Abstract
Purpose :
We recently demonstrated that both canonical and non-canonical Wnt signaling pathways promote axonal growth and RGC survival after optic nerve injury. Retinal microglia have active Wnt signaling, and Wnt3a-induced axonal regeneration is associated with increased microglia, but it is unknown whether Wnt-induced optic nerve regeneration and pro-regenerative inflammation are interrelated. In this study, we investigated whether Wnt-induced axonal regeneration is mediated by neuroinflammation.
Methods :
Mice were intravitreally injected with Wnt3a, Wnt5a or saline immediately prior to optic nerve crush (ONC) injury. BV-2 microglia cultures were treated with Wnt3a,Wnt5a, LPS or Saline. QPCR and Western Blot were used to quantify inflammatory molecules. To block inflammation, mice were injected IP with minocycline (45 mg/kg) or water on the day of ONC, then 3x/week for 2.5 weeks. Axonal regeneration was quantified by counting the number of axons at 100 micron intervals past the crush site.
Results :
Arg-1, a marker of anti-inflammatory microglia, was 15 times higher in Wnt5a injected retinas compared to saline three days after optic nerve injury (p<0.007) and was 3-fold higher in BV-2 microglia treated with Wnt5a compared to saline (p<0.01). Pro-inflammatory marker of microglia activation Nos2 was twofold higher in Wnt5a treated BV-2 cells compared to control (p<0.007) but was not changed in Wnt5a-injected retinas. Western blots showed a significant increase in key inflammatory regulators, P-JNK (p=0.0007) and P- STAT3 (p<0.0001) in Wnt5a 20ng injected eyes in comparison with PBS injected eyes. In contrast, Wnt3a did not significantly change pro- or anti-inflammatory genes in BV2 cells, while a pro-inflammatory molecule, CCL22 was threefold higher in Wnt3a treated retinas compared to saline (p<0.031). Furthermore, minocycline reduced Wnt3a-induced axonal growth (p=0.042).
Conclusions :
This study demonstrated for the first time that Wnt signaling leads to significant changes in both pro- and anti-inflammatory molecules in association with axonal regeneration both in vivo and in vitro. These findings suggest that Wnt5a-induced axonal regeneration is mediated through anti-inflammatory signaling whereas Wnt3a is mediated through pro-inflammatory mediators.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.