Recovery of Function in Skeletal Muscle Following 2 Different Contraction-Induced Injuries  Richard M. Lovering, PT, PhD, Joseph A. Roche, PT, Robert J. Bloch, PhD, Patrick G. De Deyne, MPT, PhD  Archives of Physical Medicine and Rehabilitation  Volume 88, Issue 5, Pages 617-625 (May 2007) DOI: 10.1016/j.apmr.2007.02.010 Copyright © 2007 American Congress of Rehabilitation Medicine and the American Academy of Physical Medicine and Rehabilitation Terms and Conditions

Fig 1 Gamma irradiation before injury. Animals were anesthetized before an injury was induced and 1 of the hindlimbs in the experimental group was subjected to a single localized dose of ionizing radiation (25Gy) to prevent myogenic proliferation during the recovery process. Care was taken to irradiate only the hindlimb by using a collimator and performing ion chamber dosimetry. The collimator has an adjustable opening for the hindlimb and was machined from lead with a base 2cm thick, length of 25cm, width of 8cm, and height of 10cm. Archives of Physical Medicine and Rehabilitation 2007 88, 617-625DOI: (10.1016/j.apmr.2007.02.010) Copyright © 2007 American Congress of Rehabilitation Medicine and the American Academy of Physical Medicine and Rehabilitation Terms and Conditions

Fig 2 Irradiation inhibits recovery after small strain MR injury, but not after large strain 1R injury. Maximal tetanic tension was measured from uninjured () and injured tibialis anterior muscles on the day of injury (day 0) and at days 3, 7, 14, and 21 after injury (●) to determine the recovery seen in tibialis anterior muscles that were injured (A) by multiple repetitions or (B) by a single repetition. To determine whether myogenic cell proliferation was necessary for recovery, maximal tetanic tension was measured at the same time points in tibialis anterior muscles that were irradiated before injury (▾). To confirm that the radiation dose used in this study did not have a detrimental effect on function, tibialis anterior muscles from a third group of animals received irradiation only (○). The results show that irradiation inhibits recovery after MR, but not after 1R injury (n=5 animals tested at all time points). *Significant difference in value from uninjured within groups (P<.05); †significant difference in value from injured, but not irradiated. Archives of Physical Medicine and Rehabilitation 2007 88, 617-625DOI: (10.1016/j.apmr.2007.02.010) Copyright © 2007 American Congress of Rehabilitation Medicine and the American Academy of Physical Medicine and Rehabilitation Terms and Conditions

Fig 3 Centrally nucleated fibers (CNFs) increase after the MR protocol and are absent after irradiation. Sections from each muscle were stained with hematoxylin and eosin to determine the presence of centrally nucleated fibers. (A) An increase in centrally nucleated fibers was only seen after the MR injury protocol, and was especially prominent at 14 days after the injury. (B) This increase on day 14 was completely eradicated in muscles that were irradiated before injury (MR + Irr). *Significant difference in value from uninjured (P<.05). Archives of Physical Medicine and Rehabilitation 2007 88, 617-625DOI: (10.1016/j.apmr.2007.02.010) Copyright © 2007 American Congress of Rehabilitation Medicine and the American Academy of Physical Medicine and Rehabilitation Terms and Conditions

Fig 4 Irradiation inhibits expression of myogenic markers after injury. The mRNA encoding myoD and myogenin were assayed in extracts of tibialis anterior muscle by RT-PCR at different times (3, 7, 14, and 21 days) after MR or 1R injury, with or without irradiation. Levels of both transcripts increased after MR injury and this increase was inhibited by irradiation. Smaller increases were seen after 1R injury and were similarly inhibited by irradiation. GAPDH was used as a loading control. Results confirm that irradiation effectively inhibits the proliferation of myogenic cells after injury. Archives of Physical Medicine and Rehabilitation 2007 88, 617-625DOI: (10.1016/j.apmr.2007.02.010) Copyright © 2007 American Congress of Rehabilitation Medicine and the American Academy of Physical Medicine and Rehabilitation Terms and Conditions

Fig 5 Labeling of myofibers by Evans blue dye and antibodies to dystrophin. Rats were injected with Evans blue dye and subjected to MR or 1R injury 1 day later. Frozen cross-sections were collected on the day of injury (day 0) and 7 days later, and labeled with antidystrophin antibodies and fluoresceinated secondary antibodies. Sections were examined for the presence of intracellular Evans blue dye and dystrophin at the sarcolemma. The results show that myofibers injured by the 1R protocol (eg, white arrows) retain Evans blue dye for at least 1 week after the injury, and that dystrophin that is lost immediately after injury recovers over the next week, even in fibers that still retain Evans blue dye. Evans blue dye clearly labeled fibers after the MR injury, however, Evans blue dye−positive fibers did not persist over time (lower panels). NOTE. Scale bar is 50μm. Archives of Physical Medicine and Rehabilitation 2007 88, 617-625DOI: (10.1016/j.apmr.2007.02.010) Copyright © 2007 American Congress of Rehabilitation Medicine and the American Academy of Physical Medicine and Rehabilitation Terms and Conditions

Fig 6 Quantitation of myofibers labeled by Evans blue dye (EBD) and antidystrophin after MR and 1R injuries. The results of the studies illustrated in figure 5 were quantitated. (A) The results show that the number of fibers labeled by Evans blue dye after MR injury returns to near control levels during the week after injury, and compared with the 1R injury, fewer fibers lose dystrophin over this period. (B) By contrast, the number of fibers labeled by Evans blue dye after 1R injury remains constant, even as the number of fibers that fail to label for dystrophin return to control levels. *Significant difference in value from uninjured within groups (P<.05); †significant difference in value from fibers lacking dystrophin group. Archives of Physical Medicine and Rehabilitation 2007 88, 617-625DOI: (10.1016/j.apmr.2007.02.010) Copyright © 2007 American Congress of Rehabilitation Medicine and the American Academy of Physical Medicine and Rehabilitation Terms and Conditions