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M D Grounds

Publications and source records attributed to M D Grounds.

At least 73 records · Page 4Linked to original sources
PubMed1987

Reutilisation of tritiated thymidine in studies of regenerating skeletal muscle.

Two different aspects of tritiated thymidine (3H-Tdr) reutilisation in skeletal muscle were examined. Injection of a high dose (7 microCi/g) of 3H-Tdr into mice prior to crush injury of skeletal muscle resulted in heavy labelling (grain counts) of myotube nuclei 9 d later. In contrast, myotube nuclei were essentially unlabelled when a low dose (1 microCi/g) of 3H-Tdr was injected at similar times with respect to injury. It was concluded that labelling seen after the high dose was due to reutilisation of 3H-Tdr. (Such 3H-Tdr reutilisation can account for the results of Sloper et al. (1970) which previously supported the concept of a circulating muscle precursor cell.) When replicating muscle precursors were labelled directly with 3H-Tdr 48 h after injury, the percentages of labelled myotube nuclei and the distribution of nuclear grain counts were similar with either high or low dose. We also investigated whether the light labelling seen in regenerated myotube nuclei after 9 d, when 3H-Tdr had been injected before the onset of myogenesis (as found by McGeachie and Grounds 1987), was due to 3H-Tdr reutilisation or, alternatively, to proliferation of local cells in the wound which subsequently gave rise to muscle precursors. Labelling of myotube nuclei was compared in mice injected with 3H-Tdr either 2 h before, or 2 h after injury. In another experiment, mice were injected 12 h after injury and lesions sampled 1, 12 or 36 h later, to see whether local cells were replicating 12 h after injury and what labelled cells subsequently entered to wound.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1987

A model of myogenesis in vivo, derived from detailed autoradiographic studies of regenerating skeletal muscle, challenges the concept of quantal mitosis.

We have recently shown that myogenesis following severe injury is prolonged compared with minor injury (McGeachie and Grounds 1987). In this previous autoradiographic study 44 mice were injected with tritiated thymidine at various times after muscle injury (0 to 120 h), and samples were taken 9 d after injury to determine the percentage of labelled myotube nuclei. In the present study the same experimental data are analysed in detail to reveal how many times labelled muscle precursors divided before fusing to form myotubes. Additional mice were prepared and samples removed 1 h after injection of tritiated thymidine to determine the maximum grain counts of premitotic nuclei. When a labelled premitotic nucleus divides, each of the two daughter nuclei will contain half of the original label. The grain counts of nuclei resulting from sequential divisions of a maximally labelled premitotic nucleus, forms the basis for our detailed analysis which can reveal how many times a muscle precursor has divided after labelling. Nine days after injury the autoradiographic grain counts of labelled myotube nuclei were analysed in detail. The results describe an in vivo model of myogenesis which we use to evaluate quantitatively observations derived from tissue culture studies. The analysis shows that, at the onset of myogenesis in regenerating muscle (30 h after injury), muscle precursors divide only twice before fusing to form myotubes. This observation challenges the concept of quantal mitosis as defined by the tissue culture studies of Quinn et al. (1984, 1985).

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PubMed1987

Initiation and duration of muscle precursor replication after mild and severe injury to skeletal muscle of mice. An autoradiographic study.

We test the proposal (McGeachie and Grounds 1985) that myogenesis following severe (crush) injury is prolonged compared with minor (cut) injury. Forty-four mice were injured with a cut and a crush lesion on different legs, and tritiated thymidine was injected at various times after injury (0 to 120 h), samples of regenerated muscle were taken 9d after injury and autoradiography was used to determine the initiation of muscle precursor replication, and duration of proliferation after the two different injuries. In both lesions replication of potential myoblasts was initiated 30 h after injury. Myogenesis was essentially completed in cut lesions by 96 h after injury, although the peak was finished by 60 h. In contrast, significant muscle precursor replication in crush lesions was still occurring 96 h after injury, and myogenesis was almost finished by 120 h. The pronounced difference in duration of myogenesis in different lesions strongly supports the original proposal. The extended duration of myogenesis in crush lesions, in conjunction with tritiated thymidine reutilisation, appears to account for conflicting experimental results in support of the concept of a circulating muscle precursor cell.

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PubMed1986

The development of fibre types in grafts of a slow tonic avian muscle, the dorsocutaneous latissimus dorsi.

The dorsocutaneous (DLD) and anterior (ALD) latissimus dorsii are both homogeneous slow tonic muscles. Autografts of mature DLD were attached onto the ALD of chickens to study regeneration of slow tonic muscle fibres innervated exclusively by slow tonic nerves. Fifty-three grafts were examined from 3 to 231 days after implantation for myosin ATPase, and for heavy chains of fast myosin. New muscle fibres in grafts were initially type 1 (slow) or type 2 (fast twitch). Tonic type 3 fibres were slow to differentiate and were not seen within 59 days. From 105 days many fibres were type 3A and type 1 were no longer apparent. However, type 2 fibres persisted and appeared to be present instead of type 3B fibres even after 8 months.

Adenosine Triphosphatases↗
PubMed1985

Can cells extruded from denervated skeletal muscle become circulating potential myoblasts? Implications of 3H-thymidine reutilization in regenerating muscle.

The hypothesis that satellite cells which leave denervated skeletal muscle might become circulating potential myoblasts which could participate in myogenesis in distant sites in the body has been tested. Sixteen mice had one hindlimb denervated and were given 7 daily injections of 3H-thymidine (3H-Tdr). One day later extensor digitorum longus muscle isografts from unlabelled mice were inserted into each hindlimb. As controls, the procedure was repeated in 6 non-denervated labelled mice. Fourteen days after their insertion, isografts in denervated mice contained many labelled myotubes with a labelling index of 55 +/- 4% (mean +/- SEM). In the control isografts in non-denervated ice, 38 +/- 4% of myotube nuclei were labelled. The results show that either labelled cells, or 3H-Tdr, had transferred from the host to isografts in both cases. The probability of 3H-Tdr reutilization was demonstrated in regenerating livers of 8 similarly labelled mice, where 34 +/- 3% hepatocytes adjacent to crush lesions were labelled after 14 days. This conclusion was reached because only 2-3% of normal hepatocytes incorporate 3H-Tdr under these conditions and this population is inadequate to provide sufficient labelled precursor cells for the large numbers of labelled regenerated hepatocytes. Therefore, it was concluded that 3H-Tdr reutilization is the most likely explanation for labelled myotube nuclei in the muscle isografts (rather than movement of labelled precursor cells), and that additional label for reutilization had been derived from breakdown of labelled cells in denervated muscle. The data do not support the hypothesis of a circulating precursor for skeletal muscle cells.

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PubMed1984

Improved chimaeric mouse model confirms that resident peritoneal macrophages are derived solely from bone marrow precursors.

The origin of resident peritoneal macrophages was studied in radiation mouse chimaeras with and without reconstitution of the peritoneum with viable isogeneic peritoneal cells. The selection of host and donor strains were such that the isoenzymes of glucose-6-phosphate isomerase could be used to distinguish host from donor bone marrow derived cells. It was found that the resident peritoneal macrophages were completely replaced by bone marrow donor derived cells within 5-6 weeks. There was little difference between the results from mice which had been additionally reconstituted with peritoneal cells and those which were not.

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PubMed1983

Isoenzyme studies of whole muscle grafts and movement of muscle precursor cells.

Isoenzymes of glucose-6-phosphate isomerase (GPI: E.C. 5.3.1.9) were used as markers to determine the origin of cells which give rise to new muscle formed in allografts of whole intact muscle. GPI isoenzymes were also employed to see whether host precursor cells, which have been shown to contribute to muscle formation in grafts of minced muscle, can be derived from muscle lying adjacent to grafts. Excellent muscle regeneration was found in allografts of extensor digitorum longus (EDL) muscle examined after 58 days: 12 of 16 grafts contained 80% or more new muscle. Isoenzyme analysis showed that most, and in 2 instances all, new muscle was derived from implanted donor cells; however, there was strong evidence that in 5 grafts some, or all, new muscle must have resulted from host cells moving into the graft. Although hybrid isoenzyme was not detected this was attributed to factors associated with host tolerance which appear to interfere with fusion between host and donor myoblasts. Isografts of minced muscle were placed next to whole EDL muscle allografts to see if cells from allografts moved into adjacent regenerating tissue. Unfortunately, muscle regeneration in minced isografts was poor; only 3 contained 50% or more new muscle and most contained large amounts of fibrous connective tissue. Only a single isoenzyme band was detected in 11 isografts, but in five instances, the presence of a second band showed that cells from EDL allografts were also present. As no hybrid isoenzyme was detected, it is not known whether these cells which had moved into the regenerating minced grafts were muscle precursors, fibroblasts or some other cell types.

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PubMed1983

Skeletal muscle precursors do not arise from bone marrow cells.

This paper tests the hypothesis that bone marrow stem cells can give rise to circulating muscle precursor cells. Irradiated host mice were reconstituted with bone marrow cells from a second strain of mice: the two strains were each homozygous for one of the two different isoenzyme forms of the enzyme glucose-6-phosphate isomerase, which enable cells of the two strains to be identified by different isoenzyme mobilities on starch gel electrophoresis. Isoenzyme patterns of 16 regenerated muscle isografts and 7 samples of host muscle (from 13 mice) were examined to determine whether donor bone marrow cells contributed to formation of new skeletal muscle. No evidence was found to support the hypothesis that myoblasts can arise from bone marrow precursor cells, although the possibility that muscle precursor cells derived from other tissues may circulate in the blood stream was not excluded.

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PubMed1982

Direct evidence that inflammatory multinucleate giant cells form by fusion.

Multinucleate giant cells (MGC) are believed to be formed by fusion of macrophages. In a chimaeric mouse composed of two histoincompatible strains each homozygous for one of the two isoenzymic forms of glucose-6-phosphate isomerase it was found that hybrid enzyme was produced in MGC-rich leucocytic exudates. This hybrid can only occur if nuclei of the two different strains reside within a common syncytial cytoplasm, demonstrating unequivocally that macrophage fusion occurred between cells of the two strains. Since the two strains were histoincompatible it appears that no strain specific recognition is necessary for fusion to occur.

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PubMed1981

X-linked gene loci and muscular dystrophy.

It is proposed that examination of known X-linked gene products might elucidate the gene loci and genetic defects underlying X-linked muscular dystrophies. On the basis of this hypothesis, kinetic studies were performed on the enzyme phosphoglycerate kinase (E.C. 2.7.2.3.) in blood taken from subjects with Duchenne and Becker muscular dystrophy and from controls. Although no differences were detected between enzyme from any source, it is considered that further study of X-linked gene products is warranted.

Adenosine Triphosphatases↗
PubMedDate not supplied

Fusion between myogenic cells in vivo: an ultrastructural study in regenerating murine skeletal muscle.

Fusion of myogenic cells in adult murine skeletal muscle regenerating in vivo was examined at the ultrastructural level. Fusion of myoblast to myoblast, myoblast to myotube, and myotube to myotube was observed by 4 to 5 days after injury. Fusion between myogenic cells (myoblasts or myotubes) lacking a definitive glycocalyx or external lamina (basal lamina) occurred at multiple sites. It was defined by zones of cytoplasmic confluence between apposed cells at sites where contiguous segments of the cell membranes were interrupted while their edges had united resulting in linear continuity; vesicles of varying dimensions were frequent in these areas of fusion. Myoblasts were seen invaginating the surface of myofibres and again vesicles were seen in abundance in such regions. Cilia were often observed at this junctional zone suggesting that they might play a role in fusion. In the one example of probable fusion between a myotube and a myofibre, only a single area of cytoplasmic continuity was apparent.

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PubMedDate not supplied

Specific cloning of DNA fragments unique to the dog Y chromosome.

A novel technique that enabled the specific cloning of a DNA fragment unique to the dog Y chromosome is described. The method involves competitive hybridization of DNA prepared from male dog lymphocytes with biotin-labeled DNA prepared from female dog lymphocytes. The biotinylated female-female and male-female hybrid DNA fragments were removed by capture with streptavidin-coated paramagnetic particles. Full-length double-stranded DNA was generated from the remaining fragments by using the Klenow fragment of DNA polymerase I, followed by direct cloning using a low-background ligation technique. Analysis of putative recombinant clones derived by this method has led to the identification of a fragment that hybridizes specifically to male dog DNA. The clones were selected initially on the basis of a differential signal obtained when hybridized to dilutions of male and female dog DNA immobilized on neural nylon membrane. To evaluate its suitability as a probe for trans-sexually grafted cells in transplantation studies, the fragment was labeled with digoxigenin and hybridized in situ to male and female dog tissue sections. The clone designated number 6.2 hybridized strongly to male dog nuclei. The cloning strategy employed could be extended to other studies in which competitive reassociation can be used to identify unique DNA sequences.

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PubMedDate not supplied

Problems and solutions in myoblast transfer therapy.

Duchenne muscular dystrophy is a severe X-linked neuromuscular disease that affects approximately 1/3500 live male births in every human population, and is caused by a mutation in the gene that encodes the muscle protein dystrophin. The characterization and cloning of the dystrophin gene in 1987 was a major breakthrough and it was considered that simple replacement of the dystrophin gene would ameliorate the severe and progressive skeletal muscle wasting characteristic of Duchenne muscular dystrophy. After 20 years, attempts at replacing the dystrophin gene either experimentally or clinically have met with little success, but there have been many significant advances in understanding the factors that limit the delivery of a normal dystrophin gene into dystrophic host muscle. This review addresses the host immune response and donor myoblast changes underlying some of the major problems associated with myoblast-mediated dystrophin replacement, presents potential solutions, and outlines other novel therapeutic approaches.

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PubMedDate not supplied

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.

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PubMedDate not supplied

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.

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PubMedDate not supplied

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.

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