Name: muscle velocity recovery cycles to examine muscle membrane properties
Uploaded: 2020-02-19T14:00:00.000+00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Muscle Velocity Recovery Cycles to Examine Muscle Membrane Properties at JoVE.com

This study was financially supported mainly by the two grants from Lundbeck Foundation (Grant number R191-2015-931 and Grant number R290-2018-751). Additionally, the study was financially supported by Novo Nordisk Foundation Challenge Programme (Grant number NNF14OC0011633) as part of the International Diabetic Neuropathy Consortium.

All subjects must provide written consent prior to examination, and the protocol must be approved by the appropriate local ethical review board. All methods described here were approved by the Regional Scientific Ethical Committee and Danish Data Protection Agency.
1. Preparation of the subject
<ol>
	<li>Assess subjects&#39; medical histories to ensure that they do not have any previous nervous system disorders other than the disease group that will be investigated.</li>
	<li>Inform the subject in detail about the examinations and request to obtain written consent.
	<ol>
		<li>Inform the subject about the insertion of two needles in a leg muscle and that the muscle fibers will be stimulated with weak current.</li>
		<li>Explain that the sensation may feel slightly unpleasant.</li>
		<li>Inform the subject that the stimulation can be turned off immediately at any moment during the recording in case of any discomfort.</li>
	</ol>
	</li>
	<li>Clean the subject&#39;s lower leg with alcohol.</li>
	<li>Insert the stimulating monopolar needle electrode (25 mm x 26 G) over the anterior tibial muscle and adhesive surface electrode as the anode 1 cm distal to the monopolar needle (Figure 1).</li>
	<li>Place a ground electrode distal to the anode.</li>
	<li>Insert the recording concentric needle electrode (25 mm x 30 G) about 2cm proximal to the stimulating monopolar needle electrode along the muscle fibers (Figure 1).</li>
	<li>Connect the recording concentric needle and ground electrodes to the preamplifier.</li>
	<li>Ask the subject to remain silent and avoid movement during the examination.</li>
	<li>Zero the output of the stimulator and connect the stimulating electrodes to the stimulator (Figure 1).</li>
	<li>Maintain the skin temperature between 32&#8211;36 &#176;C using a warming lamp.</li>
</ol>
2. Recording of the MVRCs
<ol>
	<li>Start the semi-automated recording software using the muscle excitability recording protocol and turn on the stimulator. Stimulations will start at 2.5 mA with 1 Hz.</li>
	<li>Increase the stimulus intensity manually by hitting the Insert key until a response is recorded (max = 10 mA).
	<ol>
		<li>Adjust the stimulating and recording needles if necessary, until recording an acceptable response with a stimulus intensity of less than 10 mA. The shape of the muscle action potential should be triphasic, if possible, and stable. Avoid large twitches of the whole muscle.</li>
		<li>Invert the muscle action potential by hitting the minus key (-) if the potential appears upside down. 
		NOTE: A magenta horizontal line appears on the screen indicating the width of the action potential.</li>
	</ol>
	</li>
	<li>Adjust the position and length of the magenta line by dragging the line with the mouse. The green horizontal line represents the baseline.</li>
	<li>Click OK to start recording the MVRCs.</li>
	<li>Select a stimulus response relationship from the main options.</li>
	<li>Increase stimulus intensity by hitting the Insert key to a max of 10 mA or tolerable.</li>
	<li>Click OK to start descending the stimulus response curve.</li>
	<li>Click OK when the test stimulus reaches zero.</li>
	<li>Set the stimulus intensity to level for stable latency.</li>
	<li>Click OK to return to the main menu.</li>
	<li>Select the option 1/2/5 conditioning stims for RC.</li>
	<li>Select a protocol from recovery cycle options (e.g., start quick recovery cycle [skip alternate delays]), which is the default. 
	NOTE: The recording continues automatically for 34 steps with decreasing inter-stimulus intervals (ISIs).</li>
	<li>Make sure that muscle action potential is stable during the recording and that the needle has not moved. The screen changes automatically to main options when the 34 steps have completed.</li>
	<li>Click on Finish recording | Close file | OK, unless a ramp-up frequency or 20 Hz s recordings is being performed.</li>
	<li>Finish the recording and save the data by clicking on the Close file and save data button.</li>
</ol>
3. MVRC analyses
<ol>
	<li>Start the analyzing software program to perform the analysis offline.</li>
	<li>Select the recording that will be analysed and click on the OK button.</li>
	<li>Click on Load parameters from the Files menu.</li>
	<li>Select MANAL9 option for the analysis. If this is not present on the list, click on Browse to find this file. Click OK to continue.</li>
	<li>When a description of MAnal9 muscle excitability analysis appears, click OK to continue.
	<ol>
		<li>Invert the muscle action potential by typing MM-1 if the potential appears upside down.</li>
		<li>Right-click the mouse to make the magenta line visible. Set the window to the base of the peak response and with a width corresponding roughly to the width of the action potential at that height. Drag with the mouse to adjust the window. The window determines the latencies within which the height and latency are measured, as indicated by the pale blue lines, and green line indicates the baseline. Click OK to continue.</li>
	</ol>
	</li>
	<li>Click OK to remeasure the latencies and peaks. This will be done automatically. 
	NOTE: In the display of the remeasured latencies, the latencies are measured to shorter delays than original ones. This is because the responses to conditioning stimuli alone were subtracted from responses to the conditioning plus the test. This ensures that conditioning stimuli do not interfere with latency measurements. As is indicated in the prompt box, single bad points can be eliminated by positioning the cursor (vertical red line) over the point and hitting the ~ key. The bad point is replaced with mean of values on either side in same channel. If there are no bad points, set DE (display end) to just after the last latency required.</li>
	<li>Click OK to create an RMC file.</li>
	<li>Ignore most of the options appearing in the &#34;Create RCC or RMC&#34; form, since these are concerned with measurements of C-fiber rather than MVRCs. Click Save and Exit to continue. After saving the RMC file, the prompt box provides different options</li>
	<li>If frequency ramp and/or repetitive stimulation data have been recorded, follow the instructions to analyse these. Otherwise, select Go straight to create MEM file option to create a MEM file. Click OK to continue.</li>
	<li>Click Save and Exit to continue.</li>
	<li>Click OK to add the RMC data to MEM file.</li>
	<li>Click Add from Input RMC file to add this data to the MEM file, then change the directory to save the composite MEM file. Then, click Save and Exit to save it.</li>
	<li>Click OK to save the remeasured QZD file to allow differentiation from the original QZD file using a # sign.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+ischaemia+in+patients+with+orthostatic+hypotension+assessed+by+velocity+recovery+cycles.">Muscle ischaemia in patients with orthostatic hypotension assessed by velocity recovery cycles.</a> Journal of Neurology, Neurosurgery and Psychiatry. 82 (12), 1394-1398 (2011).</li><li>Z'Graggen, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Velocity+recovery+cycles+of+human+muscle+action+potentials+in+chronic+renal+failure.">Velocity recovery cycles of human muscle action potentials in chronic renal failure.</a> Clinical Neurophysiology. 121 (6), 874-881 (2010).</li><li>Z'Graggen, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+membrane+dysfunction+in+critical+illness+myopathy+assessed+by+velocity+recovery+cycles.">Muscle membrane dysfunction in critical illness myopathy assessed by velocity recovery cycles.</a> Clinical Neurophysiology. 122 (4), 834-841 (2011).</li><li>Lee, J. H., Boland-Freitas, R., Liang, C., Ng, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcolemmal+depolarization+in+sporadic+inclusion+body+myositis+assessed+with+muscle+velocity+recovery+cycles.">Sarcolemmal depolarization in sporadic inclusion body myositis assessed with muscle velocity recovery cycles.</a> Clinical Neurophysiology. 19 (31205-2), 1388 (2019).</li><li>Tan, S. V., Z'Graggen, W. J., Hanna, M. G., Bostock, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+assessment+of+muscle+membrane+properties+in+the+sodium+channel+myotonias.">In vivo assessment of muscle membrane properties in the sodium channel myotonias.</a> Muscle and Nerve. 57 (4), 586-594 (2018).</li><li>Tan, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+assessment+of+muscle+membrane+properties+in+myotonic+dystrophy.">In vivo assessment of muscle membrane properties in myotonic dystrophy.</a> Muscle and Nerve. 54 (2), 249-257 (2016).</li><li>Tan, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+dysfunction+in+Andersen-Tawil+syndrome+assessed+by+velocity+recovery+cycles.">Membrane dysfunction in Andersen-Tawil syndrome assessed by velocity recovery cycles.</a> Muscle and Nerve. 46 (2), 193-203 (2012).</li><li>Tan, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride+channels+in+myotonia+congenita+assessed+by+velocity+recovery+cycles.">Chloride channels in myotonia congenita assessed by velocity recovery cycles.</a> Muscle and Nerve. 49 (6), 845-857 (2014).</li><li>Boland-Freitas, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcolemmal+excitability+in+the+myotonic+dystrophies.">Sarcolemmal excitability in the myotonic dystrophies.</a> Muscle and Nerve. 57 (4), 595-602 (2018).</li><li>Stalberg, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standards+for+quantification+of+EMG+and+neurography.">Standards for quantification of EMG and neurography.</a> Clinical Neurophysiology. 130 (9), 1688-1729 (2019).</li><li>Witt, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+velocity+recovery+cycles+in+neurogenic+muscles.">Muscle velocity recovery cycles in neurogenic muscles.</a> Clinical Neurophysiology. 130 (9), 1520-1527 (2019).</li><li>Kristensen, R. 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H.B. receives royalties from UCL for sales of his Qtrac software used in this study. The other authors have no potential conflicts of interest. All authors have approved the final article.

MVRCs, as programmed in the recording software, is a highly automated procedure, but care is needed to obtain reliable results. In the recording stage, while adjusting the needles, it is important to avoid stimulating the end-plate zone or nerve. This usually leads to large twitches of the whole muscle, which increases the risk of displacement of the stimulation and/or recording needle during recording MVRCs. To date, the method has been applied to several muscles that have better described end-plate zone; however, the endplates may be scattered (i.e., in the anterior tibial muscle). Therefore, particular attention is required.
In order to avoid stimulation of the endplate or nerve instead of muscle fibers, care should be taken when observing the muscle for twitches. The stimulating monopolar needle should be moved, as well as the recording concentric needle, to locate a site that does not cause twitches. Additionally, subjects should be asked whether or not they feel pain. MVRC recordings do not cause any unpleasantness, unless the end-plate zone or the nerve is stimulated instead of muscle fibers.
A limitation of the MVRCs method is performing the recording in only one site and examination of only a few muscle fibers, which does not necessarily represent the whole muscle. This limitation is particularly important in disorders where the pathology is not diffuse. A previous study found surprisingly no difference between patients with amyotrophic lateral sclerosis and healthy controls despite denervated muscles. This was probably because denervation activity was not recorded at the site where MVRCs were recorded23. It also cannot be excluded that the needle could have been adjusted to a healthier spot with a more optimal response.
Another limitation of MVRCs is that one may have a tendency to spot the healthy muscle fibers while adjusting the recording needle to obtain a stable response for measurements. One way to overcome this limitation may be to do the recordings from polyphasic potentials. However, this may pose problems for determining an accurate latency if there are undifferentiated peaks. Additionally, although we intend to stimulate and record from the same bundle of muscle fibers, these may not be exactly the same. The stimulated bundle may contain different fibers during ongoing experiment24.
MVRCs provide information that cannot be obtained by the conventional electrophysiological methods. Thus, there is no other method in current use that can be compared to MVRCs. The earlier report6, using single fiber needle electrodes to record at two sites from the same muscle fiber, was much more difficult. Good recordings were only obtained from 43 out of 118 muscle fiber studies, and this method has not been adopted in research labs or clinics. Another similar but unautomated approach used eight different ISIs from 20 ms to 2 ms25. The authors reported that a recording took 20&#8211;60 min, whereas this method records MVRCs with 34 ISIs in about 10 min. The analysis is also fast and highly automated.
In conclusion, MVRCs is a method that may provide invaluable information to understand the underlying mechanisms of neuromuscular disorders. For patients in which a mutation in an ion channel gene has been identified, this method also provides data on the effects of those specific mutations on muscle membrane excitability in vivo. This, together with in vitro expression studies, enables a more accurate understanding of muscle pathophysiology in these patients. This method has the potential to provide insight into the role of those channels in normal muscle physiology, thus improving the understanding of muscle disease in general. Further studies with other patient groups and larger groups are necessary. Studies recording MVRCs in different muscles are also warranted.

Nerve conduction studies (NCS) and electromyography (EMG) are the conventional electrophysiological methods used for the diagnosis of neuromuscular disorders. NCS enables detection of axonal loss and demyelination in the nerves1, while EMG can differentiate whether myopathy or neurogenic changes are present in the muscle due to nerve damage. However, NCS or EMG provide limited information about muscle fiber membrane properties and underlying disease mechanisms. This information can be achieved using intracellular electrodes in isolated muscles from muscle biopsies2,3,4. However, it is of clinical importance to use methodologies using recordings from intact muscles in patients.
The velocity of a second muscle fiber action potential changes as a function of the delay after the first5, and this velocity recovery function (or recovery cycle) has been shown to change in dystrophic or denervated muscles. The yield of such recordings from single muscle fibers was, however, too low to be of use as a clinical tool6. However, Z&#39;Graggen and Bostock later found that multi-fiber recordings, obtained by direct stimulation and recording from the same bundle of muscle fibers, provide a fast and simple method of obtaining such recordings in vivo7. A sequence of paired pulse electrical stimuli with varying interstimulus intervals (ISIs) is used in this method7,8,9,10,11.
The evaluated MVRC parameters include the following: 1) muscle relative refractory period (MRRP), which is the duration after a muscle action potential until the next action potential can be elicited; 2) early supernormality (ESN); and 3) late supernormality (LSN). ESN and LSN are the periods after the refractory period in which the action potentials are conducted along the muscle membrane faster than normal. The depolarizing afterpotential, and potassium accumulation in the t-tubules of the muscle respectively, are hypothesized as the main causes for the two periods of supernormality.
The wide applicability of MVRCs to muscle disorders has been shown in detecting membrane depolarization in ischemia7,10,12 and renal failure13, as well as providing information about muscle membrane abnormalities in critical illness myopathy14 and inclusion body myositis15. Frequency ramp and intermittent 15 Hz and 20 Hz simulation protocols have since been introduced. MVRCs, together with these additional protocols, have demonstrated the different effects on muscle membrane excitability related to loss-of-function or gain-of-function mutations in various muscle ion channels in the inherited muscle ion channelopathies (i.e., sodium channel myotonia, paramyotonia congenita16, myotonic dystrophy17, Andersen-Tawil syndrome18, and myotonia congenita19,20).
In a recent study, the applicability of MVRCs to neurogenic muscles was shown for the first time. The term &#34;neurogenic muscle&#34; refers to the secondary changes in skeletal muscles that develop as denervation and reinnervation after any injury to the anterior horn cells or motor axons. Denervation is characterized in EMG as spontaneous activity (i.e., fibrillations [fibs] and positive sharp waves [psws]), while large motor unit potentials with prolonged duration and increased amplitude present reinnervation21. EMG changes are evident in denervated muscles, but the underlying cellular changes in muscle fiber membrane potentials have only been demonstrated in experimental studies on isolated muscle tissue2,3,4. MVRCs provide further insight into in vivo human muscle membrane properties regarding the denervation process.
This paper describes the methodology of MVRCs in detail. It also summarizes the changes in neurogenic muscles in a subgroup of patients from a previously reported study22 and healthy control subjects that enables determination of whether the method is appropriate for a planned study.
The recordings are performing using a recording protocol that is part of a software program. Other equipment used is an isolated linear bipolar constant current stimulator, 50 Hz noise eliminator, isolated electromyography amplifier, and analogue-to-digital converter.

<table><tbody><tr><td>50 Hz Noise Eliminator</td><td>Digitimer Ltd</td><td></td><td>Humbug</td></tr><tr><td>Analogue-to-Digital Converter</td><td>National Instruments</td><td></td><td>NI-6221</td></tr><tr><td>Analysing software program</td><td>Digitimer Ltd (copyright Institute of Neurology, University College, London)</td><td></td><td>QtracP, MANAL9</td></tr><tr><td>Disposable concentric needle electrode, 25 mm x 30G</td><td>Natus</td><td></td><td>Dantec DCN</td></tr><tr><td>Disposable monopolar needle electrode, 25 mm x 26G</td><td>Natus</td><td></td><td>TECA elite</td></tr><tr><td>Isolated EMG amplifier</td><td>Digitimer Ltd</td><td></td><td>D440</td></tr><tr><td>Isolated linear bipolar constant-current stimulator</td><td>Digitimer Ltd</td><td></td><td>DS5</td></tr><tr><td>Software and recording protocol</td><td>Digitimer Ltd (copyright Institute of Neurology, University College, London)</td><td></td><td>QtracW software, M3REC3 recording protocol written by Hugh Bostock, Istitute of Neurology, London, UK)</td></tr></tbody></table>

muscle velocity recovery cycles to examine muscle membrane properties

Although conventional nerve conduction studies (NCS) and electromyography (EMG) are suitable for the diagnosis of neuromuscular disorders, they provide limited information about muscle fiber membrane properties and underlying disease mechanisms. Muscle velocity recovery cycles (MVRCs) illustrate how the velocity of a muscle action potential depends on the time after a preceding action potential. MVRCs are closely related to changes in membrane potential that follow an action potential, thereby providing information about muscle fiber membrane properties. MVRCs may be recorded quickly and easily by direct stimulation and recording from multi-fiber bundles in vivo. MVRCs have been helpful in understanding disease mechanisms in several neuromuscular disorders. Studies in patients with channelopathies have demonstrated the different effects of specific ion channel mutations on muscle excitability. MVRCs have been previously tested in patients with neurogenic muscles. In this prior study, muscle relative refraction period (MRRP) was prolonged, and early supernormality (ESN) and late supernormality (LSN) were reduced in patients compared to healthy controls. Thereby, MVRCs can provide in vivo evidence of membrane depolarization in intact human muscle fibers that underlie their reduced excitability. The protocol presented here describes how to record MVRCs and analyze the recordings. MVRCs can serve as a fast, simple, and useful method for revealing disease mechanisms across a broad range of neuromuscular disorders.

The following results were obtained in a subgroup of patients from a recent study22, in which there were fibs/psws in all sites showing profuse denervation activity. The results showed that changes in muscle fibers after denervation were assessed in vivo using the MVRC technique described in this protocol. MVRCs showed changes consistent with depolarization of the resting membrane potential in the neurogenic muscle fibers.
Fourteen patients were compared with 29 healthy subjects. Subject demographics are shown in Table 1. Figure 2 illustrates recordings from a healthy subject and patient. Figure 3 and Table 2 illustrate comparison of patients&#39; MVRCs with healthy subjects. MRRP was prolonged, and ESN and LSN were reduced in patients compared to healthy controls (Table 2, Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60788/60788fig01.jpg" /> 
Figure 1: Picture of MVRCs set-up. (A) Isolated linear bipolar constant-current stimulator, (B) 50 Hz noise eliminator, (C) isolated EMG amplifier, and (D) analogue-to-digital converter. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60788/60788fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60788/60788fig02.jpg" /> 
Figure 2: Examples of MVRC recordings. Recordings after one conditioning stimulus (red), two conditioning stimuli (green), and five conditioning stimuli (blue) from a (A) healthy subject and (B) patient with L5 radiculopathy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60788/60788fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60788/60788fig03.jpg" /> 
Figure 3: MVRCs with one, two, and five conditioning stimuli. (A) MVRCs in 14 patients (grey lines) compared to mean value of 29 healthy controls (filled black squares). Graphical representation of percentage change in latency is plotted against ISIs from 2&#8211;1,000 ms (logarithmic scale). (B,C): Same as (A), but with two and five conditioning stimuli. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60788/60788fig3alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Healthy controls 
			(n=29)</td>
			<td>Patients 
			(n=14)</td>
		</tr>
		<tr>
			<td>Age (years)</td>
			<td>55.7 &#177; 14.9</td>
			<td>58.9 &#177; 16.3</td>
		</tr>
		<tr>
			<td>Gender (M/F)</td>
			<td>14/15</td>
			<td>9/5</td>
		</tr>
		<tr>
			<td>Disease duration (months)</td>
			<td>-</td>
			<td>3.4 &#177; 2.7</td>
		</tr>
		<tr>
			<td>MRC score</td>
			<td>-</td>
			<td>3.0 &#177; 1.1</td>
		</tr>
		<tr>
			<td>Etiology</td>
			<td>-</td>
			<td>Peronal neuropathy (9) 
			L5 root afflication</td>
		</tr>
	</tbody>
</table>
Table 1: Demographics and clinical characteristics. Values are listed as means &#177; standard deviation. This table has been modified from Witt et al.22.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Healthy Controls 
			(n=29)</td>
			<td>Patients 
			(n=14)</td>
			<td>p-value for t-test</td>
		</tr>
		<tr>
			<td>MRRP (ms)</td>
			<td>3.5 &#177; 0.4</td>
			<td>7.6 &#177; 3.1</td>
			<td>p = 6.8-8</td>
		</tr>
		<tr>
			<td>ESN (%)</td>
			<td>11.3 &#177; 2.1</td>
			<td>7.6 &#177; 2.3</td>
			<td>p = 5.5-5</td>
		</tr>
		<tr>
			<td>ESN (ms)</td>
			<td>7.8 &#177; 1.3</td>
			<td>12.7 &#177; 2.5</td>
			<td>p = 1.6-8</td>
		</tr>
		<tr>
			<td>5ESN (%)</td>
			<td>13.7 &#177; 2.5</td>
			<td>1.0 &#177; 0.6</td>
			<td>p = 9.3-10</td>
		</tr>
		<tr>
			<td>LSN (%)</td>
			<td>4.1 &#177; 1.4</td>
			<td>2.8 &#177; 1.7</td>
			<td>p = 0.017</td>
		</tr>
		<tr>
			<td>XLSN (%)</td>
			<td>2.9 &#177; 0.7</td>
			<td>1.0 &#177; 1.6</td>
			<td>p = 1.8-10</td>
		</tr>
		<tr>
			<td>5XLSN (%)</td>
			<td>8.0 &#177; 1.4</td>
			<td>2.8 &#177; 1.6</td>
			<td>p = 2.2-11</td>
		</tr>
	</tbody>
</table>
Table 2: Comparison of MVRC parameters between healthy controls and patients. MRRP = muscle relative refractive period; ESN (%) = latency reduction of muscle action potential after one conditioning stimulus as percentage of unconditioned stimulus at ISI of &#60;15 ms. ESN (ms), ISI corresponding to ESN (%). 5ESN = peak early supernormality after five conditioning stimuli. LSN (%) = latency reduction of muscle action potential after one conditioning stimulus as percentage of unconditioned stimulus at ISI between 100&#8211;150 ms. XLSN (%) = latency reduction of muscle action potential after two conditioning stimuli as percentage of one conditioning stimulus at ISI between 100&#8211;150 ms. 5XLSN (%) = latency reduction of muscle action potential after five conditioning stimuli as percentage of one conditioning stimulus at ISI between 100&#8211;150 ms. Values are listed as means &#177; standard deviation.

Watch this Scientific Journal Video about Muscle Velocity Recovery Cycles to Examine Muscle Membrane Properties at JoVE.com

Muscle Velocity Recovery Cycles to Examine Muscle Membrane Properties

Presented here is a protocol for the recording of muscle velocity recovery cycles (MVRCs), a new method of examining muscle membrane properties. MVRCs enable in vivo assessment of muscle membrane potential and alterations in muscle ion channel function in relation to pathology, and it enables the demonstration of muscle depolarization in neurogenic muscles.

Although conventional nerve conduction studies (NCS) and electromyography (EMG) are suitable for the diagnosis of neuromuscular disorders, they provide ...

muscle-velocity-recovery-cycles-to-examine-muscle-membrane-properties

Nanyang Technological University

Research

JoVE Journal

Neuroscience

14.1K Views.  Aarhus University Hospital. Dit protocol beschrijft hoe we spiermembraanveranderingen in ontnervated spieren onderzoeken met behulp van MVRC, spiersnelheid herstelcycli. Het belangrijkste voordeel van MVRCs is dat het een snelle en eenvoudige methode voor het onderzoeken van spiermembraan eigenschappen in vivo zonder ongemak voor het onderwerp. MVRCs kunnen een potentieel hebben om een diagnostisch instrument in neuromusculaire aandoeningen in de toekomst.Verdere studies zijn echter nodig om deze mogelijkheid te ...

Video: Muscle Velocity Recovery Cycles to Examine Muscle Membrane Properties

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Biology

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

Medicine

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.

The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Bioengineering

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca2+ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

A set of protocols are presented that describe the measurement of contractile function via sarcomere length detection along with calcium (Ca2+) transient measurement in isolated rat myocytes. The application of this approach for studies in animal models of heart failure is also included.

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

Muscle Velocity Recovery Cycles to Examine Muscle Membrane Properties

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