Peripheral nerve injury leads to denervated muscle atrophy, a significant clinical concern with limited treatment options. While macrophages are known to play a role in muscle homeostasis and regeneration, the role of neutrophils, absent in normal muscle, in denervation-induced inflammation is unclear. Previous research has highlighted ROS generation as a key step in neutrophil activation and migration during infectious inflammation, and excessive ROS is linked to muscle atrophy. Denervation creates a hypoxic environment promoting ROS production and inflammation. However, the specific neutrophil subtypes involved and their chemokine mediators remain undefined. Furthermore, the timely resolution of inflammation through neutrophil apoptosis is critical for tissue repair. This study aimed to investigate the presence, activation, subtypes, function, and apoptosis of neutrophils in denervated muscle, utilizing intravital imaging, flow cytometry, transcriptome analysis, and genetic mouse models.

Existing literature demonstrates macrophages' role in maintaining skeletal muscle homeostasis and promoting regeneration. However, the involvement of neutrophils in denervated muscle, which lacks direct damage or infection, is unexplored. Studies have shown ROS's importance in neutrophil activation and migration in infectious inflammation, alongside its contribution to muscle atrophy and weakness. Denervation induces relative ischemia and hypoxia, leading to increased ROS generation and inflammatory factor production. Defining neutrophil subtypes and their chemokines is crucial, given their diverse functions. Furthermore, understanding the mechanisms regulating neutrophil apoptosis, such as the involvement of inflammatory cytokines or mediators like 15-epi-LXA4 and resolvin E1, is vital for managing inflammation.

The study used male Ly6g-DTR-GFP, P53 KO, and C57BL/6 mice. Sciatic nerve transection was performed to induce denervation. Intravital imaging with spinning-disk confocal microscopy (SD-IVM) dynamically visualized neutrophils in denervated muscle. 3D-view reconstruction analyzed the spatial distribution of neutrophils and blood vessels. Flow cytometry assessed neutrophil and ROS levels with and without ROS inhibition (L-NAC). Transcriptome analysis identified neutrophil subtypes and chemokines. Neutrophil depletion was achieved using anti-Ly6G antibody or diphtheria toxin (DT) in Ly6g-DTR-GFP mice. Muscle wet weight and fiber area, diameter, and density were measured using H&E staining. RNA-seq was used to identify differentially expressed genes (DEGs), and qPCR quantified CXCL1 expression. Statistical analyses included t-tests, ANOVA, and Friedman tests.

Neutrophil numbers increased in peripheral blood and denervated muscle after denervation, peaking at 12 hours. Increased ROS production in both blood and muscle, peaking at 12 hours, correlated with neutrophil increase. ROS inhibition reduced neutrophil numbers, indicating ROS's role in activation. Intravital imaging revealed neutrophil trafficking from circulation to muscle blood vessels, with migration into muscle tissue. The majority of neutrophils in denervated muscle were CXCR2+, recruited by CXCL1, which was upregulated after denervation. Neutrophil depletion aggravated muscle weight loss, demonstrating their protective role in delaying atrophy. P53 KO mice showed attenuated muscle atrophy and increased neutrophil numbers, indicating P53's role in neutrophil apoptosis. Neutrophils underwent P53-mediated apoptosis within 7 days after denervation.

This study provides the first visual evidence of neutrophil recruitment to denervated muscle, revealing their previously unrecognized role in this context. ROS acts as a key activator of neutrophils, triggering their recruitment via CXCL1-mediated chemotaxis. The predominantly CXCR2+ neutrophils contribute to delaying muscle atrophy, highlighting the importance of targeting specific neutrophil subsets. The findings support the hypothesis that neutrophils play a protective role in early-stage denervation-induced atrophy. The P53-mediated apoptotic pathway appears to regulate the resolution of neutrophil infiltration and subsequent muscle repair. These findings could inform the development of therapeutic strategies.

This study demonstrates that ROS-activated CXCR2+ neutrophils, recruited by CXCL1, delay denervated muscle atrophy during the initial phase of injury. These neutrophils subsequently undergo P53-mediated apoptosis. Targeting specific neutrophil subsets or manipulating the P53 pathway could offer novel therapeutic avenues for treating denervated muscle atrophy. Future studies should explore the precise mechanisms by which neutrophils delay atrophy and the potential of therapeutic interventions targeting these mechanisms.

The study primarily focused on early-stage denervation. Further investigation is needed to determine the long-term effects of neutrophil modulation. The mouse model may not perfectly replicate human denervation, requiring validation in human studies. While the study identified P53's role in neutrophil apoptosis, the precise molecular mechanisms warrant further investigation. The sample sizes in some experiments could be considered relatively small, potentially influencing statistical power.