PurposeGlaucoma is characterized by progressive loss of the retinal ganglion cells (RGCs) and their axons. Here we test an outstanding notion that microtubules (MTs) within RGC axons degrade before the loss of morphology (“MT hypothesis”).MethodsThe integrity of axonal MTs was interrogated by intrinsic second-harmonic generation (SHG) microscopy. Using DBA/2J mice as a model of glaucoma and DBA/2J-Gpnmb+ as a nonglaucomatous control, the relationship between MT disruption and morphology was quantitatively examined as a function of age and sex in the fresh retinal wholemounts.ResultsThe mean SHG density (i.e., the mean SHG intensity per thickness) was significantly lower in DBA/2J than in DBA/2J-Gpnmb+ and also depended on sex and age. The loss of SHG density, indicating MT disruption within intact RGC axons, occurred in a sectorial manner near the loss of the retinal nerve fiber bundles. The decay rate of SHG density was approximately 97% higher than that of thickness.ConclusionsCollectively, the results indicate that the breakdown of MTs is pathology of glaucoma and likely a precursor of morphological atrophy. Based on a new finding that SHG density is highly variable and spatially discrete, a new model of RGC degeneration is proposed. This study validates SHG retinal imaging for elucidating the role and mechanism of MT deficiency in the course of glaucoma pathogenesis.

We describe a novel method for visualizing the network of axons in the unlabeled fresh wholemount retina. The intrinsic radiation of second harmonic generation (SHG) was utilized to visualize single axons of all major retinal neurons, i.e. photoreceptors, horizontal cells, bipolar cells, amacrine cells, and the retinal ganglion cells. The cell types of SHG + axons were determined using transgenic GFP/YFP mice. New findings were obtained with retinal SHG imaging: Müller cells do not maintain uniformly polarized microtubules in the processes; SHG + axons of bipolar cells terminate in the inner plexiform layer (IPL) in a subtype-specific manner; a subset of amacrine cells, presumably the axon-bearing types, emits SHG; and the axon-like neurites of amacrine cells provide a cytoskeletal scaffolding for the IPL stratification. To demonstrate the utility, retinal SHG imaging was applied for testing whether the inner retina is preserved in glaucoma, using DBA/2 mice as a model of glaucoma and DBA/2-Gpnmb + as the non-glaucomatous control. It was found that the morphology of the inner retina was largely intact in glaucoma and the pre-synaptic compartments to the retinal ganglion cells were uncompromised. It proves retinal SHG imaging as a promising technology for studying the physiological and diseased retinas in 3D.

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