Microtubule Imaging Reveals Cytoskeletal Deficit Predisposing the Retinal Ganglion Cell Axons to Atrophy in DBA/2J

Abstract

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.

Imaging unlabeled axons in the mouse retina by second harmonic generation

“…SHG can be obtained from the axon because of uniformly polarized microtubules 9,10 . The nonlinear optical radiation is particularly intense from the retinal nerve fiber bundle owing to a large number of RGC axons 6,7 . Although, in principle, SHG should arise also from the axons of non-RGC retinal cells, the signal was expected to be much weaker.…”

“…Tissue preparation and pharmacology . The retinal wholemounts were prepared as previously described 6,7 . Animal was euthanized by CO 2 inhalation and the eye was enucleated.…”

“…Here we describe a new method to image single axon-like processes in the fresh retina without labeling. Endogenous SHG, previously demonstrated for imaging the retinal ganglion cell (RGC) axons 6,7 , can visualize all major neurons in the inner and outer retina. 8 (an axial projection).…”

Harmonic Generation Microscopy 2.0: New Tricks Empowering Intravital Imaging for Neuroscience

“…The pathogenic processes have been visualized by microtubule SHG, e.g., lesions near senile plaques in a mouse model of Alzheimer's disease (Kwan et al, 2009), tau-tubulin interaction (Stoothoff et al, 2008), and neuronal ischemia (Psilodimitrakopoulos et al, 2013). In our own lab, we have utilized SHG from the retinal nerve fiber bundles (i.e., the axons of the retinal ganglion cells) to obtain new findings on the glaucoma pathogenesis (Figure 1C); specifically, we found that the disruption of pseudocrystalline microtubules occurs earlier than the loss of the retinal nerve fiber bundles and that they are likely to share a common pathogenic insult (Lim and Danias, 2012a,b; Sharoukhov et al, 2018).…”

“…(C) The retinal nerve fiber bundles in the fresh wholemount retina of glaucoma-developing DBA mouse visualized by SHG. Dashed lines, characteristic sectorial degeneration of glaucoma (redrawn from Sharoukhov et al, 2018). (D,E) SHG intensity for collagen (D) and microtubules (E) , respectively, and SHG polarization anisotropy (pSHG) corresponding to the dashed lines as a function of the polarization angle (redrawn from Sharoukhov and Lim, 2016).…”