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Xueting Luo, Yadira Salgueiro, Samuel Beckerman, Vance Lemmon, Pantelis Tsoulfas, Kevin Park; Three-dimensional Evaluation of Retinal Ganglion Cell Axon Regeneration, Pathfinding and Glial Reaction in Unsectioned Tissue. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2637.
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© ARVO (1962-2015); The Authors (2016-present)
Traditional methods of assessing retinal ganglion cell (RGC) axon regeneration rely on histological sectioning. However, tissue sections provide fragmentary information about axonal trajectory and termination. Here we use tissue clearance and light sheet fluorescence microscopy (LSFM) to unequivocally evaluate regenerating RGC axons in whole optic nerve and brain without physical sectioning.
In PTEN/SOCS3 floxed mice, we induced conditional knockout of PTEN/SOCS3 genes in the adult RGCs by intravitreal delivery of adeno-associated virus (AAV)-Cre (i.e. a condition known to induce robust regeneration). Animals received intraorbital optic nerve crush, and were allowed to regenerate RGC axons up to 10 weeks. Several days prior to sacrifice, we injected cholera toxin β subunit (CTB) intravitreally to label regenerating axons. After perfusion, fixed tissues were dehydrated by incubating in tetrahydrofuran (THF) and rendered clear in BABB solution. The optic nerves and brains were imaged using an ultramicroscope (LaVision Biotec). Several hundreds of optical slices were compiled into 3D reconstruction using Imaris software (Bitplane).
In 3D visualization, we observed that RGC axons grow in meandering paths through the optic nerve, with some axons extending toward the eye (Fig 1). In the brains, many axons stall at the optic chiasm, project to the contralateral optic nerve, or grow aberrantly within the optic chiasm. Distally, projection is limited predominantly to the hypothalamus (Fig 2). Further, using transgenic mice in which glial cells are labeled with enhanced green fluorescent protein (eGFP), we assessed axon interaction with astrocytes, microglia and oligodendrocytes in damaged optic nerve.
We demonstrate the integration of tissue clearance and LSFM for comprehensive assessment of RGC axon regeneration, and unequivocally reveal significant misguidance of RGC axons following injury. Our results indicate that the adult mammalian CNS lacks the signals necessary for proper pathfinding of RGC axons, and highlight the need to investigate mechanisms that control axon guidance during regeneration in adult mammals.
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