June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Three-dimensional Evaluation of Retinal Ganglion Cell Axon Regeneration, Pathfinding and Glial Reaction in Unsectioned Tissue
Author Affiliations & Notes
  • Xueting Luo
    University of MIami, Miami, FL
  • Yadira Salgueiro
    University of MIami, Miami, FL
  • Samuel Beckerman
    University of MIami, Miami, FL
  • Vance Lemmon
    University of MIami, Miami, FL
  • Pantelis Tsoulfas
    University of MIami, Miami, FL
  • Kevin Park
    University of MIami, Miami, FL
  • Footnotes
    Commercial Relationships Xueting Luo, None; Yadira Salgueiro, None; Samuel Beckerman, None; Vance Lemmon, None; Pantelis Tsoulfas, None; Kevin Park, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 2637. doi:
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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)

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Abstract
 
Purpose
 

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.

 
Methods
 

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).

 
Results
 

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.

 
Conclusions
 

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.

 
 
Fig 1. 3D visualization of regenerating axons in the optic nerve following LSFM.
 
Fig 1. 3D visualization of regenerating axons in the optic nerve following LSFM.
 
 
Fig 2. 3D visualization of regenerating axons in the brain following LSFM and neurite tracing (ventral view on the top and lateral view on the bottom).
 
Fig 2. 3D visualization of regenerating axons in the brain following LSFM and neurite tracing (ventral view on the top and lateral view on the bottom).
 
Keywords: 687 regeneration • 551 imaging/image analysis: non-clinical • 599 microscopy: light/fluorescence/immunohistochemistry  
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