Hemoglobin induces inflammation after preterm intraventricular hemorrhage by methemoglobin formation

Abstract

BackgroundCerebral intraventricular hemorrhage (IVH) is a major cause of severe neurodevelopmental impairment in preterm infants. To date, no therapy is available that prevents infants from developing serious neurological disability following IVH. Thus, to develop treatment strategies for IVH, it is essential to characterize the initial sequence of molecular events that leads to brain damage. In this study, we investigated extracellular hemoglobin (Hb) as a causal initiator of inflammation in preterm IVH.MethodsUsing a preterm rabbit pup model, we investigated the molecular mechanisms and events following IVH. We also characterized the concentrations of cell-free Hb metabolites and pro-inflammatory mediators in the cerebrospinal fluid (CSF) of preterm human infants and rabbit pups. Finally, Hb metabolites were evaluated as causal initiators of inflammation in primary rabbit astrocyte cell cultures.ResultsFollowing IVH in preterm rabbit pups, the intraventricular CSF concentration of cell-free methemoglobin (metHb) increased from 24 to 72 hours and was strongly correlated with the concentration of TNFα at 72 hours (r2 = 0.896, P <0.001). Also, the mRNA expression of TNFα, IL-1β, and Toll-like receptor-4 and TNFα protein levels were significantly increased in periventricular tissue at 72 hours, which was accompanied by extensive astrocyte activation (that is, glial fibrillary acidic protein (GFAP)staining). Furthermore, exposure of primary rabbit astrocyte cell cultures to metHb caused a dose-dependent increase in TNFα mRNA and protein levels, which was not observed following exposure to oxyhemoglobin (oxyHb) or hemin. Finally, a positive correlation (r2 = 0.237, P <0.03) between metHb and TNFα concentrations was observed in the CSF of preterm human infants following IVH.ConclusionsFollowing preterm IVH, increased metHb formation in the intraventricular space induces expression of pro-inflammatory cytokines. Thus, the formation of metHb might be a crucial initial event in the development of brain damage following preterm IVH. Accordingly, removal, scavenging, or neutralization of Hb could present a therapeutic opportunity and plausible approach to decreasing the damage in the immature brain following preterm IVH.

Role of Red Blood Cell Lysis and Iron in Hydrocephalus after Intraventricular Hemorrhage

“…High levels of methemoglobin have been reported in CSF after IVH. 22,23 We will continue to pursue the potential role of Fe 2 þ in IVH-induced brain injury in other studies, but this is hampered by a lack of specific chelators available for ferrous iron.…”

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

“…Once Hb escapes the intra-RBC compartment, it is highly reactive and activates cytotoxic, oxidative and inflammatory pathways, eventually leading to tissue damage [8,9]. Extracellular Hb is rapidly oxidised from ferrous (Fe 2+ , denoted oxyHb) to ferric (Fe 3+ , denoted metHb) Hb [10,11], which readily releases the heme group [12]. Heme is highly lipophilic and binds to lipids intercalating into cell membranes, which results in toxic cytolytic effects [13].…”

“…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].…”

Altered Expression of Aquaporin 1 and 5 in the Choroid Plexus following Preterm Intraventricular Hemorrhage

“…When the cells reached approximately 90% confluence CSF from preterm infants with IVH, metHb and hemin (prepared immediately prior to the experiment, as described above) were added to the HCPEpiC cultures, and the cells were incubated for 4-24 h as indicated in the figure legends. The metHb and hemin concentrations used were in accordance with previously described concentrations in CSF from preterm infants with IVH [22]. After incubation, the cells were harvested using either QiazolQ Lysis reagent (for RNA extraction; QIAGEN, Germantown, Md., USA) or cell extraction buffer (for protein extraction; Invitrogen, Camarillo, Calif., USA).…”

“…Importantly, the hemorrhage is confined to the intraventricular space and results in a progressive ventricular enlargement very similar to that seen in preterm human infants [21]. In a recent study using this model and primary astrocyte cell cultures we showed that IVH is followed by a release of cell-free hemoglobin (Hb) in the intraventricular space and that accumulated Hb metabolites are causal inducers of inflammation [22]. In addition to the in vivo characterization of AQP1, 4 and 5, here we further investigated the effects of Hb metabolites on the expression of AQP1, 4 and 5 in primary human choroid plexus epithelial cells (HCPEpiC) and compared the response to that following exposure to posthemorrhagic CSF obtained from preterm human infants.…”