【作者】 胡荣；

【导师】 冯华； 徐天乐；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 随着国家经济的发展,交通、建筑及矿难等事故中导致的脊髓损伤（spinal cordinjury,SCI）已成为威胁人们健康的重要因素,关于SCI的研究因而备受国内学者的高度关注。遗憾的是,迄今为止尚无有效治疗SCI的方法和药物,促使人们重新审视SCI的病理生理过程和致病机制。SCI后主要经历急性期和慢性期两大病理生理阶段,急性期病变主要以原发损伤启动的一系列继发损伤为主,导致损伤范围的扩大和损伤程度的加重,所以,研究SCI急性期继发损伤的机制,如何尽可能地保护原发损伤周围的正常脊髓免受继发损伤的波及,是SCI治疗的首要策略。SCI慢性期则以星形胶质细胞活化、反应性增生及胶质瘢痕（glial scar）和囊腔的形成为其主要病理特点,他们成为阻挡神经再生的重要屏障,至今尚无有效的处理方法。因此,研究胶质瘢痕形成的过程、瘢痕的厚度及其与神经纤维的关系,对深入理解胶质瘢痕的特点和重新审视它在脊髓损伤与修复中的作用,具有重要的意义和价值。研究发现急性期许多致病机制参与了SCI后的继发损伤过程,包括缺血、兴奋性毒性、炎症、氧化应激、代谢能量障碍等。近年来,有不少针对上述继发损伤机制药物的基础与临床研究,但迄今未见临床疗效,提示尚有其它不为人知的继发损伤机制。新近报道的酸敏感离子通道（acid-sensing ion channel,ASICs）为SCI的研究带来了新的启发,尤其是ASICs的亚基ASIC1a。因为SCI后多种因素均可导致局部组织微环境的酸化,例如缺血缺氧可致CO₂等酸性代谢物无法带走,兴奋性氨基酸和ATP的释放（均是酸性物质）,代谢障碍致乳酸堆积,炎症亦可导致酸化。所以,SCI后的pH降低（组织酸化）可能较严重,它将激活ASIC1a通道,导致一系列病理过程。但脊髓中ASIC1a的表达分布及其在SCI后的变化,不甚清楚。所以,本课题将动态观察SCI后ASIC1a的变化特点,探讨其在SCI继发损伤中的作用及其可能机制。胶质瘢痕和囊腔的形成是SCI慢性期的重要病理变化,是导致SCI后神经再生修复失败的重要原因,研究发现即便通过神经移植桥接了囊腔坏死区,但移植物和神经纤维仍无法穿越致密的胶质瘢痕。因此,减少或去除胶质瘢痕是一项有希望的慢性期SCI治疗策略,但是有两条重要的因素决定着这一策略的效果:第一是瘢痕分布的特点如何?去除瘢痕的厚度应该是多少?囊腔周围的瘢痕厚度是否一致?如果去除瘢痕过多,会损及正常脊髓组织造成新的损伤。第二是去除胶质瘢痕的时间窗应如何选择?过早或过晚可能都存在问题。所以,本研究将动态观察胶质瘢痕形成过程、瘢痕的厚度及其与神经纤维的关系,为以上问题的回答提供实验依据。目的:1.动态观察SCI后胶质瘢痕形成规律及其与神经纤维的关系,并定量测量胶质瘢痕的厚度。2.观察ASIC1a在SCI后的表达变化特点,研究ASIC1a在SCI继发损伤中的作用及可能的分子机制方法:1.采用大鼠脊髓挫伤模型,应用组织病理学、行为学评分、诱发电位、免疫荧光技术及神经束路示踪等方法,观察SCI后组织病理变化过程、轴突再生与胶质瘢痕形成规律及相互关系,并测量瘢痕厚度。2.应用western blotting、免疫荧光及激光共聚焦显微镜、RT-PCR等方法多侧面观察SCI后ASIC1a表达变化规律,及其变化意义。3.利用体内、外损伤模型,使用TUNEL染色、电生理膜片钳记录、钙成像、鞘内置管给药、反义核酸等技术探讨ASIC1a在SCI继发损伤中的作用及其可能机制。结果:1.本研究首先制作了4种不同致伤级别的脊髓挫伤模型,应用组织病理学、行为功能评分、诱发电位、免疫荧光技术及神经束路示踪等方法,观察比较它们的病理学变化,神经功能恢复情况等,结果发现10g×50mm致伤组损伤很重,动物功能恢复较困难,10g×5mm致伤组则损伤太轻,动物基本可自行恢复到接近正常功能,10g×25mm致伤组功能恢复曲线显示早期与10g×50mm组类似,后肢瘫痪,后期类似10g×10mm致伤组。而10g×10mm致伤组动物自发功能恢复曲线稳定、独特,与其它3组区别明显。进一步,本研究比较了10g×25mm组和10g×10mm组在病理学、电生理学等方面的差异,结果显示这两种致伤级别,所导致的组织损伤程度与范围、运动/感觉诱发电位恢复等方面存在明显差别。2.本实验发现SCI后动物运动功能显著下降,以后逐渐恢复并在SCI后4w（week）左右恢复达平台。与之相应,神经电生理检查显示运动/感觉诱发电位的潜伏期在伤后明显延长,以后逐渐恢复,亦在SCI后4 w相对稳定。病理学观察表明SCI后2 w左右囊腔开始出现,到4 w时囊腔形成并较为稳定。动态观察胶质瘢痕的形成过程显示SCI后星形胶质细胞活化,胞体肥大,突起变粗并相互交联,逐渐形成胶质瘢痕,在SCI后4 w左右围绕在囊腔周围形成明显的胶质瘢痕带。以上结果提示大鼠SCI后4 w左右进入慢性期。3.应用神经束路示踪、免疫荧光双标等方法观察发现,SCI后神经纤维具有一定的再生能力,大部分走行于胶质瘢痕外,未见穿透瘢痕区达到囊腔边缘或穿透囊腔者,但也有部分轴突伸入到瘢痕外侧部一定的深度,提示胶质瘢痕是轴突再生之屏障。为将来瘢痕的去除提供了基础数据,本研究尚测量了SCI后4 w时胶质瘢痕的厚度,结果显示囊腔头尾侧瘢痕厚度与双侧壁厚度有一定差别,头尾侧厚度为107.00±20.12μm,双侧边瘢痕厚度为69.92±15.12μm。4.Western blotting和免疫荧光观察显示SCI后损伤周围区ASIC1a表达显著上调,在灰、白质均升高,并在12—24 h达高峰,然后逐渐回降,在SCI后1 w左右基本恢复到原先水平,连续观察6 w未见有变化。与此不同,SCI后损伤区ASIC1a的表达则下降,SCI后1 w左右即检测不到,应用Nissl及NeuN染色发现损伤区神经元丢失严重,可能与损伤区ASIC1a表达下降有关。免疫荧光双标显示灰质中主要为神经元表达ASIC1a,白质中则是少突胶质细胞表达ASIC1a。有趣的是,Western blotting观察发现正常脊髓ASIC2a表达水平很低,但SCI后损伤区与损伤周围区的表达均明显上升,尤其损伤周围区ASIC2a的表达升高持续到SCI后4w左右才恢复至原来水平。同样可能由于细胞的死亡,损伤区ASIC2a的表达早期升高后在24 h后迅速下降,直至检测不到。RT-PCR结果提示SCI后ASIC1a mRNA未见明显变化,而ASIC2a mRNA水平明显升高。5.应用TUNEL标记方法,发现TUNEL阳性细胞大多ASIC1a阳性,提示ASIC1a可能参与了SCI后延迟性细胞死亡的发生。相反,TUNEL阳性细胞则基本上未见ASIC2a阳性,一方面提示ASIC2a可能没有参与SCI后继发的细胞死亡,另一方面反过来说明ASIC1a与TUNEL的共标是特异的。进一步,利用体内、外损伤模型,结合TUNEL、PI/FDA染色等方法,发现分别给予ASIC1a的特异性阻断剂PcTx1和非特异性阻断剂Amiloride均可降低损伤后细胞死亡。ASIC1a特异的反义核酸knock downASIC1a的表达也可以达到同样的保护效果。6.应用电生理膜片钳记录与Ca²⁺成像技术,发现酸（pH6.0）刺激下可诱发脊髓神经元一较大的内向电流（酸电流）,亦可引起胞内Ca²⁺浓度迅速的增加,此电流和Ca²⁺内流可被ASIC1a通道特异性阻断剂阻断。在模拟的SCI后继发的缺血缺氧病理条件下,ASIC1a介导的酸电流和Ca²⁺内流均出现增强效应。7.进一步的实验显示病理条件下ASIC1a通道功能的增强可能和其磷酸化有关,Co-IP实验确实发现SCI后ASIC1a磷酸化水平显著升高,而钙/钙调素依赖性蛋白激酶Ⅱ（CaMKⅡ）可能参与了这一过程。Western blotting结果显示SCI后CaMKⅡ的表达显著升高,其表达变化的时空特点和ASIC1a具有良好的相关性,应用CaMKⅡ的特异性抑制剂KN93可显著抑制缺血缺氧诱发的ASIC1a介导的酸电流和Ca²⁺内流的增强效应,并减轻细胞损伤。8.最后,为在整体水平验证ASIC1a在SCI中的作用,应用鞘内置管技术,分别给予SCI大鼠ASIC1a的特异性阻断剂PcTx1和非特异性阻断剂Amiloride,结果发现两者均可减轻组织损伤,促进动物运动功能的恢复。ASIC1a特异的反义核酸亦具有类似的保护效果。结论:1.本研究所采用的SCI模型不仅能够将不同级别损伤区分开,且与动物行为学、电生理学和组织病理学变化想吻合。故本研究采用的模型具有较好的客观性、稳定性、相关性和重复性。2.10g×10mm致伤组动物自发功能恢复曲线稳定、独特,与其它不同级别的损伤区别明显,可较客观地反映不同SCI治疗措施和药物的疗效,故本研究采用此致伤级别。3.综合行为学、电生理学、病理学及胶质瘢痕形成观察等多方面的证据提示大鼠SCI后4 w左右进入慢性期,为人们认识和研究慢性SCI提供了重要的实验依据。4.SCI后神经纤维具有一定的再生能力,但很少能穿透胶质瘢痕,说明胶质瘢痕是阻挡轴突延伸的重要因素,提示去除胶质瘢痕可能是治疗SCI的重要策略,本研究测量了瘢痕的厚度,为将来去除瘢痕提供了重要的时空参考数据。5.SCI后ASIC1a表达上调明显,且在灰、白质均升高,在12—24 h达高峰,SCI后1 w左右回降到原先水平。免疫双标鉴定白质中表达ASIC1a的细胞类型是少突胶质细胞。ASIC1a表达的上调可能与其转录水平无关,而与翻译和/或代谢有关。6.体内外实验均表明ASIC1a确实参与了SCI后的继发性损伤,其可能过程为:SCI后出现的组织酸化可激活ASIC1a通道,引起一、二价阳离子的内流,尤其是Ca²⁺内流,导致胞内Ca²⁺蓄积,导致细胞损伤,SCI后ASIC1a表达的升高加剧了这一损伤过程。7.在SCI后继发的缺血缺氧病理条件下ASIC1a通道功能出现增强效应,此效应可能与钙/钙调素依赖性蛋白激酶Ⅱ（CaMKⅡ）介导的ASIC1a的磷酸化水平升高有关。而CaMKⅡ的激活可能与ASIC1a通道介导的Ca²⁺内流有关。8.本研究显示SCI后的组织酸化及其激活的ASIC1a通道是SCI后继发损伤的新的致病机制,为将来设计以ASIC1a为干预靶点的、特异的SCI治疗新药物,提供了重要的实验依据。综上所述,组织酸化及ASIC1a通道在SCI的继发损伤中起了重要作用,其可能机制是:SCI后组织酸化并伴随ASIC1a表达增多,H⁺激活ASIC1a通道,引起Ca²⁺内流,组织酸化持续存在导致胞内Ca²⁺的蓄积,激活CaMKⅡ,然后CaMKⅡ反作用于ASIC1a,增加其磷酸化,引起ASIC1a通道功能增强,通Ca²⁺增加,胞内Ca²⁺增加进一步激活CaMKⅡ,形成恶性循环,导致细胞Ca²⁺超载,引起细胞损伤,SCI后ASIC1a表达的升高加剧了这一损伤过程。本课题首次揭示了ASIC1a介导的酸毒在SCI继发损伤中的重要作用,加深了人们对SCI继发损伤机制的认识,为将来设计以ASIC1a为干预靶点、特异性的SCI治疗新药物和新策略,提供了重要的实验依据,具有重要的理论意义和临床应用价值。更多还原

【Abstract】 Spinal cord injury（SCI） caused by traffic,building and mine accident which become more and more with the development of economy has been a major threat to people’s health. More attention was payed to SCI research by international scientists.So far,it is a pity that there is no effective method and drug for SCI treatment.It urges people to resurvey the pathophysiological process and injury mechanisms of SCI.SCI evolves two pathophysiological stages including acute and chronic phase following injury.A variety of secondary injury mechanisms induced by primary spinal cord insult are involved in SCI during acute period,resulting in extending of the lesion size and degree.Thus,study on secondary injury mechanism and on protecting normal spinal tissue around the primary injury site is a vital strategy.Activated astrocytes,reactive gliosis with the formation of glial scar and cavity is regarded as the prominent pathophysiological feature of chronic SCI, which turn to be a major barrier to nerve regeneration.But there is no effective method to deal with.Accordingly,it is signifcant and valuable to observe the characteristics of glial scar and reestimate its role in spinal cord injury and repairing.It has been proven that many mechanisms are involved in secondary injury after SCI including ischemia,excitotoxicity,inflammation,oxidant stress,and energy failure.Several clinical and basic studies targeting to those mechanisms have been conducted over the years, but an effective pharmacologic agent has not yet been discovered,suggesting other unknown mechanism（s） responsible for secondary injury triggered by SCI.New report regarding acid-sensing ion channels（ASICs） brings us new enlightenment for SCI research, especially ASIC1a.Because a common pathological consequence which is pH falling in local tissue microenvironment following the above secondary processes.For example, acidic metabolite such as CO₂ cannot be elimiated under ischemic condition.Exitotory amino acid and ATP releasing after SCI are acidic substance.Metabolic failure causes lactic acid accumulation.Inflammation also induces acidosis.Thus,pH falling（tissue acidosis） may be serious post SCI which will activate ASIC1a channel resulting in a series of pathogenetic processes.But the expression and distribution pattern of ASIC1a in the spinal cord is unclear.In this study,we will observe the change characteristics of ASIC1a expression following SCI,and explore its role and mechanism involved in secondary injury after SCI.Glial scar and cavity formation is regarded as the prominent pathophysiological feature of chronic SCI,which is a major impediment to nerve regeneration.Studies showed that the nerve fiber or graft cannot extend through the dense glial scar,despite that neuroimplantation bridges the gap of necrotic cavity.Therefore,reducing or ablating glial scar is an hoping therapeutic strategy for chronic SCI treatment.Yet,two vital factors affect the effect of this kind of therapeutic strategy.One is what the character of distribution of glial scar is.What the thickness of glial scar needs to be ablated,and whether the thickness of glial scar around cavity is equivalent.It will cause additional injury,if ablation range is larger than the thickness of glial scar.The other one is what is the appropriate time window for the glial scar ablation.It is not benefit that glial scar ablation is carried out too late or too early.Consequently,this study will observe the process of glial scar formation,the thickness of glial scar,and the relationship between glial scar and nerve fibers,which helps to providing experimental evidence to the above questions.Objective:1.To observe the process of glial scar formation with the relationshsip between glial scar and nerve fibers,as well as quantitating the thickness of glial scar.2.To observe the change of ASIC1a expression pattern after SCI,and explore its role and possible molecular mechanism in secondary injury following SCI.Methods:1.By employing rat spinal cord contusion injury model,histopathological observation, behavioral scale,evoked potential,immunofluorescence and axonal tract tracing,we observed the histopathological process,axon regeneration,course of glial scar formation, with the relationshsip between glial scar and nerve fibers,and quantitated the thickness of glial scar.2.Using western blotting,immunofluorescence,confocal laser scanning microscope and RT-PCR methods,we examined the alterations of ASIC1a expression and the significance underlying the alterations after SCI. 3.By utilizing injury of in vivo and in vitro,TUNEL staining,electrophysiological recording,Ca²⁺ imaging,intrathecal delivery and antisense techniques,we investigated the role and underlying mechanism of ASIC1a in secondary injury of SCI.Results:1.Firstly,we made four degrees of SCI model,and compared pathological process and functional recovery among these four groups of rats by employing histopathological observation,behavioral scale,motor/sensor evoked potential,immunofluorescence and axonal tract tracing.The results showed that 10g×50mm group demonstrated difficult functional recovery due to its serious injury,but 10g×5mm group displayed good behavioral improvement due to its light injury.The recovery pattern of 10g×25mm group demonstrated faccid paralysis early after SCI like the 10g×50mm group,but it is similar to 10g×10mm late post SCI.The recovery pattern of 10g×10mm group is specific, unvariable and distinct from the other three groups.Moreover,we compared the difference in pathological and electrophysiological changes between 10g×10mm and 10g×25mm group.Results showed that the two adjacent grades of injury induced distinguishing degree and size of tissue injury,motor and sensor evoked potential.2.Behavioral performance of SCI rat decreased followed by gradually recovery after SCI,and reached the plateau around at 4 weeks after SCI（SCI 4 w）.Correspondingly,the latency of motor/sensor evoked potential prolonged after SCI followed by gradually improvement,becoming stable at SCI 4 w.Pathological data showed cavity appeared at SCI 2 w and stabilized at SCI 4 w.Observation of the process of glial scar formation demonstrated that reactive astrocytes with hypertrophic soma and cross-linked thick processes formed glial scar gradually which emerged around the cavity at SCI 4 w.These data suggested SCI evolves into chronic stage at SCI 4 w.3.By employing axonal tract tracing and double immunofluorescence,we found nerve fibers remained regenerative ability after SCI.Most fibers ran outside of glial scar,no fibers could be seen to pentrate glial scar to cavity or even through cavity.However,a few axons could be seen to regrow into the outer of layer of glial scar.These observation suggests glial scar is a barrier to axonal extension.In order to provide basic data for ablating glial scar in the future,we measured the thickness of glial scar.The results showed that the difference of thickness of glial scar between the rostral/caudal region and lateral region is distinct.The thickness of glial scar in the rostral/caudal region is 107.00±20.12μm,and 69.92±15.12μm in the lateral region.4.Examination via western blotting and immunofluorescence showed that ASIC1a expression markedly increased at peri-injury site both in gray and white matter after SCI, reached its peak at 12-24 h,then started to turn back and recovered to original level at SCI 1 w,remaining for up to SCI 6 w.On the contrary,ASIC1a expression at the injury site decreased obviously,and arrived at its rock-bottom at SCI 1 w without recovery.Nissl and NeuN staining showed that neuronal loss was serious at injury site which may cause the decrease of ASIC1a expression at injury site.Double immunofluorescent staining displayed that cells expressed ASIC1a in the gray matter were neurons,while they were oligodendrocytes in the white matter.Intriguingly,western blotting data demonstrated that ASIC2a level is very low in the normal spinal cord.But it dramatically increased afer SCI, especially in the peri-inujury site which recovered to original level untill SCI 4 w.ASIC2a expression increased early after SCI,then dramatically decreased since SCI 24 h due to neuronal loss at injury site.Data from RT-PCR showed that no obvious change were found in ASIC1a mRNA level,but ASIC2a mRNA level increased obviously after SCI.5.Double immunostaining showed that most TUNEL positive cells were ASIC1a positive,suggesting that ASIC1a may involved in delayed cell death afer SCI.On the contrary,no TUNEL positive cells displayed ASIC2a positive,suggesting ASIC2a may not be related to seondary cell death after SCI,also suggesting co-immunostaining of ASIC1a and TUNEL is specific.Moreover,by employing injury model in vivo and in vitro, TUNEL and PI/FDA staining,we found both ASIC1a specific antagonist PcTx1 and nonspecific antagonist amiloride decreased cell death induced by injury.Specific antisense targeting ASIC1a also produced the same protective effect.6.Making use of electrophysiology and Ca²⁺ iamging,we recorded that acidic stimulation evoked large transient inward currents and rapid[Ca²⁺]_i increase in cultured spinal neurons,which could be blocked by both ASIC1a specific antagonist PcTx1 and nonspecific antagonist amiloride.The acidic currents and[Ca²⁺]_i increase was enhanced by the pathological condition mimicking the ischemia/anoxia following SCI.7.Further experiment data showed that enhancement of AISC1a channel acitvity is complicated with ASIC1a phosphorylation.Co-IP data confirmed that ASIC1a phosphorylation increased after SCI which may be catalyzed by calcium/calmodulin-dependent kinaseⅡ（CaMKⅡ）.Western blotting data displayed that CaMKⅡexpression increased after SCI,sharing pertinence with the temporospatial pattern of ASIC1a expression after SCI.CaMKⅡspecific antagonist KN93 significantly inhibited enhancement of ischemia-induced ASIC1a currents and cell injury.8.Finally,in order to confirm the role of ASIC1a in SCI in the whole level in vivo,by using intrathecal delivery technique,we found both ASIC1a specific antagonist PcTx1 and nonspecific antagonist amiloride reduce tissue injury and promote functional recovery after SCI.Again,ASIC1a antisense had the similar protective effect.Conclusion:1.The SCI model used in this study can distinguish the graded injury,consistent with the results from behavior,motor/sensor evoked potential,histopathology.Thus,this SCI model possesses objectivity,stability,relativity and reproducibility.2.The spontaneous functional recovery pattern of 10g×10mm group rats is specific, unvariable and distinct from the other groups,which can objectively estimate the effect of measures and drugs used to treat SCI.Therefore,10g×10mm injury severity was employed by this study.3.All the data from behavior,electrophysiology,pathology and observation of glial scar formaiton suggests SCI evolves into chronic stage at 4 w post injury,which will provide important experimental evidence for people to recognize and study chronic SCI.4.Nerve fibers remain regenerative ability after SCI,but few of them penetrate glial scar,suggesting glial scar is an impediment for axonal extension.This also suggests glial scar ablation will be important strategy for SCI treatment.We also measured the thickness of glial scar,which offers temporospatial reference data for ablation of glial scar in the future.5.ASIC1a expression markedly increased both in gray and white matter after SCI, reached its peak at 12-24 h,then started to turn back and recovered to original level at SCI 1 w.Double immunofluorescent staining displayed that cells expressed ASIC1a are oligodendrocytes in the white matter.Upregulation of ASIC1a expression has no relativity with its transcription,but its translation and/or metabolism.6.Both data from in vivo and vitro experiments demonstrates ASIC1a is involved in the secondary injury after SCI.The process may be:tissue acidosis following SCI activates ASIC1a channel which induce mono- and bivalent cations affiux,especially Ca²⁺ intracelular flow,which leads Ca²⁺ accumulation resulting in cell injury.7.Under the ischemia/anoxia condition following SCI,ASIC1a channel function enhances,which may be related with phosphorylation of ASIC1a catalized by calcium/calmodulin-dependent kinaseⅡ（CaMKⅡ）.CaMKⅡmay be activated by Ca²⁺ intracellular flow mediated by ASIC1a channel.8.This study reveals that tissue acidosis with activated ASIC1a channel by acidosis following SCI is a new pathogenic mechanism underlying sceondary injury post SCI,which provides important experimental evidenc for designing specific drugs targeting ASIC1a for SCI treatment in the future.In conclusion,tissue acidosis with activated ASIC1a channel play a vital role in secondary injury after SCI.The following is the possible mechanism:SCI induces tisse acidosis concomitantly activates ASIC1a channel.Then,Ca²⁺ afflux mediated by ASIC1a activates CaMKⅡwhich catalyzes phosphorylation of ASIC1a,inducing enhancement of ASIC1a channel function.This enhancement will produce more Ca²⁺ afflux which further activated CaMKⅡ.Thus,a vicious circle formed which leads to Ca²⁺ accumulation resulting in cell injury.Upregulation of ASIC1a expression after SCI exacerbates this injury process.This study firstly discovers the role of acidotoxicity mediated by AISC1a in secondary injury after SCI,which will deepen our understanding of scondary injury mechanism after SCI,and provide substantial experimental data for designing specific strategy and drugs targeting ASIC1a for SCI treatment in the future.This finding implies important theoretic significance and clinical value.更多还原