Authors: Cameron Sadegh, MD; Neil Dani, PhD; Fred Shipley; Maria Lehtinen (Cambridge, MA)
Introduction: Intraventricular hemorrhage (IVH) leads to hydrocephalus in preterm infants by unclear mechanisms ranging from ependymal injury and subarachnoid scarring to choroid plexus (ChP) hypersecretion of cerebrospinal fluid (CSF). We focus on the embryonic ChP, while it is still maturing and when CSF composition is most critical to neocortical development. The earliest physiologic changes in ChP epithelial cells likely involve calcium, which is a known cellular messenger in other secretory epithelial cell populations. Our laboratory has generated a ChP epithelial calcium reporter system to enable rapid calcium imaging to evaluate whether calcium is involved and how it might be regulated. Methods: Lateral ventricle choroid plexus is dissected at embryonic day 14.5 (E14.5) from a conditional calcium-reporting transgenic mouse line (Gcamp6f, limited to expression in ChP ciliated/epithelial cells using a FoxJ1-Cre system). The choroid plexus explants are kept viable with artificial CSF, exposed to focal blood injections, and imaged for calcium responses using epifluorescence and 2 photon imaging. Results: Upon exposure to age-matched plasma or serum, embryonic ChP epithelial cells rapidly trigger a wave of calcium activity that is followed by the upregulation of high intensity, recurrent calcium transients in subsets of ChP epithelial cells. Using different conditions of calcium blockade, we identify intracellular endoplasmic reticulum storage as the primary source of calcium influx, which requires store-operated calcium channels for continued calcium release. Conclusion: Intraventricular blood products cause an immediate intracellular calcium release by the choroid plexus epithelia. This calcium release is dynamic and occurs in waves; the release machinery is characterized with inhibitors of the calcium release pathway. Combined with other data supporting the rapid early response of choroid plexus, rather than ependymal cells, these changes may predict future choroid plexus dysfunction and altered CSF production, and contribute to our understanding of IVH-induced hydrocephalus pathophysiology.