Cre-GFP Adeno-associated virus(AAV Serotype 5)

Overcoming the restricted axonal regenerative ability that limits functional repair following a central nervous system injury remains a challenge. Here, researchers report a regenerative paradigm called enriched conditioning, which combines environmental enrichment (EE) followed by a conditioning sciatic nerve axotomy that precedes a spinal cord injury (SCI). Compared with EE or a conditioning injury alone, enriched conditioning significantly increases the regenerative ability of dorsal root ganglia (DRG) sensory neurons, propelling axon growth well beyond the spinal injury site. Mechanistically, they established that enriched conditioning relies on the unique neuronal intrinsic signaling axis PKC-STAT3-NADPH oxidase 2 (NOX2), enhancing redox signaling as shown by redox proteomics in DRG. Finally, NOX2 conditional deletion or overexpression respectively blocked or phenocopied enriched conditioning-dependent axon regeneration after SCI, resulting in improved functional recovery. These studies provide a paradigm that drives the regenerative capacity of sensory neurons and offer a potential redox-dependent regeneration model for mechanistic and therapeutic discovery.

To functionally disrupt the NOX2 complex, the researchers conditionally deleted the structural subunit gp91phox (CYBB) in DRG neurons by injecting AAV5-Cre-GFP or AAV5-GFP as a control into the sciatic nerve of gp91phoxfl/fl mice. Analysis of neurite outgrowth from green fluorescent protein (GFP)-positive DRGs showed that NOX2 deficiency significantly attenuated the EE+SNA-dependent increase in outgrowth (Figure 1a, b), indicating that a functional NOX2 complex is required for the EE+SNA-dependent increase in regenerative potential. To determine whether functional NOX2 is required for EE+SNA-dependent sensory axon regeneration in vivo, mice with conditional deletion of gp91phox were exposed to EE+SNA or SH Sham and then underwent dorsal spinal cord hemisection. Six weeks later, regeneration of sensory axons was assessed using the retrograde axon tracer CTB, which was injected bilaterally into the sciatic nerve 1 week before mice were sacrificed (Figure 1c). Exposure to EE + SNA significantly increased the number of sensory axons entering and crossing the spinal cord lesion site (Figure 1d, e) without affecting the astroglial scar surrounding the lesion site. Disruption of NOX2 complex function by deleting gp91phox abolished enrichment condition (EE + SNA)-dependent axon regeneration to levels observed in SH Sham conditions, where the axon front ends retracted from the lesion border (Figure 1d, e).

Figure 1. NOX2 is required for EE + SNA-dependent increases in neurite outgrowth and axon regeneration after SCI. a Representative images of cultured DRGs from NOX2^fl/fl mice that were previously transduced in vivo with AAV5-GFP or AAV5-Cre-GFP (green) and stained with Beta-III-tubulin (red) after SH Sham or EE + SNA. b Quantification of average neurite outgrowth per neuron. c Timeline of in vivo experiments. d Representative images of dorsal column sensory axons traced by CTB (red) and DAPI (blue) after injury to identify lesion site (dashed line). e Quantification of CTB-positive regenerating axons. (De Virgiliis F, et al., 2020)