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Caspase-2 Mediates Site-Specific Retinal Ganglion Cell Death After Blunt Ocular Injury

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Caspase-2 Mediates Site-Specific Retinal Ganglion Cell Death After Blunt Ocular Injury Retinal Cell Biology Caspase-2 Mediates Site-Specific Retinal Ganglion Cell Death After Blunt Ocular Injury Chloe N. Thomas,1 Adam M. Thompson,1 Eleanor McCance,1 Martin Berry,1 Ann Logan,1 Richard J. Blanch,1,2 and Zubair Ahmed1 1Neuroscience and Ophthalmology, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United Kingdom 2Academic Department of Military Surgery and Trauma, Royal Centre for Defence Medicine, Birmingham, United Kingdom Correspondence: Zubair Ahmed, PURPOSE. Ocular trauma is common in civilian and military populations. Among other injuries, Neuroscience and Ophthalmology, closed globe blunt ocular trauma causes acute disruption of photoreceptor outer segments Institute of Inflammation and Age- (commotio retinae) and retinal ganglion cell (RGC) death (traumatic optic neuropathy ing, College of Medical and Dental [TON]), both of which permanently impair vision. Caspase-2-dependent cell death is Sciences,Room386,RobertAitken important and evidenced in models of RGC degeneration. We assessed the role of caspase-2 as Institute of Clinical Research, Uni- versity of Birmingham, Edgbaston, a mediator of RGC and photoreceptor death in a rat blunt ocular trauma model. Birmingham B15 2TT, UK; METHODS. Bilateral ballistic closed globe blunt ocular trauma was induced in female Lister- [email protected]. hooded rats and caspase-2 cleavage and localization assessed by Western blotting and RJB and ZA are joint senior authors. immunohistochemistry. Retinal caspase-2 was knocked down by intravitreal injection of Submitted: February 8, 2018 caspase-2 small interfering RNA (siCASP2). In retinal sections, RGC survival was assessed by Accepted: August 1, 2018 BRN3A-positive cell counts and photoreceptor survival by outer nuclear layer (ONL) thickness, respectively. Retinal function was assessed by electroretinography (ERG). Citation: Thomas CN, Thompson AM, McCance E, et al. Caspase-2 mediates RESULTS. Raised levels of cleaved caspase-2 were detected in the retina at 5, 24, and 48 hours site-specific retinal ganglion cell death after injury and localized to RGC but not photoreceptors. Small interfering RNA–mediated after blunt ocular injury. Invest Oph- caspase-2 knockdown neuroprotected RGC around but not in the center of the injury site. In thalmol Vis Sci. 2018;59:4453–4462. addition, caspase-2 knockdown increased the amplitude of the ERG photopic negative https://doi.org/10.1167/iovs.18-24045 response (PhNR) at 2 weeks after injury. However, siCASP2 was not protective for photoreceptors, suggesting that photoreceptor degeneration in this model is not mediated by caspase-2. CONCLUSIONS. Caspase-2 mediates death in a proportion of RGC but not photoreceptors at the site of blunt ocular trauma. Thus, intravitreally delivered siCASP2 is a possible therapeutic for the effective treatment of RGC death to prevent TON. Keywords: traumatic optic neuropathy, commotion retinae, retinal ganglion cells, caspase-2, photoreceptors cular injuries are common, occurring in up to 10% of all there are currently no effective treatments to preserve or O military casualties1 and with a lifetime prevalence of restore vision.19,20 After blunt ocular trauma, initiator 20% in civilian populations.2 Traumatic optic neuropathy caspase-9 is activated and initiates localized photoreceptor (TON) occurs with an annual incidence of 1/1,000,000 in the death, which can be attenuated in the lesion penumbra of civilian population,3 but occurs in up to 20% of military eye animal models by caspase-9 inhibition.21 However, the injuries.4 mechanisms of the accompanying RGC death in this model TON is defined as retinal ganglion cell (RGC) death and have not been defined and no treatment has been shown to axon degeneration caused by head or eye injury.5 RGCs neuroprotect RGCs after blunt ocular trauma.22 populate the inner retina and their axons form the optic RGCs die by caspase-dependent mechanisms as part of nerve (ON). RGCs are central nervous system (CNS) neurons normal development, degenerative disease, and after ON that lack an endogenous regenerative capacity. Thus, after trauma.23 Caspases are cysteine aspartate proteases that induce injury or disease, lost RGCs are not replaced, and the apoptosis through initiator and executioner family members. damaged ON does not regenerate, leading to irreversible Initiator caspases (2, 8, 9, and 10) activate executioner caspases visual loss.6–8 The ON may be injured either directly (e.g., (3, 6, and 7) through catalytic cleavage of their activation penetrating ocular injury, bony fragment damage within the domain.24–26 Caspase-2 is not part of the canonical intrinsic optic canal, and ON sheath hematomas) or indirectly after (caspase-9–mediated) or extrinsic (caspase-8–mediated) apo- traumatic head or eye injury (e.g., blunt eye injury and ptotic pathways, is highly evolutionarily conserved and can be blast).9–12 Blunt ocular trauma also damages other retinal activated by DNA damage, heat shock, endoplasmic reticulum cells, causing commotio retinae, characterized by photore- stress, and oxidative stress.27–31 Caspase-2 is activated by ceptor degeneration.13 TON can be studied using animal cleavage and subsequent dimerization; it is therefore possible models replicating blunt and blast ocular injuries14,15 and ON to use the presence of cleaved caspase-2 as a marker for its crush (ONC).16–18 TON causes permanent visual loss and activation.32 Copyright 2018 The Authors iovs.arvojournals.org j ISSN: 1552-5783 4453 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from iovs.arvojournals.org on 09/26/2021 Caspase-2 in Blunt Ocular Injury IOVS j Month 2018 j Vol. 59 j No. 0 j 4454 FIGURE 1. Experimental design of (A) in vivo protein and IHC studies and (B) in vivo siCASP2 studies. We have previously shown that RGC death after ONC is MATERIALS AND METHODS caspase-2–mediated. For example, axotomized RGC activate caspase-2, whereas pharmacological inhibition protects ~60% Experimental Design of RGC for up to 21 days after ONC and a chemically modified To determine the role of caspase-2 in RGCs and photoreceptors synthetic short interfering RNA (siRNA) against caspase-2 after blunt eye injury, groups of rats were subjected to (siCASP2) protects >95% of RGCs for up to 12 weeks.33–36 unilateral or bilateral blunt eye injury under anaesthesia as a siCASP2 also protects RGC in a mouse optic neuritis model.37 recovery procedure. To determine caspase-2 cleavage and siCASP2 (also known as QPI-1007) is currently in clinical trials protein localization, Western blotting and immunohistochem- for nonarteritic ischemic optic neuropathy (NAION) (protocol: istry (IHC) were performed at 5, 24, and 48 hours after injury QRK007 NCT01064505) and acute primary angle-closure (Fig. 1A). siCASP2 was used to knockdown caspase-2 and glaucoma (protocol: QRK208 NCT01965106) with Quark electroretinography (ERG) and cell counting in retinal sections Pharmaceuticals (Ness Ziona, Israel). Caspase-2 is also activated used to determine RGC and photoreceptor survival and and induces neuronal degeneration after spinal cord injury restitution of function (Fig. 1B). (SCI)38 and in Alzheimer’s disease.39,40 In this study, we have discriminated between the caspase-9– Animal Care and Procedures mediated photoreceptor loss and RGC death by demonstrating that caspase-2 mediates the RGC but not photoreceptor Animal procedures were licensed by the UK Home Office, degeneration in a rat blunt ocular injury model, and that approved by the University of Birmingham’s Animal Welfare siCASP2 structurally and functionally protected RGCs but not and Ethical Review Committees and conducted in accordance photoreceptors. with the ARVO Statement for the Use of Animals in Ophthalmic Downloaded from iovs.arvojournals.org on 09/26/2021 Caspase-2 in Blunt Ocular Injury IOVS j Month 2018 j Vol. 59 j No. 0 j 4455 TABLE. Antibodies Used in IHC and Western Blotting Antigen Dilution Supplier Catalog No. To Identify Caspase-2 (H-19) 1:500 (WB); Santa Cruz Biotechnology SC-623 Full-length and cleaved (Santa Cruz, CA, USA) fragments b-actin 1:10,000 (WB) Sigma (Poole, UK) A5441 Loading control Caspase-2 1:200 (IHC) Abcam (Cambridge, UK) AB2251 Full-length caspase-2 BRN3A (C-20) 1:200 (IHC) Santa Cruz SC-31984 RGC-specific transcription factor Donkey anti-goat IgG Alexa Fluor 594 1:400 (IHC) Invitrogen, (Paisley, UK) A11058 Goat IgG Donkey anti-rabbit IgG Alexa Fluor 488 1:400 (IHC) Invitrogen A-21206 Rabbit IgG Donkey anti-rabbit IgG Alexa Fluor 594 1:400 (IHC) Invitrogen A-21207 Rabbit IgG Anti-rabbit IgG, HRP-linked 1:1,000 (WB) Cell Signaling Technology 7074S Rabbit IgG (Danvers, MA, USA) Anti-mouse IgG, HRP-linked 1:1,000 (WB) Cell Signaling Technology 7076S Mouse IgG (Danvers, MA, USA) HRP, horseradish peroxidase; WB, Western blot. and Vision Research. A total of 68 animals were used in this sucrose (10%, 20%, 30%) in PBS at 48C before embedding in study (Fig. 1). Female Lister-hooded rats weighing 170 to 200 g optimum cutting temperature medium and storage at À808C. were purchased from Charles River Laboratories (Margate, Sections were cut in a plane parallel to that running between UK), kept on a 12-hour light-dark cycle with a daytime the center of the injury site and the optic disc (Supplementary luminance of 80 lux and fed and watered ad libitum. Surgery Fig. 1) at a thickness of 15 lm using a cryostat (Brights and ERG recording were performed under inhalational Instruments, Huntingdon, UK) and adhered onto Superfrost anaesthesia with 3% isoflurane in oxygen (2% for ERG studies). (Fisher Scientific, Loughborough, UK) coated glass microscope Blunt injury was induced as previously described,41 using a slides and stored at À208C until required. 0.095-g spherical plastic pellet fired using compressed air to directly impact the inferior scleral surface at a speed of 20 m/s.
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