Preterm intraventricular haemorrhage - Effects of extracellular haemoglobin

Abstract: Intraventricular haemorrhage (IVH) is the most common brain lesion in preterm infants and is most commonly seen in the sickest children, with 15-20% of very preterm infants developing IVH. The mortality of infants with severe IVH is 20-50 % in the neonatal period and over 50 % of surviving infants develop post-haemorrhagic ventricular dilatation (PHVD) and 40-80 % develop severe neurological impairment, mainly cerebral palsy and mental retardation. To date there is no available therapy to prevent infants from developing either hydrocephalus or serious neurological disability. Infants who develop hydrocephalus receive a life-long ventriculo-peritoneal shunt, which is an efficient means of preventing ventricular distension but does not reduce neurological impairment.
Mechanisms leading to brain damage and hydrocephalus following IVH are complex and incompletely understood. However, inflammation and oxidative stress have been identified as two major culprits leading to irreversible damage in the vulnerable immature periventricular brain matter. Dysfunction of cerebrospinal fluid (CSF) production contributes to the development of PHVD but the exact molecular mechanisms remain unknown. The choroid plexus, which produces the CSF, is adjacent to the origin of IVH and is the first organ to encounter the extravasated blood. The subsequent haemolysis of red blood cells causes release of cell-free haemoglobin (Hb), which will with time further degrade releasing the toxic substances hemin, free iron, and various ROS (reactive oxygen species). Metabolites of cell-free Hb have been identified as an initiator of inflammation in the context of adult cerebral haemorrhage and inflammation of the ependyma has been shown to cause alterations of the blood-brain barrier (BBB).
Our working hypothesis is that cell-free Hb and its metabolites act as causal initiators of inflammation following IVH, constituting a critical up-stream event eventually leading to periventricular cell death. We further hypothesize that inflammation of the choroid plexus alters the ability of the epithelium to maintain CSF homeostasis contributing to the formation of post-haemorrhagic hydrocephalus.
To test the hypothesis we used the rabbit pup model of preterm IVH. It is excellently suited since rabbit pups have a germinal matrix with vulnerable capillary meshwork prone to rupture and have CNS maturation comparable to a 28-30 weeks preterm human infant. The application of high-frequency ultrasound enabled accurate measurements of haemorrhagic size and distension as well as ultrasound guided intraventricular injections and CSF sampling.
Following IVH there is a release of free haemoglobin in its reduced form, oxyHb, into the intraventricular space. OxyHb autooxidises to metHb over time and the concentration of the key inflammatory cytokine TNF-α is highly correlated to that of metHb. In an astrocyte cell culture, metHb induces TNF-α production whereas oxyHb does not. We therefore conclude that the formation of metHb is a key up-stream event leading to inflammation following IVH.
Following IVH there is extensive damage to the choroid plexus epithelium, which develops over time. There is a distinct inflammatory and cellular response induced by haemoglobin metabolites. Injection or co-incubation with haptoglobin, a haemoglobin scavenger, reduces or reverses the effects of haemoglobin both in vivo and in vitro.
Aquaporins (AQP) are water transporting transmembrane proteins playing a central role in CSF production. Following IVH the expression of AQP1, the key AQP in the choroid plexus, is down-regulated whereas the expression of AQP5, not previously described in the choroid plexus, is up-regulated. This probably represents an adaptive response to insult and might be of importance in understanding the development of PHVD.
In conclusion; following IVH released cell free haemoglobin, metabolized to metHb and hemin, constitutes a causal up-stream initiator of inflammation and cellular damage. Scavenging or removal of haemoglobin might be an efficient and feasible approach to reduce brain damage following preterm IVH.