Preterm intraventricular hemorrhage in vitro: modeling the cytopathology of the ventricular zone

Fluids and Barriers of the CNS
Leandro Castaneyra-RuizDavid D Limbrick

Abstract

Severe intraventricular hemorrhage (IVH) is one of the most devastating neurological complications in preterm infants, with the majority suffering long-term neurological morbidity and up to 50% developing post-hemorrhagic hydrocephalus (PHH). Despite the importance of this disease, its cytopathological mechanisms are not well known. An in vitro model of IVH is required to investigate the effects of blood and its components on the developing ventricular zone (VZ) and its stem cell niche. To address this need, we developed a protocol from our accepted in vitro model to mimic the cytopathological conditions of IVH in the preterm infant. Maturing neuroepithelial cells from the VZ were harvested from the entire lateral ventricles of wild type C57BL/6 mice at 1-4 days of age and expanded in proliferation media for 3-5 days. At confluence, cells were re-plated onto 24-well plates in differentiation media to generate ependymal cells (EC). At approximately 3-5 days, which corresponded to the onset of EC differentiation based on the appearance of multiciliated cells, phosphate-buffered saline for controls or syngeneic whole blood for IVH was added to the EC surface. The cells were examined for the expression of EC markers of differentiat...Continue Reading

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Methods Mentioned

BETA
dissection
confocal microscopy
dissections

Software Mentioned

NIH
image lab
ImageJ

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