IP injection of THI reduces peripheral blood leukocytes and increases S1P levels in most tissues. ( A ) Leukocytes were analyzed from the peripheral blood of 1.5-MO mdx4cv mice (n = 3) before and 12 hours following treatment with THI (2 × 250 μl 0.15 mg/ml IP injections, 6 hours apart). IP administration of THI significantly reduced circulating leukocytes to values below or near age-matched wt (n = 4). The average value of each population is listed in the table below the bar graph. Values between pre and post THI, and wt were also significant by ANOVA (P <0.05) for all leukocytes except monocytes. ( B ) mdx4cv mice (n = 6, 5-MO) were treated with THI or vehicle for 3 days (2 × 250 μl 0.15 mg/ml IP injections per day) following CTX injury to assess changes in S1P muscle content. Muscles and spleens were harvested on day 4 post injury for S1P analysis by LC-MS/MS. Results indicate S1P levels in spleen and injured quadriceps (quads) were significantly elevated with THI treatment. Interestingly, uninjured quadriceps did not show a significant increase of S1P, whereas uninjured TA muscles did. *P <0.05 by student’s t-test. Error bars represent SEM. CTX, cardiotoxin; IP, intraperitoneal; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MO, month-old; S1P, sphingosine-1-phoshate; SEM, standard error of the mean; TA, tibialis anterior; THI, 2-acetyl-4(5)-tetrahydroxybutyl imidazole; wt, wild type.