Germinal Matrix Hemorrhage Affects Glutamatergic Neurogenesis

描述（由申请人提供）：脑胶质瘤出血（GMH）-脑室内出血（IVH）是早产儿的一种主要并发症，可导致脑瘫、智力低下、学习障碍、神经发育迟缓和皮质生长减少。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）中协调，并持续到孕龄28周。由于IVH通常起始于室周生发区，这可能会阻碍脑内能神经发生。因此，我们想知道IVH是否会破坏脑内巨噬细胞能神经发生和上皮层的组织（2-4），如果是的话，如何恢复。 在大脑皮层发育过程中，VZ的放射状胶质细胞（Sox 2+）发育成神经元，其经历不对称分裂以产生中间祖细胞（IPC）。IPC迁移到SVZ以进一步增殖和成熟为形成皮层层的锥体神经元。背侧SVZ中的谷氨酸能神经发生在包括Pax 6、Ngn 1/2、Emx 1/2和Insm 1的转录因子的分级表达的影响下被编排，而上皮层的神经元规格由Cux 1、Brn 2和Satb 2调节。这些转录因子由Wnt和Notch信号通路控制。这些信号级联是神经祖细胞增殖和分化的中心调节因子;这些形态发生素决定改变细胞命运的转录活性。我们的初步数据显示，IVH抑制海马能神经发生，激活Notch，下调Wnt信号。因此，我们假设：i）IVH将破坏多巴胺能神经发生和上皮层的生长，ii）Wnt或Notch通路的调节将恢复患有IVH的早产幼崽中多巴胺能神经元和皮层的发育。我们的方法是采用我们的早产兔模型甘油诱导IVH和使用尸检材料从早产儿。目标一：评价a）皮质VZ和SVZ中放射状胶质细胞（Sox 2+）和IPC（Tbr 2+）的密度、增殖和凋亡，B）皮质2-4层中锥体神经元（Cux 1+、Brn 2+）的丰度，以及c）患有IVH的早产兔幼仔与未患有IVH的幼仔的神经功能。通过在存在和不存在IVH的人类早产儿（尸检材料）中进行平行实验来验证人类中的家兔数据。目标二：确定激活Wnt/b-连环蛋白信号传导途径对a）放射状胶质细胞和IPC的密度、增殖和凋亡，B）皮质层2-4中的神经元密度，和c）前神经转录因子-Pax 6、Ngn 1/2、Emx 1/2和Insm 1，和d）IVH治疗幼仔与对照组相比的神经功能的影响。目标3：确定Notch抑制对以下方面的影响：a）放射状神经胶质的密度、增殖、凋亡以及VZ和SVZ的IPC，B）皮质层2-4中神经元的密度，c）前神经转录因子-Hes 1/5、Pax 6、Ngn 1/2和Insm 1，以及d）与载体对照相比，用IVH处理的幼仔的神经功能。

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.