【作者】 肖绪武；

【导师】 孙长凯；

【作者基本信息】 大连医科大学 ， 生理学， 2008， 博士

【摘要】 缺血性脑损伤是临床上常见的疾病之一,可引起脑神经组织细胞缺血,水肿和坏死,导致一系列的症状、体征,甚至出现不可逆的脑损害,可留下各种神经系统后遗症。为了探讨早期运动干预对缺血性脑损伤神经运动功能恢复的促进作用,本研究采取基础与临床对比研究的方法,通过观察早期运动训练,对缺血再灌注( Middle Cerebral Artery Occlusion,MCAO)模型脑缺血大鼠恢复期脑组织微管相关蛋白-2含量的影响和大鼠行为的变化,来判断早期运动对脑损伤动物功能恢复的作用;同时,以临床高危新生儿缺血性脑损伤恢复期不同阶段的智力发育指数( Mental Development Index, MDI)和运动发育指数( Psychomotive Development Index,PDI)为指标,来观察早期运动干预对早产低体重儿、缺氧缺血性脑病神经系统功能恢复的促进作用。微管相关蛋白-2(Microtuble-associated Protein 2,MAP-2)属于结构性相关蛋白家族,是一种热稳定的鳞蛋白,主要存在于神经元胞体和树突,是脑内最丰富的蛋白之一。它与微管连接促进微管的稳定,构成细胞骨架,参与神经元发育、结构稳定,突起形成和突触可塑性调节。MAP-2与缺血性脑损伤关系密切。大量研究表明,MAP-2是脑缺血的敏感指标,而且进一步证实,MAP-2不仅是神经元损伤的早期标志,也与缺血半暗带受损神经元的重建相联系。脑缺血后MAP-2丢失的机制目前还不完全清楚,可能是脑缺血后引起神经元细胞膜去极化,兴奋性氨基酸(EAA)释放,继之激活EAA受体,Ca2+通过NMDA和非NMDA受体内流从而增加细胞内游离钙水平,破坏钙稳态,Ca2+同钙调节蛋白结合,激活靶点酶,致MAP-2异常鳞酸化,从而抑制微管组装,促进解聚,最终MAP-2丢失。MAP-2主要表达在神经元胞体、树突和树突棘,与突触后膜骨架上一些信号蛋白和膜受体相连,是许多神经信号传导通路的底物,调节神经元突触可塑性以及细胞骨架动力学,对于神经功能是必须的。神经元-MAP-2的丢失涉及到神经元结构破坏和导致神经功能障碍的神经元死亡,通过检测干预后脑内MAP-2的含量,可间接了解脑神经功能的状态及干预的效果。新生儿缺血缺氧性脑病(Hypoxic Ischemic Encephalopathy,HIE)是指围产儿因宫内窒息或出生后多种心脑疾病引起的脑严重缺血缺氧性损害,其脑组织以水肿、软化和坏死出血为主要病变,临床上病情重,病死率和致残率高,可发生多种永久性神经功能缺陷如智力低下、痉挛性瘫痪和癫痫等。它不仅可引起围产期新生儿死亡,而且是新生儿期以后致残的主要原因。HIE发病机制复杂,尚未完全明了,包括脑部能量代谢障碍、再灌注损伤、细胞凋亡、钙平衡紊乱、氧自由基产生、兴奋性氨基酸增多等。本研究旨在通过基础与临床的结合对比研究,探讨早期运动干预对促进脑损伤神经系统功能恢复、降低后遗症的作用效果,为临床治疗缺血性脑疾病提供动物实验与临床依据。本研究结果显示:1、在MCAO模型动物实验中,缺血再灌注运动组大鼠行为学测试评分较单纯缺血再灌注组在术后2小时及24小时后略低,7日后有所升高;在14日和21日测试则显著增高,两组间差别具有极显著性统计学意义。2、MCAO模型制成后,脑缺血再灌注运动组及单纯缺血再灌注组在缺血中心区的MAP-2免疫阳性物质减少或缺失,缺血周边区免疫阳性物质增高。但实验至第二、第三周时,缺血再灌注运动组缺血区MAP-2免疫阳性物质显著高于单纯缺血再灌注对照组,差别有极显著性统计学意义。3、早期综合干预的早产、低出生体重高危儿组,其MDI比较在6月龄时干预组较常规组高1.6分,但差异无显著性;在12月龄、18月龄和24月龄时,干预组较常规组分别高2.2分、7.6分和7.7分,差别均有极显著性意义(P<0.01)。PDI比较在6、12、18和24月龄时干预组较常规组分别高3.8分、9.1分、12.2分和12.6分,各月龄比较差异均有极显著性意义(P<0.01)。4、早期运动干预的HIE组与对照组比较,分别于6月、12月进行MDI和PDI测查,结果显示生后6月两组比较,MDI无显著性差别,而PDI差别有显著性;生后12月两组比较,干预组MDI、PDI均较对照组有显著性增高,差别有极显著性统计学意义(P<0.01)。结论:1、早期运动训练能促进未成年MCAO大鼠的肢体功能恢复,上调脑组织缺血皮质区MAP-2的表达,从分子水平上提示大鼠的肢体功能恢复可能与神经再生有关。2、早期综合运动干预能够明显改善脑损伤高危新生儿和HIE患儿的预后,促进神经再生和脑功能的恢复,降低神经系统后遗症的发生,从而提高脑损伤新生儿患者的生存质量。3、早期综合运动干预可以明显改善早产、低出生体重儿的智能发育。通过早期动作和运动能力的强化训练,可以大大提高早产、低出生体重儿的运动发育并进一步提高其智力发育。4、早期运动干预效果确切,家庭与门诊相结合的干预模式值得在基层儿童保健部门及社区推广和应用,为提高人口素质提供保证。更多还原

【Abstract】 Ischemic brain injure is one of the common clinical diseases and often caused brain nerve tissue cell ischemic, odema, necrosis , result in serial symptoms and signs. Even appeared nonreversiber brain damage, can leave behind various kinds of nervous system sequelae. For approach the effection of early exercise intervention for ischemic brain injure nervous system functional recovery , This study take the method of contrasting experiment and clinical effect by observe early movement training for ischemic reperfusion rat model brain ischemic recovery stage brain tissue microtubule-2 content effect and rat behavioral change, to juge early movement effect for functional recovery of brain injury animal. At the same time, take the MDI, PDI for index in different stage of clinical high risk neonate ischemic brain injury cases. To observe the effect of promotion for premature and low birth weight、HIE nervous system function recovery.MAP -2 belong to structure-associated protein family clan, it is an heat-stable lipid protein, mainly reside in neuron body and dendrite. It is one of the most profused protein in the brain. It link with microtubule to promote microtubule stable, to constitute cytokelet, participate neuron development and structure stable,to form ecptoma and synapse plasticity adjustment.MAP-2 has a intimate relationship with ischemic brain injury. A lot of studies indicate that MAP-2 is a sensitive mark for brain ischemic. Moreover confirmed, MAP-2 is not only the early mark for neuron injury, but also related to re-establish of damage neuron in ischemic semidarkness band. The mechanism for MAP-2 loss after brain ischemic is not clear at present, maybe brain ischemic cause neuron membrane de-pole, excitatory amino acids(EAA) release, continue activation EAA receptor, ca++ increase endocell liberation calcium level by NMDA and non-NMDA receptor inflow, to destroy calcium homeostasis, cause MAP-2 anomaly lipid acidize, Thus inhibition microtubule construction, promote depolymerize, MAP-2 lost at finally.MAP-2 mainly expressed in pericargon, dendrite, dendritic spine, connected with signal protein and membrance receptor at postsynaptic membrance skeleton. It is substrate of conduction way for many nerves signals, adjust neuron synaptic plasticity and cystoskeleton dynamics. It is imperative for nerves function. The lost of neuron MAP-2 is related to neuron disorganization and lead to death of handicapped neuron. By detect MAP-2 content inside brain after intervention, we can get the message indirectly about cranial nerve function condition and the result of intervention.HIE is refers to serious ischemic and hypoxic brain damage because of intrauterine asphyxia or many kinds diseases of heart and brain postnatal. The main pathological changes about brain tissue is oedema, lenition and necrosis, hemorrhage. It has heavy clinical condition, high case fatality rate and mutilation rate, can cause many kinds of permanence nerves functional defect, such as mental retardation, spastic paralysis and epilepsy. It not only cause neonatal death ambibirth period, but also main causies for mutilation after neonatal period. The pathogenesis for HIE is complicated,still not completely understood. Include brain energy disbolism, reperfusion injury, apoptosis, calcium balance confused, oxygen free radical produced, excitatory amino acids increased.The purpose for this study is a contrast study by combination of animal experiment and clinical case, to approach the result of promote nervous system function recovery by early exercise intervention, and provide theory and clinical evidence for clinical therapy of ischemic brain diseases.The finding of this study shows:1. In MCAO model animal experiment, the behavious test grade for ischemic reperfusion exercise group rat is a little lower than purely ischemic reperfusion group in 2 hours and 24 hours after operation but increased 7 days later; the test grade much higher in 14 and 21 days. The difference between two groups has extremely statistical significance.2. After MCAO model finished, the MAP-2 immune positive material decreased or absence at ischemic center zone both in brain ischemic reperfusion exercise group and purely ischemic reperfusion group, but immune positive materials at ischemic perimeter zone increased. The MAP-2 immune positive materials much higher at ischemic zone in ischemic reperfusion exercise group than in purely ischemic reperfusion group. The difference between two groups has extremely statistical significance.3. In premature and low birth weight high risk infants group by early synthetic intervention, It’s MDI score 1.6 high than routine group in 6 months. There is no significant difference between two groups; Intervention group score 2.2, 7.6 and 7.7 higher than routine group during 12, 18 and 24 month age; the difference have extremely statistical significance. For PDI contrast, intervention group score 3.8, 9.1 12.2 and 12.6 higher respectively than routine group during 6, 12 18 and 24 months age. All months age compared have extremely statistical significance.(p<0.01)4. Compare early exercise intervention HIE group with control group, check their MDI and PDI in 6 and 12 months age, the results shows MDI has no significance difference in 6 months age but PDI do has. In 12 months age , both MDI and PDI in intervention group much higher than control group. The difference have extremely statistical significance.(P<0.01)Conclusion:1. Early exercise training can promote limbs function recovery for minor MCAO rats, re-regulation MAP-2 express at ischemic cortical area in brain tissue. It suggest that rat limbs function recovery maybe related to nerve regeneration in molecular level.2. Early synthetic movement intervention can obviously improve the prognosis of brain injuried high risk neonates and HIE patients, promote nerve regeneration and recovery of brain function, cut down nervous system sequel, thus elevate life quality for brain injury neonates.3. Early synthetic movement intervention can obviously improve mental development for premature and low birth weight neonates. By enrichment training of earlier start moving and motor capacity, can obviously improve motor development and enhance mental development for premature and low birth weight neonates.4. Early exercise intervention has a certain effect. It is worth spreading and utilizing in the basic level child care department and community by family connective clinic service intervention mode, and provide supports for elevation population diathesis.更多还原