Neuroprotective Effect of Sulfhydryl Reduction in a Rat Optic Nerve Crush Model

Department of Ophthalmology and Visual Science, University of Wisconsin Medical School, Madison, 53792, USA.
Investigative Ophthalmology &amp Visual Science (Impact Factor: 3.4). 11/2005; 46(10):3737-41. DOI: 10.1167/iovs.05-0155
Source: PubMed


The signaling of retinal ganglion cell (RGC) death after axotomy is partly dependent on the generation of reactive oxygen species. Shifting the RGC redox state toward reduction is protective in a dissociated mixed retinal culture model of axotomy. The hypothesis for the current study was that tris(2-carboxyethyl)phosphine (TCEP), a sulfhydryl reductant, would protect RGCs in a rat optic nerve crush model of axotomy.
RGCs of postnatal day 4 to 5 Long-Evans rats were retrogradely labeled with the fluorescent tracer DiI. At approximately 8 weeks of age, the left optic nerve of each rat was crushed with forceps and, immediately after, 4 muL of TCEP (or vehicle alone) was injected into the vitreous at the pars plana to a final concentration of 6 or 60 microM. The right eye served as the control. Eight or 14 days after the crush, the animals were killed, retinal wholemounts prepared, and DiI-labeled RGCs counted. Bandeiraea simplicifolia lectin (BSL-1) was used to identify microglia.
The mean number of surviving RGCs at 8 days in eyes treated with 60 microM TCEP was significantly greater than in the vehicle group (1250 +/- 156 vs. 669 +/- 109 cells/mm(2); P = 0.0082). Similar results were recorded at 14 days. Labeling was not a result of microglia phagocytosing dying RGCs. No toxic effect on RGC survival was observed with TCEP injection alone.
The sulfhydryl-reducing agent TCEP is neuroprotective of RGCs in an optic nerve crush model. Sulfhydryl oxidative modification may be a final common pathway for the signaling of RGC death by reactive oxygen species after axotomy.

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Available from: Christopher R Schlieve, May 01, 2014
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    • "Novel sulfhydryl reducing agents bis(3-propionic acid methyl ester)phenylphosphine borane complex (PB1) and (3-propionic acid methyl ester) diphenylphosphine borane complex (PB2) are able to protect retinal ganglion cells against apoptosis following axonal injury, a superoxide-dependent process [27], without direct scavenging of superoxide. These phosphines are alternatives to thiol drugs, and are structurally similar to the reducing agent tris(2-carboxyethyl) phosphine (TCEP), which is neuroprotective for retinal ganglion cells [17] [28] and photoreceptors [29]. PB1 and PB2 were designed to have low reactivity in the extracellular compartment, high rates of transmembrane diffusion, and a side-group that can be cleaved by intracellular enzymes, resulting in an intracellular accumulation of PB1 or PB2. "
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    ABSTRACT: Exposure to radiation can damage endothelial cells in the irradiated area via the production of reactive oxygen species. We synthesized phosphine-borane complexes that reduce disulfide bonds and had previously been shown to interfere with redox-mediated signaling of cell death. We hypothesized that this class of drugs could interfere with the downstream effects of oxidative stress after irradiation and rescue endothelial cells from radiation damage. Cultured bovine aortic endothelial cells were plated for clonogenic assay prior to exposure to varying doses of irradiation from a (137)Cs irradiator and treated with various concentrations of bis(3-propionic acid methyl ester)phenylphosphine borane complex (PB1) at different time points. The clone-forming ability of the irradiated cells was assessed seven days after irradiation. We compared the radioprotective effects of PB1 with the aminothiol radioprotectant WR1065 and known superoxide scavengers. PB1 significantly protected bovine aortic endothelial cells from radiation damage, particularly when treated both before and after radiation. The radioprotection with 1µM PB1 corresponded to a dose-reduction factor of 1.24. Radioprotection by PB1 was comparable to the aminothiol WR1065, but was significantly less toxic and required much lower concentrations of drug (1µM vs. 4mM, respectively). Superoxide scavengers were not radioprotective in this paradigm, indicating the mechanisms for both loss of clonogenicity and PB1 radioprotection are independent of superoxide signaling. These data demonstrate that PB1 is an effective redox-active radioprotectant for endothelial cells in vitro, and is radioprotective at a concentration approximately 4 orders of magnitude lower than the aminothiol WR1065 with less toxicity. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.
    06/2015; 1. DOI:10.1016/j.redox.2015.06.015
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    • "Inhibiting downstream signaling events, e.g. with redox modulating agents such as TCEP (Almasieh et al., 2011; Swanson et al., 2005) may be necessary for effective neuroprotection. "
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    ABSTRACT: Injury to retinal ganglion cell (RGC) axons within the optic nerve causes apoptosis of the soma. We previously demonstrated that in vivo axotomy causes elevation of superoxide anion within the RGC soma, and that this occurs 1-2 days before annexin-V positivity, a marker of apoptosis. Pegylated superoxide dismutase delivery to the RGC prevents the superoxide elevation and rescues the soma. Together, these results imply that superoxide is an upstream signal for apoptosis after axonal injury in RGCs. We then studied metallocorroles, potent superoxide dismutase mimetics, which we had shown to be neuroprotective in vitro and superoxide scavengers in vivo for RGCs. RGCs were retrograde labeled with the fluorescent dye 4Di-10Asp, and then axotomized by intraorbital optic nerve transection. Iron(III) 2,17-bis-sulfonato-5,10,15-tris(pentafluorophenyl)corrole (Fe(tpfc)(SO(3)H)(2)) (Fe-corrole) was injected intravitreally. Longitudinal imaging of RGCs was performed and the number of surviving RGCs enumerated. There was significantly greater survival of labeled RGCs with Fe-corrole, but the degree of neuroprotection was relatively less than that predicted by their ability to scavenge superoxide-This implies an unexpected complexity in signaling of apoptosis by reactive oxygen species.
    Experimental Eye Research 02/2012; 97(1):31-5. DOI:10.1016/j.exer.2012.02.006 · 2.71 Impact Factor
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    • "The generation of an intracellular superoxide burst is a critical molecular event underlying RGC death after axonal injury (Geiger et al. 2002, Kanamori et al. 2010, Lieven et al. 2006, Nguyen et al. 2003, Swanson et al. 2005). Superoxide increases dramatically in RGCs at the single-cell level, soon after optic nerve axotomy, and precedes RGC apoptosis (Kanamori et al. 2010). "
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    ABSTRACT: J. Neurochem. (2011) 118, 1075–1086. The reactive oxygen species (ROS) superoxide has been recognized as a critical signal triggering retinal ganglion cell (RGC) death after axonal injury. Although the downstream targets of superoxide are unknown, chemical reduction of oxidized sulfhydryls has been shown to be neuroprotective for injured RGCs. On the basis of this, we developed novel phosphine-borane complex compounds that are cell permeable and highly stable. Here, we report that our lead compound, bis (3-propionic acid methyl ester) phenylphosphine borane complex 1 (PB1) promotes RGC survival in rat models of optic nerve axotomy and in experimental glaucoma. PB1-mediated RGC neuroprotection did not correlate with inhibition of stress-activated protein kinase signaling, including apoptosis stimulating kinase 1 (ASK1), c-jun NH2-terminal kinase (JNK) or p38. Instead, PB1 led to a striking increase in retinal BDNF levels and downstream activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway. Pharmacological inhibition of ERK1/2 entirely blocked RGC neuroprotection induced by PB1. We conclude that PB1 protects damaged RGCs through activation of pro-survival signals. These data support a potential cross-talk between redox homeostasis and neurotrophin-related pathways leading to RGC survival after axonal injury.
    Journal of Neurochemistry 07/2011; 118(6):1075-86. DOI:10.1111/j.1471-4159.2011.07382.x · 4.28 Impact Factor
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