Three-dimensional evaluation of retinal ganglion cell axon regeneration and pathfinding in whole mouse tissue after injury

Exp Neurol. 2013 Sep:247:653-62. doi: 10.1016/j.expneurol.2013.03.001. Epub 2013 Mar 16.


Injured retinal ganglion cell (RGC) axons do not regenerate spontaneously, causing loss of vision in glaucoma and after trauma. Recent studies have identified several strategies that induce long distance regeneration in the optic nerve. Thus, a pressing question now is whether regenerating RGC axons can find their appropriate targets. Traditional methods of assessing RGC axon regeneration use histological sectioning. However, tissue sections provide fragmentary information about axonal trajectory and termination. To unequivocally evaluate regenerating RGC axons, here we apply tissue clearance and light sheet fluorescence microscopy (LSFM) to image whole optic nerve and brain without physical sectioning. In mice with PTEN/SOCS3 deletion, a condition known to promote robust regeneration, axon growth followed tortuous paths through the optic nerve, with many axons reversing course and extending towards the eye. Such aberrant growth was prevalent in the proximal region of the optic nerve where strong astroglial activation is present. In the optic chiasms of PTEN/SOCS3 deletion mice and PTEN deletion/Zymosan/cAMP mice, many axons project to the opposite optic nerve or to the ipsilateral optic tract. Following bilateral optic nerve crush, similar divergent trajectory is seen at the optic chiasm compared to unilateral crush. Centrally, axonal projection is limited predominantly to the hypothalamus. Together, we demonstrate the applicability of LSFM for comprehensive assessment of optic nerve regeneration, providing in-depth analysis of the axonal trajectory and pathfinding. Our study indicates significant axon misguidance in the optic nerve and brain, and underscores the need for investigation of axon guidance mechanisms during optic nerve regeneration in adults.

Keywords: Axon growth; Axon regeneration; Axotomy; PTEN; Retinal ganglion cell; SOCS3.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenoviridae / physiology
  • Animals
  • Axons / pathology*
  • Cholera Toxin
  • Cyclic AMP / genetics
  • Disease Models, Animal
  • Female
  • Functional Laterality
  • Green Fluorescent Proteins / genetics
  • Imaging, Three-Dimensional*
  • Mice
  • Mice, Transgenic
  • Microscopy, Fluorescence
  • Nerve Crush / methods
  • Nerve Regeneration / physiology*
  • Optic Nerve Diseases / pathology*
  • PTEN Phosphohydrolase / genetics
  • Retinal Ganglion Cells / pathology*
  • Retinal Ganglion Cells / physiology
  • Suppressor of Cytokine Signaling 3 Protein
  • Suppressor of Cytokine Signaling Proteins / genetics
  • Visual Pathways / pathology
  • Zymosan / genetics


  • Socs3 protein, mouse
  • Suppressor of Cytokine Signaling 3 Protein
  • Suppressor of Cytokine Signaling Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Zymosan
  • Cholera Toxin
  • Cyclic AMP
  • PTEN Phosphohydrolase
  • Pten protein, mouse