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Topical Substance P Modulates Inflammatory Responses in Healing Wounds in Nitric Oxide Synthase Knockout Mice

Introduction: Substance P (SP), a proinflammatory neuropeptide released by sensory nerves in response to cutaneous injury, increases nitric oxide (NO) production by target cells. SP upregulates three nitric oxide synthase (NOS) isoforms: neuronal (nNOS), inducible (iNOS) and endothelial (eNOS). Topical SP application improves wound healing kinetics in NOS KO mice. Since nitric oxide may be a key intermediary mediator for SP induced response to injury, this study was designed to determine whether SP promotes inflammatory cell density in early wounds of NOS KO mice. Methods: Mice of each NOS null strain and appropriate background controls (C57BL/6J – i/eNOS control;B6129SF2/J – nNOS control) were anesthetized with intraperitoneal ketamine and xylazine. After shaving, full‐thickness 1.5 × 1.5 sq. cm. dorsal excisional wounds were covered with a semi‐occlusive dressing. Mice were randomly assigned to daily topical infusion of 10−7 M SP or NS onto wound beds. Wounds were harvested at day 3 and 7 (3 mice per group). Cells were isolated by sequential digestion in dispase and hyaluronidase. Cell counts were performed to determine total cell density. Flow cytometry was performed after staining cells with FITC – labeled antibodies against F4/80 (macrophages), CD45 (pan leukocytes), CD31 (endothelial cells) or CD11c (dendritic cells). Results: Total cell number was higher at day 7 post‐wounding than at day 3 (P < 0.05) in both control and NOS null mice. Macrophage, pan‐leukocyte and dendritic cell density was greater in the SP‐treated wounds than in NS‐treated wounds at both day 3 and 7 post‐wounding (P < 0.05) in all NOS KO mice. Whereas SP increased endothelial cell number in nNOS and iNOS mice, SP had no effect on endothelial cell number in the eNOS KO mice (P > 0.05). SP did not affect cell density in background mice at either time point (P > 0.05). Conclusion: SP upregulated inflammatory cell numbers in all NOS null mice suggesting that either SP activates NOS isoforms that are not reduced by targeted deletion or that SP induces inflammatory cell migration by NO‐independent pathways. The lack of SP effect on endothelial cell number in eNOS KO wounds suggests that eNOS may mediate SP‐induced wound angiogenesis.