Inhibition of the sodium calcium exchanger NCX1 reduces secondary axotomy and improves function after experimental stretch injury to the rat sciatic nerve

Introduction: Peripheral nerve injuries (PNIs) cause significant patient morbidity. However, there exist no approved therapeutics, in part due to an incomplete understanding of the acute mechanisms of peripheral axon degeneration after injury. Here, using a clinically relevant rat model of sciatic nerve stretch injury, we seek to show that secondary axotomy – a key mechanism of delayed axonal degeneration in the injured central nervous system – occurs after stretch PNI. We also characterize the role of the sodium calcium exchanger NCX1 in axon degeneration after experimental PNI.

Methods: Male rats (~350g) were submitted to the recently described rapid stretch model of sciatic nerve injury. To characterize protein expression, nerves were submitted to immunohistochemistry and immunoblot for BAPP and NCX1. BAPP+ axonal swellings were stereologically quantified. Rats were treated with vehicle or SEA0400, a specific and potent small molecule inhibitor of NCX1. Limb function was compared between groups with accelerating Rotarod assay.

Results: To determine if secondary axotomy occurs after PNI, stretch-injured nerves were processed for immunohistochemistry against BAPP, a marker of secondary axotomy. Injured nerves showed widespread formation of BAPP+ axonal swellings, consistent with secondary axotomy. On immunolabeling and immunoblot, injured nerves exhibited expression of NCX1, which co-localized to BAPP+ axonal swellings. NCX1 was previously found to mediate axon degeneration in vitro but has not been investigated in axon degeneration in vivo. To determine if NCX1 worsens injury after PNI, rats were treated with vehicle or SEA0400, a potent and specific small molecule inhibitor of NCX1. SEA0400 reduced the incidence of BAPP+ axonal swellings on immunohistochemistry and reduced accumulation of BAPP on immunoblot after injury, consistent with a reduction in secondary axotomy. Importantly, SEA0400 also improved limb function on accelerating Rotarod assay in animals followed up to 1 month after nerve injury.

Conclusion : Our results suggest that secondary axotomy and delayed axonal degeneration may be relevant to PNI, which carries important implications for the management of PNIs. Furthermore, we identify NCX1 as a potential new druggable target to attenuate damage after PNI.