【作者】 王若冰；

【导师】 柳林； 许家军； 孙学军；

【作者基本信息】 第二军医大学 ， 眼科学， 2010， 博士

【摘要】 青光眼是全球最常见的不可逆性致盲眼病之一,青光眼视神经病变是该病致盲的根本原因。目前公认的发生机制是由于眼压升高造成视网膜神经节细胞和其它视网膜细胞发生以变性坏死为特征的原发性损害,随后又通过一系列途径诱发以视网膜神经节细胞持续性凋亡为特征的继发性损害,从而导致视神经功能进行性损伤。目前除了应用药物、手术等手段降低升高的眼内压,控制疾病的原发性损害以外,尚无有效手段防治视神经的继发性损害。视神经由RGCs的轴索组成,是间脑白质的主要成分,视神经和视网膜作为中枢神经系统在颅外的延伸,在位置上具有着中枢神经系统其他部分不可比似的易操作性,因而视神经损伤模型常被用来模拟大脑的轴突损伤；其中视神经钳夹伤模型能够较好地模拟RGCs继发性凋亡,即使视神经被完全截断后,仍有相当一部分数量的RGCs能够存活一段时间。这种钳夹伤后的神经退行性过程与青光眼视神经病变的病理过程相类似。高压氧治疗已在临床应用多年,具有确切治疗效果的疾病包括一氧化碳中毒、中风、空气栓塞及减压病等。目前大量的研究显示高压氧预处理对大脑缺血缺氧引起的迟发型损伤有确切的神经保护作用,但HBO-PC保护效应的确切机制尚未阐明,在临床应用前还需要大量的实验证据。目前,人们普遍认为HBO诱导脑神经保护作用的机制主要有以下两个方面：一是HBO预处理诱导机体组织细胞产生大量的活性氧,进而引起机体的抗氧化酶增多,增强机体抗氧化损伤能力；二是HBO预处理可诱导脑内的抗凋亡蛋白升高,增强机体的抗凋亡损伤能力。视神经钳夹伤后的视网膜神经元退行性变过程和脑损伤所引起的继发性神经元退变过程相似,因此我们采用一种临床实际工作中可行而有效的高压氧预处理方法,从病理学、形态学、视功能等各个方面验证这种预处理方法对视神经不完全损伤是否具有保护效应,并初步探讨HBO预处理发挥视神经保护作用的分子机制。第一部分大鼠不完全视神经损伤模型的建立及高压氧预处理对视神经不完全损伤的保护效应目的：建立稳定的大鼠视神经钳夹伤模型,研究高压氧预处理对大鼠视神经不完全损伤的保护效应。方法：采用2.5 ATA,100%O2,每天2次,连续2天的高压氧预处理方法,末次处理后次日采用Yasargil动脉瘤夹制作大鼠视神经钳夹伤模型。动物分为Sham组、ONC组,HBO+ONC组。分别在建模后1周或2周采用HE染色、荧光金逆行标记、闪光视觉诱发电位及TUNEL观察或检测视网膜神经节细胞的形态、数量和视神经功能的改变。结果：建模1周时Sham组视网膜节细胞层中HE着色的细胞数量为89.5±9.4个/高倍视野,2周时为92.7±9.1个／高倍视野；而1周和2周时HBO+ONC组的视网膜节细胞层中HE着色的细胞数量显著较ONC组多(69.3±7.4 vs 56.6±6.5个／高倍视野,1w,P<0.01；62.3±6.1 vs 46.1±6.6,2w,P<0.01)。建模2周时荧光金逆行标记显示,Sham组的存活视网膜节细胞的平均密度为1650±216/mm2,而HBO+ONC组的视网膜神经节细胞平均密度显著高于Sham组(995±158/mm2和478±101/mm2,P<0.01)。闪光视觉诱发电位结果显示,相对于HBO+ONC组,ONC组P1波的潜伏期延长、振幅降低。视神经钳夹伤后1周时Sham组、HBO+ONC组和ONC组P1波的潜伏期和振幅分别为90±4ms、94±4ms、108±6ms(P<0.05,HBO+ONC vs ONC)；17±2μv、18±3μv和12±2μv(P<0.01,HBO+ONC vs ONC)。2周时Sham组、HBO+ONC组和ONC组P1波的潜伏期和振幅分别为92±7ms、117±12ms、169±15ms(P<0.01,HBO+ONC vs ONC)；21±3μv、14±2μv和7±1μv(P<0.01,HBO+ONCvs ONC)。结论：HBO预处理能使视神经损伤后的视网膜节细胞存活率显著提高,同时改善视神经的功能状态,确认HBO预处理对大鼠视神经不完全损伤的保护效应。第二部分高压氧预处理抑制线粒体途径的凋亡诱导对大鼠视神经不完全损伤的保护效应目的：研究高压氧预处理对线粒体凋亡途径的影响。方法：按第一部分方法进行高压氧预处理和建模。使用TUNEL法检测RGCs凋亡数量；ELISA法检测视网膜caspase-3和caspase-9的酶活性；Western Blot检测cytochrome c、Bcl-2 BclxL、和Bax蛋白表达量。结果：HBO预处理能明显减少视神经不完全损伤后RGCs的凋亡阳性细胞数(ONC：19.1±4.2/HPF vs.HBO+ONC：7.2±1.3/HPF,P<0.01),ONC组视网膜组织的caspase-3和caspase-9酶相对活性显著高于HBO+ONC组(P>0.05)。HBO预处理显著减少了视神经损伤后视网膜组织胞浆cytochromec的蛋白水平(P<0.05),同时提高了抗凋亡蛋白Bcl-2、BclxL的水平(P<0.01,P<0.05),但对促凋亡蛋白Bax的表达影响不明显(P>0.05)。结论：高压氧预处理可能通过抑制线粒体途径的凋亡发挥对视神经不完全损伤的保护效应。第三部分高压氧预处理调控HIF/EPO通路诱导对大鼠视神经不完全损伤的保护效应目的：研究HBO预处理的保护效应是否与氧化应激诱导大鼠视网膜中HIF-1及下游神经保护性基因EPO的表达有关。方法：按第一部分方法进行高压氧预处理,末次处理后次日直接取材。动物分为对照组和HBO预处理组。使用RT-PCR方法和Western Blot检测HBO预处理对视网膜组织HIF-1及其下游基因EPO的mRNA表达和蛋白含量的影响,并检测EPO下游信号分子P-Akt和HSP-27蛋白表达的影响。结果：HBO预处理能显著上调大鼠视网膜组织中HIF-1和EPO mRNA的表达,分别是对照组的13.38±1.97倍和5.55±1.03倍(P<0.01)；同时HIF-1α和EPO蛋白表达显著增加,分别是对照组的1.86±0.24倍和3.01±0.33倍(P<0.01)。HBO预处理组视网膜组织中P-Akt和HSP-27蛋白表达也显著增加,分别是对照组的1.64±0.11倍和1.81±0.12倍(P<0.01)。结论：HBO预处理诱导增强视网膜组织中HIF-1和下游神经保护性基因EPO的表达,可能是其发挥视神经不完全损伤保护效应的机制之一。更多还原

【Abstract】 Retinal ganglion cell (RGC) death is the end result of practically all diseases of the optic nerve, including glaucomatous optic neuropathy. Optic nerve and retina are the most easily accessible parts of the central nervous system (CNS), and lying outside the skull. The optic nerve is a white matter tract of the diencephalon composed principally of the axons of RGC. Therefore, optic nerve injury has been used to model brain axonal injury. Optic nerve crush (ONC) is a well established model of delayed RGC death. The neurodegenerative process after ONC may be similar to the process of secondary degeneration described in the context of glaucomatous optic neuropathy. After ONC, a fraction of RGCs undergo early necrosis, whereas others are subjected to delayed apoptotic death and will always be relatively numerous. Even after a complete transection many RGCs are still able to survive for long periods after the insult.Hyperbaric oxygen (HBO) therapy has been traditionally applied in the treatment of carbon monoxide poisoning, stroke, air embolism and decompression sickness. Recently, HBO preconditioning (HBO-PC) has been shown to have neuroprotective effects against ischemic injury in gerbil hippocampus, rabbit spinal cord, transient brain infarct in mouse and rat or in primary cultured spinal cord neurons. However the mechanism is not fully understood and more evidence is needed for HBO treatment to be accepted clinically. The majority of studies examined HBO preconditioning effects on a delayed brain injury. HBO preconditioning should have a powerful anti-apoptotic effect as apoptosis is a dominant form of hippocampal cell death after global cerebral ischemia.In the present study, on the assumption that the processes of secondary degeneration of neurons after ONC are likely to involve pathways and mediators similar to those in brain trauma, we investigate whether the administration of HBO-PC offers neuroprotection to retinal ganglion cells by reducing ONC-induced caspase-dependent apoptosis. We hope to provide experimental evidence for the clinical application of HBO preconditioning.Part I The protective effect of HBO preconditioning on optic nerve crush injury in ratsObjective:Establishing a reliable, convenient and reproducible animal model with optic nerve crush (ONC), we investigate whether the HBO protocol is of protective effect on ONC injury.Methods:HBO preconditioning (HBO-PC) was conducted four times by given 100% oxygen at 2.5 atmosphere absolute (ATA) for 1 h every 12 h interval for 2 days prior to ONC. Rats were euthanized at 1 or 2 weeks after ONC. RGC density was counted by Hematoxylin and Eosin (HE) staining in the retina and retrograde labeling with FluoroGold application to the superior colliculus. Visual function was assessed by flash visual evoked potentials (FVEP).Results:The number of HE-stained cells in the ganglion cell layer of sham group was 89.5±9.4 cells/HPF at 1 week and 92.7±9.1 cells/HPF at 2 weeks. More HE-stained cells in the ganglion cell layer were observed in HBO-PC group than in no HBO-PC group (69.3±7.4 vs 56.6±6.5 cells/HPF at 1 week, P＜0.01; 62.3±6.1 vs 46.1±6.6 at 2 weeks, P<0.01). Therefore, HBO-PC had a statistically significant rescue effect in retinas when compared with ONC group. Moreover, we applied retrograde labeling with fluorogold to count the number of surviving RGCs at 2 weeks after ONC. The average RGC density in retinas of sham-operated group was 1650±216/mm2. At 2 weeks after ONC, the RGC densities of HBO-PC+ONC group and ONC group decreased to 995±158/mm2 and 478±101/mm2, respectively (p<0.01). The FVEP measurements illustrate that the HBO-PC+ONC group had significantly improved visual function as compared to the corresponding ONC group at both 1 and 2 weeks. The latency of the P1 wave at 1 week was 90±4ms,94±4ms, and 108±6ms for the control, HBO-PC+ONC (p>0.05 vs control), and ONC groups (p<0.01 vs control). At 2 weeks after operation, the latency of the P1 wave was 92±7ms,117±12ms,169±15ms for the control, HBO-PC+ONC (p<0.05 vs control, p＜0.001 vs ON-crushed), and ONC groups (p<0.001 vs control). The amplitude of the P1 wave was 17±2μv,18±3μv, and 12±2μv for the control, HBO-PC+ONC (p>0.05 vs control), and ONC groups (p<0.01 vs control) at 1 week,21±3μv,14±2μv, and 7±1μv for the control, HBO-PC+ONC (p<0.05 vs control, p<0.001 vs ONC), and ONC groups (p<0.001 vs control) at 2 weeks.Conclusion:The RGC density in the retinas of ONC, HBO-PC-treated rats was significantly higher than that of the corresponding ONC rats. FVEP measurements indicated a significantly better preserved latency and amplitude of the P1 wave of in the HBO-PC group. These observations demonstrate that HBO-PC appears to be neuroprotective against ONC injury.PartⅡHyperbaric oxygen preconditioning promotes survival of retinal ganglion cells by inhibition of mitochondrial apoptosis in a rat model of optic nerve crushObjective:To investigate the influence of HBO preconditioning on mitochondrial apoptosis. Methods:The animal models were made and the HBO was performed according to the protocol introduced in Part I. The terminal deoxynucleotidyl transferase-mediated Dutp nick end labeling (TUNEL) assay was performed on paraffin-embedded sections according to the manufacture instructions. The activity of caspase-3 and-9 were measured with caspase-3 and caspase-9/CPP32 Fluorometric Assay Kit. The protein expression levels of cytochourome c, Bcl-2, BclxL and Bax were detected by Western Blot.Results:At 2 weeks after operation, TUNEL assay illustrated that TUNEL-positive cells/HPF of 1.8±1.6 cells in sham operated group,7.2±1.3 cells in HBO-PC+ONC group and 19.1±4.2 cells in ONC group (p<0.01 vs HBO-PC+ONC group) in the RGC layer. Caspase-9 activity in HBO-PC+ONC group were higher than that of ONC group at indicated time points (4.15±1.33-fold vs 11.25±3.26-fold at 1 week; 2.25±0.79 vs 6.29±1.95 at 2 weeks, P＜0.01). Similarly, the measurements of caspase-3 activity showed a 2.62±0.45-fold and 3.22±0.67-fold increase in HBO-PC+ONC group and ONC group respectively at 1 week (p<0.05); a 1.72±0.34-fold and 2.82±0.51-fold increase in HBO-PC+ONC group and ONC group respectively at 2 weeks (p<0.01). Western blot analysis of retina samples at 24 h after ONC showed that HBO-PC reduced the protein expression of cytochourome c (P<0.05), HBO-PC enhanced the anti-apoptotic protein levels of Bcl-2 and BclxL.The protein level of Bax remains constant in all groups after ONC.Conclusion:The inhibition of mitochondrial apoptosis may be one of the mechanisms of neuroprotection induced by HBO preconditioning in a rat model of optic nerve crush. PartⅢMechanism of neuroprotection induced by hyperbaric oxygen preconditioning involves upregulation of hypoxia-inducible factor-1 and erythropoietin in a rat model of optic nerve crushObjective:To investigate whether the protective effect induced by HBO preconditioning involves upregulation of hypoxia-inducible factor-1 and erythropoietin in retina.Methods:HBO was performed according to the protocol introduced in Part I. RT-PCR and Western blot were used to analyze the HIF-1 and EPO mRNA level, the HIF-la, EPO, ERK and HSP70 protein level using samples taken from the retinas of the experimental groups 12 h after the last HBO pretreatment without optic nerve crush injury.Results:The HIF-1 mRNA and HIF-la protein level were significantly increased 12 h after the last HBO pretreatment (13.38±1.97-fold and 1.86±0.24-fold vs control, P＜0.01) in rat retinas, while the EPO mRNA and protein level were also significantly increased (5.55±1.03-fold and 3.01±0.33-fold vs control, P＜0.01). Moreover, EPO activates downstream effectors, such as ERK and HSP70, which protein level was also significantly increased (1.64±0.11-fold and 1.81±0.12-fold vs control, P＜0.01).Conclusion:HBO preconditioning can induce the protective effect on ONC injury, which may probably involve up-regulation of HIF-1 and its target gene EPO.更多还原