Is acid sensing ion channel 1a a viable therapeutic target for spinal cord injury?

Abstract

Traumatic spinal cord injury (SCI) can result in severe sensory and motor deficits. In the hours to days following SCI, secondary injury mechanisms are triggered that cause inflammation and cell death in surrounding tissues. A key component of this secondary damage is a reduction in blood flow at the spinal cord which initiates a well-characterised ischaemic cascade. Downstream hypoxia and acidosis activate acid sensing ion channel 1a (ASIC1a), resulting in increased ASIC1a-mediated flux of calcium and sodium ions into neurons, which in the brain triggers RIP1-kinase-mediated necroptotic cell death (Wang et al., 2015). Inhibition of ASIC1a using venom-peptide PcTx1 reduces anatomical and functional damage after ischaemic stroke (McCarthy et al., 2015) and provides moderate tissue sparing after SCI (Koehn et al., 2016). An even more potent peptidic ASIC1a inhibitor, Hi1a, was subsequently shown to provide greater improvement in tissue sparing, functional recovery and a longer clinical time window after stroke compared to PcTx1 (Chassagnon et al., 2017).With a view towards assessing Hi1a as a useful therapeutic in treatment of SCI, this thesis aimed to determine whether blockade of ASIC1a impacts cellular and functional outcomes after SCI. To this end, using SCI mouse models, this thesis utilised: (1) behavioural analyses, (2) immuno- histochemistry (IHC) and immunofluorescence (IF), and (3) whole-cell patch-clamp electrophysiology. For all the SCI-treatment studies, intravenous administration of Hi1a at 1 h post injury was used in an attempt to mimic a clinically relevant intervention. ASIC1a–/– mice were also used to explore behavioural recovery post-SCI.Pharmacological blockade of ASIC1a with Hi1a (25 µg, i.v.) at 1 day post-operation (DPO) in a contusion SCI model resulted in a transient but significant reduction in neurons rostral to the lesion site compared to saline-treated control animals (which equalised at 7 DPO). No significant changes were seen in the extent of neutrophil infiltration or blood–spinal cord barrier integrity at both 1 and 7 DPO between cohorts. However, significant myelin sparing was seen during this subacute phase, suggesting ASIC1a inhibition impacts oligodendrocytes. Behaviourally, Hi1a-treated animals showed no significant improvement in a short-term study (7 DPO) with either 2 ng/kg or 25 µg Hi1a. Consistent with the pharmacological data, we found that genetic ablation of ASIC1a led to no significant behavioural improvement in a long-term study (42 DPO) of mice that received a contusion SCI.Mice treated with Hi1a (25 µg, i.v.) after a hemisection SCI exhibited a significant reduction in half-width, rise and decay time of spontaneous excitatory post synaptic currents (sEPSCs) in a key population of deep dorsal horn interneurons. These data revealed an alteration in the time course of individual events that points towards Hi1a altering glutamatergic receptor expression in the postsynaptic membrane. However, IHC analysis revealed no significant difference in lesion volume between naive and Hi1a-treated mice at 3 DPO, consistent with the lack of behavioural improvement seen in mice that received Hi1a after spinal cord contusion injury.In summary, our genetic and pharmacological studies suggest that ASIC1a inhibitors are unlikely to be beneficial in the treatment of SCI, as Hi1a treatment or genetic ablation of ASIC1a provided no improvement in functional recovery nor reduction in lesion size after SCI.