Activated complement Bb and C5a fragments in plasma are decreased by blocking C5 activation or C5aR signaling. (A-B) After baseline pain testing, nonhyperalgesic HbSS mice were injected IV via the tail vein with control, anti-C5, or anti-C5aR mAbs (n= 4 per group; 1.2 mg/kg). Thirty minutes after mAb infusion, nonhyperalgesic mice were exposed to cold at 10°C (50°F) for 1 hour and then returned to RT. Four hours after mice were returned to RT, mice were euthanized and EDTA blood samples were collected from the heart. EDTA plasma samples were analyzed by immunoblots stained with antibodies to (A) Bb or (B) C5a activation fragments, and IgG loading controls. Zymosan-treated mouse serum, zymosan-treated heat-inactivated serum, and untreated mouse serum were analyzed on the C5a immunoblots to serve as positive and negative controls. (C) In experiments without cold exposure, HbSS mice that had ongoing hyperalgesia (PWT < 0.7) during baseline testing were injected IV via the tail vein with control or anti-C5aR mAbs (n = 4 per group; 1.2 mg/kg). EDTA blood samples were collected from the heart 4 hours after mAb infusion. EDTA plasma samples were analyzed by immunoblots stained with antibodies to Bb, C5a, and IgG. (D-G) The intensities of the Bb, C5a, and IgG light chain bands on the immunoblot images were measured using densitometry. Bb to IgG ratios in panel A are presented in panel D; C5a to IgG ratios in panel B are presented in panel E; Bb to IgG and C5a to IgG ratios in panel C are presented in panels F-G. Bars are means ± SEM. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001 (1-way ANOVA with the Tukey multiple comparison test).