Extracellular hemoglobin - mediator of inflammation and cell death in the choroid plexus following preterm intraventricular hemorrhage

Abstract

BackgroundIntraventricular hemorrhage (IVH) with post-hemorrhagic ventricular dilatation (PHVD) is a major cause of neurodevelopmental impairment and mortality in preterm infants. The mechanisms leading to PHVD and brain damage remain largely unknown. The choroid plexus and the ependyma, which constitute an essential part of the blood-brain barrier (BBB), are the first structures to encounter the damaging effects of extravasated blood. The breakdown of the BBB is a critical upstream event leading to brain damage following IVH. In this study we investigated the impact of hemorrhage and hemoglobin (Hb) metabolites on the choroid plexus epithelium.MethodsUsing a preterm rabbit pup model of IVH, the structural and functional integrity, cellular, inflammatory and oxidative response of the choroid plexus, at 24 and 72 hours following IVH + PHVD, were investigated. In order to further characterize cellular and molecular mechanisms, primary human choroid plexus epithelial cells were exposed to cerebrospinal fluid (CSF) from preterm infants with IVH as well as to Hb-metabolites. Finally, the blocking effects of the Hb-scavenger haptoglobin (Hp) were investigated both in vivo and in vitro.ResultsFollowing IVH + PHVD, an up-regulation of mRNA for the receptor-related genes TLR-4, IL1R1, FAS, the transcription factor NF-Κβ and for the pro-inflammatory and chemotactic effector molecules, IL-1β, TNFα, MCP-1, IL-8, and IL-6 was observed in the choroid plexus at 24 and 72 hours. This was associated with structural disintegration, caspase activation and cell death in the choroid plexus epithelium. In vitro characterization of choroid plexus epithelial cells, following exposure to hemorrhagic CSF and to the Hb-metabolites metHb and heme, displayed apoptotic and necrotic cell death and an up-regulation of receptor-related and inflammatory effector molecules similar to that observed in vivo following IVH + PHVD. Intraventricular injection of the Hb-scavenger Hp in vivo and co-incubation with Hp in vitro reversed or reduced the cellular activation, inflammatory response, structural damage and cell death.ConclusionHb-metabolites are important causal initiators of cell death following IVH and removal or scavenging of Hb-metabolites may present an efficient means to reduce the damage to the immature brain following IVH.

The choroid plexus as a site of damage in hemorrhagic and ischemic stroke and its role in responding to injury

“…This was accompanied by CP cell death, caspase activation and pronounced ultrastructural changes. Similar inductions in inflammatory mediators and cell death were obtained in vitro when CP epithelial cells were exposed to met-hemoglobin, heme or CSF from preterm babies with IVH [35]. The hemoglobin scavenger, haptoglobin, prevented the adverse effects of IVH in vivo and hemoglobin or IVH CSF in vitro [35].…”

“…Furthermore, Gram et al [35] found a marked inflammatory response in a rabbit preterm model of IVH at 1 and 3 days. This was accompanied by CP cell death, caspase activation and pronounced ultrastructural changes.…”

Pathophysiology of extracellular haemoglobin: use of animal models to translate molecular mechanisms into clinical significance

“…It has been shown that intracerebroventricular injection of the Hb-scavenger Hp in vivo reversed or reduced cellular activation, inflammatory response, structural damage, and cell death following IVH [38] (Fig. 3).…”

“…In a preterm rabbit pup model of IVH, it was recently shown that extracellular Hb infiltrates and is widely dispersed in large regions of the cerebral periventricular white matter, especially in areas with high extracellular plasticity [37]. Furthermore, metHb was shown to be a potent inducer of pro-inflammation, displaying a strong correlation with TNF protein levels in intraventricular CSF and increased mRNA levels for TNF, IL-1β and of HO-1 in periventricular brain tissue [10,38].…”

Hemoglobin-induced neuronal degeneration in the hippocampus after neonatal intraventricular hemorrhage

“…A recent finding suggests that intraventricular Hb induces cytotoxic markers in astrocytes and can cause significant cell death and inflammatory activation in the choroid plexus (Gram et al, 2013, 2014). It is known that the heme moiety released by Hb interacts with intracellular heme oxygenase (HO) proteins (such as HO-1 in macrophages and HO-2 in neurons), releasing bilirubin, carbon monoxide, and iron (Fe) cations able to participate in Fenton and Haber-Weiss reactions (Lee et al, 2010; Lok et al, 2011; Wagner et al, 2003; Wang and Dore, 2007).…”