This work was supported by a University of Texas at Arlington Interdisciplinary Research Program grant.

This protocol follows the guidelines of the institutional review board at the University of Texas at Arlington and conforms to the standards set by the latest version of the Declaration of Helsinki. Accordingly, written informed consent was (and should be) obtained prior to commencement of research procedures.
1. Instrumentation
NOTE: The following instrumentation description is based on the near-infrared (NIR) spectrometer and data acquisition system used in our lab (see Table of Materials). Thus, the instructions include steps that are necessary for the optimal function of these devices. These steps include the calibration of the NIR probe using the accompanying software and calibration phantom, and the application of a dark cloth to exclude ambient light. In the event that different data collection hardware and/or software are used, investigators should consult their own specific user manuals for calibration and ambient light considerations. Figure 1 illustrates the experimental set-up and instrumentation described immediately below.
<ol>
	<li>Instruct the subject to lie supine with their legs inside a lower body negative pressure (LBNP) chamber (Figure 1A), so that their belt line is approximately even with the opening to the LBNP box. For instructions on how to build a LBNP chamber, see References17.</li>
	<li>Place three electrocardiogram electrodes on the subject: two in an inferior, mid-clavicular location and one on the subject&#39;s left side medial to the iliac crest. This configuration provides the best results due to limited access to the lower limbs, instrumentation of the upper limbs, and arm movement during hand grip exercise.</li>
	<li>Place a non-invasive blood pressure monitor module on the subject&#39;s dominant wrist. Place the finger blood pressure cuffs on each finger and connect them to the module (Figure 1B). Ensure the finger blood pressure cuffs are properly calibrated according to the user&#39;s manual accompanying your device.</li>
	<li>Instruct the subject to grasp a hand grip dynamometer (HGD) with their non-dominant arm in a slightly abducted position. The arm should be comfortably positioned on a bedside table. The distance and angle of the HGD should be adjusted to allow for optimal grip strength with minimal arm movement (Figure 1C).</li>
	<li>Secure the HGD to a bedside table.</li>
	<li>Measure the maximum voluntary contraction (MVC) of the participant. Tell the participant that, when prompted, they must squeeze the HGD as hard as possible while only utilizing the muscles in the hand and forearm. Instruct the subject that they must refrain from recruiting their upper arm, chest, shoulder, or abdominal muscles when performing the maximum grip.</li>
	<li>Repeat Step 1.6 three times, separated by at least 60 s. Record the maximum force achieved (best of 3). This maximum force will be used to calculate the exercise intensity for skeletal muscle oxidative capacity and neurovascular coupling (below).</li>
	<li>Place a rapid-inflation cuff around the upper arm of the exercising hand. Connect the airline from the rapid inflation controller to the cuff.</li>
	<li>Identify the flexor digitorum profundus. Use a skin marker to demarcate the borders of the palpable muscle.</li>
	<li>Ensure that the NIR spectrometer is properly calibrated according to the user&#39;s manual included with your device. Clean the skin over which the NIR probe will be positioned with an alcohol prep wipe.</li>
	<li>Place the NIR probe over the center of the belly of the muscle (flexor digitorum profundus) and affix it securely to the forearm.</li>
	<li>Wrap the probe and forearm with dark cloth, minimizing interference from ambient light (Figure 1C, Figure 1D).</li>
	<li>When ready to perform the functional sympatholysis portion of the study, seal the subject into the LBNP chamber.</li>
</ol>
2. Skeletal Muscle Oxidative Capacity
NOTE: A representative data tracing illustrating the experimental procedure for measuring skeletal muscle oxidative capacity is depicted in Figure 2. This experimental approach has previously been validated against in vivo phosphorus MRS18 and in situ muscle respirometry19, and is gaining widespread acceptance20.
<ol>
	<li>Instrument the subject as indicated above (Instrumentation).</li>
	<li>Instruct the subject to lie still for 2 min while monitoring deoxyhemoglobin (HHb) and oxyhemoglobin (HbO2) via the NIR probe. 
	NOTE: This rest period allows the subject to recover from any movement artifact associated with the instrumentation process, and ensures stable baseline measurements. If after 2 min no significant fluctuations have occurred, the subject may be considered at a steady state, or resting baseline.</li>
	<li>Prior to cuff occlusion, notify your subject that you will be inflating the cuff. Inflate the upper arm cuff at least 30 mmHg above systolic blood pressure for 5 min (i.e., suprasystolic). Instruct the subject to keep their arm as still and relaxed as possible both during cuff inflation and following cuff deflation. 
	NOTE: This 5 min brachial artery cuff occlusion protocol closely reflects the currently accepted clinical standard for vascular occlusion tests21,22,23,24,25.</li>
	<li>Record the initial/baseline value (prior to cuff occlusion) and the nadir value of tissue saturation (StO2) during the cuff occlusion and determine the midpoint between these two values. 
	<img alt="Equation 1" src="/files/ftp_upload/57317/57317eq1.jpg" /></li>
	<li>Allow the subject to recover from the cuff occlusion and return to the resting baseline values. Once the subject has maintained a resting baseline for at least 1 full min, continue to the next step.</li>
	<li>Instruct subject to squeeze and maintain an isometric hand grip at 50% of their MVC. Encourage the subject to maintain their isometric contraction until the tissue desaturates by 50%. Upon achieving this value, tell the subject to relax their hand and inform them that no more exercise or movement is needed.</li>
	<li>Within 3 - 5 s following exercise cessation, administer the following rapid cuff occlusion series (one series = 1 inflation + 1 deflation), as previously established18: 
	Series #1 - 6: 5 s on/5 s off 
	Series #7 - 10: 7 s on/10 s off 
	Series #11 - 14: 10 s on/15 s off 
	Series #15 - 18: 10 s on/20 s off</li>
	<li>After completing the 18th inflation/deflation series, instruct the subject to rest, allowing tissue saturation to return to initial baseline values. After these values have remained consistent for at least 2 min, repeat steps 2.4 and 2.5.</li>
	<li>Calculating Skeletal Muscle Oxidative Capacity
	<ol>
		<li>Calculate the slope of change in the StO2for each of the individual 18 cuff occlusions, forming the monoexponential recovery points illustrated in Figure 2C.</li>
		<li>Fit the calculated data from 2.7 to the following monoexponential curve18,19,26 
		y = End - &#916; x e-kt 
		NOTE: &#39;y&#39; is the relative muscle oxygen consumption rate (mV&#775;O2) during cuff inflation, &#39;End&#39; represents the mV&#775;O2 immediately following the cessation of exercise; delta (&#39;&#916;&#39;) signifies the change in mV&#775;O2 from rest to the end of exercise; &#39;k&#39; is the fitting rate constant; &#39;t&#39; is time. Tau is calculated as 1/k.</li>
	</ol>
	</li>
</ol>
3. Reactive Hyperemia
NOTE: A representative data tracing illustrating the experimental procedure for measuring reactive hyperemia is depicted in Figure 3.
<ol>
	<li>With the subject lying supine and instrumented as described above (Instrumentation), instruct the subject to lie as still as possible.</li>
	<li>Once the subject has achieved a consistent resting state, continue to record at least 1 min of baseline data and then rapidly inflate a blood pressure cuff on the upper arm to a suprasystolic pressure (30 mmHg above systolic blood pressure).</li>
	<li>At the 5 min mark, rapidly deflate the cuff while recording the hyperemic response.</li>
	<li>Continue recording for at least 3 min to capture the subject&#39;s recovery.</li>
	<li>Calculating Reactive Hyperemia 
	NOTE: The NIRS parameters calculated are depicted in Figure 3.
	<ol>
		<li>Calculate baseline StO2&#160;as the average StO2&#160;over 1 full min prior to the onset of arterial cuff occlusion.</li>
		<li>Determine the resting skeletal muscle metabolic rate as the desaturation rate (i.e., average slope) during cuff occlusion (defined as Slope 1)27,28.</li>
		<li>Calculate reactive hyperemia as follows: 
		a) the average upslope following cuff release (i.e., reperfusion rate, defined as slope 2), calculated from the moment of cuff release through the linearly increasing phase of the rebound trace; 
		b) the highest StO2&#160;value reached after cuff release (denoted as StO2max); 
		c) the reactive hyperemia area under the curve (AUC); calculated from the time of cuff release to 1-, 2- and 3-min post cuff-occlusion (AUC 1-min, AUC 2-min, and AUC 3-min, respectively); and 
		d) the hyperemic reserve, calculated as the change in StO2&#160;above baseline and reported as a percent (%) change. This value is calculated as the highest saturation achieved during the post-occlusive rebound minus the average saturation calculated in step 3.5.1 (see above). 
		NOTE: Large differences in baseline data will greatly affect the interpretation of the hyperemic reserve.</li>
	</ol>
	</li>
</ol>
4. Functional Sympatholysis
NOTE: A representative data tracing illustrating the experimental procedure for measuring functional sympatholysis is depicted in Figure 4.
<ol>
	<li>Instrument the subject as indicated above (Instrumentation).</li>
	<li>Ensure an airtight seal in the LBNP chamber.</li>
	<li>With the subject lying still and at rest, collect 3 min of baseline data.</li>
	<li>At the 3 min mark, turn on the vacuum. Adjust the vacuum so that the pressure inside the LBNP chamber is between -20 and -30 mmHg. Allow the vacuum to run for 2 min while monitoring the subject&#39;s response.</li>
	<li>At the 5 min mark, turn off the vacuum and allow the subject to rest for 3 min.</li>
	<li>At the 8 min mark, initiate the voice prompt guiding the subject through the rhythmic hand grip exercise (20% MVC).</li>
	<li>Confirm that the subject is maintaining their squeeze throughout the entirety of each gripping phase and relaxing completely during between each repetition. Monitor their force output and confirm that they are achieving 20% MVC with each grip. Continue exercise until the 11 min mark.</li>
	<li>At the 11 min mark, turn on the vacuum encouraging the subject to continue their rhythmic exercise. Allow the vacuum to run from 11 - 13 min, then turn it off.</li>
	<li>Have the subject continue performing rhythmic hand grip exercise at 20% of their MVC for an additional 2 min. Upon exercise cessation, have the subject rest quietly and lie still.</li>
	<li>Calculating Functional Sympatholysis
	<ol>
		<li>Normalize the change in oxyhemoglobin with LBNP to the total labile signal (TLS), determined during 5 min cuff occlusion: 
		<img alt="Equation 2" src="/files/ftp_upload/57317/57317eq2.jpg" /> 
		<img alt="Equation 3" src="/files/ftp_upload/57317/57317eq3.jpg" /></li>
		<li>Calculate each event as the final 20 min average of each event.</li>
		<li>Calculate the exercise-induced attenuation of the oxyhemoglobin reduction: 
		<img alt="Equation 4" src="/files/ftp_upload/57317/57317eq4.jpg" /></li>
	</ol>
	</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=Relationship+between+brachial+artery+blood+flow+and+total+[hemoglobin+myoglobin]+during+post-occlusive+reactive+hyperemia.">Relationship between brachial artery blood flow and total [hemoglobin+myoglobin] during post-occlusive reactive hyperemia.</a> Microvasc Res. 91, 37-43 (2014).</li><li>Willingham, T. B., Southern, W. M., McCully, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+reactive+hyperemia+in+the+lower+limb+using+near-infrared+spectroscopy.">Measuring reactive hyperemia in the lower limb using near-infrared spectroscopy.</a> J Biomed Opt. 21 (9), 091302 (2016).</li><li>Kragelj, R., Jarm, T., Erjavec, T., Presern-Strukelj, M., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parameters+of+postocclusive+reactive+hyperemia+measured+by+near+infrared+spectroscopy+in+patients+with+peripheral+vascular+disease+and+in+healthy+volunteers.">Parameters of postocclusive reactive hyperemia measured by near infrared spectroscopy in patients with peripheral vascular disease and in healthy volunteers.</a> Ann Biomed Eng. 29 (4), 311-320 (2001).</li><li>Gurley, K., Shang, Y., Yu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+optical+quantification+of+absolute+blood+flow,+blood+oxygenation,+and+oxygen+consumption+rate+in+exercising+skeletal+muscle.">Noninvasive optical quantification of absolute blood flow, blood oxygenation, and oxygen consumption rate in exercising skeletal muscle.</a> J Biomed Opt. 17 (7), 075010 (2012).</li></ol>

The authors declare that they have no competing financial interests.

The methods described herein enable non-invasive, clinical evaluation of reactive hyperemia, neurovascular coupling, and skeletal muscle oxidative capacity in a single clinic or laboratory visit.
Critical Considerations
Although NIRS is relatively robust and easy to use, collection of these data require careful placement of the optodes directly over the muscle belly, secured tightly in place to avoid movement artifact, and covered with a bl...

The hyperemic response to a brief period of tissue ischemia has emerged as a key non-invasive measure of (micro)vascular function. During occlusion of a conduit artery, downstream arterioles dilate in an effort to offset the ischemic insult. Upon release of the occlusion, the decreased vascular resistance results in hyperemia, the magnitude of which is dictated by one&#39;s ability to dilate the downstream microvasculature. While reactive hyperemia is a strong independent predictor of cardiovascular events1,2&#160;and therefore a clinically significant endpoint, its functional significance to exercise tolerance and quality of life is less clear.
Indeed, dynamic exercise represents a major cardiovascular stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. For example, skeletal muscle blood flow can increase nearly 100-fold during isolated muscle contractions3, which would overwhelm the pumping capacity of the heart if such a hemodynamic response were extrapolated to whole-body exercise. Accordingly, to avoid severe hypotension, sympathetic (i.e., vasoconstrictor) nervous activity increases to redistribute cardiac output away from inactive and visceral tissues and towards active skeletal muscle4. Sympathetic outflow is also directed to the exercising skeletal muscle5; however, local metabolic signaling attenuates the vasoconstrictor response in order to ensure adequate tissue oxygen delivery6,7,8,9,10,11. Collectively, this process is termed functional sympatholysis12, and is imperative to the normal regulation of skeletal muscle blood flow during exercise. Since skeletal muscle blood flow is a key determinant of aerobic capacity &#8212; an independent predictor of quality of life and cardiovascular disease morbidity and mortality13&#8212; understanding the control of skeletal muscle blood flow and tissue oxygen delivery during exercise is of great clinical significance.
Oxygen delivery is only half of the Fick equation, however, with oxygen utilization satisfying the other half of the equation. Among the major determinates of oxygen utilization, mitochondrial oxidative phosphorylation plays an essential role in supplying adequate energy for cellular processes both at rest and during exercise. Indeed, impairments in muscle oxidative capacity can limit functional capacity and quality of life14,15,16. Various measures are commonly used to provide an index of muscle oxidative capacity, including invasive muscle biopsies and expensive and time-consuming magnetic resonance spectroscopy (MRS) techniques.
Here, we propose a novel, non-invasive approach, using near-infrared spectroscopy (NIRS), to assess each of these three major clinical endpoints (reactive hyperemia, sympatholysis, and muscle oxidative capacity) in a single clinic or laboratory visit. The major advantages of this approach are three-fold: First, this technique is easily portable, relatively low cost, and easy to perform. Current Doppler ultrasound approaches for measuring reactive hyperemia are highly operator-dependent &#8212; requiring extensive skill and training &#8212; and require sophisticated, high-cost, data acquisition hardware and post-processing software. Moreover, this could conceivably be introduced into the clinic and/or large clinical trials for bedside monitoring or testing therapeutic efficacy. Second, by virtue of the methodology, this technique focuses specifically on the skeletal muscle microvasculature, increasing the overall specificity of the technique. Alternative approaches using Doppler ultrasound focus entirely on upstream conduit vessels and infer changes downstream, which can dampen the signal. Third, this technique is completely non-invasive. Skeletal muscle oxidative capacity is traditionally assessed with invasive and painful muscle biopsies, and functional sympatholysis may be assessed with intra-arterial injection of sympathomimetics and sympatholytics. This approach avoids these requirements all together.

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			<td height="42" style="height:42px;width:328px;">Dual-channel OxiplexTS Near-infrared spectroscopy machine</td>
			<td style="width:223px;">Iss Medical</td>
			<td align="right" style="width:119px;">101</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">NIRS muscle sensor</td>
			<td style="width:223px;">Iss Medical</td>
			<td align="right" style="width:119px;">201.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">E20 Rapid cuff inflation system</td>
			<td style="width:223px;">Hokanson</td>
			<td style="width:119px;">E20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">AG101 Air Source</td>
			<td style="width:223px;">Hokanson</td>
			<td style="width:119px;">AG101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Smedley Handgrip dynometer (recording)</td>
			<td style="width:223px;">Stolting</td>
			<td align="right" style="width:119px;">56380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Powerlab 16/35, 16 Channel Recorder</td>
			<td style="width:223px;">ADInstruments</td>
			<td style="width:119px;">PL3516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Human NIBP Set</td>
			<td style="width:223px;">ADInstruments</td>
			<td style="width:119px;">ML282-SM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Bio Amp</td>
			<td style="width:223px;">ADInstruments</td>
			<td style="width:119px;">FE132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Quad Bridge Amp</td>
			<td style="width:223px;">ADInstruments</td>
			<td style="width:119px;">FE224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Connex Spot Monitor</td>
			<td style="width:223px;">Welch Allyn</td>
			<td style="width:119px;">71WX-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Origin(Pro) graphing software</td>
			<td style="width:223px;">OrignPro</td>
			<td style="width:119px;">Pro</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:328px;">Lower body negative pressure chamber</td>
			<td style="width:223px;">Physiology Research Instruments</td>
			<td style="width:119px;">standard unit</td>
			<td></td>
		</tr>
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skeletal muscle neurovascular coupling, oxidative capacity, and microvascular function with 'one stop shop' near-infrared spectroscopy

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. Reactive hyperemia (in response to a brief period of tissue ischemia) is an independent predictor of cardiovascular events and provides important insight into vascular health and vasodilatory capacity. Skeletal muscle oxidative capacity is equally important in health and disease, as it determines the energy supply for myocellular processes. Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess each of these major clinical endpoints (reactive hyperemia, neurovascular coupling, and muscle oxidative capacity) during a single clinic or laboratory visit. Unlike Doppler ultrasound, magnetic resonance images/spectroscopy, or invasive catheter-based flow measurements or muscle biopsies, our approach is less operator-dependent, low-cost, and completely non-invasive. Representative data from our lab taken together with summary data from previously published literature illustrate the utility of each of these end-points. Once this technique is mastered, application to clinical populations will provide important mechanistic insight into exercise intolerance and cardiovascular dysfunction.

Skeletal muscle oxidative capacity
Figure 2 illustrates a representative participant response during a NIRS-derived skeletal muscle oxidative capacity assessment. Panel A shows the tissue saturation profile during a 5 min arterial cuff occlusion protocol, handgrip exercise, and intermittent arterial occlusion during recovery from exercise. Panel B illustrates the expected t...

Watch this Scientific Journal Video about Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy at JoVE.com

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia, neurovascular coupling and skeletal muscle oxidative capacity in a single clinic or laboratory visit.

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...

skeletal-muscle-neurovascular-coupling-oxidative-capacity

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12.4K Views.  University of Texas at Arlington. Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia, neurovascular coupling and skeletal muscle oxidative capacity in a single clinic or laboratory visit. 

Video: Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

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 ...

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

A New Best Practice for Validating Tail Vein Injections in Rat with Near-infrared-Labeled Agents

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part of many experimental procedures where reagents need to be introduced directly into the bloodstream. Unwittingly, the injection can be subcutaneous, possibly altering the scientific outcomes. Utilizing a nanoemulsion-based biological probe with an incorporated near-infrared fluorescent (NIRF) dye, this method offers the capability of imaging a successful tail vein injection in vivo. With the use of a NIRF imager, images are taken before and after the injection of the agent. An acceptable IV injection is then qualitatively or quantitatively determined based on the intensity of the NIRF signal at the site of injection.

Here we present a method to validate tail vein injections in rats by utilizing near-infrared fluorescence imaging data from dyes incorporated into agents or biological probes. The tail is imaged before and after the injection, the fluorescent signal is quantified, and an assessment of the injection quality is made.

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part ...

How to Administer Near-Infrared Spectroscopy in Critically ill Neonates, Infants, and Children

Near infrared spectroscopy (NIRS) calculates regional tissue oxygenation (rSO2) using the different absorption spectra of oxygenated and deoxygenated hemoglobin molecules. A probe placed on the skin emits light that is absorbed, scattered, and reflected by the underlying tissue. Detectors in the probe sense the amount of reflected light: this reflects the organ-specific ratio of oxygen supply and consumption - independent of pulsatile flow. Modern devices enable the simultaneous monitoring at different body sites. A rise or dip in the rSO2 curve visualizes changes in oxygen supply or demand before vital signs indicate them. The evolution of rSO2 values in relation to the starting point is more important for interpretation than are absolute values.
A routine clinical application of NIRS is the surveillance of somatic and cerebral oxygenation during and after cardiac surgery. It is also administered in preterm infants at risk for necrotizing enterocolitis, newborns with hypoxic ischemic encephalopathy and a potential risk of impaired tissue oxygenation. In the future, NIRS could be increasingly used in multimodal neuromonitoring, or applied to monitor patients with other conditions (e.g., after resuscitation or traumatic brain injury).

This protocol is designed to assist clinicians to measure regional tissue oxygenation at different body sites in infants and children. It can be used in situations where tissue oxygenation is potentially compromised, particularly during cardiopulmonary bypass, when using non-pulsatile cardiac-assist devices, and in critically ill neonates, infants and children.

Near infrared spectroscopy (NIRS) calculates regional tissue oxygenation (rSO2) using the different absorption spectra of oxygenated and deoxygenated ...