M. Oria1, R. L. Figueira1, F. Scorletti1, L. Sbragia1, F. Y. Lim1, J. L. Peiro1 1Cincinnati Children’s Hospital Medical Center,Center For Fetal, Cellular And Molecular Therapy,Cincinnati, OH, USA
Introduction:
Spina bifida aperta is the most common permanently disabling birth defects. However, the pathophysiological mechanism by astrocyte proliferation with reactive astrogliosis and neuroinflammation related to microglial activation are poorly understood. We hypothesized that microglia are activated in the exposed neural tissue in spina bifida, and this activation may be associated with impairment of CD200-CD200R-mediated microglia silencing in the exposed spinal cord, which could lead to the irreversible neurological alterations after birth.
Methods:
Spinal cord exposed and spinal cord non-exposed to the amniotic fluid in spina bifida pups were collected at day 20 of gestation in retinoic acid-induced spina bifida rats and spinal cord from sham-treated pups (control) (n=6 moms/group). RNA was isolated and pellets were partially re-extracted, precipitated, DNAseI-digested and cleaned. A 1-µg cDNA sample was used to set up RT-qPCR using TaqManR gene expression assay. CD200 and CD200R expression were assessed by flow cytometry. Protein expression were analyzed by immunofluorescence, western blot for target proteins and multiplex technology was used for cytokine tissue expression (IL1β , IL6, and INFγ).
Results:
Spina bifida samples present typical alterations with open posterior arc, dysraphic spinal cord, lack of dura and, exposed spinal cord to the amniotic fluid. Exposed neural tissue shows reactive astrocytes and activated microglia (p<0.05) located in the exposed external layers. These activated microglia exhibited disruption of the inhibitory immune ligand-receptor system (CD200-CD200R) in the lesion with neural tissue loss down-expressing CD200 and stimulating neuroinflammation up-regulating CD200R (p<0.05). The spinal cord lesion induced neuroinflammation with increased tissue water content compared wiith the non-exposed spinal cord (p<0.05) and cytokine production (IL1β, IL6, and INFγ)(p<0.05).
Conclusion:
Our study analyzed the spinal cord alterations in RA-induced spina bifida aperta in rats. For first time, we discern the relationship of the activate microgliosis by disruption of the endogenous inhibitory system (CD200-CD200R) and neural tissue loss in the spinal cord exposed to the amniotic fluid in spina bifida.