ABSTRACT
Radiation therapy (RT) is a crucial treatment modality for central nervous system (CNS) tumors but toxicity to healthy CNS tissues remains a challenge. Additionally, environmental exposure to radiation during nuclear catastrophes or space travel presents a risk of CNS toxicity. However, the underlying mechanisms of radiation-induced CNS toxicity are not fully understood. Neural progenitor cells (NPCs) are highly radiosensitive, resulting in decreased neurogenesis in the hippocampus. This study aimed to characterize a novel platform utilizing rat NPCs cultured as 3D neurospheres (NSps) to screen the safety and efficacy of experimental drugs with and without radiation exposure. The effect of radiation on NSp growth and differentiation was assessed by measuring sphere volume and the expression of neuronal differentiation markers Nestin and GFAP and proliferation marker Ki67. Radiation exposure inhibited NSp growth, decreased proliferation, and increased GFAP expression, indicating astrocytic differentiation. RNA sequencing analysis supported these findings, showing upregulation of Notch, BMP2/4, S100b, and GFAP gene expression during astrogenesis. By recapitulating radiation-induced toxicity and astrocytic differentiation, this single-NSp culture system provides a high-throughput preclinical model for assessing the effects of various radiation modalities and evaluates the safety and efficacy of potential therapeutic interventions in combination with radiation.
ABSTRACT
During embryonic spinal cord development, neural progenitor cells (NPCs) generate three major cell lines: neurons, oligodendrocytes, and astrocytes at precise times and locations within the spinal cord. Recent studies demonstrate early astrogenesis in animal models of spina bifida, which may play a role in neuronal dysfunction associated with this condition. However, to date, the pathophysiological mechanisms related to this early astrocytic response in spina bifida are poorly understood. This study aimed to characterize the development of early astrogliosis over time from Pax6+, Olig2+, or Nkx2.2+ NPCs using a retinoic acid-induced spina bifida rat model. At three gestational ages (E15, E17, and E20), spinal cords from fetuses with retinoic acid-induced spina bifida, their healthy sibling controls, or fetuses treated with the vehicle control were analyzed. Results indicated that premature astrogliosis and astrocytic activation were associated with an altered presence of Pax6+, Olig2+, and Nkx2.2+ NPCs in the lesion compared to the controls. Finally, this response correlated with an elevation in genes involved in the Notch-BMP signaling pathway. Taken together, changes in NPC patterning factor expression with Notch-BMP signaling upregulation may be responsible for the altered astrogenesis patterns observed in the spinal cord in a retinoic acid-induced spina bifida model.
ABSTRACT
A better understanding of the transcriptomic modifications that occur in spina bifida may lead to identify mechanisms involved in the progression of spina bifida in utero and the development of new therapeutic strategies that aid in spinal cord regeneration after surgical interventions. In this study, RNA-sequencing was used to identify differentially expressed genes in fetal spinal cords from rats with retinoic acid-induced spina bifida at E15, E17, and E20. Gene ontology, KEGG, and protein-protein interaction analysis were conducted to predict pathways involved in the evolution of the disease. Approximately 3000, 1000 and 300 genes were differentially expressed compared to the control groups at E15, E17 and E20, respectively. Overall, the results suggest common alterations in certain pathways between gestational time points, such as upregulation in p53 and sonic hedgehog signaling at E15 and E17 and downregulation in the myelin sheath at E17 and E20. However, there were other modifications specific to gestational time points, including skeletal muscle development at E15, downregulated glucose metabolism at E17, and upregulated inflammation at E20. In conclusion, this work provides evidence that gestational age during spina bifida repair may be a significant variable to consider during the development of new regenerative therapeutics approaches.
ABSTRACT
Spina bifida aperta is a congenital malformation characterized by the failure of neural tube closure resulting in an unprotected fetal spinal cord. The spinal cord then undergoes progressive damage, likely due to chemical and mechanical factors related to exposure to the intrauterine environment. Astrogliosis in exposed spinal cords has been described in animal models of spina bifida during embryonic life but its relationship with neuroinflammatory processes are completely unknown. Using a retinoic acid-induced rat model of spina bifida we demonstrated that, when exposed to amniotic fluid, fetal spinal cords showed progressive astrogliosis with neuronal loss at mid-gestation (E15) compared to unexposed spinal cords. The number of microglial cells with a reactive phenotype and activation marker expression increased during gestation and exhibited progressive disruption in the inhibitory immune ligand-receptor system. Specifically we demonstrate down-regulation of CD200 expression and up-regulation of CD200R. Exposed spinal cords demonstrated neuroinflammation with increased tissue water content and cytokine production by the end of gestation (E20), which correlated with active Caspase3 expression in the exposed layers. Our findings provide new evidence that microglia activation, including the disruption of the endogenous inhibitory system (CD200-CD200R), may participate in the pathogenesis of spina bifida through late gestation.
