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

Leandro Castaneyra-Ruiz,James P Mcallister,Diego M Morales,Diego M Morales,Albert M Isaacs,David D Limbrick,David D Limbrick,Steven L Brody,David D Limbrick

doi:10.1186/s12987-020-00210-7

Abstract

BackgroundSevere 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.MethodsMaturing 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 differentiation and maturation to qualitatively and quantitatively assess the effect of blood exposure on VZ transition from neuroepithelial cells to EC.DiscussionThis protocol will allow investigators to test cytopathological mechanisms contributing to the pathology of IVH with high temporal resolution and query the impact of injury to the maturation of the VZ. This technique recapitulates features of normal maturation of the VZ in vitro, offering the capacity to investigate the developmental features of VZ biogenesis.

Highlights

Intraventricular hemorrhage (IVH) is a severe neurological disorder of preterm infants, affecting about 20 percent of the preterm infants born at or below 32 gestational weeks [1]
We have recently reported on the effects of syngeneic blood on the ventricular zone (VZ) using an in vitro model [32], but the details of our procedures have not been published
The current effort describes the (See figure on page.) Fig. 2 In vitro ependymal cells (EC) differentiation and characteristic markers: a, b Representative cell culture at day 5 and 7 respectively, labeled with anti-βIV tubulin and anti-GFAP, in which a remarkable increase in βIV tubulin expression occurs at day 7. a′, b′

Summary

Introduction

Intraventricular hemorrhage (IVH) is a severe neurological disorder of preterm infants, affecting about 20 percent of the preterm infants born at or below 32 gestational weeks [1]. An understanding of the complex effects of the blood on the VZ is needed to develop preventive treatments for neurological sequelae of IVH/ PHH. This protocol describe an in vitro experimental strategy that provides the ability to control the environment of murine VZ development. 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

Objectives

Methods

Results

Conclusion