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G. Chidlow, J. P. M. Wood, R. J. Casson; Visualisation of Optic Nerve Damage in Rat Models of Retinal Ganglion Cell Injury. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4352.
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Optic nerve damage in experimental models of retinal ganglion cell injury is traditionally identified by light microscopic evaluation of transverse sections of the optic nerve. This methodology is labour intensive and primarily affords information relating to axon number. In the current study, we made use of three distinct animal models of retinal ganglion cell death with the aim of identifying reliable, unambiguous, qualitative markers of optic nerve injury using longitudinal sections of the optic nerve head and optic nerve. Of particular interest was the usefulness of the fluorescent dye fluoro-jade C (FJC), which stains degenerating neurons in the brain.
Adult rats received one of the following insults: (1) intravitreal injection of 30nmol NMDA into the right eye. The left eye served as a control; (2) permanent bilateral occlusion of the common carotid arteries. Sham animals received the same operation without occlusion of the vessels; (3) Optic nerve transection of the right eye. The left eye served as a control. Rats were killed at various time points. Eyes with optic nerve attached were carefully dissected, fixed in buffered formalin, embedded in paraffin and 5µm thick sections taken. Sections were then processed for immunohistochemistry and FJC visualisation using standard methodologies.
In the retina, FJC proved a useful tool for identifying degenerating neurons following all three injuries; for example, after NMDA administration cell bodies and dendrites within the ganglion cell layer and inner plexiform layer were labelled within 6h. In the optic nerve, however, FJC was ineffective for visualisation of injured axons. Significant amounts of non-specific signal were evident within the tissue sections, emanating from extracellular matrix or glial components, which masked any specific fluourescence. In contrast to FJC, immunolocalisation of amyloid precursor protein, and axonal proteins, such as non-phosphorylated and phosphorylated neurofilament heavy, neurofilament light, internexin and beta-tubulin, specifically and reliably demonstrated axonal abnormalities, including reduced axonal transport, axonal swelling and degradation. Moreover, immunolocalisation of nestin and heat shock protein 27 revealed glial cell activation following these injuries.
Immunolocalisation of a panel of proteins expressed by components of the optic nerve provides sensitive, specific and reliable information regarding the health of the optic nerve. FJC proved useful for identifying retinal injury but, in our study, was ineffective at visualising optic nerve damage.
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