Therapeutic Strategies for Neonatal Hypoxic-Ischemic Encephalopathy

描述（由申请方提供）：将使用仔猪窒息性心脏骤停的创新模型，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（HI）脑病，这是人类新生儿发病率和死亡率的主要原因。虽然低温有希望作为一种治疗方法，但在临床试验中典型的低温启动延迟可能无法保护纹状体，其中神经元可能在复氧后6小时内死亡。该提案的重点是针对快速纹状体神经保护的治疗方法，并且可以延长低温治疗窗口，以保护其他区域的延迟神经变性。新生儿HI中纹状体特异性损伤的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，纹状体苍白球神经元富含腺苷A2 A受体，两者都通过PKA和磷酸化调节蛋白DARPP-32起作用。D1受体激活通过NR 1磷酸化放大NMDA通道钙电流，并降低Na，K-ATP酶活性。在先前的工作中，D1多巴胺受体拮抗剂治疗在恢复3小时时改善了HI诱导的DARPP-32、NR 1和Na，K-ATP酶上PKA敏感位点的磷酸化，提高了Na，K-ATP酶活性，并选择性地保护了仔猪纹状体中的D1神经元。初步数据表明，A2 A拮抗剂后处理保护一部分纹状体神经元，并减弱DARPP-32、NR 1和Na，K-ATP酶上PKA敏感位点处HI诱导的磷酸化。花生四烯酸代谢物20-HETE也已知降低Na，K-ATP酶活性，但通过PKC依赖性磷酸化。20-HETE合成抑制剂治疗后的初步数据表明部分神经保护和选择性阻断Na，K-ATP酶和NR 1上PKC敏感位点的磷酸化。通过靶向参与兴奋性毒性和多种细胞类型的关键蛋白的不同磷酸化位点，20-HETE合成抑制可以补充D1和A2 A拮抗作用。在目的1中，将研究在HI心脏复苏后用A2 A拮抗剂处理对DARPP-32、NR 1和Na，K-ATP酶磷酸化、Na，K-ATP酶活性、超氧化物产生和氧化应激标志物、神经元活力和仔猪纹状体中A2 A免疫反应性的保留的影响。目的2将确定后处理与组合A2 A和D1拮抗剂提供不同的神经元群体在纹状体的附加保护。目的3将确定用20-HETE合成抑制剂后处理是否减少NR 1和Na，K-ATP酶上PKC敏感位点的磷酸化，提高Na，K-ATP酶活性，并保护D1和A2 A纹状体神经元。目的4将确定是否与目的1 - 3中发现有效的药物联合后处理延长了延迟性低温的治疗窗口。这些新的神经保护研究，使用一个大型动物模型的全身窒息，将呈现独特的机制的见解，在不成熟的基底神经节和治疗，可以很容易地转化为新生儿HI脑病的治疗快速神经退行性变的原因。公共卫生相关性： 可用的治疗方法仅限于经历低氧期的新生儿，这些低氧期会在分娩和分娩期间以及出生后损害他们的大脑，并导致长期残疾，如痉挛性肌肉控制，认知缺陷，癫痫发作和发育迟缓。损伤的机制是多因素的，并且在未成熟脑的特定区域之间存在差异。使用窒息性心脏骤停的新生动物模型来模拟足月人类新生儿的脑损伤，本申请的目标是在选择性脆弱的脑区域中研究这些机制，并制定涉及药物和冷却身体的组合疗法的合理设计，以改善导致终身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.