Mechanisms of neonatal erythropoietin neuroprotection

描述(申请人提供)：早产儿容易出现认知迟缓、行为异常、癫痫和脑瘫。药物和手术可以部分改善运动功能和癫痫发作控制，但没有任何干预措施直接解决最常见的缺陷-认知和行为问题，尽管这些障碍对这些儿童成为有生产力的成年人构成了最大的障碍。了解早期脑损伤如何扰乱大脑发育的机制，以及干预措施如何恢复大脑功能，将指导有希望的治疗方法的适应症和时机。早产所致的中枢神经系统(CNS)损伤通常是由全身性产前缺氧缺血(HI)和/或炎症性损伤引起的。在妊娠后期，亚板神经元引导和完善大脑皮质回路的发育，特别是在大脑皮层IV层。亚板神经元是通过阳离子-氯共转运体KCC2的时空上调和GABAAR亚单位的成熟而在发育的IV层启动GABA能抑制。通过挤压氯化物，增加KCC2的表达调节GABA反应从兴奋性到抑制性的发育开关，并随着GABAAR的成熟，引导有效的皮质回路形成。我们假设与早产相关的中枢神经系统损伤会导致板下神经元的过早丢失，从而损害皮质IV层GABAAR亚单位的成熟和功能性KCC2的表达，这是大脑皮层发育的重要组成部分。此外，我们预测，损伤后神经保护性促红细胞生成素(EPO)治疗可以通过限制GABAAR亚单位和KCC2成熟的改变，以及通过促进神经恢复来缓解受损的皮质发育。利用我们的临床相关模型-胚胎第18天(E18)啮齿动物短暂的全身性缺氧-缺血(TSHI)模型以及人类极早产相关的全局中枢神经系统损伤模型，我们提出了下列目标：1)测试出生前TSHI后亚板的过早丢失是否损害体内大脑皮层的成熟以及采用机械亚板消融的脑片培养；2)测试产前TSHI是否通过BDNF/Calain介导的机制限制KCC2在皮质IV层的表达；3)测试体内和体外新生EPO治疗是否促进E18 TSHI后大脑皮层IV层KCC2和GABAAR的恢复。我们预测，出生后EPO治疗可以最大限度地减少过早的亚板退化，增加KCC2的表达，并恢复GABAAR亚单位的成熟。胎儿期促红细胞生成素或赋形剂治疗后，将测试幼年成年大鼠的认知和行为功能，以验证我们的预测，即促红细胞生成素可以恢复大脑皮层发育。总之，这些研究将阐明胎儿期整体缺氧缺血后早期大脑皮质发育的亚板调控机制，并为如何通过延迟促红细胞生成素治疗逆转产前损伤提供见解，目的是改善产前损伤后的认知和行为结果。

DESCRIPTION (provided by applicant): Infants who are born very preterm are prone to cognitive delay, behavioral abnormalities, epilepsy and cerebral palsy. Medications and surgery can partially improve motor function and seizure control, but no interventions directly address the most common deficits - cognitive and behavioral problems, even though these impairments pose the biggest obstacles to these children becoming productive adults. Understanding the mechanisms of how early brain injury disrupts cerebral development, and moreover, how interventions restore cerebral function, will guide the indications and timing of promising therapies. Central nervous system (CNS) injury from very preterm birth often results from a global prenatal hypoxic-ischemic (HI) and/or inflammatory insult. Late in gestation subplate neurons guide and refine cerebral cortical circuit development, especially in cortical layer IV. Subplate neurons are essential for initiation of GABAergic inhibition in developing layer IV via spatiotemporal upregulation of the cation-chloride co-transporter KCC2 and maturation of GABAAR subunits. By extruding chloride, increasing KCC2 expression regulates the developmental switch of GABA responses from excitatory to inhibitory, and along with GABAAR maturation, directs effective cortical circuit formation. We hypothesize that CNS injury associated with preterm birth causes premature subplate neuron loss, and thus impairs maturation of cortical layer IV GABAAR subunits and functional KCC2 expression, essential components of cerebral cortical development. Further, we predict that post-injury neuroprotective erythropoietin (EPO) treatment can mitigate compromised cortical development by limiting alterations in GABAAR subunits and KCC2 maturation, and by promoting neurological recovery. Using our clinically-relevant model of prenatal transient systemic hypoxia-ischemia (TSHI) in rodents on embryonic day 18 (E18) that models the global CNS injury associated with very preterm birth in humans, we propose these Aims: 1) to test that premature loss of subplate following prenatal TSHI in rats impairs cerebral cortical maturation in vivo and in slice cultures with mechanical subplate ablation, 2) to test that prenatal TSHI limits KCC2 membrane expression in cortical layer IV via BDNF/calpain-mediated mechanisms, and 3) to test that post-injury neonatal EPO treatment promotes cortical layer IV KCC2 and GABAAR recovery after E18 TSHI in vivo and in vitro. We predict postnatal EPO treatment minimizes premature subplate regression, increases KCC2 expression and restores GABAAR subunit maturation. After prenatal TSHI followed by neonatal EPO or vehicle treatment, cognitive and behavioral function will be tested in young adult rats to test our prediction that EPO can restore cerebral cortical development. Together, these studies will elucidate mechanisms of subplate regulation of early cerebral cortical development following prenatal global HI, and provide insights into how prenatal injury is reversed with delayed EPO treatment, with the goal of improving cognitive and behavioral outcomes after prenatal injury.