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Harnessing in and ex vivo imaging to investigate motor nerve terminal injury and recovery in a mouse model of Guillain-Barré syndrome

Rupp, Angelika Frances (2012) Harnessing in and ex vivo imaging to investigate motor nerve terminal injury and recovery in a mouse model of Guillain-Barré syndrome. PhD thesis, University of Glasgow.

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Abstract

The peripheral nerve disorder Guillain-Barré syndrome (GBS) accounts for the most common cause of acute acquired paralysis in the Western World. Circulating anti-ganglioside antibodies (Abs) are considered important mediators of this disease. Research conducted in a mouse model of GBS has the revealed the neuromuscular junction (NMJ) as a potential site of anti-ganglioside Ab-binding, due to this structure lying outside the blood-nerve barrier. The ganglioside composition of the neural and glial components of the NMJ determines which of these structures are bound and in the following subjected to complement-mediated injury. Some patients suffering from the acute motor axonal neuropathy (AMAN) forms of GBS recovery very rapidly from paralysis; it has been proposed that in these patients the injury was restricted to the distal motor axons and nerve terminals (NTs), which are able to regenerate over a short time-frame. To test this hypothesis, the mouse model of GBS was combined with in and ex vivo imaging of the NMJ in the ventral neck muscles of mice expressing cytosolic fluorescent proteins in their axons (cyan fluorescent protein: CFP) and Schwann cells (green fluorescent protein: GFP). Following confirmation of the stability of NMJs in these mice over time, optimisation of in vivo imaging procedures and determination of the most advantageous Abs for these kinds of investigations, 45 mice were subjected to a single in vivo topical application of anti-ganglioside Ab followed by a source of complement. Group A (n=15) received Ab that selectively bound to the NTs, group B (n=15) received Abs that bound both to the NTs and the perisynaptic Schwann cells (pSCs) and group C (control animals; n=15) only received complement. Evolution of the injury was documented by in vivo imaging, and following euthanasia the muscles were reimaged ex vivo both quantitatively and qualitatively, either immediately, or after 1, 2, 3 or 5 days of regeneration (each n=3 per group). Within 15 minutes of complement application, a rapid loss of CFP overlying the NMJ could be seen; in group A, the GFP signal remained unchanged, whereas in group B the GFP signal was also lost. In group C no changes to either CFP or GFP were observed. At 24h, 6% of the superficial NMJs in group A and 12% of the NMJs in group B exhibited CFP; the CFP-loss extended proximally until the axons formed little bundles. In both groups, CFP returned within the next five days (group A: 93.5%, group B: 94%; p=0.739), with the recovery of CFP being preceded by a return of GFP-positive cells overlying the NMJ in group B. Both in groups A and B, the pSCs exhibited processes which extended beyond the normal NMJ boundaries and very occasionally accompanied by axonal sprouts. This behavior was similar to that of pSCs challenged by traumatic denervation of their NMJ, albeit the changes observed in the Ab-mediated injury were lower in frequency and less dramatic when compared to those observed following traumatic denervation. The rate of motor NT-regeneration corresponds well to rates observed following the application of spider or snake toxins, which mediate selective injury to the NTs. Auxiliary investigations revealed that the loss of CFP at the NMJ correlated with a loss of axonal architectural proteins (neurofilament (NF) heavy and light) and a return of CFP at the NMJ was accompanied by a return of NF heavy. Ultrastructurally, the injured NTs resembled NTs undergoing degeneration following a traumatic denervation of the endplate and following five days of regeneration, the NMJs exhibited a physiological morphology. The results described above indicate that following a single anti-ganglioside Ab-mediated and complement-mediated attack, independent of whether there are healthy and mature pSCs overlying the NMJ, the murine NT is capable of recovering its architectural, axolemmal and ultrastructural integrity very rapidly. This data supports the notion that an equivalent mechanism may account for the rapid recovery seen in some clinical cases of AMAN.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Publications linked to this thesis: a.) Experimental Neurology 233 (2012): 836-848 b.) The Journal of Clinical Investigation 122 (2012): 1037-1061
Keywords: Guillain-Barre syndrome, autoimmune neuropathy, mouse model, fluorescent mouse, in vivo imaging, ex vivo imaging, gangliosides, antibody, complement, neuromuscular junction, motor nerve terminal, perisynaptic Schwann cell, injury, regeneration
Subjects: R Medicine > RZ Other systems of medicine
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Neuroscience and Psychology
Supervisor's Name: Willison, Prof. Hugh J.
Date of Award: 2012
Depositing User: DR Angelika F Rupp
Unique ID: glathesis:2012-3364
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 May 2012
Last Modified: 10 Dec 2012 14:06
URI: http://theses.gla.ac.uk/id/eprint/3364

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