Haptoglobin as a novel treatment for subarachnoid haemorrhage

Morton, Matthew (2018) Haptoglobin as a novel treatment for subarachnoid haemorrhage. University of Southampton, Doctoral Thesis, 216pp.

Record type: Thesis (Doctoral)

Abstract

Subarachnoid haemorrhage (SAH) is a severe neurological condition caused by rupture of an intracranial aneurysm. A clot forms in the subarachnoid space and erythrocyte lysis causes haemoglobin (Hb) to accumulate in the CSF. In patients surviving the initial bleed, secondary complications such as vasospasm and delayed ischaemic neurological deficits (DIND) can result in poor long term outcome. Hb is neurotoxic through oxidative mechanisms and has also been implicated in the pathogenesis of vasospasm. Management of SAH patients is aimed at securing the aneurysm, inhibiting vasospasm and diversion of CSF to treat hydrocephalus; high morbidity means novel treatments to improve long term outcome are needed and there are none targeting Hb toxicity.

Haptoglobin (Hp) is responsible for binding to cell free Hb, facilitating scavenging by CD163-positive macrophages. Hp inhibits free Hb’s pro-oxidative potential by shielding the redox active iron contained in the haem group, rendering Hb oxidatively inert. Two co-dominant HP alleles (HP1 and HP2) exist in humans as a result of an intragenic duplication event. This lead to the production of dimers in HP1 homozygotes (HP1-1), dimers and trimers in heterozygotes (HP2-1), and finally tetramers and large polymers in HP2 homozygotes (HP2-2). Hp isoforms differ in their expression levels, their ability to inhibit Hb’s oxidative potential, and when in complex with Hb, their affinity for CD163 and uptake by macrophages.

In this study I hypothesized that: haptoglobin affects outcome after subarachnoid haemorrhage through its role in haemoglobin scavenging and haptoglobin supplementation after SAH is of therapeutic benefit. The aims were to: 1) investigate if HP genotype influences outcome after SAH via its role in Hb scavenging, 2) characterise the kinetics of Hb scavenging in the two week period after SAH, 3) establish whether Hp can reverse Hb neurotoxicity in vitro, and 4) establish and characterise an in vivo model of SAH to translate the results of the in vitro experiments.

Due to differential Hb scavenging function, it was hypothesized that HP genotype impacts on outcome after SAH. 1299 SAH patients were typed for their HP duplication genotype and the single nucleotide polymorphism rs2000999, the largest genetic contributor to Hp expression level. Logistic regression analysis of long term outcome identified no effect of rs2000999 but the Hp duplication affected outcome, implying mediation through Hp function rather than expression level. HP2-2 was associated with favourable outcome in high Fisher grade patients (grades III and IV, Odds Ratio (OR): 2.4, 95% Confidence Intervals (CI) 1.4-.4.3, p=0.007).
To seek further mechanistic evidence for the effect of Hp on outcome after SAH, the kinetics of Hb and Hp was investigated in serial cerebrospinal fluid (CSF) samples from 44 Fisher grade III-IV SAH patients during a two week period post-ictus. Total Hb levels increased gradually, reaching a plateau of 11.5µM (median between days 11 and 13, interquartile range (IQR): 2.1-16.2µM); the majority of this Hb was found to be uncomplexed (median 96.3%, (IQR): 83.3-99.4%, from third day onwards). There was a trend towards an association of high uncomplexed Hb levels with worse outcome (modified Rankin Scale (mRS) at 6 months post-ictus) (OR: 0.7, 95% CI 0.5-1.0, p=0.064). Auto-oxidation of Hb can cause oxidative damage which inhibits complex formation by Hp. Exogenous Hp was added to the CSF, enough to saturate all Hb, to investigate how much Hb was still able to bind Hp; 90.6% (IQR: 65.8- 96.5%) of the uncomplexed Hb was able to form complexes with exogenous Hp. The Hb scavenging ability of Hp isoforms was investigated with linear mixed modelling. Significantly lower levels of uncomplexed Hb were associated with the Hp2-2 phenotype (p=0.03, vs Hp1-1), in keeping with improved Hb scavenging due to Hp2-2’s higher valency for Hb.

To test if Hp can protect against Hb neurotoxicity, primary hippocampal neurones from C57BL/6 mouse pups were cultured and treated with Hb, Hb+Hp, Hp or vehicle. Following a week in culture, viable neurones were counted. Hb produced a dose dependent cytotoxic effect (pSignificantly lower levels of uncomplexed Hb were associated with the Hp2-2 phenotype (p=0.03, vs Hp1-1), in keeping with improved Hb scavenging due to Hp2-2’s higher valency for Hb. To test if Hp can protect against Hb neurotoxicity, primary hippocampal neurones from C57BL/6 mouse pups were cultured and treated with Hb, Hb+Hp, Hp or vehicle. Following a week in culture, viable neurones were counted. Hb produced a dose dependent cytotoxic effect (p=0.003). No adverse effects on neuronal viability were observed with Hp alone.

To translate the in vitro findings, I established in our laboratory an in vivo murine model of SAH, involving a stereotaxic injection of blood into the pre-chiasmatic cistern of C57BL/6 mice. The model was optimized to refine the technique, to prepare for future experiments testing Hp as a therapeutic agent after experimental SAH.