Direct administration of S1P promotes muscle regeneration following acute injury. ( A ) Experimental schematic of S1P and PBS (vehicle) injected daily for the first 72 hours into TAs of 3-MO mdx4cv:Myf5nlacZ/+ mice (n = 3, left TAs injected S1P, right TAs injected PBS) following CTX injury. ( B ) Top row: X-gal staining reveals an increased number of β-galactosidase+ nuclei at the sites of injury in S1P-treated TA muscles compared to vehicle controls. Bottom row: staining for eMyHC with DAB reveals a significant increase in the number of newly regenerated muscle fibers in S1P-treated TA muscles. Scale bars = 50 μm. ( C ) Left graph: quantification of β-galactosidase+ nuclei indicates the number of Myf5+ cells is significantly increased at the site of injury in S1P-treated compared to untreated muscles. Middle graph: a significant increase in β-galactosidase+ nuclei was also observed over the entire CSA of each S1P-treated TA muscle. Right graph: quantification of the number of eMyHC fibers within areas of regeneration was significantly greater with S1P treatment. *P <0.05 by student’s t-test. Error bars represent SEM. CSA, cross-sectional area; CTX, cardiotoxin; DAB, 3,3'-diaminobenzidine; eMyHC, embryonic myosin heavy chain; MO, month-old; S1P, sphingosine-1-phoshate; SEM, standard error of the mean; TA, tibialis anterior.