Retinal Ganglion Cells Die by Necroptotic Mechanisms in a Site-Specific Manner in a Rat Blunt Ocular Injury Model.

Chloe N. Thomas,Adam M. Thompson,Richard J. Blanch,Zubair Ahmed

doi:10.3390/cells8121517

Abstract

Closed-globe injury can cause visual loss in military and civilian populations, with retinal cell death, including retinal ganglion cell (RGC) degeneration, leading to irreversible blindness. RGC and optic nerve (ON) degeneration after eye or head injury is termed traumatic optic neuropathy (TON). There are currently no treatments for RGC loss, therefore novel therapeutics to prevent RGC death or promote axonal regeneration are a priority. We investigated necroptotic signaling mechanisms in a rat blunt ocular injury model. After bilateral blunt trauma, protein expression and retinal localization of necroptosis pathway members (receptor interacting protein kinase 1, RIPK1; receptor interacting protein kinase 3, RIPK3; and mixed lineage kinase domain like pseudokinase, MLKL) were assessed by Western blot and immunohistochemistry (IHC), and potent necroptosis inhibitor Necrostatin-1s (Nec-1s) was delivered by intravitreal injection to one eye and vehicle to the contralateral eye. RGC and photoreceptor survival were assessed by cell counting and outer nuclear layer (ONL) thickness measurements on histology. The neuroprotective effects of Nec-1s were assessed in primary retinal culture by βIII-tubulin+ RGC cell counts. MLKL protein expression were upregulated at 48 h after injury and MLKL immunolocalised to retinal binding protein with multiple splice (RBPMS)+ RGC, inner nuclear cells and ONL cells, specifically at the retinal injury site. RIPK3 expression did not increase but RIPK3 co-immunolocalised with RBPMS+ RGC in intact and injured retinae. In vitro, a Nec-1s concentration of 0.01 pg/µL was RGC neuroprotective. In the blunt ocular injury rat model, Nec-1s prevented RGC death at the center of the impact site but did not protect against ONL thinning or provide functional restitution. RGC degeneration in our blunt ocular injury model is site-specific, with necroptosis driving death at the center of the focal impact site.

Highlights

Ocular injuries are common, occurring in 10% of all military casualties [1] and have a lifetime prevalence of 20% in the civilian population [2]
We first determined if the expression of key necroptotic proteins, Receptor-interacting protein kinase 1 (RIPK1) and receptorinteracting interactingprotein proteinkinase kinase 33 (RIPK3) as well as downstream mixed lineage kinase domain-like (MLKL), were altered in our blunt ocular trauma model by performing bilateral blunt ocular injury collecting retinal tissue for immunofluorescent staining and Western blot at 5, 24 and 48 h after injury, as well as from separate intact control animals
We further show that treatment with a necroptosis inhibitor, Nec-1s, was neuroprotective to retinal ganglion cells (RGC) in vitro and prevented RGC loss at the center of the impact site in a rat model of closed-globe blunt ocular injury in vivo

Summary

Introduction

Ocular injuries are common, occurring in 10% of all military casualties [1] and have a lifetime prevalence of 20% in the civilian population [2]. The retina is an extension of the central nervous system (CNS), which means that if retinal cells, including photoreceptors and RGC, are injured and lost through disease or injury, they are not endogenously replaced. Their preservation is important to prevent irreversible loss of vision. Traumatic optic neuropathy (TON) can be caused by head or eye injury and is associated with RGC and axonal degeneration, causing irreversible blindness [3,4]. TON can be studied using animal models replicating blunt and blast ocular injuries [12,13,14,15] and ON crush (ONC) [16,17,18]

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