Neuromuscular junction endplate morphology, acetylcholine receptor aggregation and accessory protein co-localisation during regeneration of a skeletal muscle crush injury

Faulmann, T. S. (2019-03)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

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.

AFRIKAANSE OPSOMMING: Perifere senuwese bewaar die skeletspier-motoriese eenhede by die neuromuskulêre-aansluiting (NMA) om neurale transmissie te herleef. Die asetielcholienreseptore (AChRs) is geleë op die post-sinaptiese membraan en het hulp-na-sinaptiese proteïene wat benodig word vir die kompleks. Rapsyn, MuSK, LRP4 en Dok7 is almal betrokke om die NMJ-funksies behoorlik te verseker. Ontwrigting van hierdie proteïene, die AChRs en die interaksies tussen hulle vind plaas tydens verskeie gevalle van endogene of eksogene komplikasies. Die huidige studie het 'n kontusie-beserings model gebruik en daarop gemik om kwalitatiewe morfologie van die skeletspierweefsel en die post-sinaptiese NMJ in gesonde volwasse muise te vestig. Veranderinge wat plaasgevind het as gevolg van geïnduseerde besering, was beoordeel. 'n Tydlyn van spierfunksie is ook geassesseer op voor- en verskeie na-beserings tydspunte. Muise is verdeel in kontrolegroep (D0) of een van drie beseringsgroepe wat op verskillende tydspunte na besering geoffer is. Groepe is geoffer op 3 (D3), 7 (D7) of 14 (D14) dae na besering. Spierkrag/stimulasie frekwensietoetsing is by basislyn uitgevoer en onmiddellik gevolg deur die spierverliesbesering in die beserings eksperimentele groepe. Spierkragtoetsing is weer uitgevoer op die onderskeie tydspunte net voor opoffering. Nadat die aorta gesny is, is bloedmonsters versamel deur die torakale holte te dreineer, en plasma geïsoleer vir MuSK ELISA-analise. Die gastrocnemius spiermonsters is geoes en dwarsdeursnit of lengte gemonteer op kurk. Dit is gevries in vloeibaar stikstofgekoelde isopentaan en dan in afdelings gesny. Die afdelings is gekleur met H & E om die weefselmorfologie te assesseer. Op al vier tydspunte is immunohistochemie (IHC) met kombinasies van teenliggaampies gebruik om die AChR (α-Btx) saamgekleur met elkeen van die geassosieerde post-sinaptiese proteïene hierbo genoem. Fluorescentie beelde is verkry met behulp van ‘n konfokale mikroskoop. Seleksiekriteria is toegepas op beelde om 'n gesig NMJ te identifiseer om te analiseer. Beeldontleding met behulp van ImageJsagteware het die totale uiteenlopende area (TUA), totale gekleurde area (TGA), vlekintensiteit (VI) en ko-lokalisering beoordeel. Besering het 'n algemene afname in kragproduksie veroorsaak wat nog beduidend laer was by D14 (P<0,0001). H & E-kleuring het bevestig dat die kontusie-besering spiervernietiging veroorsaak. Plasma MuSK konsentrasies het eksponensieel gestyg in reaksie op besering en beriek by D14 (P<0,0001). Dit bevestig skade aan die post-sinaptiese NMJ. IHC-kleuring het duidelike mede-voorkoms en korrelasie tussen die AChR en sy gepaardgaande postsinaptiese proteïene by D0 gevestig. Kolokalisering van die AChR met die postsynaptiese proteïene is ernstig geraak deur besering en het beduidend afgeneem by D3. Herstel het by D7 begin. Al vier postsinaptiese hulpproteïene het gereageer op beserings met uitgebreide verspreiding vanaf hul basislyn (beide TOA en TSA) en 'n verlies aan struktuur. Verspreiding was die ergste vir LRP4 en MuSK, en die minste vir rapsyn. By D14 was daar merkbare verbeteringe in die proteïene-subgroepe, maar die minste verbetering in LRP4. Ten slotte het die kontusie-beserings negatiewe uitwerkings op beide die strukturele integriteit en funksionele kapasiteit van die NMJ gehad. Slegs gedeeltelike herstel is deur D14 behaal, en nie alle hulpproteïene het dieselfde tydskursus gevolg nie.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105717
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