Therapeutic Strategies for Neonatal Hypoxic-Ischemic Encephalopathy

描述(由申请人提供)：将使用一种创新的仔猪窒息心脏骤停模型，其中选择性神经元易损性模式模拟足月新生儿缺氧缺血性脑病，这是导致新生儿发病率和死亡率的主要原因。尽管低温有望作为一种治疗方法，但临床试验中典型的启动低温的延迟可能不会保护纹状体，那里的神经元可以在复氧后6小时内死亡。这项建议的重点是为快速纹状体神经保护量身定做的疗法，这些疗法可以延长低温的治疗窗口，以保护其他区域的延迟性神经变性。新生儿缺氧缺血性脑病特定的纹状体损伤机制尚未得到很好的研究。纹状体黑质神经元富含多巴胺D1受体，纹状体苍白质神经元富含腺苷A2a受体，两者均通过PKA和磷酸化调节蛋白DARPP-32发挥作用。D1R的激活通过NR1的磷酸化放大NMDA通道钙电流，降低Na，K-ATPase的活性。在以前的工作中，D1多巴胺受体拮抗剂处理改善了HI诱导的仔猪纹状体内DARPP-32、NR1和Na，K-ATPase上PKA敏感部位的磷酸化，提高了Na，K-ATPase的活性，并选择性地保护了D1区神经元。初步数据表明，A2A拮抗剂后处理保护部分纹状体神经元，并减弱HI诱导的DARPP-32、NR1和Na，K-ATPase上PKA敏感部位的磷酸化。花生四烯酸代谢产物20-HETE也可降低Na，K-ATPase活性，但依赖于PKC的磷酸化。治疗后使用20-HETE合成抑制剂的初步数据显示，部分神经保护和选择性阻断Na，K-ATPase和NR1上PKC敏感部位的磷酸化。20-HETE合成抑制通过靶向参与兴奋性毒性的关键蛋白的不同磷酸化位点和多种细胞类型，可以补充D1和A2A的拮抗作用。目的1研究缺氧缺血复苏后应用A2A拮抗剂对仔猪纹状体内DARPP-32、NR1和Na，K-ATPase磷酸化、Na，K-ATPase活性、超氧化物歧化产物和氧化应激标志物、神经元活性以及A2A免疫反应性保存的影响。目的2将确定联合使用A2A和D1拮抗剂的后处理是否对纹状体中不同的神经元群体提供额外的保护。目的3将确定20-HETE合成抑制剂的后处理是否减少了NR1和Na，K-ATPase上PKC敏感部位的磷酸化，提高了Na，K-ATPase的活性，并保护了D1和A2A纹状体神经元。AIM 4将确定与AIMS 1 3中被发现有效的药物联合治疗是否延长了延迟性低温的治疗窗口。这些新的神经保护研究，使用全身窒息的大型动物模型，将对未成熟基底节快速神经变性的原因提供独特的机械见解，并提供易于转化为治疗新生儿缺氧缺血性脑病的治疗方法。与公共卫生相关：对于在分娩和分娩期间以及出生后经历低氧合时期的新生儿，可用的治疗措施有限，这会导致长期残疾，如痉挛肌肉控制、认知缺陷、癫痫发作和发育迟缓。损伤的机制是多因素的，而且在未成熟脑的特定区域有所不同。使用窒息心脏骤停的新生动物模型来模拟足月儿的脑损伤，本应用的目标是在选择性脆弱的脑区研究这些机制，并制定合理的组合疗法设计，包括药物和降温，以改善导致终生破坏性后果的脑损伤的进展。

DESCRIPTION (provided by applicant): An innovative model of asphyxic cardiac arrest in piglets will be used in which the pattern of selective neuronal vulnerability simulates term neonatal hypoxic-ischemic (HI) encephalopathy, a major cause of morbidity and mortality in human newborns. Although hypothermia holds promise as a treatment, the delay in initiating hypothermia typical in clinical trials may not protect striatum, where neurons can die within 6 h of reoxygenation. This proposal focuses on therapies that are tailored for rapid striatal neuroprotection and that can extend the therapeutic window for hypothermia to protect delayed neurodegeneration in other regions. Mechanisms of injury specific to striatum have not been well studied in neonatal HI. Striatonigral neurons are enriched with dopamine D1 receptors, and striatopallidal neurons are enriched with adenosine A2A receptors, both of which act via PKA and the phosphorylation regulatory protein DARPP-32. D1 receptor activation amplifies NMDA channel calcium currents by NR1 phosphorylation and decreases Na,K-ATPase activity. In previous work, D1 dopamine receptor antagonist treatment ameliorated HI-induced phosphorylation at PKA- sensitive sites on DARPP-32, NR1 and Na,K-ATPase at 3 h of recovery, improved Na,K-ATPase activity, and selectively protected D1 neurons in piglet striatum. Preliminary data indicate that A2A antagonist post- treatment protects a portion of striatal neurons and attenuates HI-induced phosphorylation at PKA-sensitive sites on DARPP-32, NR1, and Na,K-ATPase. The arachidonic acid metabolite 20-HETE is also known to decrease Na,K-ATPase activity, but by PKC-dependent phosphorylation. Preliminary data with 20-HETE synthesis inhibitor post-treatment indicate partial neuroprotection and selective blockage of phosphorylation at PKC-sensitive sites on Na,K-ATPase and NR1. By targeting different phosphorylation sites of key proteins involved in excitotoxicity and multiple cell types, 20-HETE synthesis inhibition could complement D1 and A2A antagonism. In Aim 1, the effect of treatment with an A2A antagonist after cardiac resuscitation from HI will be studied on DARPP-32, NR1, and Na,K-ATPase phosphorylation, Na,K-ATPase activity, superoxide production and markers of oxidative stress, neuronal viability, and preservation of A2A immunoreactivity in piglet striatum. Aim 2 will determine whether post-treatment with combined A2A and D1 antagonists provides additive protection of distinct neuronal populations in striatum. Aim 3 will determine if post-treatment with a 20-HETE synthesis inhibitor reduces phosphorylation at PKC-sensitive sites on NR1 and Na,K-ATPase, improves Na,K-ATPase activity, and protects both D1 and A2A striatal neurons. Aim 4 will determine whether combined post-treatment with drugs found to be effective in Aims 1 3 extends the therapeutic window for delayed hypothermia. These novel neuroprotective studies, using a large animal model of whole body asphyxia, will render both unique mechanistic insights into the cause of rapid neurodegeneration in immature basal ganglia and therapies that can be readily translated for treatment of neonatal HI encephalopathy. PUBLIC HEALTH RELEVANCE: Available treatments are limited for newborns who experience periods of low oxygenation that damages their brains during labor and delivery, and after birth, and that leads to long-term disabilities, such as spastic muscle control, cognitive deficits, seizures, and developmental delays. The mechanisms of injury are multifactorial and differ among the specific regions of immature brain. Using a newborn animal model of asphyxic cardiac arrest to simulate the brain injury in term human newborns, the goal of this application is to investigate these mechanisms in selectively vulnerable brain regions and to formulate a rational design of combination therapies that involve drugs and cooling the body for ameliorating the progression of brain injury that leads to life-long devastating consequences.