Mechanisms of neonatal erythropoietin neuroprotection

描述（申请人提供）：非常早产的婴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控制，但没有干预直接解决最常见的缺陷——认知和行为问题，尽管这些缺陷对这些孩子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破坏大脑发育的机制，以及干预如何恢复大脑功能，将指导有希望的治疗的适应症和时机。极早产引起的中枢神经系统（CNS）损伤通常是由全身性产前缺氧缺血性（HI）和/或炎症性损伤引起的。妊娠后期，亚板神经元引导和完善大脑皮层回路的发育，特别是在皮层第四层。亚板神经元通过时空上调阳离子-氯共转运体KCC2和GABAAR亚基的成熟，在发育第四层时启动gaba能抑制是必不可少的。通过挤出氯化物，KCC2表达的增加调节GABA反应从兴奋性到抑制性的发育转换，并随着GABAAR的成熟，指导有效的皮质回路的形成。我们假设，与早产相关的中枢神经系统损伤会导致底板神经元过早丢失，从而损害大脑皮层发育的重要组成部分——皮层IV层GABAAR亚基的成熟和功能性KCC2的表达。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亚基和KCC2成熟的改变以及促进神经恢复来减轻受损的皮质发育。利用我们的临床相关的啮齿动物胚胎第18天（E18）的产前短暂全身性缺氧缺血（TSHI）模型，模拟与人类非常早产相关的全身中枢神经系统损伤，我们提出以下目标：1)验证大鼠产前TSHI后亚板过早丢失是否会损害体内和机械亚板消融片培养中的大脑皮层成熟；2)验证产前TSHI通过BDNF/calpain介导的机制限制皮质IV层KCC2膜的表达；3)验证新生儿损伤后EPO治疗促进E18 TSHI后皮质IV层KCC2和GABAAR的体内和体外恢复。我们预测，产后EPO治疗可使亚板过早退化最小化，增加KCC2表达并恢复GABAAR亚基成熟。在产前TSHI和新生儿EPO或载体治疗后，将在年轻成年大鼠中测试认知和行为功能，以验证我们关于EPO可以恢复大脑皮层发育的预测。总之，这些研究将阐明产前全脑HI后早期大脑皮质发育的亚板调节机制，并为产前损伤如何通过延迟EPO治疗逆转提供见解，目的是改善产前损伤后的认知和行为结局。

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.