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.
In the United States, about 12,000 premature infants develop bleeding in and around the ventricle (cavity) of the brain each year, which results in mental retardation, neurodevelopmental delay, and reduced brain growth. Since formation of neurons (brain cells) continues in fetuses until 28 weeks of pregnancy, bleeding in the ventricle might suppress the generation of neurons and brain maturation in preterm infants. In this proposal, we will determine whether the brain hemorrhage affects the formation of neurons and will test strategies to restore the development of neurons and organization of brain layers in our preterm rabbit model of brain hemorrhage.
