Regenerative Index: a method to assess muscle regeneration in patients with Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating disease manifested in skeletal muscle by repetitious myonecrosis and regeneration. Because the regenerative process is closely linked to the cumulative severity of muscle damage, which is variably distributed within and between muscle groups, accurately quantifying muscle regeneration has remained a significant challenge. Myofibers are delineated by immunostaining for laminin, and subsequent image analysis employed to generate a masked outline precisely within each myofiber boundary. Morphometric parameters including minimal Feret’s diameter, cross-sectional area, and circularity were measured for each myofiber. In addition, the number of Pax7-expressing satellite cells were quantified. To evaluate regenerative activity, newly formed myofibers were identified by immunostaining for expression of embryonic myosin heavy chain (eMHC). Necrotic myofibers were enumerated by immunofluorescent detection of immunoglobulin G (IgG) infiltration. The Regenerative Index (RI) was calculated as the number of regenerating (eMHC + ) myofibers divided by the number of necrotic (IgG + ) myofibers. Determination of RI was performed on muscle biopsies obtained from 10 boys with DMD and 3 age-matched non-DMD controls. A trend toward an increasing minimal Feret’s diameter, cross-sectional area and circularity was observed with increasing age in DMD boys, with circularity showing the strongest trend. Furthermore, compared to DMD boys 7- to 8-years old, the boys 9- to 11-years old had increased myofiber circularity. Pax7-expressing cells per myofiber were elevated in DMD boys compared to control boys of similar ages, without any observation of age-related changes. The Regenerative Index in DMD boys exhibited a decline between 7 and 11 years of age, with an inverse correlation between RI and age. The use of eMHC and IgG immunostaining to calculate RI appears to provide a way to assess regeneration across biopsies that differ in histopathologic severity. Using this approach, RI showed a negative correlation with age in DMD boys aged 7 to 11 years which requires further investigation.