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

Abstract

Injured retinal ganglion cell (RGCs) 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 toward 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.

Cell type-specific Nogo-A gene ablation promotes axonal regeneration in the injured adult optic nerve

“…In order to assess the pattern of growing axons, a three-dimensional analysis of the regenerating fibers was carried out in whole-mounted optic nerves after tissue clearing using a modified protocol previously described. 37,38 In the optic nerve segments situated between 100 and 300 mm past the lesion site, branched and unbranched axons could be distinguished, as well as axons that formed U-turns within this region (Figures 2e and f). We found that the proportion of branching axons did not significantly vary between Cnp-Cre þ / À xRtn4 flox/flox and Rtn4 flox/flox control mice (Figure 2g).…”

“…47,48 Acting at two levels, the neuronal growth program activation with Zymosan and the blockade of Nogo-A signaling potentiated Our previous three-dimensional analyses revealed axonal guidance and patterning defects reflected by increased axonal U-turns and branching after growth induction in the injured optic nerve. 34,37 In the present study, we hypothesized that the myelin Nogo-A could have a role in axon guidance after optic nerve lesion. However, by examining individual growing axons in 3D, we found no significant difference in the proportion of branching or U-turn-forming axons between mouse genotypes.…”

“…To visualize CTB-A594-labeled axons in the whole Targeted Nogo-A ablation promotes axonal outgrowthinjured optic nerve, the tissue was cleared following the adapted protocol from. 37 After intracardial perfusion with PBS and 4% PFA, optic nerves were further fixed overnight, then rinsed with PBS and stored at 4 1C. The samples were dehydrated in baths of increasing concentrations of tetrahydrofuran (THF, Sigma-Aldrich, Buchs, Switzerland) (50, 80, and 100%) for 30 min each and for additional 30 min in 100% THF at room temperature under constant agitation.…”

Hyper-IL-6: a potent and efficacious stimulator of RGC regeneration

“…42 Therefore, sustained mTOR activity upon cytokine treatment is seemingly not necessarily required for switching RGCs into, but rather for maintaining the regenerative state. 17,42 Overall, the effects of PTEN knockout on PI3K/AKT activation, neuroprotection and optic nerve regeneration are much stronger compared with IS or direct cytokine application, 36,43,44 indicating a contribution of further targets beyond mTOR in these processes. Despite the remarkable effects of intrinsic PTEN or SOCS3 depletion on RGCs survival and regeneration, such approaches are, however, unfavorable from a clinical point of view due to their high cancerogenic potential and the requirement for pre-injury neuronal transduction to achieve optimal results.…”

Boosting Central Nervous System Axon Regeneration by Circumventing Limitations of Natural Cytokine Signaling

“…6 Ancillary activation of gp130 signaling upstream of PTEN with either CNTF bolus injections or IS further increases the number and length of regenerating axons of PTEN-depleted RGCs. [23][24][25] However, this approach is unfavorable as therapeutic treatment, since it requires neuronal transduction weeks prior to CNS injury and might increase the risk of cancer development post-treatment. [17][18][19] Instead of genetically deleting intracellular inhibitors, we hypothesized that it might be possible to boost axon regeneration by directly enhancing the cytokine-induced stimulus.…”