Abstract
purpose. 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.
methods. 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 μL of TCEP (or vehicle alone) was injected into the vitreous at the pars plana to a final concentration of 6 or 60 μM. 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.
results. The mean number of surviving RGCs at 8 days in eyes treated with 60 μM TCEP was significantly greater than in the vehicle group (1250 ± 156 vs. 669 ± 109 cells/mm2; 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.
conclusions. 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.
Diseases of the optic nerve (optic neuropathies) are a cause of blindness in millions of people. Optic neuropathies as diverse as glaucoma, optic neuritis, and traumatic optic neuropathy have as a common feature the eventual death of retinal ganglion cells (RGCs) in the eye.
1 Death commonly occurs through apoptosis, an orderly cell suicide process, and, in most cases, optic neuropathies are initiated by damage to RGC axons.
2 3 4 RGC axotomy results in apoptosis.
2 3 4 5 Results from our laboratory suggest that reactive oxygen species (ROS) are part of the signaling pathway in cell death after axonal injury (Lieven CJ, et al.
IOVS 2003;44:ARVO E-Abstract 835).
6 7 8 9 We previously demonstrated that RGC survival after axotomy depends critically on the redox state of the cell and that shifting the redox state toward mild reduction is protective in a dissociated mixed retinal culture model. In particular, tris(2-carboxyethyl)phosphine (TCEP), a sulfhydryl reductant that does not contain oxidizable sulfhydryls, maintained long-term survival of RGCs as potently as neurotrophic factors in mixed retinal culture.
8 Because of these findings, we hypothesized that the oxidative modification of sulfhydryl-containing proteins by ROS is a mechanism for signaling apoptosis. However, it is also possible that sulfhydryl reduction neuroprotection arises from the mechanical effects of enzymatic dissociation in the preparation of mixed retinal cultures, not from axotomy. To distinguish these possibilities, we tested whether the potent reducing agent TCEP would protect RGCs in a rat optic nerve crush model of axotomy. We found that not only was TCEP neuroprotective in an in vivo model of RGC axonal damage, but it also prevented sulfhydryl oxidation in a cell-free assay.
The fluorescent tracer 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) was obtained from Molecular Probes (Eugene, OR). Balanced saline solution and triple antibiotic ophthalmic ointment were obtained from Wilson Ophthalmic (Mustang, OK). Fluorescein-conjugated Bandeiraea simplicifolia lectin I (BSL-I) was obtained from Vector Laboratories (Burlingame, CA). Paraformaldehyde and Triton X-100 were obtained from Fisher Scientific (Pittsburgh, PA). TCEP, along with all other reagents unless otherwise noted, was obtained from Sigma-Aldrich (St. Louis, MO).
Effect of Intravitreal Injection of TCEP on the Survival of RGCs after Optic Nerve Crush Compared with Vehicle Alone