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.

Chronic deficiency of vitamin B is the only nutritional deficiency definitively proved to cause optic neuropathy and loss of vision. The mechanism by which this occurs is unknown. Optic neuropathies are associated with death of retinal ganglion cells (RGCs), neurons that project their axons along the optic nerve to the brain. Injury to RGC axons causes a burst of intracellular superoxide, which then signals RGC apoptosis. Vitamin B (cobalamin) was recently shown to be a superoxide scavenger, with a rate constant similar to superoxide dismutase. Given that vitamin B deficiency causes an optic neuropathy through unknown mechanisms and that it is a potent superoxide scavenger, we tested whether cobalamin, a vitamin B vitamer, would be neuroprotective in vitro and in vivo. We found that cobalamin scavenged superoxide in neuronal cells in vitro treated with the reduction-oxidation cycling agent menadione. In vivo confocal scanning laser ophthalmoscopy demonstrated that optic nerve transection in Long-Evans rats increased superoxide levels in RGCs. The RGC superoxide burst was significantly reduced by intravitreal cobalamin and resulted in increased RGC survival. These data demonstrate that cobalamin may function as an endogenous neuroprotectant for RGCs through a superoxide-associated mechanism.

Axonal degeneration is a pathophysiological mechanism common to several neurodegenerative diseases. The slow Wallerian degeneration (Wld S ) mutation, which results in reduced axonal degeneration in the central and peripheral nervous systems, has provided insight into a redox-dependent mechanism by which axons undergo self-destruction. We studied early molecular events in axonal degeneration with single-axon laser axotomy and time-lapse imaging, monitoring the initial changes in transected axons of purified retinal ganglion cells (RGCs) from wild-type and Wld S rat retinas using a polarity-sensitive annexin-based biosensor (annexin B12-Cys101,Cys260-N,NЈ-dimethyl-N-(iodoacetyl)-NЈ-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylenediamine). Transected axons demonstrated a rapid and progressive change in membrane phospholipid polarity, manifested as phosphatidylserine externalization, which was significantly delayed and propagated more slowly in axotomized Wld S RGCs compared with wild-type axons. Delivery of bis(3-propionic acid methyl ester)phenylphosphine borane complex, a cell-permeable intracellular disulfide-reducing drug, slowed the onset and velocity of phosphatidylserine externalization in wild-type axons significantly, replicating the Wld S phenotype, whereas extracellular redox modulation reversed the Wld S phenotype. These findings are consistent with an intra-axonal redox mechanism for axonal degeneration associated with the initiation and propagation of phosphatidylserine externalization after axotomy.

Purpose. Optic nerve injury causes loss of retinal ganglion cells (RGCs) and their axons. The reduction in RGC counts over time in axonal injury is well studied, but the correlation with the timing of anterograde and retrograde axonal degeneration is less clear. The authors longitudinally imaged RGC axons stained with a chloromethyl derivative of fluorescein diacetate (CMFDA) in live rats after optic nerve injury. Methods. Optic nerves were transected. Three days later CMFDA was intravitreously injected. Confocal scanning laser ophthalmoscopy was performed daily, and mean fluorescence intensity and the number of CMFDA bundles were calculated. RGC soma survival was studied after retrograde fluorescence labeling. Retinal nerve fiber layer (RNFL) thickness was evaluated histologically. Results. CMFDA-positive RGC axon bundles could be imaged in vivo. Axons lost 68% +/- 29% of their fluorescence by 7 days after transection compared with 25% +/- 21% in nontransected eyes. The number of labeled axon bundles decreased by 61% +/- 28% at 7 days after transection compared with 26% +/- 9% in nontransected eyes. The number of retrograde-labeled RGCs detected in vivo declined by 53% at 7 days and by 76% at 14 days after transection. RGC soma and CMFDA axon counts decreased most rapidly between 5 and 7 days after transection. Histologic examination demonstrated a reduction in RNFL thickness 7 days after transection. Conclusions. Intravitreal CMFDA can be used to longitudinally monitor RGC axons within the RNFL in vivo. Imaging the disappearance of retrograde-labeled RGC somas and axons indicates that axonal and somal degeneration occur in parallel after axotomy.

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 neuro-protective 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)(SO3H)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.