M. Oria1, B. Pathak1, K. Bakri1, J. L. Peiro1 1Cincinnati Children’s Hospital Medical Center (CCHMC),Division Of Pediatric General And Thoracic Surgery,Cincinnati, OH, USA
Introduction: During spinal cord development, neural progenitor cells (NPC) generate three major cell lines; neurons, oligodendrocytes and astrocytes at precise time and position. Normally, neurogenesis occurs in early embryonic stage and astrocytes and oligodendrocytes later. However there have been studies indicating of astrocyte differentiation in early embryonic stages of spina bifida aperta. To date the pathophysiological mechanisms related to astrocyte differentiation with reactive astrogliosis of spina bifida aperta are poorly understood. In our study, we characterize the development of reactive astrocytes that differentiates from Pax6 and Olig2 neural progenitor cells.
Methods: Twenty fetal rat spinal cords from retinoic-acid (RA) induced spina bifida and other 6 from sham controls were analyzed at three gestational times (E15, E17, and E20).
Differentially expressed genes were determined using standard RNA sequencing approach and PT-qPCR for not only the transcription factors Pax6, Olig2 and Nkx2.2, but also the HLH transcription factors, Mash1, Ngn2, ID1 and ID2 that regulate the spatiotemporal neurogenesis and gliogenesis.
Histological analysis using specific antibodies for NPC (Pax6, NKX2.2 and Olig2) and astrocytes (GFAP) were used to quantify the NPC and to identify the NPC differentiating into astrocytes
Results: Patterning factor Pax6, NKX2.2 and Olig2 were downregulated in spina bifida animals at the 3 gestational times (E15, E17 and E20) in the RNAseq data and validated by RT-qPCR compared to non-spina bifida animals. GFAP gene expression was upregulated in spina bifida animals compared to normal animals during gestation. We have identified the presence of Olig2+ and Pax6+ multipotent progenitor cells in GFAP+ reactive astrocytes during gestation and increased numbers in the spinal cord tissue exposed to amniotic fluid in the spina bifida rats. Downregulation of patterning factors in the NPC and upregulation of astrogenic signaling pathway via BMP2 and 4 suggest early differentiation of the NPC into astrocytes in spina bifida aperta.
Conclusion: Together, our observations demonstrate altered premature astrogenesis affecting normal neurogenesis and oligodendrogenesis contributing to injury, and neural loss in spina bifida aperta after birth.