Experimental procedures

The ovariectomized mice were divided into control (OC, n = 18) and estradiol (OE, n = 18) groups. Those two groups were then divided into three euthanasia time point groups, targeting 32 h, 96 h (4 d), and 192 h (8 d) post-crush and representing different phases of muscle recovery. Before the mice underwent the crush injury procedure, pellets were implanted four to nine weeks after ovariectomy (day of placebo or estrogen treatment). The OC mice were administered a 60-day-release placebo pellet (Innovative Research of America, Sarasota, FL, USA; 0.18 mg total), and the OE mice received a 60-day-release 17-β estradiol pellet (Innovative Research of America, Sarasota, FL, USA; 0.18 mg total).

On the eighth day of placebo or estrogen treatment, the crush injury procedure was performed. Mice were administered buprenorphine (Reckitt Benckiser Pharmaceuticals, Richmond, VA, USA), approximately 0.10 mg/kg subcutaneously, 1--5% inhaled isoflurane to effect and 100% oxygen. The crush injury was then applied to each mouse. The apparatus piston is in direct contact with the skin over the gastrocnemius and quadriceps muscles on the right side. Pressure was applied for 30 s at 45 psi for two to four applications. For a full description of the muscle crush model, see Dobek et al.³ A second dose of buprenorphine was administered 10–12 h from the first dose.

Approximately 22–24 h following the crush injury procedure, the mice were exposed to hypobaria (565 ± 5 torr) for approximately 8–9 h, which was to a similar protocol as described in St. Pierre Schneider et al.³³