Macrophage function in the elderly and impact on injury repair and cancer.

Older age is associated with deteriorating health, including escalating risk of diseases such as cancer, and a diminished ability to repair following injury. This rise in age-related diseases/co-morbidities is associated with changes to immune function, including in myeloid cells, and is related to immunosenescence. Immunosenescence reflects age-related changes associated with immune dysfunction and is accompanied by low-grade chronic inflammation or inflammageing. This is characterised by increased levels of circulating pro-inflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1ß and IL-6. However, in healthy ageing, there is a concomitant age-related escalation in anti-inflammatory cytokines such as transforming growth factor-ß1 (TGF-ß1) and IL-10, which may overcompensate to regulate the pro-inflammatory state. Key inflammatory cells, macrophages, play a role in cancer development and injury repair in young hosts, and we propose that their role in ageing in these scenarios may be more profound. Imbalanced pro- and anti-inflammatory factors during ageing may also have a significant influence on macrophage function and further impact the severity of age-related diseases in which macrophages are known to play a key role. In this brief review we summarise studies describing changes to inflammatory function of macrophages (from various tissues and across sexes) during healthy ageing. We also describe age-related diseases/co-morbidities where macrophages are known to play a key role, focussed on injury repair processes and cancer, plus comment briefly on strategies to correct for these age-related changes.

Effects of loaded voluntary wheel exercise on performance and muscle hypertrophy in young and old male C57Bl/6J mice.

This study compared the capacity of young and old male C57Bl/6J mice to exercise with increasing resistance over 10 weeks, and its impact on muscle mass. Young mice (aged 15-25 weeks) were subjected to low (LR) and high (HR) resistance exercise, whereas only LR was used for old mice (107-117 weeks). Weekly patterns of voluntary wheel activity, food consumption and body weights were measured. Running patterns changed over time and with age, with two peaks of activity detected for young, but only one for old mice: speed and distance run was also less for old mice. The mass for six limb muscles was measured at the end of the experiment. The most pronounced increase in mass in response to exercise was for the soleus in young and old mice, and also quadriceps and gastrocnemius in young mice. Soleus and quadriceps muscles were analyzed histologically for myofiber number and size. A striking feature was the many small myofibers in response to exercise in young (but not old) soleus, whereas these were not present after exercise in young or old quadriceps. Overall, there was a striking difference in response to exercise between muscles and this was influenced by age.

Lifelong exercise and locally produced insulin-like growth factor-1 (IGF-1) have a modest influence on reducing age-related muscle wasting in mice.

The age-related loss of skeletal muscle mass and function is termed sarcopenia and has been attributed to a decline in concentrations of insulin-like growth factor-1 (IGF-1). We hypothesized that constitutively expressed IGF-1 within skeletal muscles with or without exercise would prevent sarcopenia. Male transgenic mice that overexpress IGF-1 Ea in skeletal muscles were compared with wild-type littermates. Four-month-old mice were assigned to be sedentary, or had access to free-running wheels, until 18 or 28 months of age. In wild-type mice, the mass of the quadriceps muscles was reduced at 28 months and exercise prevented such loss, without affecting the diameter of myofibers. Conversely, increased IGF-1 alone was ineffective, whereas the combination of exercise and IGF-1 was additive in maintaining the diameter of myofibers in the quadriceps muscles. For other muscles, the combination of IGF-1 and exercise was variable and either increased or decreased the mass at 18 months of age, but was ineffective thereafter. Despite an increase in the diameter of myofibers, grip strength was not improved. In conclusion, our data show that exercise and IGF-1 have a modest effect on reducing aged-related wasting of skeletal muscle, but that there is no improvement in muscle function when assessed by grip strength.

Impact of fasting on the rhythmic expression of myogenic and metabolic factors in skeletal muscle of adult mice.

Circadian rhythms and metabolism are tightly integrated, and rhythmic expression of metabolic factors is common in homeostatic processes. We measured the temporal changes in the expression of myogenic regulatory factors and expression and activity level of molecules involved in protein metabolism in skeletal muscles and livers in mice and examined the impact of fasting. Tissues were collected over 24 h (at zeitgeber times ZT1, ZT5, ZT9, ZT13, ZT17, ZT21, and ZT1 the following day) from adult male C57Bl/6J mice that had been either freely fed or fasted for 24 h. In skeletal muscle, there was a robust rise in the mRNA expression of the myogenic regulatory factors MyoD and myogenin during dark hours which was strongly suppressed by fasting. Circadian pattern was observed for mRNA of MuRF1, Akt1, and ribosomal protein S6 in muscles in fed and fasted mice and for Fbxo32 in fed mice. Activity (phosphorylation) levels of Akt(Ser473) displayed temporal regulation in fasted (but not fed) mice and were high at ZT9. Fasting caused significant reductions in phosphorylation for both Akt and S6 in muscles, indicative of inactivation. Hepatic phosphorylated Akt(Ser473) and S6(Ser235/236) proteins did not exhibit daily rhythms. Fasting significantly reduced hepatic Akt(473) phosphorylation compared with fed levels, although (unlike in muscle) it did not affect S6(Ser235/236) phosphorylation. This in vivo circadian study addresses for the first time the signaling activities of key molecules related to protein turnover and their possible cross-regulation of expression of genes related to protein degradation.

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.

Duchenne muscular dystrophy: focus on pharmaceutical and nutritional interventions.

Duchenne muscular dystrophy is a lethal X-linked muscle disease resulting from a defect in the muscle membrane protein dystrophin. The absence of dystrophin leads to muscle membrane fragility, muscle death (necrosis) and eventual replacement of skeletal muscle by fat and fibrous connective tissue. Extensive muscle wasting and respiratory failure results in premature death often by the early 20s. This short review evaluates drug and nutritional interventions designed to reduce the severity of muscular dystrophy, while awaiting the outcome of research into therapies to correct the fundamental gene defect. Combinations of dietary supplementation with amino-acids such as creatine, specific anti-inflammatory drugs and perhaps drugs that target ion channels might have immediate realistic clinical benefits although rigorous research is required to determine optimal combinations of such interventions.

Rskalpha-actin/hIGF-1 transgenic mice with increased IGF-I in skeletal muscle and blood: impact on regeneration, denervation and muscular dystrophy.

Human IGF-I was over-expressed in skeletal muscles of C57/BL6xCBA mice under the control of the rat skeletal alpha-actin gene promoter. RT-PCR verified expression of the transgene in skeletal muscle but not in the liver of 1- and 21-day old heterozygote transgenic mice. The concentration of endogenous mouse IGF-I, measured by an immunoassay which does not detect human IGF-I, was not significantly different between transgenic mice and wild-type littermates (9.5 +/- 0.8 and 13.3 +/- 1.9 ng/g in muscle; 158.3 +/- 18.6 and 132.9 +/- 33.1 ng/ml in plasma, respectively). In contrast, quantitation with antibodies to human IGF-I showed an increase in IGF-I of about 100 ng/ml in plasma and 150 ng/g in muscle of transgenic mice at 6 months of age. Transgenic males, compared to their age matched wild-type littermates, had a significantly higher body weight (38.6 +/- 0.53 g vs. 35.8 +/- 0.64 g at 6 months of age; P < 0.001), dry fat-free carcass mass (5.51 +/- 0.085 vs. 5.08 +/- 0.092 g; P < 0.001) and myofibrillar protein mass (1.62 +/- 0.045 vs. 1.49 +/- 0.048 g; P < 0.05), although the fractional content of fat in the carcass was lower (167 +/- 7.0 vs. 197 +/- 7.7 g/kg wet weight) in transgenic animals. There was no evidence of muscle hypertrophy and no change in the proportion of slow type I myofibres in the limb muscles of Rskalpha-actin/hIGF-I transgenic mice at 3 or 6 months of age. Phenotypic changes in Rskalpha-actin/hIGF-I mice are likely to be due to systemic as well as autocrine/paracrine effects of overproduction of IGF-I due to expression of the human IGF-I transgene. The effect of muscle specific over-expression of Rskalpha-actin/hIGF-I transgene was tested on: (i) muscle regeneration in auto-transplanted whole muscle grafts; (ii) myofibre atrophy following sciatic nerve transection; and (iii) sarolemmal damage and myofibre necrosis in dystrophic mdx muscle. No beneficial effect of muscle specific over-expression of Rskalpha-actin/hIGF-I transgene was seen in these three experimental models.

The onset of myogenesis in denervated mouse skeletal muscle regenerating after injury.

Denervation of skeletal muscle stimulates increased turnover of muscle nuclei and connective tissue cells. The present investigation tests whether denervation "primes" myogenic precursor cells, so that the onset of DNA synthesis in muscle precursors after traumatic injury occurs earlier than in innervated muscle. The left legs of 29 male BALBc mice were denervated, and 1 week later small incisions were made in the tibialis anterior muscles of both legs (denervated and innervated). At specific times after injury each mouse was injected once with tritiated thymidine to label replicating muscle precursors. Muscle lesions were sampled 10 days after injury (when all precursors had fused to form myotubes) and prepared for auto-radiography. The presence of labelled myotube nuclei showed that muscle precursors had been synthesizing DNA at the time when [3H]thymidine had been injected. Our data suggest that very few precursors were proliferating in denervated muscle within 30 h after injury, and the onset of myogenesis at 30 h was essentially the same in denervated and innervated muscle. The retrospective analysis indicates that there were also similar proportions of muscle precursors proliferating at different times after injury, in regenerating lesions of both denervated and innervated muscle. Thus, denervation does not stimulate an earlier regenerative response in injured skeletal muscle.

The role of tumor necrosis factor-alpha (TNF-alpha) in skeletal muscle regeneration. Studies in TNF-alpha(-/-) and TNF-alpha(-/-)/LT-alpha(-/-) mice.

The role of tumor necrosis factor-alpha (TNF-alpha), an important mediator of the inflammatory response after injury, was investigated in regenerating skeletal muscle. The pattern of expression of TNF-alpha during muscle regeneration was examined by immunohistochemistry in tissue sections of crush-injured or transplanted muscle autografts and in primary cultures of adult skeletal muscle. TNF-alpha was highly expressed in injured myofibers, inflammatory cells, endothelial cells, fibroblasts, and mast cells. Myoblasts and myotubes also expressed TNF-alpha in primary muscle cultures and tissue sections. The essential role of TNF-alpha and its homologue lymphotoxin-alpha (LT-alpha) during muscle regeneration was assessed by basic histology in TNF-alpha(-/-) and TNF-alpha(-/-)/LT-alpha(-/-) mice. No difference was apparent in the onset or pattern of muscle regeneration (i.e., inflammatory response, activation and fusion of myoblasts) between the two strains of null mice or between nulls and normal control mice. However, both strains of null mice appeared more prone to bystander damage of host muscle and regeneration distant from the site of injury/transplantation. Although expression of TNF-alpha may play an important role in muscle regeneration, the studies in the null mice show that redundancy within the cytokine system (or some other response) can effectively compensate for the absence of TNF-alpha in vivo.

Nonspecific binding of nucleic acid probes to Paneth cells in the gastrointestinal tract with in situ hybridization.

Nonspecific binding of a number of unrelated nucleic acid probes to cells in the crypts of Lieberkuhn was observed in the small intestine of mice with the in situ hybridization technique. Hybridization signal was localized to cells which, by virtue of their histological position, represented Paneth cells. This signal could not be removed by RNAse, DNAse, or proteinase K treatment, and was not removed after high-stringency washing conditions. This report indicates that caution must be exercised in the interpretation of in situ hybridization data when looking for nucleic acid sequences in the gastrointestinal tract.

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.

Survival and migration of transplanted male glia in adult female mouse brains monitored by a Y-chromosome-specific probe.

A Y-chromosome-specific probe and in situ hybridization technology have been used to monitor the survival and migration of neonatal male glia isografted to the left cerebral hemisphere of adult female mice. More than 95% of the cultured donor glia were glial fibrillary acidic protein (GFAP)-positive astrocytes. By 4 weeks, large numbers of transplanted glia were found in both cerebral hemispheres; the extent of glial migration was greatest in white matter tracts. This method provides a new way of identifying all surviving donor cells within the brains of immunologically compatible hosts.

Age-associated changes in the response of skeletal muscle cells to exercise and regeneration.

This paper looks at the effects of aging on the response of skeletal muscle to exercise from the perspective of the behavior of muscle precursor cells (widely termed satellite cells or myoblasts) and regeneration. The paper starts by outlining the ways in which skeletal muscle can respond to damage resulting from exercise or other trauma. The age-related changes within skeletal muscle tissue and the host environment that may affect the proliferation and fusion of myoblasts in response to injury in old animals are explored. Finally, in vivo and in vitro data concerning the wide range of signaling molecules that stimulate satellite cells and other aspects of regeneration are discussed with respect to aging. Emphasis is placed on the important role of the host environment, inflammatory cells, growth factors and their receptors (particularly for FGF-2), and the extracellular matrix.

Exposure to tissue culture conditions can adversely affect myoblast behavior in vivo in whole muscle grafts: implications for myoblast transfer therapy.

The effects of tissue culture conditions on the viability of myoblasts in whole muscles transplanted in vivo were investigated. Whole male (SJL/J) donor muscles were exposed to various tissue culture reagents and proteolytic enzymes, and allografted into female (SJL/J) host mice. Desmin immunohistochemistry was used to assess the numbers of myogenic cells (as an index of myoblast viability and the extent of regeneration) in tissue sections of whole-muscle grafts sampled on days 7 and 14. DNA quantitation with a Y-chromosome-specific probe was used to determine the total Y-1 sequence DNA (as an index of myoblast survival and proliferation) in whole-muscle grafts sampled on days 1, 3, and 7. In grafts exposed to serum-free medium, there was a delay in myoblast fusion at 7 days that was recovered by 14 days, but exposure to serum (10% or 20%) had a prolonged adverse effect on myotube formation at 14 days. DNA quantitation demonstrated that either serum-free culture medium or 10% serum enhanced the number of male cells within whole-muscle grafts at 7 days. Proteolytic digestion (even for 5 min) of whole muscles prior to grafting was extremely detrimental to myoblast survival and viability at 7 and 14 days. The unexpected finding of adverse effects of tissue culture conditions on the regeneration of whole-muscle grafts in vivo appears to parallel the major problem of the rapid death of isolated cultured donor myoblasts after injection in myoblast transfer therapy. The use of whole-muscle grafts provides an alternative and sensitive model to analyze the crucial effects of various tissue culture components on the subsequent survival and proliferation of myogenic cells in vivo.

A potential alternative strategy for myoblast transfer therapy: the use of sliced muscle grafts.

Excellent long-term survival (up to 1 yr) of donor skeletal muscle cells was demonstrated using a mouse Y-chromosome specific probe, following the transplantation of grafts of whole muscles from male "normal" C57B1/10Sn mice into dystrophic muscles of female host mice. After the transplantation of equivalent sliced muscle grafts there was extensive movement of the male donor cells and fusion with host myofibres. This contrasts with the extremely poor survival of isolated myoblasts after injection into the same mouse model for Duchenne muscular dystrophy. The use of sliced muscle grafts may therefore represent a potential alternative approach to myoblast transfer therapy.

Why do cultured transplanted myoblasts die in vivo? DNA quantification shows enhanced survival of donor male myoblasts in host mice depleted of CD4+ and CD8+ cells or Nk1.1+ cells.

Overcoming the massive and rapid death of injected donor myoblasts is the primary hurdle for successful myoblast transfer therapy (MTT), designed as a treatment for the lethal childhood myopathy Duchenne muscular dystrophy. The injection of male myoblasts into female host mice and quantification of surviving male DNA using the Y-chromosome-specific (Y1) probe allows the speed and extent of death of donor myoblasts to be determined. Cultured normal C57BL/10Sn male donor myoblasts were injected into untreated normal C57BL/10Sn and dystrophic mdx female host mice and analyzed by slot blots using a 32P-labeled Y1 probe. The amount of male DNA from donor myoblasts showed a remarkable decrease within minutes and by 1 h represented only about 10-18% of the 2.5 x 10(5) cells originally injected (designated 100%). This declined further over 1 week to approximately 1-4%. The host environment (normal or dystrophic) as well as the extent of passaging in tissue culture (early "P3" or late "P15-20" passage) made no difference to this result. Modulation of the host response by CD4+/CD8+ -depleting antibodies administered prior to injection of the cultured myoblasts dramatically enhanced donor myoblast survival in dystrophic mdx hosts (15-fold relative to untreated hosts after 1 week). NK1.1 depletion also dramatically enhanced donor myoblast survival in dystrophic mdx hosts (21-fold after 1 week) compared to untreated hosts. These results provide a strategic approach to enhance donor myoblast survival in clinical trials of MTT.

The migration and intermixing of donor and host glia on nitrocellulose polymers implanted into cortical lesion cavities in adult mice and rats.

The fate of neonatal glia (mostly glial fibrillary acidic protein-positive astrocytes), cultured on nitrocellulose papers and implanted into cortical lesion cavities, was examined in adult mice and rats. In mice, a Y-chromosome-specific probe and in situ hybridization techniques were used to identify male cells. Male-female grafts allowed visualization of donor glia and their behaviour after transplantation; female-male grafts allowed an analysis of how host cells responded to the presence of the implants. There was substantial intermixing of cells, with many donor glia migrating away from the implants and host cells migrating onto both sides of the nitrocellulose paper. In rats, donor glia were labelled with fluorescein-conjugated latex microspheres prior to transplantation on nitrocellulose polymers. The rat data were broadly consistent with those obtained from the mouse; moreover, immunohistochemical studies in rats suggested that the majority of host cells migrating onto the previously cell-coated papers were astrocytes. In a number of studies, glia-coated polymers have been used in an attempt to promote the regrowth of axons across lesion sites in the brain and spinal cord. The present work suggests that both transplanted and host glia may influence the regenerative growth seen in such implants.

Age-related changes in replication of myogenic cells in mdx mice: quantitative autoradiographic studies.

Cell replication in muscle was measured by tritiated thymidine (3H-TdR) incorporation and autoradiography, in mdx mice from 2-44 weeks of age. Pre-mitotic labelling (within 1 h of 3H-TdR injection) was determined in 16 mice aged from 15 to 300 days. In 30 further mdx mice, one leg was irradiated 1 h after 3H-TdR injection to block DNA synthesis. Post-mitotic labelling was measured in both legs 10-15 days later. Between 20 and 60 days of age a very high proportion (up to 2%) of muscle (satellite cell) nuclei were replicating pre-mitotically; from 80-300 days cell replication was detectable but at much lower levels. Centrally placed nuclei within muscle fibres appeared at 24 days, increased rapidly to 50% by 50-100 days, declining thereafter to 25% at 300 days. In post-mitotic samples, labelled myotubes and labelled peripheral muscle nuclei (satellite cell nuclei and myonuclei) appeared at 28 days and were present in the mdx muscles through to 310 days, indicating continued cell replication and muscle regeneration. Myogenic cell replication was both retarded and inhibited by irradiation. These data demonstrate that muscle cell replication in mdx mice commences at about 3 weeks of age, is maximal at 4-8 weeks, but continues at lower levels until at least 44 weeks.

Muscle precursor replication after repeated regeneration of skeletal muscle in mice.

We propose that when skeletal muscle regenerates after injury, myogenic precursor cell replication commences earlier if the muscle has undergone a recent previous cycle of regeneration. To test this a series of muscles in mice were crush-injured and allowed to regenerate. Then either 7 or 28 days after the initial injury, the muscles were reinjured. The onset of DNA synthesis in myogenic precursor cells was determined by injecting each mouse once with tritiated thymidine at a specific time after the second injury. All reinjured muscles were left for 10 days to regenerate to the myotube stage. The presence of autoradiographically labelled myotube nuclei indicated that these nuclei were the progeny of myogenic precursors labelled at the time of injection of tritiated thymidine. Thus the onset of muscle precursor cell replication was determined. A similar series of experiments was conducted on muscle autotransplants, injured at 28 days after transplantation and sampled 10 days later. In none of these situations did muscle precursor cell replication occur earlier than in control muscles, which were injured only once, where myogenesis commenced between 24 and 30 h after injury. The autoradiographic data do not support our hypothesis. We conclude that the increased numbers of muscle precursors reported (by various authors) after repeated cycles of regeneration, are not due to the earlier initiation of DNA synthesis in myogenic precursor cells.

Towards understanding skeletal muscle regeneration.

Factors which effect proliferation and fusion of muscle precursor cells have been studied extensively in tissue culture, although little is known about these events in vivo. This review assesses the tissue culture derived data with a view to understanding factors which may control the regeneration of mature skeletal muscle in vivo. The following topics are discussed in the light of recent developments in cell and molecular biology: 1) Injury and necrosis of mature skeletal muscle fibres 2) Phagocytosis of myofibre debris 3) Revascularisation of injured muscle 4) Activation and proliferation of muscle precursor cells (mpc) in vivo Identification of mpcs; Satellite cell relationships; Extracellular matrix; Growth factors; Hormones; Replication. 5) Differentiation and fusion of muscle precursor cells in vivo Differentiation; Fusion; Extracellular matrix; Cell surface molecules: Growth factors and prostaglandins 6) Myotubes and innervation.