconditioning) does not play a role in the induction of immediate adaptive mechanisms for hypoxia.     

Changes in cPKC isoform-specific membrane translocation and protein expression in the brain of hypoxic preconditioned mice

Previous studies have shown that the level of total conventional protein kinase C (cPKC) membrane translocation (activation) was increased in the brain of hypoxic preconditioned mice. In order to find out which isoform of cPKC may participate in the development of cerebral hypoxic preconditioning (HPC), we used Western bolt and immunohistochemistry to observe the effects of repetitive hypoxic exposure (H1-H6, n = 6 for each group) on the level of cPKC isoform-specific protein expression and its membrane translocation in the cortex and hippocampus of mice. We found that the levels of cPKC betaII and gamma membrane translocation were increased significantly (p < 0.05 versus normoxic H0 group, n = 6) in response to repetitive hypoxic exposure (H1-H4) at an early phase of hypoxic preconditioning, but no significant changes of cPKC alpha and betaI membrane translocation were found during cPKC alpha, betaI, betaII and gamma protein expression both in hippocampus and cortex. In addition, an extensive subcellular redistribution of cPKC betaII and gamma was detected by immunohistochemistry staining in the cortex after repetitive hypoxic exposures (H3). However, a significant decrease in the expression of cPKC gamma protein (p < 0.05 versus H0 group) was found only in the cortex of delayed hypoxic preconditioned mice (H5-H6). These results suggest that the activation of cPKC betaII and gamma may be involved in the early phase of cerebral hypoxic preconditioning and the changes in cPKC gamma protein expression may participate in the development of the late phase of cerebral hypoxic preconditioning as well as selective vulnerability to hypoxia both in cortex and hippocampus.     

Decreased phosphorylation and protein expression of ERK1/2 in the brain of hypoxic preconditioned mice

Accumulated reports have suggested that activation of protein kinase C (PKC) isoforms may involve the activation of extracellular signal-regulated kinases 1/2 (ERK1/2) in the neuronal response to hypoxic stimuli. We have previously demonstrated that the membrane translocation or activation of conventional PKC (cPKC) betaII, gamma and novel PKC (nPKC) varepsilon are increased in the early phase of cerebral hypoxic preconditioning in mice. However, the role of ERK1/2 in the development of cerebral hypoxic preconditioning is unclear. In the current study, we used Western blot analysis to investigate the effects of repetitive hypoxic exposure (H0-H6, n=6 for each group) on the levels of phosphorylation and protein expression of ERK1/2 in the frontal cortex and the whole hippocampus of mice. We found that the levels of phosphorylated ERK1/2, not protein expression of ERK1/2, decreased significantly in both cortex and hippocampus of the early hypoxic preconditioned mice (H1-H4), when compared to that of the normoxic group (p<0.05). In addition, a significant decrease (p<0.05) in the ERK1/2 protein expression, not the phosphorylated form of ERK1/2, was found both in the frontal cortex and hippocampus of mice followed hypoxia with previous hypoxia (H5 and H6). These results suggest that the decreased phosphorylation and downregulation of protein expression of ERK1/2 might be involved in the development of hypoxic preconditioning.     

低氧预适应对小鼠海马神经元中TSC1表达的影响

目的:在体视显微镜下分割ICR小鼠海马CA1区和CA3区,研究结节性硬化症因子1(TSC1)在小鼠海马低氧中的神经保护作用。方法:ICR小鼠分为对照组(control)、低氧对照组(hypoxia)及低氧预适应组(HPC),在体视显微镜下观察海马形态并分割CA1区和CA3区;采用real time RT-PCR和Western Blot的方法分别检测小鼠海马组织TSC1 mRNA和蛋白的表达;采用免疫荧光检测小鼠海马组织TSC1荧光强度。结果:大脑冠状切片清晰显示出海马CA1区、CA3区和DG区; CA1区TSC1 mRNA在低氧组降低而在低氧预适应组增高;Western Blot和组织免疫荧光显示:与对照组相比,低氧预适应组CA1区TSC1表达增加;而CA3区TSC1在低氧组和低氧预适应组均增加。结论:TSC1的差异性表达可能提示TSC1可能参与了低氧预适应对低氧敏感的CA1区神经细胞的保护。     

Hypoxia preconditioning attenuates bladder overdistension-induced oxidative injury by up-regulation of Bcl-2 in the rat

We explored whether hypoxic preconditioning minimizes oxidative injury induced by overdistension/emptying in the rat bladder. For hypoxic preconditioning, female Wistar rats were placed in a hypobaric chamber (380 Torr) 15 h day⁻¹ for 28 days. Overdistension was induced by infusion of two times the threshold volume of saline into the bladder and was maintained for 1 or 2 h, followed by drainage/emptying. During overdistension (ischaemia) and emptying (reperfusion) periods, a bursting increase of reactive oxygen species (ROS) from the bladder was originated from the large numbers of infiltrating leucocytes and scattered resident cells, including urothelial, submucosal, and smooth muscle cells. ROS impaired the voiding function by a reduction of bladder afferent and efferent nerve activity and bethanecol- or ATP-induced detrusor contraction. ROS enhanced pro-apoptotic mechanisms, including increases in the Bax/Bcl-2 ratio, CPP32 expression, and poly(ADP-ribose) polymerase (PARP) fragments with subsequent apoptotic cell formation in the insulted bladders. Hypoxia preconditioning up-regulated Bcl-2 expression in the bladder and significantly reduced the levels of ROS and apoptosis detected in the overdistension/emptying bladders and preserved partial voiding function. Bcl-2 up-regulation by hypoxia preconditioning contributes protection against overdistension/emptying-induced oxidative stress and injury in the bladder.