Neurite plasticity is a critical aspect of functional recovery after stroke. Emerging data suggest that Rac1 plays a central role in neurite regeneration in the injured brain, specifically by stimulating neuronal intrinsic growth and counteracting the growth inhibitory signaling that leads to growth cone collapse. We assessed the hypothesis that Rac1 is an important stimulator of neurite regeneration after stroke.
Transient focal ischemia was induced by middle cerebral artery occlusion for 90 minutes in 7 to 8 week old male wild type mice. A specific Rac1 inhibitor, NSC 23766 (NSC) and a selective inhibitor (IPA 3) of PAK1, a downstream kinase of Rac1, were injected starting 7 days after stroke. Pellet reaching test (success rate) was used to assess the integrity of sensorimotor function.
Treatment with NSC worsened recovery assessed by pellet reaching test from day 14 to day 28 after stroke (Day 28: Vehicle 0.39 ±0.03 vs. NSC 0.18±0.03, n=7-9, p<0.05). It additionally reduced axonal density (neurofilament staining, NFL) in the peri-infarct zone (PIZ) assessed on 28 days after stroke (Vehicle 1.00±0.12 vs. NSC 0.50±0.04, n=4, p<0.05), with no effect on brain cavity size. Treatment with IPA 3 reduced the success rate in the pellet reaching test after ischemia (Day 28: Vehicle 0.40±0.03 vs. IPA 3 0.17 ±0.03, n=7-9, p<0.05) and NFL staining in PIZ 28 days after stroke (Vehicle 1.00±0.07 vs. IPA 3 0.52 ±0.10, n=4, p<0.05). LIM domain Kinase (LIMK1) is a key component of the intrinsic capacity of neurite regeneration. Interestingly, NSC reduced LIMK1 phosphorylation (Vehicle 1.47±0.09 vs. NSC 0.75±0.12, n=4, p<0.05) day 14 after stroke, while it increased levels of glial fibrillary acidic protein (GFAP), a signal of neurite growth inhibition (Day 14: Vehicle 1.00±0.24 vs. NSC 1.66±0.11, n=4, p<0.05).
Conclusions: Inhibition of Rac1 signaling worsened functional recovery and reduced axonal density. This was associated with decreased activation of intrinsic pro-regenerative molecules and increased growth inhibitory signaling. Our data suggested that Rac1 signaling could enhance neurite regeneration and improve post-stroke functional recovery.