Germinal Matrix Hemorrhage Affects Glutamatergic Neurogenesis

描述(申请人提供)：生发基质出血(GMH)-脑室出血(IVH)是早产儿的主要并发症，会导致脑瘫、智力低下、学习障碍、神经发育迟缓和皮质生长障碍。谷氨酸能神经发生-谷氨酸能神经元的形成-在大脑皮层的背侧脑室(VZ)和脑室下区(SVZ)协调，一直持续到28周。由于IVH通常起始于脑室周围生发区，这可能会阻碍谷氨酸能神经发生。因此，我们问IVH是否扰乱了谷氨酸能神经发生和皮层上层的组织(2-4)，如果是的话，如何恢复。在大脑皮质发育过程中，谷氨酸能神经元由VZ的放射状胶质细胞(Sox2+)发育而来，经过不对称分裂产生中间前体细胞(IPC)。IPC迁移到SVZ，进一步增殖和成熟为构成皮质各层的锥体神经元。背侧SVZ的谷氨酸能神经发生受转录因子Pax6、Ngn1/2、Emx1/2和Insm1的分级表达的影响，而皮质上层的神经元规格受CUX1、Brn2和Satb2的调节。这些转录因子受Wnt和Notch信号通路的控制。这些信号级联是神经前体细胞增殖和分化的中心调节器；这些形态原决定了改变细胞命运的转录活动。我们的初步数据显示，IVH抑制谷氨酸能神经发生，激活Notch，并下调Wnt信号。因此，我们假设：1)IVH将干扰谷氨酸能神经发生和皮质上层的生长；2)Wnt或Notch通路的调节将恢复IVH早产幼鼠的谷氨酸能神经元和皮质层的发育。我们的方法是使用我们的甘油诱导的IVH的早产兔模型，并使用早产儿的尸检材料。目的1.观察IVH早产幼兔VZ和SVZ皮质神经胶质细胞(Sox2+)和IPC(Tbr2+)的密度、增殖和凋亡，皮质2-4层锥体神经元(CuX1+，Brn2+)的丰度，以及神经功能。通过在有和没有IVH的人类早产儿(尸检材料)中进行平行实验，在人类中验证兔子的数据。目的：研究激活Wnt/b-catenin信号通路对IVH仔鼠神经功能的影响：a)治疗组与对照组相比，1)放射状胶质细胞和IPC的密度、增殖和凋亡；b)皮质2-4层神经元密度；c)神经原转录因子Pax6、Ngn1/2、Emx1/2和Insm1；目的：研究Notch抑制对IVH组仔鼠神经功能的影响：a)VZ区和SVZ区神经胶质细胞和IPC的密度、增殖、凋亡，b)皮质2-4层神经元密度，c)神经原转录因子Hes1/5、Pax6、Ngn1/2和Insm1，d)神经功能。

DESCRIPTION (provided by applicant): Germinal matrix hemorrhage (GMH)-intraventricular hemorrhage (IVH) is a major complication of premature infants that results in cerebral palsy, mental retardation, learning disabilities, neurodevelopmental delay, and reduced cortical growth. Glutamatergic neurogenesis-formation of glutamatergic neurons-is orchestrated in the dorsal ventricular (VZ) and subventricular zones (SVZ) of the cerebral cortex, and continues until 28 weeks of gestational age. As IVH typically initiates in the periventricular germinal zone, this might hamper glutamatergic neurogenesis. Therefore, we ask whether IVH disrupts glutamatergic neurogenesis and the organization of the upper cortical layers (2-4) and if so, how this can be restored. During the development of the cerebral cortex, glutamatergic neurons develop from radial glia (Sox2+) of the VZ, which undergo asymmetric division to generate intermediate progenitors (IPC). IPC migrate to the SVZ to further proliferate and mature into pyramidal neurons that form the layers of cortex. Glutamatergic neurogenesis in the dorsal SVZ is orchestrated under the influence of graded expression of transcription factors including Pax6, Ngn1/2, Emx1/2, and Insm1, while neuronal specification of upper cortical layers is regulated by Cux1, Brn2, and Satb2. These transcription factors are controlled by Wnt and Notch signaling pathways. These signaling cascades are the central regulators of proliferation and differentiation of neural progenitor cells; and these morphogens determine the transcriptional activity that modifies cell fate. Our preliminary data revealed that IVH suppressed glutamatergic neurogenesis, activated Notch, and downregulated Wnt signaling. Therefore, we hypothesize that i) IVH will disrupt glutamatergic neurogenesis and the growth of the upper cortical layers, and that ii) modulation of the Wnt or Notch pathway will restore the development of glutamatergic neurons and cortical layers in preterm pups with IVH. Our approach is to employ our preterm rabbit model of glycerol-induced IVH and use autopsy materials from preterm infants. Aim #1: Evaluate a) the density, proliferation, and apoptosis of radial glia (Sox2+) and IPC (Tbr2+) in the cortical VZ and SVZ, b) the abundance of pyramidal neurons (Cux1+, Brn2+) in cortical layers 2-4, and c) neurological function in premature rabbit pups with IVH vs. pups without IVH. Validate rabbit data in humans by performing parallel experiments in human premature infants (autopsy materials) with and without IVH. Aim #2: Determine the effect of activating Wnt/b-catenin signaling pathways on a) the density, proliferation, and apoptosis of radial glia and IPC, b) neuronal density in cortical layers 2-4, and c) proneural transcription factors--Pax6, Ngn1/2, Emx1/2 and Insm1, and d) neurological function in treated pups with IVH vs. controls. Aim #3: Determine the effect of Notch inhibition on a) the density, proliferation, an apoptosis of radial glia and IPC of the VZ and SVZ, b) the density of neurons in cortical layers 2-4, c) proneural transcription factors-- Hes1/5, Pax6, Ngn1/2, and Insm1, and d) neurological function in treated pups with IVH vs. vehicle controls.