Browsing by Author "Louw, Elizabeth Adrienne"

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    Changes in acetylcholine receptor expression : neuromuscular junction morphology and associated myonuclei in BALB/C mice following muscle contusion injury
    (Stellenbosch : Stellenbosch University, 2015-03) Louw, Elizabeth Adrienne; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Contusion injuries cause significant muscle damage, that effects skeletal muscle in its entirety, including the innervating motorneuron and the myofibre’s neuromuscular junction (NMJ). Upon injury, the acetylcholine receptors (AChR) scatter and disintegrate yet reaggregate over time to re-create an optimally functioning motor end-plate. This process involves the upregulation of the receptor subunits’ – α, β, γ, δ, ε – expression to varying degrees. Satellite cells are key role players in muscle regeneration, but studies linking the regenerative roles of satellite cells to the rehabilitating NMJ are limited. Moreover, the majority of studies on acetylcholine receptors investigate the effects of a denervation event rather than an injury that affects the muscle tissues in their entirety. Bromodeoxyuridine (BrdU) is a useful tool in labelling and tracking proliferated satellite cells. Two experimental groups (referred to as PCR and BrdU group) of male BALB/C mice were subjected to a hind limb contusion injury induced with the mass-drop technique. Alzet® mini-osmotic pumps delivering BrdU (50 mM, 1.0 μl.h−1 release rate) were inserted into the BrdU group prior to injury. Animals were sacrificed at days 1, 3, 5, 7, 10 and 14 post injury. Both injured and contralateral, non-injured gastrocnemius muscles were collected. qPCR was performed for AChR-γ and AChR-ε mRNA expression on the muscles of the PCR mice. Muscles from the BrdU group were cryo-sectioned in longitudinal orientation and stained with 1) H&E and 2) immunohistochemically with α-bungarotoxin to visualise AChRs; and also with antibodies against laminin and BrdU. Images were obtained by light microscopy (1) to detect and describe contusion injury in longitudinal section and confocal microscopy (2) to observe the form, prevalence and arrangement of NMJs along both the injured and non-injured muscle. AChRs position themselves into junctional folds that adopted a coral-like appearance – identifiable as a NMJ. A 3D z-stack image at 40x magnification revealed myonuclei residing beneath the NMJ in intimate connection. These NMJ were arranged along the muscle in central band; however contusion injury resulted in a disintegration of part of or the entire junctional complex. Super-resolution microscopy revealed in depth structural arrangement in the intact NMJ. This became jagged and dispersed following contusion injury, by 7 days. Robust, regenerating NMJs were detected in muscle sections at 14 days post injury. Surface area and volume were measured and revealed a trend towards a decrease in NMJ size at 7 days post injury, followed by an exaggerated increase in NMJ size by day 14 post injury. A two-step staining procedure exposed BrdU+ cells residing beneath the neuromuscular junction at 14 days post injury. The results of this study show that NMJ morphology is indeed affected by muscle contusion injury, and repairs itself by increasing its AChR subunit production. We explored novel techniques for analysis of neuromuscular morphology and its changes after injury and during regeneration. We have also ascertained the migration of satellite cells to beneath the NMJ following contusion injury. These findings lay the foundation for future research to better understand the role players involved in neuromuscular regeneration.