Limits...
Endomicroscopy and electromyography of neuromuscular junctions in situ.

Brown R, Dissanayake KN, Skehel PA, Ribchester RR - Ann Clin Transl Neurol (2014)

Bottom Line: We show that an integrated EMG/CEM probe is effective in longitudinal evaluation of functional and morphological changes that take place over a 7-day period during axotomy-induced, slow neuromuscular synaptic degeneration.EMG amplitude declined in parallel with overt degeneration of motor nerve terminals.EMG/CEM was safe and effective when nerve terminals and motor endplates were selectively stained with vital dyes.

View Article: PubMed Central - PubMed

Affiliation: Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh Hugh Robson Building, George Square, Edinburgh, EH8 9XD, United Kingdom.

ABSTRACT

Objective: Electromyography (EMG) is used routinely to diagnose neuromuscular dysfunction in a wide range of peripheral neuropathies, myopathies, and neuromuscular degenerative diseases including motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Definitive neurological diagnosis may also be indicated by the analysis of pathological neuromuscular innervation in motor-point biopsies. Our objective in this study was to preempt motor-point biopsy by combining live imaging with electrophysiological analysis of slow degeneration of neuromuscular junctions (NMJs) in vivo.

Methods: We combined conventional needle electromyography with fiber-optic confocal endomicroscopy (CEM), using an integrated hand-held, 1.5-mm-diameter probe. We utilized as a test bed, various axotomized muscles in the hind limbs of anaesthetized, double-homozygous thy1.2YFP16: Wld (S) mice, which coexpress the Wallerian-degeneration Slow (Wld(S)) protein and yellow fluorescent protein (YFP) in motor neurons. We also tested exogenous vital stains, including Alexa488-α-bungarotoxin; the styryl pyridinium dye 4-Di-2-Asp; and a GFP conjugate of botulinum toxin Type A heavy chain (GFP-HcBoNT/A).

Results: We show that an integrated EMG/CEM probe is effective in longitudinal evaluation of functional and morphological changes that take place over a 7-day period during axotomy-induced, slow neuromuscular synaptic degeneration. EMG amplitude declined in parallel with overt degeneration of motor nerve terminals. EMG/CEM was safe and effective when nerve terminals and motor endplates were selectively stained with vital dyes.

Interpretation: Our findings constitute proof-of-concept, based on live imaging in an animal model, that combining EMG/CEM may be useful as a minimally invasive precursor or alternative to motor-point biopsy in neurological diagnosis and for monitoring local administration of potential therapeutics.

No MeSH data available.


Related in: MedlinePlus

NMJ can be visualized using CEM after local injection of fluorochromes. (A) Combined CEM/EMG and hypodermic needle probe (1 mL syringe) used to exogenously administer fluorochromes in vivo. (B) EMG recordings following instillation of Alexa488-α-BTX. As expected, the labeled toxin produced progressive and eventually complete neuromuscular block. (C) CEM image of fluorescent motor endplates after local infusion of Alexa488-α-BTX. (D) Standard confocal microscopic image of NMJ labeled by infusion with Alexa488-α-BTX for comparison. (E) Intramuscular axons in a thy1.2YFP16: WldS mouse 5 days after axotomy. Axons were pseudocolored yellow. (F) The same group of intramuscular axons reimaged after infusion of Alexa488-α-BTX. Endplates were masked and then pseudocolored green, to differentiate their denervated state, denuded of overlying motor nerve terminals. Compare, for example, with Figures 1H and 2A. EMG, electromyography; NMJ, neuromuscular junctions; CEM, confocal endomicroscopy.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4265058&req=5

fig03: NMJ can be visualized using CEM after local injection of fluorochromes. (A) Combined CEM/EMG and hypodermic needle probe (1 mL syringe) used to exogenously administer fluorochromes in vivo. (B) EMG recordings following instillation of Alexa488-α-BTX. As expected, the labeled toxin produced progressive and eventually complete neuromuscular block. (C) CEM image of fluorescent motor endplates after local infusion of Alexa488-α-BTX. (D) Standard confocal microscopic image of NMJ labeled by infusion with Alexa488-α-BTX for comparison. (E) Intramuscular axons in a thy1.2YFP16: WldS mouse 5 days after axotomy. Axons were pseudocolored yellow. (F) The same group of intramuscular axons reimaged after infusion of Alexa488-α-BTX. Endplates were masked and then pseudocolored green, to differentiate their denervated state, denuded of overlying motor nerve terminals. Compare, for example, with Figures 1H and 2A. EMG, electromyography; NMJ, neuromuscular junctions; CEM, confocal endomicroscopy.

Mentions: Translating CEM/EMG recording into a clinical context will require labeling of NMJs with high-contrast fluorochromes, without recourse to transgenic expression of fluorescent proteins. We first tested the feasibility of this in six mice by local infusion of Alexa488-α-BTX, to label postsynaptic acetylcholine receptors (Fig. 3). As expected and indicated by simultaneous EMG recording, labeling AChR with this irreversible toxic ligand blocked synaptic transmission within 20 min of application, both in vivo and in vitro (Fig. 3B; Video S3). Bright, high-contrast fluorescent endplates were subsequently discernible in both mice (Fig. 3C and D) and rats (data not shown). Alexa488-α-BTX was also applied to muscles in thy1.2YFP16: WldS mice, 5 days after sciatic nerve section. The fluorescence intensity of Alexa488-α-BTX-labeled endplates was similar to that of YFP fluorescence of the axons. Alexa488-α-BTX staining also confirmed that several axons had lost their motor nerve terminals (Fig. 3E and F; compare with Fig. 2).


Endomicroscopy and electromyography of neuromuscular junctions in situ.

Brown R, Dissanayake KN, Skehel PA, Ribchester RR - Ann Clin Transl Neurol (2014)

NMJ can be visualized using CEM after local injection of fluorochromes. (A) Combined CEM/EMG and hypodermic needle probe (1 mL syringe) used to exogenously administer fluorochromes in vivo. (B) EMG recordings following instillation of Alexa488-α-BTX. As expected, the labeled toxin produced progressive and eventually complete neuromuscular block. (C) CEM image of fluorescent motor endplates after local infusion of Alexa488-α-BTX. (D) Standard confocal microscopic image of NMJ labeled by infusion with Alexa488-α-BTX for comparison. (E) Intramuscular axons in a thy1.2YFP16: WldS mouse 5 days after axotomy. Axons were pseudocolored yellow. (F) The same group of intramuscular axons reimaged after infusion of Alexa488-α-BTX. Endplates were masked and then pseudocolored green, to differentiate their denervated state, denuded of overlying motor nerve terminals. Compare, for example, with Figures 1H and 2A. EMG, electromyography; NMJ, neuromuscular junctions; CEM, confocal endomicroscopy.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4265058&req=5

fig03: NMJ can be visualized using CEM after local injection of fluorochromes. (A) Combined CEM/EMG and hypodermic needle probe (1 mL syringe) used to exogenously administer fluorochromes in vivo. (B) EMG recordings following instillation of Alexa488-α-BTX. As expected, the labeled toxin produced progressive and eventually complete neuromuscular block. (C) CEM image of fluorescent motor endplates after local infusion of Alexa488-α-BTX. (D) Standard confocal microscopic image of NMJ labeled by infusion with Alexa488-α-BTX for comparison. (E) Intramuscular axons in a thy1.2YFP16: WldS mouse 5 days after axotomy. Axons were pseudocolored yellow. (F) The same group of intramuscular axons reimaged after infusion of Alexa488-α-BTX. Endplates were masked and then pseudocolored green, to differentiate their denervated state, denuded of overlying motor nerve terminals. Compare, for example, with Figures 1H and 2A. EMG, electromyography; NMJ, neuromuscular junctions; CEM, confocal endomicroscopy.
Mentions: Translating CEM/EMG recording into a clinical context will require labeling of NMJs with high-contrast fluorochromes, without recourse to transgenic expression of fluorescent proteins. We first tested the feasibility of this in six mice by local infusion of Alexa488-α-BTX, to label postsynaptic acetylcholine receptors (Fig. 3). As expected and indicated by simultaneous EMG recording, labeling AChR with this irreversible toxic ligand blocked synaptic transmission within 20 min of application, both in vivo and in vitro (Fig. 3B; Video S3). Bright, high-contrast fluorescent endplates were subsequently discernible in both mice (Fig. 3C and D) and rats (data not shown). Alexa488-α-BTX was also applied to muscles in thy1.2YFP16: WldS mice, 5 days after sciatic nerve section. The fluorescence intensity of Alexa488-α-BTX-labeled endplates was similar to that of YFP fluorescence of the axons. Alexa488-α-BTX staining also confirmed that several axons had lost their motor nerve terminals (Fig. 3E and F; compare with Fig. 2).

Bottom Line: We show that an integrated EMG/CEM probe is effective in longitudinal evaluation of functional and morphological changes that take place over a 7-day period during axotomy-induced, slow neuromuscular synaptic degeneration.EMG amplitude declined in parallel with overt degeneration of motor nerve terminals.EMG/CEM was safe and effective when nerve terminals and motor endplates were selectively stained with vital dyes.

View Article: PubMed Central - PubMed

Affiliation: Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh Hugh Robson Building, George Square, Edinburgh, EH8 9XD, United Kingdom.

ABSTRACT

Objective: Electromyography (EMG) is used routinely to diagnose neuromuscular dysfunction in a wide range of peripheral neuropathies, myopathies, and neuromuscular degenerative diseases including motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Definitive neurological diagnosis may also be indicated by the analysis of pathological neuromuscular innervation in motor-point biopsies. Our objective in this study was to preempt motor-point biopsy by combining live imaging with electrophysiological analysis of slow degeneration of neuromuscular junctions (NMJs) in vivo.

Methods: We combined conventional needle electromyography with fiber-optic confocal endomicroscopy (CEM), using an integrated hand-held, 1.5-mm-diameter probe. We utilized as a test bed, various axotomized muscles in the hind limbs of anaesthetized, double-homozygous thy1.2YFP16: Wld (S) mice, which coexpress the Wallerian-degeneration Slow (Wld(S)) protein and yellow fluorescent protein (YFP) in motor neurons. We also tested exogenous vital stains, including Alexa488-α-bungarotoxin; the styryl pyridinium dye 4-Di-2-Asp; and a GFP conjugate of botulinum toxin Type A heavy chain (GFP-HcBoNT/A).

Results: We show that an integrated EMG/CEM probe is effective in longitudinal evaluation of functional and morphological changes that take place over a 7-day period during axotomy-induced, slow neuromuscular synaptic degeneration. EMG amplitude declined in parallel with overt degeneration of motor nerve terminals. EMG/CEM was safe and effective when nerve terminals and motor endplates were selectively stained with vital dyes.

Interpretation: Our findings constitute proof-of-concept, based on live imaging in an animal model, that combining EMG/CEM may be useful as a minimally invasive precursor or alternative to motor-point biopsy in neurological diagnosis and for monitoring local administration of potential therapeutics.

No MeSH data available.


Related in: MedlinePlus