The effect of decamethonium bromide induced postsynaptic membrane blockade on the development of the musculoskeletal system in the embryo of the domestic fowl (gallus gallus domesticus).
Macharia, R G
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The effects of post-synaptic membrane blockade in the neuromuscular junctions have been investigated in the developing chick embryo. Decamethonium bromide (DeB), a non competitive blocker of acetylcholine that binds strongly to the postsynaptic membrane of the neuromuscular junctions was applied at 0.2% solution in normal saline on the chorioallantoic membrane for the experimental group while the controls received normal saline. Body weights, skeletal muscle histology and ultrastructure were compared between age-matched control and experimental embryos from day 7 to day 20 of incubation. Bone histology of the tibia and the general ossification pattern was similarly observed from day 7 to 14. Immunohistochemical fibre reactions were carried out on serial cryostat sections from control and experimental embryo leg muscles associated with the tibia. The sections were stained with F59 antibody, which stains myosin heavy chains in fast fibres, S46 antibody which stains myosin heavy chain in slow fibres and anti desmin which stains desmin at the periphery of the Z-line of muscle fibres. The control and experimental embryo muscles were also studied by Electron and Light microscopy to show both ultrastructure and general histology respectively. Serial sections from the tibia were stained with Masson's trichrome, Alcian blue and Hematoxylin to show bone formation, cartilage and general structure respectively. Alizarin red and Alcian blue staining was performed on whole embryos to determine cartilage and bone development. The experimental embryos showed massive subcutaneous oedema, reduced body weights and high mortality compared with the controls, which never had any oedema. Muscle development was retarded and immunohistochemical reaction XIV revealed delayed muscle patterning as well as degeneration in the experimental embryos. There was almost complete skeletal muscle degeneration in the experimental group of embryos by 14th day of incubation. Electron microscopy showed well-developed myofibrils in the controls while the same did not form in the experimental group. In both the control and experimental embryos, the muscle membrane appeared intact however, the cytoplasm was dense and vacuolated in the experimental group. Histological sections showed that most of the degeneration process in the muscles in the experimental embryos started after the application of second dose of DCB. Bone histology showed that timing and onset of ossification of the tibia was not affected by postsynaptic blockade in the experimental embryos although the size of tibia was reduced by about 30% in comparison to the controls. There was fusion of the intervertebral joints in the cervical region and also the femorotibial joint. The lowerjaw was found to protrude beyond the upper jaw in the experimental embryos. The body weights of the control embryos were found to be significantly higher (p<0.05) than in the experimental group and this was more pronounced from day 16 of incubation. Decamethonium bromide induced blockade produced complete paralysis in the experimental embryos and interfered with normal myosin heavy chain formation, which leads to disturbed muscle patterning and degeneration. The degenerative changes in muscles in turn resulted in subcutaneous fluid accumulation due to reduced muscle contraction and slowed venous return. Paralysis in the embryos resulted in poor joint formation and in some cases, joint fusion due to lack of skeletal movements around the articular surfaces. On the other hand DCB did not to have any influence on the ossification process of the long bones, which appears to be under an xv independent genetic program. Skeletal muscle paralysis caused reduced long bone size by 30%. Bone size development is affected by the intermittent cycles of compression and tension from muscles, which leads to calcification, remodelling and shaping of the bones. Paralysis counteracted the beneficial effect of muscle contraction thereby leading to lower bone sizes in the experimental embryos. In conclusion, DCB induced synaptic blockade caused severe skeletal muscle degeneration, disturbed muscle patterning, joint fusion between bones and reduced size of the long bones in the chick embryo. However DCB did not affect the timing of bone ossification.