Abstract Germinal matrix hemorrhage (GMH) is the bleeding from the thin-walled immature blood vessels in the germinal matrix of pre‑term infants. Post‑hemorrhagic hydrocephalus is a common but severe consequence from GMH. However, there is no effective treatment for this so far but surgical shunting, which causes a huge socioeconomic burden. Thus, to characterize the underlying mechanisms and identify the potential therapeutic targets are of the utmost importance. Cerebrospinal fluid (CSF) is mainly produced from the choroid plexus and reabsorbed by subarachnoid villi and to a greater extent in neonates, through the glymphatic system. GMH results in breakdown of blood products, inflammation and astrogliosis, which can damage periventricular tissues. Tissues responsible for maintaining normal CSF flow dynamics may be injured following GMH, contributing to post-hemorrhagic hydrocephalus. The choroid plexus is specialized for CSF production and regulating the blood-CSF barrier. Iron toxicity from lysed red blood cells after GMH may lead to increased expression of slc4a10, a sodium bicarbonate co-transporter responsible for CSF secretion, and consequent CSF over-secretion at the choroid plexus. Furthermore, glymphatic CSF drainage is postulated to play a great role in neonates, since subarachnoid villi are sparsely distributed, and its function may also be compromised following GMH. The glymphatic system involves astrocyte-mediated CSF-interstitial fluid (ISF) exchange in Virchow-Robin space, which is driven by astrocytic aquaporin-4. In addition, inwardly rectifying potassium channel 4.1 (Kir4.1) works in conjunction with aquaporin-4 in astrocytes for regulating osmotic gradients and consequent water flow, yet its role in glymphatic CSF-ISF exchange has not been established. Astrogliosis from GMH may alter aquaporin-4 and Kir4.1 expression or function, disrupting glymphatic CSF-ISF exchange and consequently reducing CSF reabsorption. Our overall hypothesis is that, following GMH, acute iron overload contributes to CSF overproduction at the choroid plexus by inducing slc4a10 and long-term astrogliosis impairs normal CSF reabsorption through the glymphatic system, leading to post- hemorrhagic hydrocephalus in neonates. To test this hypothesis, we will conduct our study in two specific aims. Specific Aim 1: Determine the role of iron-induced expression of slc4a10 at the choroid plexus after GMH, leading to increased CSF secretion. Specific Aim 2: Determine the role of GMH-induced astrogliosis and consequent Kir4.1 and aquaporin-4 expression in disrupting CSF-ISF exchange and CSF clearance through the glymphatic system.

Narrative There is no effective treatment for hydrocephalus after germinal matrix hemorrhage in neonates other than surgical shunting, which causes significant side effects and economic burden. In this proposal, we aim to study the underlying mechanisms of short-term cerebrospinal fluid (CSF) overproduction due to the upregulation of sodium bicarbonate transporter slc4a10 and reduced CSF reabsorption in the long term due to the impaired glymphatic system. The completion of this project may reveal novel therapeutic targets for post-hemorrhagic hydrocephalus in neonates.