Marilyn Davies

Evans Blue Dye as an in vivo marker of myofibre damage: optimising parameters for detecting initial myofibre membrane permeability

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 µm 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.

Reduced necrosis of dystrophic muscle by depletion of host neutrophils, or blocking TNFα function with Etanercept in mdx mice

Necrosis of skeletal muscle fibres in the lethal childhood myopathy Duchenne Muscular Dystrophy results from deficiency of the cell membrane associated protein, dystrophin. We test the hypothesis in dystrophin-deficient mice, that the initial sarcolemmal breakdown resulting from dystrophin deficiency is exacerbated by inflammatory cells, specifically neutrophils, and that cytokines, specifically Tumour Necrosis Factor alpha (TNFalpha), contribute to myofibre necrosis. Antibody depletion of host neutrophils resulted in a delayed and significantly reduced amount of skeletal muscle breakdown in young dystrophic mdx mice. A more striking and prolonged protective effect was seen after pharmacological blockade of TNFalpha bioactivity using Etanercept. The extent of exercise induced myofibre necrosis in adult mdx mice after voluntarily wheel exercise was also reduced after Etanercept administration. These data show a clear role for neutrophils and TNFalpha in necrosis of dystrophic mdx muscle in vivo. Etanercept is a highly specific anti-inflammatory drug, widely used clinically, and potential application to muscular dystrophies is suggested by this reduced breakdown of mdx skeletal muscle.

Myotube Formation is Delayed but not Prevented in MyoD-deficient Skeletal Muscle: Studies in Regenerating Whole Muscle Grafts of Adult Mice:

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.

Reduced muscle necrosis and long-term benefits in dystrophic mdx mice after cV1q (blockade of TNF) treatment

Tumour necrosis factor (TNF) is a potent inflammatory cytokine that appears to exacerbate damage of dystrophic muscle in vivo. The monoclonal murine specific antibody cV1q that specifically neutralises murine TNF demonstrated significant anti-inflammatory effects in dystrophic mdx mice. cV1q administration protected dystrophic skeletal myofibres against necrosis in both young and adult mdx mice and in adult mdx mice subjected to 48 h voluntary wheel exercise. Long-term studies (up to 90 days) in voluntarily exercised mdx mice showed beneficial effects of cV1q treatment with reduced histological evidence of myofibre damage and a striking decrease in serum creatine kinase levels. However, in the absence of exercise long-term cV1q treatment did not reduce necrosis or background pathology in mdx mice. An additional measure of well-being in the cV1q treated mice was that they ran significantly more than control mdx mice.

Age influences the early events of skeletal muscle regeneration: Studies of whole muscle grafts transplanted between young (8 weeks) and old (13-21 months) mice.

Injured skeletal muscle generally regenerates less efficiently with age, but little is understood about the effects of ageing on the very early inflammatory and neovascular events in the muscle repair process. This study used a total of 174 whole muscle grafts transplanted within and between young and old mice to analyse the effects of ageing on the early inflammatory response in two strains of mice (BALB/c and SJL/J). There was a very slight delay in the early inflammatory response, and in the appearance of myotubes at day 4 in BALB/c muscle grafted into an old host environment (implicating systemic events). In SJL/J mice, the initial speed of the inflammatory response was slightly delayed with old muscle grafts regardless of host age (implicating muscle-derived factors), while an old host environment transiently affected myogenesis (myotube formation). The slight delays in inflammatory and neovascular responses in old mice did not dramatically impact on the overall formation of new muscle. The neovascular response to injured young and old muscle tissue was further analysed using the corneal micropocket assay. This showed a very clear 1-2 day delay in angiogenesis induced by old versus young BALB/c muscle tissue implanted into the young rat cornea, indicating that new blood vessel formation is at least partly determined by muscle-derived factors. Taken together these results indicate that, while there are slight age-associated delays in inflammation and neovascularisation in response to injured muscle, there is no detrimental effect on myogenesis in the mouse model used in this study.

Insulin-like growth factor I slows the rate of denervation induced skeletal muscle atrophy

Loss of the nerve supply to skeletal muscle results in a relentless loss of muscle mass (atrophy) over time. The ability of insulin-like growth factor-1 to reduce atrophy resulting from denervation was examined after transection of the sciatic nerve in transgenic MLC/mIGF-1 mice that over-express mIGF-1 specifically in differentiated myofibres. The cross sectional area (CSA) of all types of myofibres and specifically type IIB myofibres was measured in tibialis anterior muscles from transgenic and wild-type mice at 28 days after denervation. There was a marked myofibre atrophy ( approximately 60%) in the muscles of wild-type mice over this time with increased numbers of myofibres with small CSA. In the muscles of MLC/mIGF-1 mice, over-expression of mIGF-1 reduced the rate of denervation induced myofibre atrophy by approximately 30% and preserved myofibres with larger CSA, compared to wild-type muscles. It is proposed that the protective effect of mIGF-1 on denervated myofibres might be due to reduced protein breakdown.

Early Regeneration of Whole Skeletal Muscle Grafts Is Unaffected by Overexpression of IGF-1 in MLC/mIGF-1 Transgenic Mice

Early myogenic events in regenerating whole muscle grafts were compared between transgenic MLC/mIGF-1 mice with skeletal muscle-specific overexpression of the Exon-1 Ea isoform of insulin-like growth factor-1 (mIGF-1) and control FVB mice, from day 3 to day 21 after transplantation. Immunocytochemistry with antibodies against desmin showed that skeletal muscle-specific overexpression of IGF-1 did not affect the pattern of myoblast activation or proliferation or the onset and number of myotubes formed in regenerating whole muscle grafts. Hypertrophied myotubes were observed in MLC/mIGF grafts at day 7 after transplantation, although such hypertrophy was transient, and the transgenic and control grafts had a similar appearance at later time points (days 10, 14, and 21). Immunostaining with antibodies to platelet endothelial cell adhesion molecule-1, which identifies endothelial cells, demonstrated no difference in the formation of new vascular network in grafts of transgenic and control mice. Skeletal muscle-specific overexpression of mIGF-1 does not appear to stimulate the early events associated with myogenesis during regeneration of whole muscle grafts. (J Histochem Cytochem 52:873–883, 2004)

Enhancement of Neovascularization in Regenerating Skeletal Muscle by the Sustained Release of Erucamide from a Polymer Matrix

The angiogenic agent erucamide (cis-13-docosenamide), incorporated into a polymeric biomaterial (Elvax® 40P, a copolymer of ethylene and vinyl acetate), was used to determine whether angiogenesis can be increased in the regenerating skeletal muscle, and whether the enhanced revascularization improves the new muscle formation. The angiogenic nature of this lipid was confirmed in a rat cornea-micropocket assay, prior to insertion of small strips of the polymer containing either 3 μg, 30 μg, 300 μg erucamide or only polymer as a control into the mid-region of crush-injured tibialis anterior (TA) muscles of forty-five adult male BALB/c mice. All TA muscles were sampled ten days after injury and analyzed morphometrically. Statistical analyses of the mean blood vessel area density in lesions from twelve perfused TA muscles (three from each of the erucamide-treated or control group), revealed a dose-dependent angiogenic effect of erucamide: a dosage of 3 μg increased mean blood vessel area density to 5.1% compared to 2.0% in controls, due to numerous large caliber, thin-walled vessels, whereas the mean vessel area density in both the 30-μg (3.5%) and 300-μg (1.5%) doses were similar to controls. However, at all three doses tested, erucamide did not significantly alter the degree of new muscle formation, connective tissue deposition, or removal of necrotic debris.

Insulin-like growth factor-1 overexpression in cardiomyocytes diminishes ex vivo heart functional recovery after acute ischemia

BACKGROUND Acute insulin-like growth factor-1 administration has been shown to have beneficial effects in cardiac pathological conditions. The aim of the present study was to assess the structural and ex vivo functional impacts of long-term cardiomyocyte-specific insulin-like growth factor-1 overexpression in hearts of transgenic αMHC-IGF-1 Ea mice. METHODS Performance of isolated transgenic αMHC-IGF-1 Ea and littermate wild-type control hearts was compared under baseline conditions and in response to 20-min ischemic insult. Cardiac desmin and laminin expression patterns were determined histologically, and myocardial hydroxyproline was measured to assess collagen content. RESULTS Overexpression of insulin-like growth factor-1 did not modify expression patterns of desmin or laminin but was associated with a pronounced increase (∼30%) in cardiac collagen content (from ∼3.7 to 4.8 μg/mg). Baseline myocardial contractile function and coronary flow were unaltered by insulin-like growth factor-1 overexpression. In contrast to prior evidence of acute cardiac protection, insulin-like growth factor-1 overexpression was associated with significant impairment of acute functional response to ischemia-reperfusion. Insulin-like growth factor-1 overexpression did not modify ischemic contracture development, but postischemic diastolic dysfunction was aggravated (51±5 vs. 22±6 mmHg in nontransgenic littermates). Compared with wild-type control, recovery of pressure development and relaxation indices relative to baseline performance were significantly reduced in transgenic αMHC-IGF-1 Ea after 60-min reperfusion (34±7% vs. 62±7% recovery of +dP/dt; 35±11% vs. 57±8% recovery of -dP/dt). CONCLUSIONS Chronic insulin-like growth factor-1 overexpression is associated with reduced functional recovery after acute ischemic insult. Collagen deposition is elevated in transgenic αMHC-IGF-1 Ea hearts, but there is no change in expression of the myocardial structural proteins desmin and laminin. These findings suggest that sustained cardiac elevation of insulin-like growth factor-1 may not be beneficial in the setting of an acute ischemic insult.

An evaluation of leukaemia inhibitory factor as a potential therapeutic agent in the treatment of muscle disease

The exogenous delivery of growth factors and cytokines is a potential therapeutic strategy to alleviate the degenerative effects of primary inherited myopathies such as Duchenne muscular dystrophy. The mdx mouse diaphragm is a model for examining the progressive degeneration of dystrophic muscle. We have delivered leukaemia inhibitory factor to the mdx diaphragm using slow release alginate gels. Previous studies have reported an improvement in the histology of mdx diaphragms after delivery of leukaemia inhibitory factor in a similar manner, but little attention has been paid to the mechanism by which leukaemia inhibitory factor acts. We have used autoradiography to examine cell proliferation, Evans Blue Dye to examine myofibre damage, and morphometric analysis to examine histology in leukaemia-inhibitory-factor-treated diaphragms and compared them with untreated mdx and normal C57Bl10/ScSn diaphragms. Autoradiography showed that although myoblast proliferation was significantly increased in leukaemia inhibitory factor-treated mdx diaphragms, leukaemia inhibitory factor did not reduce myofibre damage and no histological improvement was observed. The data presented here, while demonstrating a role for leukaemia inhibitory factor in myoblast proliferation, do not support a strong and consistent benefit of leukaemia inhibitory factor on dystrophic muscle in vivo as a means of alleviating the effects of chronic dystrophic muscle degeneration.