Animals were humanely euthanized by cervical dislocation while under anesthesia. The tibialis anterior muscle was harvested and immediately frozen in 2 methyl-butane pre-cooled in liquid nitrogen, and stored at -80 °C. Serial cross-sections (10 μm) were obtained and mounted onto slides. Statistical analyses were performed using standard statistical software packages (SPSS, v19.0 software). First, Levene's test was used to assess whether there was equality of variances. Independent samples t-test was then performed to investigate differences between NMES and Control groups.

Hematoxylin and Eosin (H&E) stains were performed to investigate whether the NMES protocol would result in increased injury in the dystrophic muscle. One section was selected, and pictures were obtained using a light microscope (Nikon Eclipse E800; Nikon, Japan). The total number of fibers and the number of fibers with centrally located nuclei were manually counted using the National Institutes of Health (NIH) - developed image analysis software, Image J. There was no significant increase in the regeneration index (number of centrally nucleated fibers/total number of fibers) in the animals submitted to NMES, as compared to controls (p=0.802; Figure 1). This suggests that the application of NMES does not increase the degeneration-regeneration cascade observed in dystrophic animals, and is therefore not likely to be inducing an increased muscle injury.

Immunofluorescent staining for CD31 was performed to investigate the effect of the 4-week NMES protocol on muscle vascularity. Briefly, muscle sections were fixed in 4% formalin and blocked using 5% Horse Serum (HS). Sections were incubated with a rat anti-mouse primary antibody (1:300 dilution in 5% HS) and treated with a 555-labeled goat anti-rat secondary antibody (1:300 dilution in 5% HS). To quantify the total number of CD31 positive cells, one section was selected, and photographed using fluorescent microscopy (Nikon Eclipse E800; Japan). The total number of capillaries was manually counted using Northern Eclipse Software (Empix Imaging Inc.). There was a significant increase in the number of CD31 positive cells in the animals submitted to NMES when compared to controls (p<0.01; Figure 2), indicating that the NMES protocol as described promotes skeletal muscle angiogenesis.

In situ contractile testing: Four weeks after completion of an NMES protocol, contractile testing of the anterior compartment muscles was performed using an in situ testing apparatus (Model 809B, Aurora Scientific Inc, Canada), stimulator (Model 701C, Aurora Scientific Inc, Canada), and force transducer (Aurora Scientific Inc, Canada). Briefly, the peroneal nerve of anesthetized animals was isolated through a small incision lateral to the knee. Mice were then placed supine on a platform and the foot being tested was positioned on the footplate. The hindlimb used for testing was stabilized with cloth tape on the knee and foot. Muscles were stimulated using a hook electrodes inserted beneath the skin. Muscle tetanic contraction was tested at a frequency of 150Hz for 350ms to investigate whether the 4-week NMES protocol would induce improvements in muscle force. Results were normalized to wet muscle weight. There was an approximately 30% increase in the tetanic contraction of animals submitted to NMES, when compared to controls (p=0.005) (Figure 3), suggesting the protocol described improves skeletal muscle strength in mdx/SCID animals.

Figure 1. Hematoxylin & Eosin skeletal muscle staining of dystrophic (mdx)/ immunodeficient mice (4-5 months)(10x magnification): A) untreated controls, B) after 4 weeks of NMES.

Figure 2. CD31 immunofluorescence (red), as a marker of skeletal muscle vascularity, in the skeletal muscle of dystrophic (mdx)/ immunodeficient mice (4-5 months)(20x magnification) A) untreated controls, B) after 4 weeks of NMES.