Premature infants with intraventricular hemorrhage (IVH) are at high risk of neurobehavioral disorders, including inattention, hyperactivity, major depressive disorders, and seizures. These disorders can be attributed to the defective development and function of cortical interneurons. However, the effect of IVH on the generation and maturation of cortical interneuron is unknown, even though the window of interneuron neurogenesis overlaps with the period when infants develop IVH (23-28 weeks of gestation). Interneurons are produced in the medial and caudal ganglionic eminence (MGE and CGE). MGE gives rise to parvalbumin+ (PV) and somatostatin+ (SST) interneurons, whereas CGE produces calretinin+ and NRY+ interneurons. The production of interneurons in the MGE is regulated by a number of transcription factors, including Nkx2.1, Dlx1/2, Lhx6/8, and Mash1, which are primarily controlled by Sonic Hedgehog (Shh) signaling pathways. As the post-mitotic interneurons migrate from MGE to the cortical layers, Sox6 plays critical roles in their specification and maturation into PV+ and SST+ interneurons. This production and maturation of interneurons would be affected by IVH, because it initiates in the MGE/CGE and induces oxidative cell injury and death. Our preliminary studies in rabbits with IVH show reduced neurogenesis in the MGE, deficit in PV+ and SST+ interneurons in the upper cortical layers, and a decline in Shh and Sox6 levels relative to controls. To ameliorate neurogenesis in the MGE and restore cortical interneuron deficit, our goal is to a) restore Shh > Nkx2.1 > Sox6 signaling and b) reduce oxidative stress to minimize cell injury and death. Since Nrf2 transcription factor is a master redox switch to turn on several antioxidant pathways, we will activate Nrf2 to minimize oxidative stress in our animal model of IVH. Therefore we hypothesize: i) IVH disrupts interneuron neurogenesis and differentiation in the MGE and CGE resulting in interneuron deficits in the cortical layers of preterm humans & rabbits, and ii) alleviating oxidative stress (Nrf2 stimulation) or activating interneuron production (lenti-mDlx-Shh) and maturation (lenti-mDlx-Sox6) will ameliorate PV and SST deficits and neurological function in rabbits with IVH.
In Aim #1, we will determine the effect of IVH on a) apoptosis, proliferation, and density of interneuron progenitors in the MGE (Nkx2.1+, Dlx1/2+) and CGE (Dlx2+ & Coup-TFII+), b) the density of mature interneurons?PV, SST and others--in the upper and lower cortical layers, and c) transcriptional changes in the MGE & CGE. Additionally, we will validate rabbit data in humans by analyzing autopsy materials from preterm infants with and without IVH.
In Aim #2 and # 3, we will assess the effect of alleviating oxidative stress (Ad-Nrf2-GFP or sulforaphane) or activating production of Shh (lenti-mDlx-Shh-GFP) and Sox6 (lenti-mDlx-Sox6-GFP) specifically in the interneuron progenitors on a) the density of progenitors in the MGE, b) population of PV+ and SST+ neurons in the cortical layers, c) transcriptional changes in MGE, d) neurobehavioral function in rabbits with IVH. The proposed studies will hasten development of new therapies to prevent neurobehavioral disorders in infants with IVH.
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 neuropsychiatric complications in the survivors including mental retardation, attention deficit, hyperactivity, major depressive episodes, and epilepsy. These disorders are attributed to abnormal development and function of interneurons in the brain. Brain bleeds in premature infants might suppress the generation and maturation of interneurons. In this proposal, we will determine whether the brain hemorrhage affects the formation of interneurons and test strategies to restore the development of interneurons and organization of brain layers in our preterm rabbit model of brain hemorrhage.
