EphA4-ephrinA3系统在大鼠短暂前脑缺血/再灌注引起的海马区神经元迟发性死亡中的作用研究
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摘要
研究背景和目的:
脑卒中已成为当今第三大死亡原因及首位致残原因。据统计我国脑卒中发病率高达120/10万,致残率高达75%。缺血性脑卒中(脑梗死)占全部脑卒中的60%~80%。长时间的脑缺血将导致广泛的、非选择性的神经元坏死。而短暂的脑缺血事件后,虽然血供随之恢复,但在人类和一些动物模型都可以引起选择性的神经元死亡,主要影响海马CA1区的锥体神经元,这种死亡甚至可发生于再灌注28天后,称为神经元迟发性死亡(Delayed neuronal death,DND)。脑卒中致残的程度与DND密切相关,而DND的发生发展是一个复杂的,多种病理机制参与的过程,其机制尚未能完全阐明,因而缺乏理想的治疗手段。
DND的发生机制虽未被充分了解,但研究发现神经凋亡在其中扮演了重要的角色。Pulsinelli建立的大鼠短暂性前脑缺血/再灌注模型是目前国际上研究缺血性脑损伤后继发性神经元死亡机制普遍采用的模型之一,先行通过手术阻断椎动脉,次日短暂夹闭颈动脉(15分钟)进行缺血/再灌注,由于通过Willis环的血供仍然保留,对脑干功能无明显影响。在这一模型中,再灌注后大鼠海马发生特异性病理学改变:CA1区锥体细胞发生迟发性神经元死亡(即脑缺血/再灌注2～3天后光镜下才见到CA1区锥体细胞的死亡),而CA3区的锥体细胞和齿状回颗粒细胞却几乎不受损害。而CA1区锥体细胞发生迟发性神经元死亡中,凋亡发挥了重要作用。
ephrin(配体)-Eph(受体)系统属于受体酪氨酸激酶家族。Eph-ephrin系统的受体分为EphA与EphB两个亚族,相应的配体也分为ephrinA (A1～A5 )与ephrinB (B1～B3 )两个亚族。ephrinA配体具有一个胞外保守的受体结合区,通过糖基磷脂酰肌醇(glycosylphosphatidylinositol,GPI)形式锚定于细胞膜。Eph受体胞外域含配体结合区、半胱氨酸富集区与两个纤连蛋白Ⅲ型区,胞内域包括酪氨酸激酶区、SAM及PDZ结合区。EphA受体优先结合ephrinA, EphB优先结合ephrinB,但EphA4除与ephrinA结合外也可与ephrinB1和ephrinB2相结合。Eph-ephrin信号传递依赖复合物的寡聚化,游离的单体不具有生理作用。该系统在神经系统发育以及突触可塑性调控中起重要的作用。近期研究发现它们可能也与缺血损伤有关。研究显示ephrinA3是成年海马区最为丰富的Eph配体,主要位于GFAP阳性的星形胶质细胞上, EphA4分布于海马神经元特别是CA1区的锥体神经元上。而在病理情况下,CA1、CA3区及DG的锥体细胞层均可表现出强烈的缺血诱导的ephrinA3的表达。在未成年的大鼠,缺血缺氧可使EphA4下降,也有研究发现慢性脑缺血时EphA4上调,但EphA4-ephrinA3在缺血损伤中的作用尚不明确,此外它是否参与神经元的凋亡及其信号通路亦不明确。
ephrin配体活化Eph受体的信号传递包括MAPK,PI3激酶等多种下游信号通路。已有研究发现MAPKs,包括ERK1/2,JNK和p38 MAPK,广泛参与了神经元存活和凋亡调控过程,包括在缺血/再灌注损伤后神经元存活与死亡的调控。那么缺血/再灌注后通过EphA4-ephrinA3活化的MAPKs是否参与损伤后神经元凋亡亦不明确。
除上述信号通路参与对细胞增殖及凋亡的调节外,细胞的容积变化也在细胞的增殖、凋亡过程发挥作用。参与容积调节的主要是离子通道包括K+通道,Cl-通道等。近年来的研究表明,容积调节性Cl-通道通过对细胞容积、膜电位的调节在细胞凋亡过程中发挥了重要作用。但氯离子通道活动的调控机制尚未完全阐明,有研究提示蛋白酪氨酸磷酸化信号参与了容积调节性氯通道的调节,应用氯通道阻断剂或酪氨酸激酶抑制剂可明显减少短暂脑缺血/再灌注后海马神经元的凋亡。由于EphA4-ephrinA3属于酪氨酸激酶家族,它们是否可以通过影响氯通道功能在缺血/再灌注损伤中发挥作用呢?
综上所述,我们拟通过建立短暂前脑缺血/再灌注模型进行以下研究:1.通过检测缺血/再灌注诱导的凋亡及EphA4-ephrinA3在海马区的表达变化,观察EphA4-ephrinA3系统在缺血/再灌注后神经元凋亡过程发挥的作用; 2.通过记录氯通道电流及应用相应的激动剂、拮抗剂,观察EphA4-ephrinA3是否通过对海马CA1区氯通道的调控参与了神经元凋亡的过程;3.通过检测MAPKs信号通路中各途径(ERK,JNK、P38 MAPK)的表达,分析EphA4-ephrinA3参与缺血/再灌注损伤的信号转导通路。
一、EphA4-ephrinA3在缺血/再灌注损伤后大鼠脑海马神经元迟发性死亡的作用观察
目的:
建立大鼠全脑短暂缺血/再灌注模型,观察大鼠海马组织CA1区神经元的损伤(凋亡)情况,以及在此过程EphA4-ephrinA3表达的变化。为进一步研究EphA4-ephrinA3对于缺血/再灌注后CA区神经元凋亡的作用,分别通过侧脑室给予EphA4受体激动剂ephrinA3-Fc,以及受体阻断剂EphA4-Fc,观察EphA4-ephrinA3系统在脑缺血/再灌注诱导的CA1区神经元凋亡过程的作用。
方法:
1选取成年雄性Wistar大鼠,随机分为以下各组:
伪手术(sham)组(各时间点n=6),先行灼闭双侧椎动脉,次日打开颈部切口但不夹闭颈动脉;
缺血/再灌注组(各时间点n=6),先行灼闭双侧椎动脉,次日夹闭双侧颈动脉15分钟后松开动脉夹实现再灌注,6、24、48小时取材检测;
Vehicle+缺血/再灌注组(各时间点n=6),侧脑室给予无菌PBS10μl,于再灌注后6、24、48小时取材检测;
ephrinA3-Fc+缺血/再灌注组(各时间点n=6),给予ephrinA3-Fc,再灌注后6、24、48小时取材检测;
EphA4-Fc+缺血/再灌注组(各时间点n=6),给予EphA4-Fc,再灌注后6、24、48小时取材检测;
2建立大鼠前脑短暂缺血/再灌注模型,药物干预组缺血前经侧脑室内给予可溶性融合蛋白ephrinA3-Fc以激活EphA4受体,或者给予可溶性融合蛋白EphA4-Fc阻断体内EphA4-ephrinA3相互作用。留取海马组织,通过克紫染色观察海马神经元损伤情况;分别采用原位末端标记法(TUNEL)、Caspase 3活性测定法检测大鼠海马组织中神经神经元凋亡的情况;以Western blot方法检测大鼠海马组织中EphA4与ephrinA3的表达水平变化。从而了解EphA4与ephrinA3在大鼠脑缺血/再灌注后神经元损伤过程中的表达变化。
结果:
1.短暂脑缺血/再灌注可以引起海马CA1区神经元迟发性死亡。
在再灌注后不同时间点取海马组织进行尼氏染色,观察到进行性的CA1区神经元丢失。至缺血/再灌注后7天,CA1区细胞带消失,提示CA1区神经元大量死亡。Caspase 3活性在缺血/再灌注后6小时即有升高,随着时间推移在24小时及48小时继续升高。各时间点(OD值分别为2.3±0.19,2.2±0.28,1.9±0.015)与sham组(OD值为1.1±0.017)比较均具有显著性差异(P<0.05)。TUNEL染色的结果与Caspase3的结果类似,在三个时间点TUNEL染色的阳性率分别为24.5±5.36,22.3±4.76,23.8±4.17),与sham组(5.33±3.33)比较均具有统计学意义(P<0.05)。上述结果提示,短暂脑缺血/再灌注可以引起海马CA1区神经元凋亡增加
2.短暂脑缺血/再灌注后大鼠海马组织EphA4与ephrinA3蛋白表达均显著增加
实验结果显示,与对照组相比,海马缺血/再灌注后6小时海马CA1区组织中ephrin A3的表达水平显著增高(IOD值为2.05±0.12 vs 0.78±0.02,P<0.05),随后明显减少,但在再灌注24 h与对照相比仍有显著差异,再灌注48 h逐渐回复至对照组水平。EphA4也出现了由缺血诱导的增高,再灌后6小时与对照组相比即有明显增高(2.21±0.12 vs 0.72±0.03, P<0.05 ),之后逐渐下降,再灌注后24小时与对照相比仍有明显差异,至48小时与对照组相比无明显差异。
3.侧脑室内给予EphA4受体激动剂促进神经元的凋亡
当侧脑室内给予激动剂ephrinA3-Fc后,Caspase 3的活性较单纯缺血/再灌注组有明显升高(6 h: 2.76±0.15 vs. 2.28±0.28,P<0.05;24 h:2.24±0.24 vs. 2.20±0.19,P<0.05;48 h:2.04±0.44 vs. 1.90±0.29,P<0.05),同时TUNEL染色阳性细胞亦有增加(6 h: 31.8±4.40% vs. 26.33±5.32%,P<0.05;24 h:25.50±4.03% vs. 23.8±5.56%,P<0.05;48 h:22.17±4.52% vs. 19.0±4.34%,P<0.05)。
4.侧脑室给予EphA4受体阻断剂抑制神经元的凋亡
当侧脑室内给予阻断剂EphA4-Fc后,Caspase 3的活性较单纯再灌注组降低(6 h:1.73±0.30 vs. 2.28±0.28;24 h:1.52±0.26 vs. 2.20±0.19;48 h:1.43±0.23 vs. 1.90±0.29,P<0.05),而TUNEL染色阳性细胞亦明显减少(6 h:17.5±4.97% vs. 26.33±5.32%;24 h:15.0±4.47% vs. 23.8±5.56%;48 h:12.0±3.46% vs. 19.0±4.34%,P<0.05),提示EphA4-ephrinA3系统的活化可能参与了再灌注后神经元凋亡的发生。
小结:
1.短暂缺血/再灌注可导致海马CA1区神经元的迟发性死亡;
2.缺血/再灌注引起的神经元凋亡是导致海马CA1区神经元迟发性死亡的重要原因,而且神经元凋亡主要是Caspase依赖性的;
3.短暂脑缺血/再灌注可诱导海马CA1区EphA4与ephrinA3的表达明显增高,其时间过程与Caspase增高的时间过程一致,提示EphA4与ephrinA3的表达增高在CA1区神经元损伤过程中发挥重要作用。
4. EphA4与ephrinA3参与了缺血/再灌注后海马区神经元的凋亡过程,激活EphA4可导致神经元的凋亡增加,而阻断EphA4的作用可部分抑制再灌注后海马区神经元的凋亡。
二、EphA4-ephrinA3系统通过调控外向整流氯离子通道在缺血/再灌注损伤中发挥作用
目的:
已有研究显示,在中枢神经系统,氯通道的活动与神经元损伤及凋亡有密切关系。海马CA1区锥体神经元缺血损伤后外向整流氯离子通道(ORCC)功能活动增强,可能通过改变细胞容积引起神经元的凋亡。而应用氯离子通道阻断剂可以抑制脑缺血后引起的迟发性神经元死亡。另有研究显示ORCC的活化可由酪氨酸激酶受体介导。EphA4为在海马区表达丰富的酪氨酸激酶受体,设想也可能对氯通道功能产生影响,从而参与神经元凋亡过程。本实验通过制备缺血/再灌注大鼠海马脑片,并对锥体神经元进行全细胞脑片膜片钳记录,利用具有相对完整神经元和胶质细胞网络的大脑切片中,分别给予EphA4受体阻断剂EphA4-Fc和激动剂ephrinA3-Fc,观察EphA4-ephrinA3系统通过对氯通道功能的影响在缺血/再灌注损伤中发挥的作用。
方法:
选择出生后21- 25天健康雄性Wistar大鼠。体重180 ~ 250 g。
伪手术组(n=6),进行伪手术并制备脑片后正常人工脑脊液孵育并行电生理记录;
缺血/再灌注6小时组(n=6),制备缺血/再灌注模型,并在再灌注后6小时制备脑片,进行电生理记录;
缺血/再灌注24小时组(n=6),制备缺血/再灌注模型,并在再灌注后6小时制备脑片,进行电生理记录;
EphA4-Fc组+伪手术组(n=6):伪手术后6小时制备脑片,在记录前给予EphA4-Fc孵育10分钟;
EphA4-Fc组+再灌注组:再灌注后6小时制备脑片,在记录前给予EphA4-Fc孵育10分钟;
ephrinA3-Fc组+伪手术组(n=6):伪手术后6小时制备脑片,在记录前给予ephrinA3-Fc孵育10分钟;
ephrinA3-Fc组+再灌注组(n=6):再灌注后6小时制备脑片,在记录前给予ephrinA3-Fc孵育10分钟;
ephrinA3-Fc及EphA4-Fc以PBS配制高浓度贮存液,使用时以人工脑脊液稀释使终浓度为10μg/ml。
结果:
1.短暂脑缺血后大鼠海马CA1区锥体神经元外向整流氯通道的功能增强,表现为氯电流增加。
短暂脑缺血后大鼠海马CA1区锥体神经元外向整流氯通道电流持续增强。缺血后6小时和24小时的全细胞电流从正常的9.11±1.2pA/pF (-80mV)和64.2±6.9pA/pF (80mV)分别增强为15.80±1.70、17.70±1.93 pA/pF (-80mV)和120.16±12.3、129.82±11.83 pA/pF (+80 mV) (n=15,p <0.05 ),与对照组相比电流明显增强。
2.EphA4受体激动剂ephrinA3-Fc增强外向整流氯通道电流
在正常脑片上给予EphA4受体激动剂ephrinA3-Fc后,氯通道电流有轻微升高,但与正常对照相比,差异无显著性。但在缺血/再灌注后6小时的脑片上,激动剂ephrinA3-Fc可使大鼠海马CA1区锥体神经元外向整流氯通道的功能明显增强。以EphA4-Fc孵育脑片10分钟后进行全细胞膜片钳记录,与单纯缺血/再灌注时记录到的电流(15.80±1.70 pA/pF)相比有显著差异。结果显示,ephrinA3-Fc可使外向整流氯通道电流进一步增加,分别为16.980±1.9pA/pF (-80mV)和132.35±10.33 pA/pF (80mV),与缺血/再灌注组相比,差异具有显著性。。
3.EphA4受体阻断剂EphA4-Fc抑制外向整流氯通道电流
在正常脑片上给予EphA4受体阻断剂EphA4-Fc后,同样未观察到氯通道电流的显著变化。但在缺血/再灌注后6小时的脑片上,EphA4受体阻断剂EphA4-Fc可使外向整流氯通道电流下降。以EphA4-Fc孵育脑片10分钟后进行全细胞膜片钳记录,与单纯缺血/再灌注时记录到的电流(15.80±1.70 pA/pF)相比有显著差异。结果显示,EphA4-Fc可使外向整流氯通道电流下降,分别为13.30±1.3pA/pF (-80mV)和113.35±7.33 (80mV),与缺血/再灌注组相比,差异具有显著性。
小结:
1.脑缺血后大鼠海马CA1区锥体神经元外向整流氯通道功能活动持续性增强。
2.给予EphA4受体激动剂和阻断剂可影响缺血/再灌注后外向整流氯通道的电流。激动剂使通道电流进一步增加,而阻断剂可部分抑制通道电流。提示EphA4-ephrinA3系统可通过对氯通道影响在缺血/再灌注损伤中发挥作用。
三、EphA4-ephrinA3系统在脑缺血再灌后神经元迟发性死亡过程中的信号通路-MAPKs通路研究
目的:
ephrin配体活化Eph受体的信号传递包括Rho GTPases,MAPK,Src激酶和PI3激酶等多种下游信号通路。其中MAPKs包括ERK1/2,JNK和p38 MAPK,在多种病理情况下参与了神经元存活和凋亡的调控过程。因此,我们拟通过建立短暂前脑缺血/再灌注模型并给予EphA4-Fc或ephrinA3-Fc,观察在大鼠脑海马缺血/再灌注损伤后MAPKs信号通路中P38、ERK1/2及JNK的变化,及其与EphA4-ephrinA3系统表达变化的关系,以分析MAPKs信号通路各途径在EphA4-ephrinA3系统中的作用。
方法:
1.选取成年雄性Wistar大鼠,分组同第二部分。
2.建立大鼠全脑短暂缺血/再灌注模型,在缺血前侧脑室内给予可溶性融合蛋白ephrinA3-Fc以激活EphA4受体,或者给予可溶性融合蛋白EphA4-Fc阻断体内EphA4-ephrinA3相互作用,在再灌注后不同时间点留取海马组织,以Western blot方法检测大鼠海马组织中中P38、ERK1/2及JNK的表达变化,并分析其意义。
结果:
1.缺血/再灌注可引起p-P38在CA1区表达增高,再灌注6小时即与对照组有显著差异(0.83±0.18 vs 0.45±0.23,P<0.05)。给予ephrinA3-Fc后缺血/再灌注诱导的p-P38表达升高更为显著(分别为1.23±0.14,1.65±0.13,2.09±0.07),而EphA4-Fc对再灌注后p-P38的表达未产生明显改变。
2缺血/再灌注可诱导海马CA1区ERK1/2的活化,在各检测的时间点表达逐渐增高,但侧脑室给予EphA4-Fc及ephrinA3-Fc均不影响缺血/再灌注所诱导的表达升高。
3缺血/再灌注可诱导海马CA1区p-JNK的活化,在再灌注后6小时有明显升高,再灌注后48小时恢复正常水平,侧脑室给予EphA4-Fc及ephrinA3-Fc均不影响缺血/再灌注所诱导的表达升高。
小结:
1. EphA4-ephrinA3系统参与缺血/再灌注损伤的作用与与MAPK通路的活化密切相关,尤以P38途径的作用重要,但给予阻断剂EphA4-Fc未明显抑制P38的活化,其原因有待进一步研究;
2. ERK1/2与JNK途径参与了缺血/再灌注损伤的过程(缺血/再灌注均可使其活化),但在EphA4-ephrinA3系统中可能不发挥作用。
结论:
1、短暂缺血/再灌注引起海马CA1区神经元的迟发性死亡,以Caspase依赖的神经元凋亡为主。
2、在缺血/再灌注诱导的神经元凋亡过程EphA4-ephrinA3系统发挥了重要作用,该系统由此参与缺血/再灌注诱导的神经元损伤。
3、在缺血/再灌注引起的神经损伤过程,EphA4-ephrinA3加强了外向整流氯通道的活动使氯通道电流增加,这可能是EphA4-ephrinA3引起神经元凋亡的重要因素之一。
4、MAPKs通路介导了EphA4-ephrinA3在缺血/再灌注损伤中的作用,其中P38途径的作用尤为重要。
Background and purpose:
Stroke has become the third largest cause of death and first cause of disability. According to statistics, the incidence of stroke as high as 120/10 million,and disability up to 75%. Among them ischemic stroke (cerebral infarction) in total is 60% to 80%. Transient ischemic attack is cerebral ischemic cause by bloke of one or more blood vessels, which causes focal brain dysfunction, focal neurological signs and symptoms continue from a few minutes to several hours, and may relieve without treatment. But 1/3 of the TIA patients will develop ischemic stroke, while the onset of stroke often relapse. Prolonged cerebral ischemia will lead to broad, non-selective neuronal necrosis. And transient ischemic attacks, although the blood supply has resumed, but in humans and some animal models can lead to selective neuronal death, mainly affects pyramidal neurons in hippocampal CA1 region. This kind of death can occur even 28 days after reperfusion, called delayed neuronal death (Delayed neuronal death, DND). The degree of disability is closely related with the DND. But the occurrence and development of DND is a complex process involving a variety of pathological mechanisms, which mechanisms are not yet fully elucidated, and thus lack of a better therapy strategy.
The mechanism of DND has not yet been fully understood, but the study found that apoptosis plays an important role in this processing. Pulsinelli established rat transient forebrain ischemia/reperfusion model, it became a commonly used model to study the post-ischemic brain injury and secondary neuronal death. The vertebral artery were permanently occluded by surgery, the next day carotid artery was closed by clips for a short time (15 minutes) and them released to reperfusion, because the blood supply through the Willis ring is retained, no significant effect on the brain stem. In this model, specific pathological changes occured after reperfusion in hippocampus: delayed neuronal death in CA1 pyramidal cells (ie, only pyramidal cells death in CA1 area can be seen under the light microscope 2 to 3 days after reperfusion), while the pyramidal cells in CA3 region and granule cells in dentate gyrus are almost intact. Many studies have found that in experimental cerebral ischemia, the neuronal apoptosis plays an important role in the hippocampal delayed neuronal death.
Eph-ephrin system is a member of receptor tyrosine kinase family. Eph receptors include total of 16 members, divided into two subtribes: EphA and EphB, the corresponding ephrin ligands are divided into ephrinA (A1 ~ A5) and ephrinB (B1 ~ B3). ephrinA has a conserved extracellular binding region ,and anchored to the membrane by glycosyl phosphatidyl inositol (GPI). Eph receptors contain the extracellular ligand binding domain, cysteine-rich region and two fibronectin typeⅢarea, including the intracellular tyrosine kinase domain, SAM, and PDZ binding. EphA receptor priority binding ephrinA, EphB priority with ephrinB, but EphA4 and ephrinB2 may also be combined with the ephrinB1. Eph-ephrin signaling complex depend on oligomerization, free monomer does not have a physiological role. In neural development and synaptic plasticity this system plays an important role. Recent studies have found that they may involved in ischemic injury. Study found that ephrinA3 is the most abundant Eph ligands in adult hippocampus, mainly located in the GFAP-positive astrocytes, EphA4 distributed in the hippocampus, especially in pyramidal neurons of the CA1 region. In pathological conditions, CA1, CA3 area and DG of the pyramidal cell layer showed a strong ischemia-induced expression of ephrinA3. In the neonatal rats, ischemia and hypoxia decreased EphA4, but other study also found that chronic cerebral ischemia induced EphA4 increases, but what is the role of EphA4-ephrinA3 in these processes, whether they involved in neuronal apoptosis and what are its signaling pathways and not clear.
Signaling involved in the activation of Eph receptor including MAPK, PI3 kinase and other downstream signaling pathways. Among them, the large number of studies focused on that MAPKs, which including ERK1/2, JNK and p38 MAPK, these molecules are widely involved in the regulation of neuronal survival and apoptosis, including ischemia / reperfusion injury. So if MAPKs involved in the ischemia/reperfusion induced activation of EphA4-ephrinA3 and modulates neuronal apoptosis is also not clear.
Variety of factors involved in the regulation of cell proliferation and apoptosis. Cell proliferation, apoptosis, and some basic physiological processes are accompanied by changes in cell volume. Ion channels, such as K + channels and Cl-channel, are involved in volume regulation of cell volume. Recent studies show that the volume regulation of Cl-channels play an important role in cell volume, membrane potential, and other mechanisms involved in the regulation of apoptosis. But the mechanism of regulation of chloride channel is not fully understood, studies suggest protein tyrosine phosphorylation signal involved in the volume regulation of chloride channels, and in the application of chloride channel blockers or tyrosine kinase inhibitors can significantly reduce the transient cerebral ischemia / reperfusion induced apoptosis of hippocampal neurons. EphA4-ephrinA3 belongs to receptor tyrosine kinase family and whether they involved in the regulation of neuronal apoptosis by chloride channels?
In summary, by establishment of transient forebrain ischemia / reperfusion model, we performed the following studies: 1. Observe the expression of EphA4-ephrinA3 in ischemia / reperfusion injury, and cell apoptosis related assays were performed simultaneous; 2. The effects of EphA4-ephrinA3 in ischemia/reperfusion induced neuronal apoptosis; 3. whether EphA4-ephrinA3 involved in the ischemia / reperfusion induced apoptosis in hippocampal CA1 neuronal by the regulation of chloride channels. 4. By observing the alterations of MAPKs expression, to investigate if MAPKs involved in EphA4-ephrinA3 signal pathway after reperfusion injury.
PartⅠIschemia/reperfusion induced delayed neuronal death and expression of EphA4 and ephrinA3 in rat hippocampus
Objective
To investigate the role of EphA4 and ephrinA3 in ischemia/reperfusion induced apoptosis of hippocampus CA1 neurons, rat transient cerebral ischemia/reperfusion model was established, intraventricular administration of ephrinA3-Fc, the EphA4 receptor agonist, or EphA4-Fc, the antagonist of EphA4 receptor, was performed respectively. The neuronal damage of hippocampus CA1 area and the change of EphA4-ephrinA3 expression were observed.
Methods
1. The adult male Wistar rats were divided into the following groups at random:
control group (n=6), normal healthy rats;
Vertebral artery occlusion group (n=6), only occlusion of vertebral artery was performed; Sham group (at each point, n=6), bilateral vertebral arteries occlusion was done first, the neck incision was opened the next day, but carotid occlusion was not performed;
Ischemia / reperfusion group (at each point, n=6), bilateral vertebral arteries occlusion was done first, at the following day after 15 minutes of bilateral carotid artery occlusion by clips, them clips were released to achieve reperfusion, detection were performed 6, 24,48 hours after reperfusion;
Vehicle + ischemia / reperfusion group (at each point, n=6), intraventricular injection of sterile PBS 10μl, detection were performed 6, 24, 48 hours after reperfusion;
ephrinA3-Fc + ischemia / reperfusion group (at each point, n=6), intraventricular injection of ephrinA3-Fc, detection were performed 6, 24, 48 hours after reperfusion;
EphA4-Fc + ischemia / reperfusion group (at each point, n=6), intraventricular injection of e EphA4-Fc, detection were performed 6, 24, 48 hours after reperfusion;
2. The transient cerebral ischemic/reperfusion were made by 4-VO model. Soluble fusion protein ephrinA3-Fc was given intraventricular to active EphA4 receptor, or EphA4-Fc was given intraventricular to block EphA4 receptor. Corresponding drug administration was performed before vertebral artery occlusion. The hippocampi were removed. Hippocampus neurons damage was observed by Cresyl violet staining; neuronal apoptosis was determined by in situ TdT mediated dUTP nick end labeling (TUNEL) and measurement of Caspase 3 activity in hippocampus; ephrinA3 EphA4 expression changes were measured by Western blot.
Results
1. A transient cerebral ischemia / reperfusion can lead to hippocampal area CA1 delayed neuronal death.
At different time points after ischemia, progressive loss of hippocampal CA1 neurons was observed by Nissl staining. 7 days after reperfusion, neuron soma in CA1 area almost disappeared. This means most of the ca1 pyramidal cells were dead. Caspase 3 activity increased to peak 6 hours after reperfusionthen decreased, but after 48 hours reperfusion it did not recover to normal levels (OD: 2.3±0.19,2.2±0.28,1.9±0. 15, vs sham: 1.1±0.017, P<0.05). The results showed that transient cerebral ischemia/reperfusion can lead to Caspase 3 activity increased significantly in hippocampal CA1 area; TUNEL detection also indicate progressive neuronal apoptosis.
2. Transient cerebral ischemia / reperfusion in rat hippocampus ephrinA3 and EphA4 expression were significantly increased
Western-blot assay by the hippocampus ephrinA3 and EphA4 expression, the results show that compared with normal control group, 6 hours after reperfusion in hippocampal CA1 area ephrinA3 expression was significantly higher (IOD: 2.05±0.12 vs 0.78±0.02, P<0.05), 6 hours after ischemia the expression a peak, then decreased gradually, Until 48 hours after there is not significant defference compared with controls. EphA4 expression in CA1 area has increased 6 hours after reperfusion (2.21±0.12 vs 0.72±0.03, P<0.05), and then decreased gradually.
3. Pretreatment by ephrinA3-Fc before ischemia may induce increasing of Caspase 3 activity and it is significantly higher than ischemia/reperfusion group without ephrinA3-Fc treatment. Neuronal apoptosis have also increased, suggesting the occurrence of apoptosis after reperfusion may be enhanced by activation of EphA4.
4. After intraventricular injection of EphA4-Fc, the blocker of EphA4, Caspase 3 activity decreased, and neuronal apoptosis are significantly reduced, suggesting that activation of EphA4 is involved in neuronal apoptosis after reperfusion. Summary
1. Transient ischemic / reperfusion lead to delayed neuronal death of hippocampal CA1 neurons.
2. Caspase 3 activity significantly increased 6 hours after reperfusion, then decreased, but not back to normal level until 48h reperfusion which indicate that caspase dependent apoptosis may be an important factor of delayed neuronal deaths.
3. Transient cerebral ischemia / reperfusion induced ephrinA3 and EphA4 significant upregulation in hippocampal CA1, the time course consistent with the increasing of Caspase, TUNEL detection also confirmed the results. It suggesting that increased EphA4 and ephrinA3 expression in CA1 neurons may play important role in reperfusion injury.
4. EphA4 and ephrinA3 involved in the reperfusion induced neuronal apoptosis, activation of EphA4 can lead to increased neuronal apoptosis, and blocking of EphA4 partially inhibited reperfusion induced neuronal apoptosis.
PartⅡThe effect of EphA4-ephrinA3 system to outwardly rectifying chloride channel in hippocampal CA1 neurons
Objective
Studies showed that in the central nervous system, the chloride channel activity and neuronal damage, apoptosis are closely related. after ischemic injury outwardly rectifying chloride channel (ORCC) of hippocampal CA1 pyramidal neurons enhanced, caused neuronal apoptosis possibly by changes of cell volume. The application of chloride channel blockers can inhibit brain ischemia induced delayed neuronal death. Studies have shown that of ORCC can be activated by tyrosine kinase receptors. EphA4 is one of tyrosine kinase receptor abundant in the hippocampus, Present study performed on hippocampal slices obstained from ischemia / reperfusion model and normal rat. The ORCC of pyramidal neurons was recorded by whole cell patch clamp, treated with EphA4 receptor blocker EphA4-Fc and EphA4 receptor agonist ephrinA3-Fc, we attempted to confirmed the effects of EphA4-ephrinA3 system to the chloride channel in the ischemic / reperfusion injury.
Methods
25-21 day postnata healthy male Wistar rats were choosed. Weight: 180 ~ 250g. randomly grouped as follows:
Pseudo-operation group, n=6;
Ischemia / reperfusion 6 hours group, n=6, the recording was performed 6 houes after reperfusion, without other treatment;
Ischemia / reperfusion 24 hours group, n=6, the recording was performed 24 houes after reperfusion, without other treatment;
EphA4-Fc + sham group6 hours, n=6, the recording was performed 6 hours after operation, the slice was incubate in 10μg/ml EphA4-Fc for 10 minutes;
EphA4-Fc group + ischemia / reperfusion 6 hours group, n=6. he recording was performed 6 hours after reperfusion, the slice was incubate in 10μg/ml EphA4-Fc for 10 minutes;
ephrinA3-Fc + sham group 6 hours + sham group, n=6, the recording was performed 6 hours after operation, the slice was incubate in 10μg/ml ephrinA3-Fc for 10 minutes;
ephrinA3-Fc + ischemia / reperfusion 6 hours group, n=6, the recording was performed 6 hours after reperfusion, the slice was incubate in 10μg/ml ephrinA3-Fc for 10 minutes.
Results
1. Transient cerebral ischemia may induce enhance of outwardly rectifying chloride channels in rat hippocampal CA1 pyramidal neurons.
Transient cerebral ischemia induces outwardly rectifying chloride currents in rat hippocampus CA1 pyramidal neurons increasing. Whole-cell current 6 hours and 24 hours after ischemia/reperfusion were increased from the sham 9.11±1.2pA/pF (-80mV) and 64.2±6.9pA/pF (80mV) to: 15.80±1.70, 17.70±1.93 pA / pF ( -80mV) and 120.16±12.3,129.82±11.830 pA / pF (+80 mV) (n = 15, p <0.05), compared with the control gro the increasing was significantly.
2. Effects of ephrinA3-Fc and EphA4-Fc on ORCC of hippocampal CA1 pyramidal neurons
10 minutes pre-incubated with ephrinA3-Fc enhance the increased ORCC current induced by ischemic/reperfusion, respectively, 16.980±1.9pA/pF (- 80mV) and 132.35±10.33 (80mV).
10 minutes pre-incubated with EphA4--Fc decrease the ORCC enhanced by ischemic/reperfusion 13.30±1.3pA/pF (-80mV) and 113.35±7.33 (80mV), the difference was significant compared with ischemia / reperfusion group.
ephrinA3-Fc pre-incubated to brain slice from sham may slightly increase chloride current, but compared with normal controls, the difference was not significant. EphA4-Fc pre-incubated to brain slice from sham were not affect the chloride currents
Summary
1. Cerebral ischemia/reperfusion induced outwardly rectifying chloride channel activity enhancement in rat hippocampal CA1 pyramidal neurons.
2. Given EphA4 receptor agonists and antagonists can affect the ischemia / reperfusion induced increasing of chloride current, given EphA4-Fc can inhibit the ischemia/reperfusion induced increasing of outwardly rectifying chloride currents, and ephrinA3-Fc can increase the outward rectifying chloride currents, suggesting EphA4-ephrinA3 system may involve ischemia / reperfusion injury through regulation of chloride channel.
PartⅢThe downstream signaling pathway of EphA4-ephrinA3 system involved in the ischemia/reperfusion induced neuronal apoptosis–investigate of MAPKs pathway
Objective
The signaling way involved in ephrin activate Eph receptor including Rho GTPases, MAPK, PI3 kinase and other downstream signaling pathways. MAPKs, including ERK1 / 2, JNK and p38 MAPK, involved in a variety of pathological conditions and regulate neuronal survival and apoptosis after injur.
Therefore, by establishment of transient forebrain ischemia / reperfusion model and administrate of EphA4-Fc or ephrinA3-Fc, we proposed to observed the expression alteration of MAPKs signaling pathways in P38, ERK1 / 2 and JNK, to analysis is MAPKs signal pathway is involved in the signal transduction of EphA4-ephrinA3 system.
Methods
1.Adult male Wistar rats were selected, grouped seemas partⅡ
2.Cerebral transient ischemic/reperfusion model were established, soluble fusion protein ephrinA3-Fc was given intraventricular to active EphA4 receptor, or EphA4-Fc was given intraventricular to block EphA4 receptor. At different time points after reperfusion, the hippocampi were removed. Then P38, ERK1/2 and JNK expressions were determined and analyzed by western blot.
Results
1 ischemia / reperfusion can increase p-P38 expression in the CA1 area. Treatment with EphrinA3-Fc increase ischemia / reperfusion-induced p-P38 expression, and EphA4-Fc does not affect expression of p-P38 after reperfusion.
2 ischemia / reperfusion induced ERK1 / 2 activation in hippocampal CA1 area, but given EphA4-Fc and lateral ephrinA3-Fc did not affect this ischemia / reperfusion induced upregulation..
3 ischemia / reperfusion induced p-JNK activation in hippocampal CA1 area. It was significantly increased 6 hours after reperfusion and then decreased gradually, returned to normal levels at 48 hours after reperfusionr, and treatment with EphA4-Fc or ephrinA3-Fc did not affect this ischemia / reperfusion-induced increasing.
Summary
EphrinA3-EphA4 system is involved in ischemia / reperfusion injury and associated with MAPKs, especially p-P38 activation, activation of the receptor can be lead to activation of p-P38 was significantly increased, but blcoked EphA4 by EphA4-Fc did not inhibited the activation of p-P38 significantly, it is also need further study.
Conclusions:
1. Transient ischemic / reperfusion can lead to delayed death of hippocampal CA1 neurons. In this process, ephrinA3 and EphA4 expression in the hippocampal CA1 area was significantly increased, the time course is consistent with the increasing of Caspase and TUNEL, suggesting that ephrinA3 with increased expression of EphA4 in the CA1 area may be play an important role in neuronal ischemia/reperfusion injury.
2. EphA4 and ephrinA3 involved in reperfusion induced neuronal apoptosis, activation of EphA4 can lead to increased neuronal apoptosis, and EphA4 blocking may partially inhibited the reperfusion induced neuronal apoptosis.
3 Cerebral ischemia / reperfusion in rat hippocampus CA1 pyramidal neurons may lead to enhancement of outwardly rectifying chloride channel activity. Given EphA4 receptor agonists and antagonists can affect the ischemia / reperfusion enhanced chloride channel currents, ephrinA3-Fc activation of EphA4 receptor can increase neurons outward rectifying chloride currents after ischemia and reperfusion. Giving EphA4-Fc block EphA4 receptor can inhibit the ischemia/ reperfusion induced increasing of outwardly rectifying chloride currents, suggesting EphA4-ephrinA3 system may be affecte neuronal apoptosis after ischemia / reperfusion by regulation of chloride channel.
4. EphrinA3-EphA4 system may be involved in ischemia / reperfusion in hippocampal CA1 neurons through the P38 pathway, activation of the receptor can lead to significant increase in p-P38, but EphA4-Fc block the receptor did not alter the expression of p-P38, it is needs further study.
脑卒中已成为当今第三大死亡原因及首位致残原因。据统计我国脑卒中发病率高达120/10万,致残率高达75%。缺血性脑卒中(脑梗死)占全部脑卒中的60%~80%。长时间的脑缺血将导致广泛的、非选择性的神经元坏死。而短暂的脑缺血事件后,虽然血供随之恢复,但在人类和一些动物模型都可以引起选择性的神经元死亡,主要影响海马CA1区的锥体神经元,这种死亡甚至可发生于再灌注28天后,称为神经元迟发性死亡(Delayed neuronal death,DND)。脑卒中致残的程度与DND密切相关,而DND的发生发展是一个复杂的,多种病理机制参与的过程,其机制尚未能完全阐明,因而缺乏理想的治疗手段。
DND的发生机制虽未被充分了解,但研究发现神经凋亡在其中扮演了重要的角色。Pulsinelli建立的大鼠短暂性前脑缺血/再灌注模型是目前国际上研究缺血性脑损伤后继发性神经元死亡机制普遍采用的模型之一,先行通过手术阻断椎动脉,次日短暂夹闭颈动脉(15分钟)进行缺血/再灌注,由于通过Willis环的血供仍然保留,对脑干功能无明显影响。在这一模型中,再灌注后大鼠海马发生特异性病理学改变:CA1区锥体细胞发生迟发性神经元死亡(即脑缺血/再灌注2～3天后光镜下才见到CA1区锥体细胞的死亡),而CA3区的锥体细胞和齿状回颗粒细胞却几乎不受损害。而CA1区锥体细胞发生迟发性神经元死亡中,凋亡发挥了重要作用。
ephrin(配体)-Eph(受体)系统属于受体酪氨酸激酶家族。Eph-ephrin系统的受体分为EphA与EphB两个亚族,相应的配体也分为ephrinA (A1～A5 )与ephrinB (B1～B3 )两个亚族。ephrinA配体具有一个胞外保守的受体结合区,通过糖基磷脂酰肌醇(glycosylphosphatidylinositol,GPI)形式锚定于细胞膜。Eph受体胞外域含配体结合区、半胱氨酸富集区与两个纤连蛋白Ⅲ型区,胞内域包括酪氨酸激酶区、SAM及PDZ结合区。EphA受体优先结合ephrinA, EphB优先结合ephrinB,但EphA4除与ephrinA结合外也可与ephrinB1和ephrinB2相结合。Eph-ephrin信号传递依赖复合物的寡聚化,游离的单体不具有生理作用。该系统在神经系统发育以及突触可塑性调控中起重要的作用。近期研究发现它们可能也与缺血损伤有关。研究显示ephrinA3是成年海马区最为丰富的Eph配体,主要位于GFAP阳性的星形胶质细胞上, EphA4分布于海马神经元特别是CA1区的锥体神经元上。而在病理情况下,CA1、CA3区及DG的锥体细胞层均可表现出强烈的缺血诱导的ephrinA3的表达。在未成年的大鼠,缺血缺氧可使EphA4下降,也有研究发现慢性脑缺血时EphA4上调,但EphA4-ephrinA3在缺血损伤中的作用尚不明确,此外它是否参与神经元的凋亡及其信号通路亦不明确。
ephrin配体活化Eph受体的信号传递包括MAPK,PI3激酶等多种下游信号通路。已有研究发现MAPKs,包括ERK1/2,JNK和p38 MAPK,广泛参与了神经元存活和凋亡调控过程,包括在缺血/再灌注损伤后神经元存活与死亡的调控。那么缺血/再灌注后通过EphA4-ephrinA3活化的MAPKs是否参与损伤后神经元凋亡亦不明确。
除上述信号通路参与对细胞增殖及凋亡的调节外,细胞的容积变化也在细胞的增殖、凋亡过程发挥作用。参与容积调节的主要是离子通道包括K+通道,Cl-通道等。近年来的研究表明,容积调节性Cl-通道通过对细胞容积、膜电位的调节在细胞凋亡过程中发挥了重要作用。但氯离子通道活动的调控机制尚未完全阐明,有研究提示蛋白酪氨酸磷酸化信号参与了容积调节性氯通道的调节,应用氯通道阻断剂或酪氨酸激酶抑制剂可明显减少短暂脑缺血/再灌注后海马神经元的凋亡。由于EphA4-ephrinA3属于酪氨酸激酶家族,它们是否可以通过影响氯通道功能在缺血/再灌注损伤中发挥作用呢?
综上所述,我们拟通过建立短暂前脑缺血/再灌注模型进行以下研究:1.通过检测缺血/再灌注诱导的凋亡及EphA4-ephrinA3在海马区的表达变化,观察EphA4-ephrinA3系统在缺血/再灌注后神经元凋亡过程发挥的作用; 2.通过记录氯通道电流及应用相应的激动剂、拮抗剂,观察EphA4-ephrinA3是否通过对海马CA1区氯通道的调控参与了神经元凋亡的过程;3.通过检测MAPKs信号通路中各途径(ERK,JNK、P38 MAPK)的表达,分析EphA4-ephrinA3参与缺血/再灌注损伤的信号转导通路。
一、EphA4-ephrinA3在缺血/再灌注损伤后大鼠脑海马神经元迟发性死亡的作用观察
目的:
建立大鼠全脑短暂缺血/再灌注模型,观察大鼠海马组织CA1区神经元的损伤(凋亡)情况,以及在此过程EphA4-ephrinA3表达的变化。为进一步研究EphA4-ephrinA3对于缺血/再灌注后CA区神经元凋亡的作用,分别通过侧脑室给予EphA4受体激动剂ephrinA3-Fc,以及受体阻断剂EphA4-Fc,观察EphA4-ephrinA3系统在脑缺血/再灌注诱导的CA1区神经元凋亡过程的作用。
方法:
1选取成年雄性Wistar大鼠,随机分为以下各组:
伪手术(sham)组(各时间点n=6),先行灼闭双侧椎动脉,次日打开颈部切口但不夹闭颈动脉;
缺血/再灌注组(各时间点n=6),先行灼闭双侧椎动脉,次日夹闭双侧颈动脉15分钟后松开动脉夹实现再灌注,6、24、48小时取材检测;
Vehicle+缺血/再灌注组(各时间点n=6),侧脑室给予无菌PBS10μl,于再灌注后6、24、48小时取材检测;
ephrinA3-Fc+缺血/再灌注组(各时间点n=6),给予ephrinA3-Fc,再灌注后6、24、48小时取材检测;
EphA4-Fc+缺血/再灌注组(各时间点n=6),给予EphA4-Fc,再灌注后6、24、48小时取材检测;
2建立大鼠前脑短暂缺血/再灌注模型,药物干预组缺血前经侧脑室内给予可溶性融合蛋白ephrinA3-Fc以激活EphA4受体,或者给予可溶性融合蛋白EphA4-Fc阻断体内EphA4-ephrinA3相互作用。留取海马组织,通过克紫染色观察海马神经元损伤情况;分别采用原位末端标记法(TUNEL)、Caspase 3活性测定法检测大鼠海马组织中神经神经元凋亡的情况;以Western blot方法检测大鼠海马组织中EphA4与ephrinA3的表达水平变化。从而了解EphA4与ephrinA3在大鼠脑缺血/再灌注后神经元损伤过程中的表达变化。
结果:
1.短暂脑缺血/再灌注可以引起海马CA1区神经元迟发性死亡。
在再灌注后不同时间点取海马组织进行尼氏染色,观察到进行性的CA1区神经元丢失。至缺血/再灌注后7天,CA1区细胞带消失,提示CA1区神经元大量死亡。Caspase 3活性在缺血/再灌注后6小时即有升高,随着时间推移在24小时及48小时继续升高。各时间点(OD值分别为2.3±0.19,2.2±0.28,1.9±0.015)与sham组(OD值为1.1±0.017)比较均具有显著性差异(P<0.05)。TUNEL染色的结果与Caspase3的结果类似,在三个时间点TUNEL染色的阳性率分别为24.5±5.36,22.3±4.76,23.8±4.17),与sham组(5.33±3.33)比较均具有统计学意义(P<0.05)。上述结果提示,短暂脑缺血/再灌注可以引起海马CA1区神经元凋亡增加
2.短暂脑缺血/再灌注后大鼠海马组织EphA4与ephrinA3蛋白表达均显著增加
实验结果显示,与对照组相比,海马缺血/再灌注后6小时海马CA1区组织中ephrin A3的表达水平显著增高(IOD值为2.05±0.12 vs 0.78±0.02,P<0.05),随后明显减少,但在再灌注24 h与对照相比仍有显著差异,再灌注48 h逐渐回复至对照组水平。EphA4也出现了由缺血诱导的增高,再灌后6小时与对照组相比即有明显增高(2.21±0.12 vs 0.72±0.03, P<0.05 ),之后逐渐下降,再灌注后24小时与对照相比仍有明显差异,至48小时与对照组相比无明显差异。
3.侧脑室内给予EphA4受体激动剂促进神经元的凋亡
当侧脑室内给予激动剂ephrinA3-Fc后,Caspase 3的活性较单纯缺血/再灌注组有明显升高(6 h: 2.76±0.15 vs. 2.28±0.28,P<0.05;24 h:2.24±0.24 vs. 2.20±0.19,P<0.05;48 h:2.04±0.44 vs. 1.90±0.29,P<0.05),同时TUNEL染色阳性细胞亦有增加(6 h: 31.8±4.40% vs. 26.33±5.32%,P<0.05;24 h:25.50±4.03% vs. 23.8±5.56%,P<0.05;48 h:22.17±4.52% vs. 19.0±4.34%,P<0.05)。
4.侧脑室给予EphA4受体阻断剂抑制神经元的凋亡
当侧脑室内给予阻断剂EphA4-Fc后,Caspase 3的活性较单纯再灌注组降低(6 h:1.73±0.30 vs. 2.28±0.28;24 h:1.52±0.26 vs. 2.20±0.19;48 h:1.43±0.23 vs. 1.90±0.29,P<0.05),而TUNEL染色阳性细胞亦明显减少(6 h:17.5±4.97% vs. 26.33±5.32%;24 h:15.0±4.47% vs. 23.8±5.56%;48 h:12.0±3.46% vs. 19.0±4.34%,P<0.05),提示EphA4-ephrinA3系统的活化可能参与了再灌注后神经元凋亡的发生。
小结:
1.短暂缺血/再灌注可导致海马CA1区神经元的迟发性死亡;
2.缺血/再灌注引起的神经元凋亡是导致海马CA1区神经元迟发性死亡的重要原因,而且神经元凋亡主要是Caspase依赖性的;
3.短暂脑缺血/再灌注可诱导海马CA1区EphA4与ephrinA3的表达明显增高,其时间过程与Caspase增高的时间过程一致,提示EphA4与ephrinA3的表达增高在CA1区神经元损伤过程中发挥重要作用。
4. EphA4与ephrinA3参与了缺血/再灌注后海马区神经元的凋亡过程,激活EphA4可导致神经元的凋亡增加,而阻断EphA4的作用可部分抑制再灌注后海马区神经元的凋亡。
二、EphA4-ephrinA3系统通过调控外向整流氯离子通道在缺血/再灌注损伤中发挥作用
目的:
已有研究显示,在中枢神经系统,氯通道的活动与神经元损伤及凋亡有密切关系。海马CA1区锥体神经元缺血损伤后外向整流氯离子通道(ORCC)功能活动增强,可能通过改变细胞容积引起神经元的凋亡。而应用氯离子通道阻断剂可以抑制脑缺血后引起的迟发性神经元死亡。另有研究显示ORCC的活化可由酪氨酸激酶受体介导。EphA4为在海马区表达丰富的酪氨酸激酶受体,设想也可能对氯通道功能产生影响,从而参与神经元凋亡过程。本实验通过制备缺血/再灌注大鼠海马脑片,并对锥体神经元进行全细胞脑片膜片钳记录,利用具有相对完整神经元和胶质细胞网络的大脑切片中,分别给予EphA4受体阻断剂EphA4-Fc和激动剂ephrinA3-Fc,观察EphA4-ephrinA3系统通过对氯通道功能的影响在缺血/再灌注损伤中发挥的作用。
方法:
选择出生后21- 25天健康雄性Wistar大鼠。体重180 ~ 250 g。
伪手术组(n=6),进行伪手术并制备脑片后正常人工脑脊液孵育并行电生理记录;
缺血/再灌注6小时组(n=6),制备缺血/再灌注模型,并在再灌注后6小时制备脑片,进行电生理记录;
缺血/再灌注24小时组(n=6),制备缺血/再灌注模型,并在再灌注后6小时制备脑片,进行电生理记录;
EphA4-Fc组+伪手术组(n=6):伪手术后6小时制备脑片,在记录前给予EphA4-Fc孵育10分钟;
EphA4-Fc组+再灌注组:再灌注后6小时制备脑片,在记录前给予EphA4-Fc孵育10分钟;
ephrinA3-Fc组+伪手术组(n=6):伪手术后6小时制备脑片,在记录前给予ephrinA3-Fc孵育10分钟;
ephrinA3-Fc组+再灌注组(n=6):再灌注后6小时制备脑片,在记录前给予ephrinA3-Fc孵育10分钟;
ephrinA3-Fc及EphA4-Fc以PBS配制高浓度贮存液,使用时以人工脑脊液稀释使终浓度为10μg/ml。
结果:
1.短暂脑缺血后大鼠海马CA1区锥体神经元外向整流氯通道的功能增强,表现为氯电流增加。
短暂脑缺血后大鼠海马CA1区锥体神经元外向整流氯通道电流持续增强。缺血后6小时和24小时的全细胞电流从正常的9.11±1.2pA/pF (-80mV)和64.2±6.9pA/pF (80mV)分别增强为15.80±1.70、17.70±1.93 pA/pF (-80mV)和120.16±12.3、129.82±11.83 pA/pF (+80 mV) (n=15,p <0.05 ),与对照组相比电流明显增强。
2.EphA4受体激动剂ephrinA3-Fc增强外向整流氯通道电流
在正常脑片上给予EphA4受体激动剂ephrinA3-Fc后,氯通道电流有轻微升高,但与正常对照相比,差异无显著性。但在缺血/再灌注后6小时的脑片上,激动剂ephrinA3-Fc可使大鼠海马CA1区锥体神经元外向整流氯通道的功能明显增强。以EphA4-Fc孵育脑片10分钟后进行全细胞膜片钳记录,与单纯缺血/再灌注时记录到的电流(15.80±1.70 pA/pF)相比有显著差异。结果显示,ephrinA3-Fc可使外向整流氯通道电流进一步增加,分别为16.980±1.9pA/pF (-80mV)和132.35±10.33 pA/pF (80mV),与缺血/再灌注组相比,差异具有显著性。。
3.EphA4受体阻断剂EphA4-Fc抑制外向整流氯通道电流
在正常脑片上给予EphA4受体阻断剂EphA4-Fc后,同样未观察到氯通道电流的显著变化。但在缺血/再灌注后6小时的脑片上,EphA4受体阻断剂EphA4-Fc可使外向整流氯通道电流下降。以EphA4-Fc孵育脑片10分钟后进行全细胞膜片钳记录,与单纯缺血/再灌注时记录到的电流(15.80±1.70 pA/pF)相比有显著差异。结果显示,EphA4-Fc可使外向整流氯通道电流下降,分别为13.30±1.3pA/pF (-80mV)和113.35±7.33 (80mV),与缺血/再灌注组相比,差异具有显著性。
小结:
1.脑缺血后大鼠海马CA1区锥体神经元外向整流氯通道功能活动持续性增强。
2.给予EphA4受体激动剂和阻断剂可影响缺血/再灌注后外向整流氯通道的电流。激动剂使通道电流进一步增加,而阻断剂可部分抑制通道电流。提示EphA4-ephrinA3系统可通过对氯通道影响在缺血/再灌注损伤中发挥作用。
三、EphA4-ephrinA3系统在脑缺血再灌后神经元迟发性死亡过程中的信号通路-MAPKs通路研究
目的:
ephrin配体活化Eph受体的信号传递包括Rho GTPases,MAPK,Src激酶和PI3激酶等多种下游信号通路。其中MAPKs包括ERK1/2,JNK和p38 MAPK,在多种病理情况下参与了神经元存活和凋亡的调控过程。因此,我们拟通过建立短暂前脑缺血/再灌注模型并给予EphA4-Fc或ephrinA3-Fc,观察在大鼠脑海马缺血/再灌注损伤后MAPKs信号通路中P38、ERK1/2及JNK的变化,及其与EphA4-ephrinA3系统表达变化的关系,以分析MAPKs信号通路各途径在EphA4-ephrinA3系统中的作用。
方法:
1.选取成年雄性Wistar大鼠,分组同第二部分。
2.建立大鼠全脑短暂缺血/再灌注模型,在缺血前侧脑室内给予可溶性融合蛋白ephrinA3-Fc以激活EphA4受体,或者给予可溶性融合蛋白EphA4-Fc阻断体内EphA4-ephrinA3相互作用,在再灌注后不同时间点留取海马组织,以Western blot方法检测大鼠海马组织中中P38、ERK1/2及JNK的表达变化,并分析其意义。
结果:
1.缺血/再灌注可引起p-P38在CA1区表达增高,再灌注6小时即与对照组有显著差异(0.83±0.18 vs 0.45±0.23,P<0.05)。给予ephrinA3-Fc后缺血/再灌注诱导的p-P38表达升高更为显著(分别为1.23±0.14,1.65±0.13,2.09±0.07),而EphA4-Fc对再灌注后p-P38的表达未产生明显改变。
2缺血/再灌注可诱导海马CA1区ERK1/2的活化,在各检测的时间点表达逐渐增高,但侧脑室给予EphA4-Fc及ephrinA3-Fc均不影响缺血/再灌注所诱导的表达升高。
3缺血/再灌注可诱导海马CA1区p-JNK的活化,在再灌注后6小时有明显升高,再灌注后48小时恢复正常水平,侧脑室给予EphA4-Fc及ephrinA3-Fc均不影响缺血/再灌注所诱导的表达升高。
小结:
1. EphA4-ephrinA3系统参与缺血/再灌注损伤的作用与与MAPK通路的活化密切相关,尤以P38途径的作用重要,但给予阻断剂EphA4-Fc未明显抑制P38的活化,其原因有待进一步研究;
2. ERK1/2与JNK途径参与了缺血/再灌注损伤的过程(缺血/再灌注均可使其活化),但在EphA4-ephrinA3系统中可能不发挥作用。
结论:
1、短暂缺血/再灌注引起海马CA1区神经元的迟发性死亡,以Caspase依赖的神经元凋亡为主。
2、在缺血/再灌注诱导的神经元凋亡过程EphA4-ephrinA3系统发挥了重要作用,该系统由此参与缺血/再灌注诱导的神经元损伤。
3、在缺血/再灌注引起的神经损伤过程,EphA4-ephrinA3加强了外向整流氯通道的活动使氯通道电流增加,这可能是EphA4-ephrinA3引起神经元凋亡的重要因素之一。
4、MAPKs通路介导了EphA4-ephrinA3在缺血/再灌注损伤中的作用,其中P38途径的作用尤为重要。
Background and purpose:
Stroke has become the third largest cause of death and first cause of disability. According to statistics, the incidence of stroke as high as 120/10 million,and disability up to 75%. Among them ischemic stroke (cerebral infarction) in total is 60% to 80%. Transient ischemic attack is cerebral ischemic cause by bloke of one or more blood vessels, which causes focal brain dysfunction, focal neurological signs and symptoms continue from a few minutes to several hours, and may relieve without treatment. But 1/3 of the TIA patients will develop ischemic stroke, while the onset of stroke often relapse. Prolonged cerebral ischemia will lead to broad, non-selective neuronal necrosis. And transient ischemic attacks, although the blood supply has resumed, but in humans and some animal models can lead to selective neuronal death, mainly affects pyramidal neurons in hippocampal CA1 region. This kind of death can occur even 28 days after reperfusion, called delayed neuronal death (Delayed neuronal death, DND). The degree of disability is closely related with the DND. But the occurrence and development of DND is a complex process involving a variety of pathological mechanisms, which mechanisms are not yet fully elucidated, and thus lack of a better therapy strategy.
The mechanism of DND has not yet been fully understood, but the study found that apoptosis plays an important role in this processing. Pulsinelli established rat transient forebrain ischemia/reperfusion model, it became a commonly used model to study the post-ischemic brain injury and secondary neuronal death. The vertebral artery were permanently occluded by surgery, the next day carotid artery was closed by clips for a short time (15 minutes) and them released to reperfusion, because the blood supply through the Willis ring is retained, no significant effect on the brain stem. In this model, specific pathological changes occured after reperfusion in hippocampus: delayed neuronal death in CA1 pyramidal cells (ie, only pyramidal cells death in CA1 area can be seen under the light microscope 2 to 3 days after reperfusion), while the pyramidal cells in CA3 region and granule cells in dentate gyrus are almost intact. Many studies have found that in experimental cerebral ischemia, the neuronal apoptosis plays an important role in the hippocampal delayed neuronal death.
Eph-ephrin system is a member of receptor tyrosine kinase family. Eph receptors include total of 16 members, divided into two subtribes: EphA and EphB, the corresponding ephrin ligands are divided into ephrinA (A1 ~ A5) and ephrinB (B1 ~ B3). ephrinA has a conserved extracellular binding region ,and anchored to the membrane by glycosyl phosphatidyl inositol (GPI). Eph receptors contain the extracellular ligand binding domain, cysteine-rich region and two fibronectin typeⅢarea, including the intracellular tyrosine kinase domain, SAM, and PDZ binding. EphA receptor priority binding ephrinA, EphB priority with ephrinB, but EphA4 and ephrinB2 may also be combined with the ephrinB1. Eph-ephrin signaling complex depend on oligomerization, free monomer does not have a physiological role. In neural development and synaptic plasticity this system plays an important role. Recent studies have found that they may involved in ischemic injury. Study found that ephrinA3 is the most abundant Eph ligands in adult hippocampus, mainly located in the GFAP-positive astrocytes, EphA4 distributed in the hippocampus, especially in pyramidal neurons of the CA1 region. In pathological conditions, CA1, CA3 area and DG of the pyramidal cell layer showed a strong ischemia-induced expression of ephrinA3. In the neonatal rats, ischemia and hypoxia decreased EphA4, but other study also found that chronic cerebral ischemia induced EphA4 increases, but what is the role of EphA4-ephrinA3 in these processes, whether they involved in neuronal apoptosis and what are its signaling pathways and not clear.
Signaling involved in the activation of Eph receptor including MAPK, PI3 kinase and other downstream signaling pathways. Among them, the large number of studies focused on that MAPKs, which including ERK1/2, JNK and p38 MAPK, these molecules are widely involved in the regulation of neuronal survival and apoptosis, including ischemia / reperfusion injury. So if MAPKs involved in the ischemia/reperfusion induced activation of EphA4-ephrinA3 and modulates neuronal apoptosis is also not clear.
Variety of factors involved in the regulation of cell proliferation and apoptosis. Cell proliferation, apoptosis, and some basic physiological processes are accompanied by changes in cell volume. Ion channels, such as K + channels and Cl-channel, are involved in volume regulation of cell volume. Recent studies show that the volume regulation of Cl-channels play an important role in cell volume, membrane potential, and other mechanisms involved in the regulation of apoptosis. But the mechanism of regulation of chloride channel is not fully understood, studies suggest protein tyrosine phosphorylation signal involved in the volume regulation of chloride channels, and in the application of chloride channel blockers or tyrosine kinase inhibitors can significantly reduce the transient cerebral ischemia / reperfusion induced apoptosis of hippocampal neurons. EphA4-ephrinA3 belongs to receptor tyrosine kinase family and whether they involved in the regulation of neuronal apoptosis by chloride channels?
In summary, by establishment of transient forebrain ischemia / reperfusion model, we performed the following studies: 1. Observe the expression of EphA4-ephrinA3 in ischemia / reperfusion injury, and cell apoptosis related assays were performed simultaneous; 2. The effects of EphA4-ephrinA3 in ischemia/reperfusion induced neuronal apoptosis; 3. whether EphA4-ephrinA3 involved in the ischemia / reperfusion induced apoptosis in hippocampal CA1 neuronal by the regulation of chloride channels. 4. By observing the alterations of MAPKs expression, to investigate if MAPKs involved in EphA4-ephrinA3 signal pathway after reperfusion injury.
PartⅠIschemia/reperfusion induced delayed neuronal death and expression of EphA4 and ephrinA3 in rat hippocampus
Objective
To investigate the role of EphA4 and ephrinA3 in ischemia/reperfusion induced apoptosis of hippocampus CA1 neurons, rat transient cerebral ischemia/reperfusion model was established, intraventricular administration of ephrinA3-Fc, the EphA4 receptor agonist, or EphA4-Fc, the antagonist of EphA4 receptor, was performed respectively. The neuronal damage of hippocampus CA1 area and the change of EphA4-ephrinA3 expression were observed.
Methods
1. The adult male Wistar rats were divided into the following groups at random:
control group (n=6), normal healthy rats;
Vertebral artery occlusion group (n=6), only occlusion of vertebral artery was performed; Sham group (at each point, n=6), bilateral vertebral arteries occlusion was done first, the neck incision was opened the next day, but carotid occlusion was not performed;
Ischemia / reperfusion group (at each point, n=6), bilateral vertebral arteries occlusion was done first, at the following day after 15 minutes of bilateral carotid artery occlusion by clips, them clips were released to achieve reperfusion, detection were performed 6, 24,48 hours after reperfusion;
Vehicle + ischemia / reperfusion group (at each point, n=6), intraventricular injection of sterile PBS 10μl, detection were performed 6, 24, 48 hours after reperfusion;
ephrinA3-Fc + ischemia / reperfusion group (at each point, n=6), intraventricular injection of ephrinA3-Fc, detection were performed 6, 24, 48 hours after reperfusion;
EphA4-Fc + ischemia / reperfusion group (at each point, n=6), intraventricular injection of e EphA4-Fc, detection were performed 6, 24, 48 hours after reperfusion;
2. The transient cerebral ischemic/reperfusion were made by 4-VO model. Soluble fusion protein ephrinA3-Fc was given intraventricular to active EphA4 receptor, or EphA4-Fc was given intraventricular to block EphA4 receptor. Corresponding drug administration was performed before vertebral artery occlusion. The hippocampi were removed. Hippocampus neurons damage was observed by Cresyl violet staining; neuronal apoptosis was determined by in situ TdT mediated dUTP nick end labeling (TUNEL) and measurement of Caspase 3 activity in hippocampus; ephrinA3 EphA4 expression changes were measured by Western blot.
Results
1. A transient cerebral ischemia / reperfusion can lead to hippocampal area CA1 delayed neuronal death.
At different time points after ischemia, progressive loss of hippocampal CA1 neurons was observed by Nissl staining. 7 days after reperfusion, neuron soma in CA1 area almost disappeared. This means most of the ca1 pyramidal cells were dead. Caspase 3 activity increased to peak 6 hours after reperfusionthen decreased, but after 48 hours reperfusion it did not recover to normal levels (OD: 2.3±0.19,2.2±0.28,1.9±0. 15, vs sham: 1.1±0.017, P<0.05). The results showed that transient cerebral ischemia/reperfusion can lead to Caspase 3 activity increased significantly in hippocampal CA1 area; TUNEL detection also indicate progressive neuronal apoptosis.
2. Transient cerebral ischemia / reperfusion in rat hippocampus ephrinA3 and EphA4 expression were significantly increased
Western-blot assay by the hippocampus ephrinA3 and EphA4 expression, the results show that compared with normal control group, 6 hours after reperfusion in hippocampal CA1 area ephrinA3 expression was significantly higher (IOD: 2.05±0.12 vs 0.78±0.02, P<0.05), 6 hours after ischemia the expression a peak, then decreased gradually, Until 48 hours after there is not significant defference compared with controls. EphA4 expression in CA1 area has increased 6 hours after reperfusion (2.21±0.12 vs 0.72±0.03, P<0.05), and then decreased gradually.
3. Pretreatment by ephrinA3-Fc before ischemia may induce increasing of Caspase 3 activity and it is significantly higher than ischemia/reperfusion group without ephrinA3-Fc treatment. Neuronal apoptosis have also increased, suggesting the occurrence of apoptosis after reperfusion may be enhanced by activation of EphA4.
4. After intraventricular injection of EphA4-Fc, the blocker of EphA4, Caspase 3 activity decreased, and neuronal apoptosis are significantly reduced, suggesting that activation of EphA4 is involved in neuronal apoptosis after reperfusion. Summary
1. Transient ischemic / reperfusion lead to delayed neuronal death of hippocampal CA1 neurons.
2. Caspase 3 activity significantly increased 6 hours after reperfusion, then decreased, but not back to normal level until 48h reperfusion which indicate that caspase dependent apoptosis may be an important factor of delayed neuronal deaths.
3. Transient cerebral ischemia / reperfusion induced ephrinA3 and EphA4 significant upregulation in hippocampal CA1, the time course consistent with the increasing of Caspase, TUNEL detection also confirmed the results. It suggesting that increased EphA4 and ephrinA3 expression in CA1 neurons may play important role in reperfusion injury.
4. EphA4 and ephrinA3 involved in the reperfusion induced neuronal apoptosis, activation of EphA4 can lead to increased neuronal apoptosis, and blocking of EphA4 partially inhibited reperfusion induced neuronal apoptosis.
PartⅡThe effect of EphA4-ephrinA3 system to outwardly rectifying chloride channel in hippocampal CA1 neurons
Objective
Studies showed that in the central nervous system, the chloride channel activity and neuronal damage, apoptosis are closely related. after ischemic injury outwardly rectifying chloride channel (ORCC) of hippocampal CA1 pyramidal neurons enhanced, caused neuronal apoptosis possibly by changes of cell volume. The application of chloride channel blockers can inhibit brain ischemia induced delayed neuronal death. Studies have shown that of ORCC can be activated by tyrosine kinase receptors. EphA4 is one of tyrosine kinase receptor abundant in the hippocampus, Present study performed on hippocampal slices obstained from ischemia / reperfusion model and normal rat. The ORCC of pyramidal neurons was recorded by whole cell patch clamp, treated with EphA4 receptor blocker EphA4-Fc and EphA4 receptor agonist ephrinA3-Fc, we attempted to confirmed the effects of EphA4-ephrinA3 system to the chloride channel in the ischemic / reperfusion injury.
Methods
25-21 day postnata healthy male Wistar rats were choosed. Weight: 180 ~ 250g. randomly grouped as follows:
Pseudo-operation group, n=6;
Ischemia / reperfusion 6 hours group, n=6, the recording was performed 6 houes after reperfusion, without other treatment;
Ischemia / reperfusion 24 hours group, n=6, the recording was performed 24 houes after reperfusion, without other treatment;
EphA4-Fc + sham group6 hours, n=6, the recording was performed 6 hours after operation, the slice was incubate in 10μg/ml EphA4-Fc for 10 minutes;
EphA4-Fc group + ischemia / reperfusion 6 hours group, n=6. he recording was performed 6 hours after reperfusion, the slice was incubate in 10μg/ml EphA4-Fc for 10 minutes;
ephrinA3-Fc + sham group 6 hours + sham group, n=6, the recording was performed 6 hours after operation, the slice was incubate in 10μg/ml ephrinA3-Fc for 10 minutes;
ephrinA3-Fc + ischemia / reperfusion 6 hours group, n=6, the recording was performed 6 hours after reperfusion, the slice was incubate in 10μg/ml ephrinA3-Fc for 10 minutes.
Results
1. Transient cerebral ischemia may induce enhance of outwardly rectifying chloride channels in rat hippocampal CA1 pyramidal neurons.
Transient cerebral ischemia induces outwardly rectifying chloride currents in rat hippocampus CA1 pyramidal neurons increasing. Whole-cell current 6 hours and 24 hours after ischemia/reperfusion were increased from the sham 9.11±1.2pA/pF (-80mV) and 64.2±6.9pA/pF (80mV) to: 15.80±1.70, 17.70±1.93 pA / pF ( -80mV) and 120.16±12.3,129.82±11.830 pA / pF (+80 mV) (n = 15, p <0.05), compared with the control gro the increasing was significantly.
2. Effects of ephrinA3-Fc and EphA4-Fc on ORCC of hippocampal CA1 pyramidal neurons
10 minutes pre-incubated with ephrinA3-Fc enhance the increased ORCC current induced by ischemic/reperfusion, respectively, 16.980±1.9pA/pF (- 80mV) and 132.35±10.33 (80mV).
10 minutes pre-incubated with EphA4--Fc decrease the ORCC enhanced by ischemic/reperfusion 13.30±1.3pA/pF (-80mV) and 113.35±7.33 (80mV), the difference was significant compared with ischemia / reperfusion group.
ephrinA3-Fc pre-incubated to brain slice from sham may slightly increase chloride current, but compared with normal controls, the difference was not significant. EphA4-Fc pre-incubated to brain slice from sham were not affect the chloride currents
Summary
1. Cerebral ischemia/reperfusion induced outwardly rectifying chloride channel activity enhancement in rat hippocampal CA1 pyramidal neurons.
2. Given EphA4 receptor agonists and antagonists can affect the ischemia / reperfusion induced increasing of chloride current, given EphA4-Fc can inhibit the ischemia/reperfusion induced increasing of outwardly rectifying chloride currents, and ephrinA3-Fc can increase the outward rectifying chloride currents, suggesting EphA4-ephrinA3 system may involve ischemia / reperfusion injury through regulation of chloride channel.
PartⅢThe downstream signaling pathway of EphA4-ephrinA3 system involved in the ischemia/reperfusion induced neuronal apoptosis–investigate of MAPKs pathway
Objective
The signaling way involved in ephrin activate Eph receptor including Rho GTPases, MAPK, PI3 kinase and other downstream signaling pathways. MAPKs, including ERK1 / 2, JNK and p38 MAPK, involved in a variety of pathological conditions and regulate neuronal survival and apoptosis after injur.
Therefore, by establishment of transient forebrain ischemia / reperfusion model and administrate of EphA4-Fc or ephrinA3-Fc, we proposed to observed the expression alteration of MAPKs signaling pathways in P38, ERK1 / 2 and JNK, to analysis is MAPKs signal pathway is involved in the signal transduction of EphA4-ephrinA3 system.
Methods
1.Adult male Wistar rats were selected, grouped seemas partⅡ
2.Cerebral transient ischemic/reperfusion model were established, soluble fusion protein ephrinA3-Fc was given intraventricular to active EphA4 receptor, or EphA4-Fc was given intraventricular to block EphA4 receptor. At different time points after reperfusion, the hippocampi were removed. Then P38, ERK1/2 and JNK expressions were determined and analyzed by western blot.
Results
1 ischemia / reperfusion can increase p-P38 expression in the CA1 area. Treatment with EphrinA3-Fc increase ischemia / reperfusion-induced p-P38 expression, and EphA4-Fc does not affect expression of p-P38 after reperfusion.
2 ischemia / reperfusion induced ERK1 / 2 activation in hippocampal CA1 area, but given EphA4-Fc and lateral ephrinA3-Fc did not affect this ischemia / reperfusion induced upregulation..
3 ischemia / reperfusion induced p-JNK activation in hippocampal CA1 area. It was significantly increased 6 hours after reperfusion and then decreased gradually, returned to normal levels at 48 hours after reperfusionr, and treatment with EphA4-Fc or ephrinA3-Fc did not affect this ischemia / reperfusion-induced increasing.
Summary
EphrinA3-EphA4 system is involved in ischemia / reperfusion injury and associated with MAPKs, especially p-P38 activation, activation of the receptor can be lead to activation of p-P38 was significantly increased, but blcoked EphA4 by EphA4-Fc did not inhibited the activation of p-P38 significantly, it is also need further study.
Conclusions:
1. Transient ischemic / reperfusion can lead to delayed death of hippocampal CA1 neurons. In this process, ephrinA3 and EphA4 expression in the hippocampal CA1 area was significantly increased, the time course is consistent with the increasing of Caspase and TUNEL, suggesting that ephrinA3 with increased expression of EphA4 in the CA1 area may be play an important role in neuronal ischemia/reperfusion injury.
2. EphA4 and ephrinA3 involved in reperfusion induced neuronal apoptosis, activation of EphA4 can lead to increased neuronal apoptosis, and EphA4 blocking may partially inhibited the reperfusion induced neuronal apoptosis.
3 Cerebral ischemia / reperfusion in rat hippocampus CA1 pyramidal neurons may lead to enhancement of outwardly rectifying chloride channel activity. Given EphA4 receptor agonists and antagonists can affect the ischemia / reperfusion enhanced chloride channel currents, ephrinA3-Fc activation of EphA4 receptor can increase neurons outward rectifying chloride currents after ischemia and reperfusion. Giving EphA4-Fc block EphA4 receptor can inhibit the ischemia/ reperfusion induced increasing of outwardly rectifying chloride currents, suggesting EphA4-ephrinA3 system may be affecte neuronal apoptosis after ischemia / reperfusion by regulation of chloride channel.
4. EphrinA3-EphA4 system may be involved in ischemia / reperfusion in hippocampal CA1 neurons through the P38 pathway, activation of the receptor can lead to significant increase in p-P38, but EphA4-Fc block the receptor did not alter the expression of p-P38, it is needs further study.
引文
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