Necroptosis in a murine repeated primary blast injury model

Abstract

Purpose : Repeated primary blast injury (rPBI) causes visual dysfunction and degeneration. Necroptosis (regulated necrosis) promotes neuronal death in trauma, neurodegeneration and in ocular disease and involves interactions between receptor interacting protein kinase (RIPK)-1,-3 and mixed lineage kinase domain-like (MLKL). RIPK1 and RIPK3 are elevated after a single 26 psi blast and necroptosis promotes retinal ganglion cell (RGC) death in blunt ocular injury. We aimed to determine if necroptosis promotes RGC death and ON degeneration in a murine rPBI model and if inhibition using Necrostatin-1 stable (Nec-1s) was protective.

Methods : We exposed the left eye of anesthetised C57Bl/6 mice to repeated blast of overpressure air (2x15 psi blasts in quick succession, 24 hours apart over 3 days). Sham mice were exposed to anaesthetics but not the blast wave. Nec-1s (2µl of 3.6mM Nec-1s in DMSO and 0.09% methyl-β-cyclodextrin) or vehicle control were intravitreally injected 48 hours before blast and repeated every 7 days and mice culled at 28 days post injury (dpi) (n=10 per group). Eyes were cryosectioned and RBPMS⁺ immunostained RGC quantified. Far proximal ON was resin embedded, sectioned, and stained with p-phenylenediamine and toluidine blue. Total healthy and degenerating axons were counted. The remaining ON was cryosectioned longitudinally and immunostained. Retinal optical coherence tomography (OCT) imaging was performed throughout the experiment and % of eyes with vitreal inflammation quantified.

Results : There were no differences in RBPMS⁺ RGC between sham and rPBI, but a reduction with combined rPBI and intravitreal injections. Nec-1s provided neuroprotection compared to vehicle (13.08 ± 0.81 compared to 10.29 ± 0.37 RBPMS⁺ cells/mm of retina). OCT images showed more vitreal inflammation in eyes receiving blast and injection combination (0% in sham and rPBI compared to 83% in vehicle and 88% in Nec-1s). The direct blast left eye had more inflammation compared to contralateral eye (33% in vehicle and 11% in Nec-1s). There were increased degenerating axons after rPBI which were not affected by Nec-1s. ED1⁺ macrophages in the ON increased after rPBI and remained elevated with Nec-1s.

Conclusions : Necroptosis might have a role in RGC death after rPBI. Intravitreal injections in combination with direct blast exposure increased vitreal inflammation and RGC death, suggesting an alternative administration for therapy would be preferable.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.