Evans Blue Dye (EBD) is widely used to study cellular membrane permeability and has recently been utilised in mdx mice to identify permeable skeletal myofibres that have become damaged as a result of muscular dystrophy. EBD has the potential to be a useful vital stain of myofibre permeability in other models of skeletal muscle injury and membrane-associated fragility. The parameters for its use for such purposes were optimised in the present study.Of particular interest is the use of EBD to identify the onset of muscle damage. This study compared intravenous vs. intraperitoneal injection; tissue fixation; volume of EBD; time of availability in tissue; and persistence after injection in mdx mice (with endogenous muscle damage) and control mice. Satisfactory labelling of permeable myofibres was seen in frozen sections viewed with fluorescence microscopy when intraperitoneal injection of a 1% EBD solution injected at 1% volume relative to body mass was administered between 16 and 24 h prior to tissue sampling. EBD labelling was then assessed in three mouse models of experimental injury and repair -cut injury, whole muscle grafts, and exercise-induced muscle damage. These experiments demonstrated that (i) following a cut injury across myofibres, EBD penetrated up to 150 µ m from the injury site over a 20-h period; (ii) EBD was present throughout myofibres of avascular whole muscle graft by one day after transplantation; and (iii) damaged myofibres were detected within 20 min after controlled lengthening-contraction exercise. This simple and inexpensive technique has sensitivity for the detection of increased myofibre permeability and/or sublethal damage that has advantages over other traditional histological techniques at the light microscopy level.
The ability of very old animals to make new muscle after injury remains controversial. This issue has major implications for the regenerative potential of damaged geriatric human muscle, to age-related loss of muscle mass (sarcopenia) and to the proposed need for muscle stem cell therapy for the aged. To further address issues of inherent myogenic capacity and the role of host systemic factors in new muscle formation, whole muscle grafts were transplanted between geriatric (aged 27-29 months) and young (3 months) C57Bl/6J mice and compared with autografts in geriatric and young mice. Grafts were sampled at 5 and 10 days for histological analysis. Inflammation and formation of new myotubes was strikingly impaired at 5 days in the geriatric muscle autografts. However, there was a strong inflammatory response by the geriatric hosts to young muscle grafts and geriatric muscles provoked an inflammatory response by young hosts at 5 days. At 10 days, extensive myotube formation in geriatric muscle autografts (equivalent to that seen in young autografts and both other groups) confirmed excellent intrinsic capacity of myogenic (stem) cells to proliferate and fuse. The key conclusion is that a weaker chemotactic stimulus by damaged geriatric muscle, combined with a reduced inflammatory response of old hosts, results in delayed inflammation in geriatric muscle autografts. This delay is transient. Once inflammation occurs, myogenesis can proceed. The presence of well developed myotubes in old muscle autografts at 10 days confirms a very good inherent myogenic response of geriatric skeletal muscle.
We compared the time course of myogenic events in vivo in regenerating whole muscle grafts in MyoD(-/-) and control BALB/c adult mice using immunohistochemistry and electron microscopy. Immunohistochemistry with antibodies to desmin and myosin revealed a striking delay by about 3 days in the formation of myotubes in MyoD(-/-) autografts compared with BALB/c mice. However, myotube formation was not prevented, and autografts in both strains appeared similar by 8 days. Electron microscopy confirmed myotube formation in 8- but not 5-day MyoD(-/-) grafts. This pattern was not influenced by cross-transplantation experiments between strains examined at 5 days. Antibodies to proliferating cell nuclear antigen demonstrated an elevated level of replication by MyoD(-/-) myoblasts in autografts, and replication was sustained for about 3 days compared with controls. These data indicate that the delay in the onset of differentiation and hence fusion is related to extended proliferation of the MyoD(-/-) myoblasts. Overall, although muscle regeneration was delayed it was not impaired in MyoD(-/-) mice in this model.
Skeletal muscle regeneration in SJL/J and BALB/c mice subjected to identical crush injuries is markedly different: in SJL/J mice myotubes almost completely replace damaged myofibres, whereas BALB/c mice develop fibrotic scar tissue and few myotubes. To determine the cellular changes which contribute to these differential responses to injury, samples of crushed tibialis anterior muscles taken from SJL/J and BALB/c mice between 1 and 10 days after injury were analysed by light and electron microscopy, and by autoradiography. Longitudinal muscle sections revealed about a 2-fold greater total mononuclear cell density in SJL/J than BALB/c mice at 2 to 3 days after injury. Electron micrographs identified a similar proportion of cell types at 3 days after injury. Autoradiographic studies showed that the proportions of replicating mononuclear cells in both strains were similar: therefore greater absolute numbers of cells (including muscle precursors and macrophages) were proliferating in SJL/J muscle. Removal of necrotic muscle debris in SJL/J mice was rapid and extensive, and by 6 to 8 days multinucleated myotubes occupied a large part of the lesion. By contrast, phagocytosis was less effective in BALB/c mice, myotube formation was minimal, and fibrotic tissue conspicuous. These data indicate that the increased mononuclear cell density, more efficient removal of necrotic muscle, together with a greater capacity for myotube formation in SJL/J mice, contribute to the more successful muscle regeneration seen after injury.
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