Antibody array analysis of peripheral and blood cytokine levels in rats after masseter inflammation

Abstract

This study was undertaken to evaluate the changes in cytokine levels in response to orofacial deep tissue inflammation. Inflammation was induced by injecting complete Freund's adjuvant (CFA, 0.05 ml 1:1 oil/saline suspension) into the masseter of the male Sprague–Dawley rat under brief halothane anesthesia. At 30 min, 5 h and 24 h after CFA injection ( n = 3–4/time point), tissues were dissected from masseter and total proteins isolated. Rat Cytokine Antibody Array 1.1 (RayBiotech) coated with 19 specific cytokine antibodies were probed with protein samples and the relative cytokine levels were compared. Compared to saline-injected rats, there were significant increases ( p < 0.05–0.01) in the levels of seven cytokines in the masseter tissue after CFA, including interleukin (IL)-1β (5 h), IL-6 (5 h), tumor necrosis factor-α (5 h), monocyte chemoattractant protein-1 (5 h, 24 h), cytokine-induced neutrophil chemoattactant-2 and -3 (5 h, 24 h), and tissue inhibitor of metalloproteinase-1 (5 h, 24 h). All 19 cytokines were detected in the blood samples, but they did not show significant changes after inflammation. Masseter hyperalgesia and allodynia occurred at 30 min and persisted at 5–24 h after inflammation, as assessed by probing the skin above the masseter with von Frey filaments. The present results indicate selective localized cytokine responses to masseter inflammation. Although different cytokines exist in the blood, their levels did not mirror, nor did not appear to depend on, the local cytokine levels. The findings provide specific targets for further studying the involvement of cytokines in orofacial inflammation and hyperalgesia.