Blood Exposure Causes Ventricular Zone Disruption and Glial Activation In Vitro

Abstract

Intraventricular hemorrhage (IVH) is the most common cause of pediatric hydrocephalus in North America but remains poorly understood. Cell junction-mediated ventricular zone (VZ) disruption and astrogliosis are associated with the pathogenesis of congenital, nonhemorrhagic hydrocephalus. Recently, our group demonstrated that VZ disruption is also present in preterm infants with IVH. On the basis of this observation, we hypothesized that blood triggers the loss of VZ cell junction integrity and related cytopathology. In order to test this hypothesis, we developed an in vitro model of IVH by applying syngeneic blood to cultured VZ cells obtained from newborn mice. Following blood treatment, cells were assayed for N-cadherin-dependent adherens junctions, ciliated ependymal cells, and markers of glial activation using immunohistochemistry and immunoblotting. After 24-48 hours of exposure to blood, VZ cell junctions were disrupted as determined by a significant reduction in N-cadherin expression (p < 0.05). This was also associated with significant decrease in multiciliated cells and increase in glial fibrillary acid protein-expressing cells (p < 0.05). These observations suggest that, in vitro, blood triggers VZ cell loss and glial activation in a pattern that mirrors the cytopathology of human IVH and supports the relevance of this in vitro model to define injury mechanisms.

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

“…Established IHC routines can be followed to perform IHC, see (32) and (30). The list of antibodies used are available in Table 1.…”

“…VZ cells in vitro progressively differentiate from monociliated NSC to multiciliated EC (30,32). To de ne the development of the EC and monitor the validity this IVH in vitro model, we used well-known VZ/EC markers ( Table 1).…”

“…Despite the importance of the VZ in brain development, there are few reported methods to differentiate EC from brain neural stem cells (NSC) (30,(41)(42)(43). We have recently reported on the effects of syngeneic blood on the VZ using an in vitro model (32), but the details of our procedures have not been published.…”

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

“…The methods detailed herein are uniquely applied to IVH, PHH, or hydrocephalus more generally. While other groups have cultured ependymal cells (EC) previously [30,31], the notion of using VZ cultures to study the effects of IVH is a novel strategy reported from our group [32]. This protocol is a major step forward in IVH/PHH research, as it enables rigorously controlled mechanistic experiments, with the added flexibility of mimicking various human physiological parameters.…”

“…Despite the importance of the VZ in brain development, there are few reported methods to differentiate EC from brain neural stem cells (NSC) [30,[41][42][43]. We have recently reported on the effects of syngeneic blood on the VZ using an in vitro model [32], but the details of our procedures have not been published. cortical hemispheres, as well as the brainstem and the olfactory bulbs by cutting in the midline using a single-edge razor blade (Fig 1).…”