Department of Physiological Sciences

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Browsing Department of Physiological Sciences by Subject "Acetylcholine -- Receptors"

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    Neuromuscular junction endplate morphology, acetylcholine receptor aggregation and accessory protein co-localisation during regeneration of a skeletal muscle crush injury
    (Stellenbosch : Stellenbosch University, 2019-03) Faulmann, T. S.; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: At the neuromuscular junction (NMJ), peripheral nerves innervate the skeletal muscle to relay neural transmission. The acetylcholine receptors (AChRs) are located on the post-synaptic membrane and have auxiliary post-synaptic proteins required for the complex. Rapsyn, MuSK, LRP4 and Dok7 are all involved in ensuring the NMJ functions appropriately. Disruptions to these proteins, the AChRs and the interactions between them occur during various instances of endogenous or exogenous complications. The current study utilised a contusion injury model and aimed to establish morphology of the skeletal muscle tissue and the post-synaptic NMJ in healthy adult mice before qualitatively and quantitatively assessing the changes that occurred in response to injury. A timeline of muscle function was also assessed at pre- and several post-injury time points. Mice were split into control (D0) or one of three injury groups that were sacrificed at different time points post-injury, namely after 3 (D3), 7 (D7) or 14 (D14) days. Muscle force/stimulation frequency testing was conducted at baseline and followed immediately by induction of the muscle crush injury in the injury experimental groups. Muscle force testing was conducted again at the respective time points prior to sacrifice. After severing the aorta, blood samples were collected by draining the thoracic cavity, and plasma isolated for MuSK ELISA analysis. Gastrocnemius muscle samples were harvested, mounted on cork either cross-sectionally or longitudinally, and frozen in liquid nitrogen cooled isopentane. Samples were cryo-sectioned and initially stained with haematoxylin and eosin (H&E) to assess morphology. At all four time points, immunohistochemistry (IHC) with combinations of antibodies was used to identify the AChR (α-Btx) co-stained with each associated post-synaptic protein mentioned above. Fluorescence images were acquired using a confocal microscope and selection criteria were applied to images to identify en face NMJs to analyse. Image analysis using ImageJ software assessed the total outlined area (TOA), total stained area (TSA), staining intensity (SI) and co-localisation. Injury caused a general decrease in force production that was still significantly lower at D14 (P < 0.0001). H&E staining confirmed that the contusion injury resulted in substantial destruction to the muscle tissue. Plasma MuSK concentrations rose exponentially in response to injury, peaking at D14 (P < 0.0001), confirming damage to the post-synaptic NMJ. IHC staining established clear co-occurrence and correlation between the AChR and its associated postsynaptic proteins at D0. Co-localisation of the AChR with the post-synaptic proteins was affected severely by injury, with a general trend for a nadir at D3 (P < 0.01), before a return to baseline was initiated by D7. Although all four post-synaptic auxiliary proteins responded to injury with widespread dispersion from baseline (both TOA and TSA) and a loss of structure, this was the most severe for LRP4 and MuSK, and least severe for rapsyn. By D14 there was noticeable improvement across protein subgroups, but least improvement in LRP4. In conclusion, the contusion injury affected both the structural integrity and functional capacity of the NMJ negatively. Only partial recovery was achieved by D14, and not all auxiliary proteins followed the same time course.