Haemoglobin neurotoxicity, haptoglobin scavenging and synaptic function in subarachnoid haemorrhage

Abstract

During subarachnoid haemorrhage, blood spreads into the subarachnoid space and surrounding tissues. Slow release of haemoglobin (Hb) from red blood cell lysis causes oxidative damage and cell death. High rates of disability and cognitive decline in SAH survivors is attributed to loss of neurons and functional connections during secondary brain injury. Haptoglobin (Hp) sequesters free Hb for clearance, but this scavenging system is overwhelmed in a haemorrhage. Hp infusion has been shown to attenuate cytotoxic effects of haemoglobin on neurons in both in vitro and in vivo models of SAH, and other haemorrhagic conditions. The functional effects of clinically relevant and sub-lethal Hb concentrations on surviving neurons, and whether cellular function can be protected with Hp treatment, remain unclear.
In this thesis, the effects of a one week exposure to Hb are analysed in primary hippocampal neuron cultures. Our results demonstrate reduced ATP levels and neurite beading at a range of concentrations of haemolysate. At a sub-lethal concentration of 10 μM free Hb, which is the average Hb peak in the CSF after SAH in humans, intrinsic membrane properties are normal. However, we found a reduction in AMPA receptor-mediated current amplitude in the absence of presynaptic alterations, indicating fewer AMPA receptors at the synapse, and an overall reduction in GluA1 subunit expression. By scavenging one-third of free Hb with Hp in vitro, synaptic AMPA receptor impairment can be partially rescued and is indistinguishable from control, indicating that functional rescue can be achieved by Hp even without 1:1 stoichiometric neutralization of Hb. At higher concentrations of free Hb, higher levels of saturation with Hp can protect from ATP deficits and neurite beading. Hp in itself does not alter pre- or post-synaptic measures of synaptic neurotransmission, and has no effect on intrinsic membrane properties at 10μM. Our data highlight a role for Hb in modifying synaptic function after SAH, which may link to impaired cognition or plasticity, and the potential of haptoglobin as a therapy for subarachnoid haemorrhage.

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Identifiers

ORCID for Mariana Vargas-Caballero: orcid.org/0000-0003-2326-4001

ORCID for Diederik Bulters: orcid.org/0000-0001-9884-9050

ORCID for Ian Galea: orcid.org/0000-0002-1268-5102

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