Changes in acetylcholine receptor expression : neuromuscular junction morphology and associated myonuclei in BALB/C mice following muscle contusion injury

Louw, Elizabeth Adrienne (2015-03)

Thesis (MSc)--Stellenbosch University, 2015.

Thesis

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.

AFRIKAANSE OPSOMMING: Kontusie-beserings veroorsaak beduidende spierskade wat skeletspiere as geheel beïnvloed, insluitend die innerverende motorneuron en die spierweefsel se neuromuskulêre-aansluiting (NMA). Tydens besering disintegreer en spat die asetielcholienreseptore (AChR) uitmekaar, en hergroepeer tog oor tyd om 'n optimaal-funksionerende motor-eindplaat te herskep. Hierdie proses behels die opregulering van die reseptor-subeenhede – α, β, γ, δ, ε – uitdrukking tot verskillende grade. Satellietselle is die sleutelrolspelers in hergenerering van spierweefsel, maar min studies wat die verband tussen hergenererende funksies van satellietselle en die rehabilitasie van NMA ondersoek is beskikbaar. Verder behandel die meerderheid van die studies wat asetielcholienreseptore ondersoek die gevolge van 'n senuwee-onderbreking, in plaas van beserings wat die spierweefsel as geheel beïnvloed. Bromodeoksi-uridine (BrdU) is 'n nuttige hulpmiddel in die merking en die monitering van prolifereerde satellietselle. Twee eksperimentele groepe (naamlik PCR en BrdU groepe) van manlike BALB/C muise was onderworpe aan kontusie-besering van agterste ledemate, wat met die gewigimpaktegniek veroorsaak is. Alzet® mini-osmotiese-pompe wat BrdU lewer (50 mM, 1.0 μl.h-1 vrylatingstempo) is voor die besering in die BrdU groep ingeplant. Diere is geslag en die beseerde en kontralaterale, nie-beseerde gastrocnemius-spiere is ingesamel. qPCR is gedoen vir AChR-γ en AChR-ε mRNA uitdrukking op die spiere van die PCR-muise. ‘n Lengte-kriodeursnee van spiere van die BrdU groep was geneem en is met 1) H&E gekleur en 2) immunohistochemies gekleur met α-bungarotoksien om AChRs te visualiseer; en ook met teenliggaampies gekleur vir laminin en BrdU. Beelde is verkry deur ligmikroskopie (1) om kontusie besering in lengtesnee te observeer en te beskryf, en konfokale mikroskopie (2) om die vorm, voorkoms en rangskikking van NMAs langs beide die beseerde en nie-beseerde spiere te observeer. AChRs het hulself in aansluitingsvoue posisioneer en 'n koraal-agtige voorkoms aangeneem - wat as 'n NMA identifiseer kan word. 'n 3D z-stapel beeld op 40x vergroting het gewys dat myonukleusse in ‘n noue verband onder die neuromuskulêre aansluiting teenwoordig is. Hierdie neuromuskulêre-aansluitings is gereël langs die spiere in sentrale band; hoewel kontusie-besering gelei tot 'n verbrokkeling van 'n gedeelte of van die hele aansluitingskompleks. Super-resolusie mikroskopie het ‘n in-diepte strukturele rangskikking getoon in ongeskonde NMA. Dit het binne 7 dae gekronkel en versprei na kontusie-besering. Robuuste, hergenererende NMAs is 14 dae na die besering in spierlengtesnee waargeneem. Oppervlakte en volume is gemeet en het getoon dat NMA grootte 7 dae na besering neig om af te neem, gevolg deur 'n oordrewe toename in NMA grootte op die 14de dag na besering. 'n Twee-stap verkleuring-prosedure het 14 dae na die besering BrdU+ selle onder die neuromuskulêre aansluiting blootgestel. Die resultate van hierdie studie toon dat NMA morfologie inderdaad deur kontusie-besering van spiere beïnvloed, en herstel self deur die van die AChR subeenheid produksie te verhoog. Ons verken nuwe tegnieke vir analisering van neuromuskulêre morfologie en veranderinge na 'n besering en tydens hergenerering. Ons het ook vasgestel dat migrasie van satelliet selle onder die NMA na kontusie-besering plaasvind. Hierdie bevindinge lê die grondslag vir toekomstige navorsing om die rolspelers betrokke by neuromuskulêre hergenerering beter te verstaan.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/96718
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