Optic neuropathy is the leading cause of irreversible blindness, and a paradigm for central nervous system axonal disease. The primary event is damage to retinal ganglion cell axons, with subsequent death of the cell body by apoptosis. Trials of neuroprotection for these and other neuronal diseases have mostly failed, primarily because mechanisms of neuroprotection in animals do not necessarily translate to humans. We developed a methodology for imaging an intracellular transduction pathway that signals neuronal death in the living animal. Using longitudinal confocal scanning multilaser ophthalmoscopy, we identified the production of superoxide within retrograde-labelled rat retinal ganglion cells after optic nerve transection. Superoxide was visualized by real-time imaging of its reaction product with intravitreally administered hydroethidine and confirmed by differential spectroscopy of the specific product 2-hydroxyethidium. Retinal ganglion cell superoxide increased within 24 h after axotomy, peaking at 4 days, and was not observed in contralateral untransected eyes. The superoxide signal preceded phosphatidylserine externalization, indicating that superoxide generation was an early event and preceded apoptosis. Intravitreal pegylated superoxide dismutase blocked superoxide generation after axotomy and delayed retinal ganglion cell death. Together, these results are consistent with superoxide being an upstream signal for retinal ganglion cell apoptosis after optic nerve injury. Early detection of axonal injury with superoxide could serve as a predictive biomarker for patients with optic neuropathy.

Corroles are tetrapyrrolic macrocycles that have come under increased attention because of their unique capabilities for oxidation catalysis, reduction catalysis, and biomedical applications. Corrole-metal complexes (metallocorroles) can decompose certain reactive oxygen species (ROS), similar to metalloporphyrins. We investigated whether Fe-, Mn-, and Ga-corroles have neuroprotective effects on neurons and correlated this with superoxide scavenging activity in vitro and in vivo. Apoptosis was induced in retinal ganglion cell-5 neuronal precursor cells by serum deprivation. Cell death was measured with sodium 3¢-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene-sulfonic acid hydrate and calcein-AM/propidium iodide assays. Fe-and Mn-corroles, but not the non-redox-active Ga-corrole used as control, reduced RGC-5 cell death after serum deprivation.Serum deprivation caused increased levels of intracellular superoxide, detected by an increase in the fluorescence intensity of 2-hydroxyethidium, and this was blocked by Feand Mn-corroles, but not Ga-corrole. In vivo real-time confocal imaging of retinas after optic nerve transection assessed the superoxide production within individual rat retinal ganglion cells. Fe-and Mn-corroles, but not Ga-corrole, scavenged neuronal superoxide in vivo. Given that the neuroprotective activity of metallocorroles correlated with superoxide scavenging activity, Fe-and Mn-corroles could be candidate drugs for delaying neuronal death after axonal injury in optic neuropathies, such as glaucoma. Keywords: metallocorroles, neuroprotection, neurons, superoxide, optic neuropathy. J. Neurochem. (2010) 114, 488-498. JOURNAL OF NEUROCHEMISTRY | 2010 | 114 | 488-498 doi: 10.1111/j.1471-4159.2010 et al. 2010). Over the past decade they have received increasing attention, following the major discoveries for their facile synthesis (Gross and Galili 1999;Gryko 2002). Porphyrins have been used in numerous applications, including the use of metalloporphyrins as neuroprotective agents that work via decomposition of ROS (Cuzzocrea et al. 2001). We predicted that metallocorroles, some of which can be highly specific ROS scavengers (reviewed in Table 1), might be useful small molecule drugs for inhibiting ROS-mediated signaling of cell death.To study this, we investigated the neuroprotective effect of iron (Fe), manganese (Mn), and gallium (Ga) corroles ( Fig. 1) in both in vitro and in vivo models of neuronal death. For the in vitro studies, we used serum-deprived neuronal precursor retinal ganglion cell (RGC)-5 cells, which when differentiated with low levels of staurosporine, have several features similar to that of mature neurons (Frassetto et al. 2006). For in vivo studies, we used confocal scanning laser ophthalmoscopy (CSLO) coupled with intravitreal fluorescent dyes to demonstrate that certain metallocorroles function as SOD mimetics. MethodsMaterials RGC-5 cells were a generous gift of Neeraj Agarwal, Ph.D.; Dulbecco's modified Eagle's medium and fetal bovine s...

Axonal injury and degeneration are pivotal pathological events in diseases of the nervous system. In the past decade, it has been recognized that the process of axonal degeneration is distinct from somal degeneration and that axoprotective strategies may be distinct from those that protect the soma. Preserving the cell body via neuroprotection cannot improve function if the axon is damaged, because the soma is still disconnected from its target. Therefore, understanding the mechanisms of axonal degeneration is critical for developing new therapeutic interventions for axonal disease treatment. We combined in vivo imaging with a multilaser confocal scanning laser ophthalmoscope and in vivo axotomy with a diode-pumped solid-state laser to assess the time course of Wallerian and retrograde degeneration of unmyelinated retinal ganglion cell axons in living rats for 4 weeks after intraretinal axotomy. Laser injury resulted in reproducible axon loss both distal and proximal to the site of injury. Longitudinal polarization-sensitive imaging of axons demonstrated that Wallerian and retrograde degeneration occurred synchronously. Neurofilament immunostaining of retinal whole-mounts confirmed axonal loss and demonstrated sparing of adjacent axons to the axotomy site. In vivo fluorescent imaging of axonal transport and photobleaching of labeled axons demonstrated that the laser axotomy model did not affect adjacent axon function. These results are consistent with a shared mechanism for Wallerian and retrograde degeneration.