Abstract
Abstract: :
Purpose: Free radical regulation is essential for retinal ganglion cell (RGC) survival. A key mitochondrial anti–oxidant, manganese superoxide dismutase (MnSOD), is critical for neuronal management of oxidative stress. Our previous work has demonstrated that injury to optic nerve up–regulates MnSOD mRNA in RGCs. Changes in MnSOD protein expression with respect to retinal ganglion axonal crush–injury is presently investigated. Methods: Litters of postnatal day 3 (P3) neonatal Sprague–Dawley rat pups were anesthetized before undergoing survival surgery, adhering to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Half of the litter was used as control (no crush) and the other half as experimental (optic nerve crush). Animals were allowed to survive for five days. On P8, the crush–injured neonatal rat pup group along with the controls were sacrificed. Optic nerves were then collected and fixed with 4% paraformadehyde. Immunohistochemistry using antibodies specific for mammalian MnSOD and neurofilament was carried out on 8um sections. Sections were visualized using confocal microscopy and the images were recorded digitally for analysis. Results: The crush–injury of optic nerve induced a generalized upregulation of MnSOD protein localization in RGC axons, as compared to normals. Furthermore, MnSOD signal was intense at discrete foci throughout the RGC axons as demarcated by neurofilament staining. Conversely, MnSOD in the normal group exhibited fairly uniform low intensity signal throughout the length of the optic nerve, without the intense foci seen in the crush–injury group. Meanwhile, the crush–injury group showed an overall reduction in neurofilament staining, compared to the control group. This reduction may indicate the onset of neuronal response to injury. Conclusions: In RGC axons of the optic nerve, MnSOD protein expression exhibits sensitivity to nerve injury. The changes in MnSOD expression seem to complement previously reported increases and subsequent decreases in MnSOD gene transcription in the days following the crush injury. The intense foci of MnSOD localization in the crused axons may suggest either regenerating RGC axons struggling to survive or dying RGC axons in the process of succumbing to apoptosis. Lending support to an ongoing hypothesis, these findings suggest that a critical role exists for MnSOD function in RGC survival after injury.
Keywords: neuro–ophthalmology: optic nerve • ganglion cells • microscopy: light/fluorescence/immunohistochemistry