Name: method to measure tone of axial and proximal muscle
Uploaded: 2011-12-14T13:00:00
Duration: 10 min 41 s
Description: Watch this Scientific Journal Video about Method to Measure Tone of Axial and Proximal Muscle at JoVE.com

We would like to thank Eugene Gurfinkel and Mark Chapman for their roles in the design and fabrication of Twister. The development of Twister was funded by National Institutes of Health grants R01 AR-31017 to P. Cordo and V. Gurfinkel and F32 HD-008520 to T. Cacciatore. T. Cacciatore would also like to acknowledge the Medical Research Council, UK for support to write this manuscript.

1. Introduction
Twister is a servo-controlled device for quantifying postural tone in axial and proximal body regions during active, upright stance. The original device was constructed at Oregon Health &amp; Science University and a similar device is under construction for the University of Southampton, UK. In this report, we describe the function and rationale for Twister and its various uses. We then provide a detailed description to facilitate its reproduction and show how it can be used to investigate the control of postural tone.
2. Overview
Twister consists of a rigid steel frame, rotating platform, torque sensor, counterbalanced suspension system, upper and lower fixations, and body attachments (Fig 1), as well as a servo control system for regulating platform rotation. Twister quantifies postural tone in standing subjects by rotating lower body segments about the vertical axis relative to upper segments. This twists the region in between, changing the length of muscles within. Upper and lower fixations can be attached to impart twisting to the neck, trunk or hip regions (Fig 2). Because Twister does not provide postural support, tonic activity of skeletal muscle is necessary to counteract gravitational torques. This tonic regulation is studied by the torsional resistance to twisting as well as electromyography from relevant muscles. Resistance to twisting is assessed by a torque sensor within the upper fixation and reflects the state of all muscles undergoing length changes. Twister uses several different platform rotation profiles to study tonic control, including a triangle profile, step profile, and triangle profile of increasing magnitude (Fig 3). These rotate the platform at a constant slow velocity, which minimizes inertial effects on the subject and measurement. 
We use a twisting perturbation about the vertical axis because it: 1) changes the length of axial and proximal muscles, as these structures are oriented obliquely and have broad anatomic origins and insertions; 2) does not alter the relationship of the whole body and its parts to gravity; 3) rotates the body around an axis of minimal moment of inertia2; 4) corresponds to a neutral zone3,4, so that resistance from small displacements from the forward facing position reflects muscular rather than osteo-ligamentous forces; and 5) naturally occurs in everyday activities3,5.
Twister is a flexible device that can be used to address various aspects of tonic control. These include: 1) stiffness related to tonic activity6-8; 2) tonic responses to muscular length changes6,8. 3) the effect of twisting on remote body regions; 4) tonic effects from kinesthetic information9; 5) the effects of disease on postural tone7,10; and 6) perceptual thresholds to slow rotation11. 
3. Detailed description of device 
We detail the components of Twister below.
<ol>
 <li>Rotating platform 
 
 Subjects stand on a platform that rotates &plusmn;20&deg;on a bearing about the vertical axis (Figs 1, 4A). An electric motor powers this rotation at a drive ratio that achieves platform speeds between 0.5&deg;/s and 5&deg;/s and high torque. Twister rotates the lower body in space, rather than the upper body to eliminate vestibular signals that could disrupt quiet stance.</li>
 <li> A belt and pulley system is used for drive reduction, which dampens vibration and eliminates lash that can interfere with torque measurement. Vibration is minimized because it can cue the subject about platform motion. </li>
 <li>For safety, hard stops are used to limit maximal platform displacement. </li>
 <li>An optical encoder (Hewlett-Packard HEDS-5540) fixed to the platform shaft reports rotational displacement for both servo-control and data-analysis. 
 </li>
 <li>Frame 
 A rigid, steel frame (1.5m x 1.5m x 3m) with diagonal cross bracing creates high torsional stiffness between the platform assembly and torque sensor, necessary for accurate torque measurement. 
 </li>
 <li>Upper fixation and suspension system 
 The upper fixation and lightweight, counterbalanced suspension system connect the upper margin of the twisted region to the frame (Fig 4B). A torque sensor (Futek TFF220, Irvine, CA) positioned within the upper fixation measures a subject's resistance to rotation. </li>
 <li>The suspension system consists of four rectangular aluminum plates that are alternately hinged along the anterior-posterior and mediolateral axes. This creates a high stiffness for rotation about the vertical axis (590 Nm/&deg;), in order to accurately measure torque, without restricting movement in other dimensions. In particular, the low stiffness for translation in x, y, and z directions (0.25 N/cm) ensures subjects maintain postural stability themselves and prevents the upper fixation from providing a spatial reference. This also allows each individual to maintain their own, unique vertical posture without affecting postural motion in the horizontal plane. </li>
 <li>Springs act to counteract the weight of the suspension system. </li>
 <li>A vertical bearing assembly (Fig 1, 4B) is used to adjust the upper fixation to subject height. 
 </li>
 <li>Lower fixation 
 
 A lower fixation connects the lower margin of the twisted region to the rotating platform. Body segments below the lower fixation rotate with the platform. </li>
 <li> The lower fixation consists of a lightweight telescoping bar that is connected to the rotating platform. A hinge connects the telescoping bar to the platform to allow anterior-posterior postural sway. 
 </li>
 <li>Body Attachments 
Three attachments are used with Twister: a lightweight helmet, a shoulder harness and a pelvis orthotic, which can each be securely fixed to the body (Fig 2). </li>
 <li>To twist the neck, attach the helmet above and the shoulders below. </li>
 <li>To twist the trunk attach the shoulders above and the pelvis below </li>
 <li>To twist the hips attach the pelvis above. In this case, twisting is localized to internal and external hip rotation as the feet, shank and thigh rotate with the platform. 
 </li>
 <li>External fixation 
 A third, external fixation can be used to twist one body region while measuring torque produced by another. Because the latter segment is stationary, the measured torque is not resistive but originates from muscular forces within the segment, potentially induced from remote twisting. </li>
 <li>The external fixation consists of a lightweight telescoping bar that prevents the connected segment from rotating about the vertical axis. A hinge joint between the bar and frame allows anterior-posterior postural sway. </li>
 <li>Fig 4C shows the configuration for measuring neck torque during trunk twisting. Alternatively, neck torque in response to hip twisting can be assessed by connecting the pelvis to the external fixation. </li>
 <li>A standard forceplate can be placed between the subjects feet and rotating platform, to simultaneously measure resistive torque in the twisted segment. This forceplate can also be used to quantify postural sway during twisting. 
 </li>
 <li>Servo-control of platform rotation 
 A custom built real-time servo-system controls platform rotation. This hardware PID controller outputs a motor drive signal based on a platform position signal from the optical encoder and the desired rotation (see Fig 7). A custom PC program interfaces with the hardware controller to specify the desired temporal profile of platform rotation and initiate a trial. </li>
 <li>The controller generates three profiles for platform rotation. Select the triangle profile to alternate between constant-speed clockwise and counter-clockwise rotation (Fig. 3, trace 1). Use the step profile to achieve discontinuous rotation (Fig. 3, trace 2). Rotation can also be driven with a triangle profile that increases in amplitude across cycles (Fig. 3, trace 3). </li>
 <li>For all profiles, rotation is smoothed to limit acceleration to 12 &deg;/s2 during movement initiation and directional changes. </li>
</ol>
4. Experimental protocol
A typical experimental is run as follows:
<ol>
 <li>Place body attachments (i.e. helmet, shoulder harness or pelvis orthotic) on the desired segments, ensuring they are snug and there is no torsional play. </li>
 <li>Adjust the height of the linear bearing so that the upper fixation is at the same height as the corresponding body attachment.</li>
 <li> Adjust the lower fixation using the telescoping bar to correspond to the height of the lower body attachment. </li>
 <li>Instruct the subject to stand on the rotating platform, facing forward. </li>
 <li>Attach the upper and lower fixations to the corresponding body attachments, positioning adjustments so zero torque is applied to the subject in the pre-trial position.</li>
 <li>Blindfold the subject. </li>
 <li>Instruct the subject to stand relaxed and not to intervene. </li>
 <li>Select an amplifier gain for the torque sensor according to which body region is twisted, in order to maximize the dynamic range of this signal. </li>
 <li>Reset the bias on the torque sensor. </li>
 <li>Begin surface oscillation in yaw and data recording. Torque and platform rotation signals are typically recorded at 50 Hz using Spike 2 acquisition software (Cambridge Electronic Devices, Cambridge, UK). </li>
 <li>Initiate twisting with the desired platform rotation profile. In general movement should be slow and smooth enough so that subjects do not accurately perceive twisting. </li>
</ol>
5. Representative results
Resistive torque typically increases with platform excursion, however the increase slows with larger excursion. Overall resistance is typically quantified by peak-to-peak torque, averaged across cycles. Fig 5A shows single trial responses across subjects for torsional resistance of constant speed ramp for the trunk. We have observed data reproducible over months within a subject (Fig 5B; interclass correlation coefficient = 0.89). Mean resistance to twisting differs across body segments, and has been reported to be 0.54 &plusmn; 0.24 Nm for the neck, 5.11 &plusmn; 1.94 Nm for the trunk and 3.23 &plusmn; 1.67 Nm for the hips6 (Fig 6). It is important that platform rotation is smooth and there is no lash. Absence of lash is indicated by smooth changes in torque during directional changes and a rapid change in torque at the onset, presumably due to short-range stiffness of muscle (see Fig 3A in Gurfinkel et al.6).
The measured torque reflects both the dynamic changes in tone with twisting as well as the distribution of baseline tonic activity (which includes co-contraction). Because of the slow speed of twisting, the increase in torque per degree is equivalent to intrinsic stiffness12 only when muscle activity is constant. Note that because active structures contribute to the measured resistance Twister technically assesses pseudostiffness.
In general, two types of responses are observed that correspond to constant or modulated tonic activity within the twisted region. The former is characterized by low cycle-to-cycle variation in torque, high peak-to-peak torque magnitude, and relatively constant EMG. In contrast, dynamic modulation is characterized by high cycle-to-cycle variability, low torsional resistance and EMG modulation coherent with twisting. On angle vs. torque plots unmodulated subjects exhibit a regular hysteresis loop while modulated subjects have an irregular pattern that can reverse direction (see Fig 3A in Gurfinkel et al.6 Dynamic modulation generally consists of increasing tonic activity during muscle shortening and decreasing activity during lengthening (i.e. Sherrington's lengthening and shortening reactions13), which are opposite in sign to the stretch reflex. An integrative measure of the extent of modulation can be obtained by the shift in a subject's neutral (zero-torque) position within a cycle, referred to as torque phase advance6,8.
<img src="/files/ftp_upload/3677/3677fig1.jpg" alt="Figure 1" /> 
Figure 1. Schematic of Twister from the side. Components are labeled as follows: 1) rotating platform; 2) telescoping bar for lower fixation; 3) hinge joint between lower telescoping bar and rotating platform; 4) helmet attached to upper fixation; 5) torque sensor and counterbalanced suspension system; 6) locking vertical linear bearing; 7) external fixation for measuring induced torque; 8) hinge joint analogous to 3; 9) rigid frame; 10) diagonal cross bracing for rigid frame.
<img src="/files/ftp_upload/3677/3677fig2.jpg" alt="Figure 2" /> 
Figure 2. Twisting applied to axial and proximal levels. Subjects stand on a rotating platform (yellow) with upper and lower body attachments affixed to impart twisting to the desired body region. The upper attachment is connected via a suspension system (zigzag lines) to the torque sensor (T), which is fixed with regard to rotation around the vertical axis. The lower attachment connects to the rotating platform via a hinge joint (black circle) that allows rotation in the sagittal plane of the subject. A: Neck twisting is achieved by attaching a helmet to the torque sensor and the shoulders to the platform. B: Trunk twisting is achieved by attaching the shoulders to the torque sensor and the pelvis to the platform. C: Hip twisting is achieved by attaching the pelvis to the torque sensor.
<img src="/files/ftp_upload/3677/3677fig3.jpg" alt="Figure 3" /> 
Figure 3. Different twisting profiles. Various profiles can be used to study specific aspects of tonic control. The output of the optical encoder specifying platform rotation is shown in Volts. Upward deflection corresponds to counter-clockwise platform rotation when viewed from above. 1) Triangle profile: In this case the rotational velocity, maximum excursion and number of cycles are specified. Two cycles of 12&deg; are shown. 2) Discontinuous, step profile: Amplitude, velocity, and holding time a step are specified. Two cycles 12&deg; rotations, consisting of four, 3&deg; deg steps are shown. 3) Increasing amplitude triangle waves: two cycles of each of 3&deg;, 6&deg; and 9&deg; rotations are shown. In this example the rate of platform rotation is constant for all conditions.
<img src="/files/ftp_upload/3677/3677fig4.jpg" alt="Figure 4" /> 
Figure 4. Photograph of Twister from the side. A: Configuration for trunk twisting with components labeled as follows: 1) rotating platform; 2) motor and servo-control assembly; 3) hinge joint between lower telescoping bar and the rotating platform; 4) lower fixation and pelvis orthotic; 5) upper fixation and shoulder harness; 6) bar connecting suspension to upper fixation; 7) torque sensor and counterbalanced suspension system; 8) external fixation for measuring induced torque; 9) rigid frame. B: Closeup of torque sensor and suspension system labeled as follows: 1) torque sensor; 2-5) lightweight hinged aluminum plates. The hinge between plates 2 and 3 rotates about the anterior-posterior axis, while the hinge between plates 4 and 5 is oriented around the mediolateral axis. 8) locking vertical linear bearing; 9) lightweight helmet and upper attachment. C: Configuration for twisting the trunk but measuring the torsional effect on the neck. In this configuration the pelvis is fixed to the rotating platform (1) and the shoulders are connected to the external fixation (2), which prevents the shoulders, neck and head from rotating, restricting twisting to the trunk. The head is also attached to the upper fixation (3) so that any induced neck torque is applied to the torque sensor.
<img src="/files/ftp_upload/3677/3677fig5.jpg" alt="Figure 5" /> 
Figure 5. Torsional resistance of the trunk. A) Torque traces from individual trials from different subjects. Three cycles of 10&deg;, 1&deg;/s triangle waves were used. Subjects have consistent torque behavior across cycles, with large variation in resistance between subjects. Traces with highest resistance are typical of unmodulated behavior, while traces with least resistance are typical of high modulation. B) Inter-subject repeatability in torsional resistance across time. Two measurements from 7 subjects separated by one month. Peak-to-peak trunk torque shows consistent within-subject behavior across testing sessions but wide inter-subject variation. 
<img src="/files/ftp_upload/3677/3677fig6.jpg" alt="Figure 6" /> 
Figure 6. Restive torque from different axial levels. The resistive torque to 10&deg;, 1&deg;/s triangle waves for the neck, trunk and hip levels. Single trials from a representative subject are shown. Note the different magnitude and timecorse across levels.
<img src="/files/ftp_upload/3677/3677fig7.jpg" alt="Figure 7" /> 
Figure 7. Schematic of servo-control. The control loop consists of a PID (proportional, integral, derivative) controller, which receives input from an optical encoder attached to the platform shaft. The controller determines the motor drive current. Custom software running on a PC is used for selecting the desired platform trajectory, which then downloads this information to the controller.

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	<li>Foster, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Text+Book+of+Physiology.">A Text Book of Physiology.</a> The Central Nervous System. Vol. III, (1892).</li><li>Zatsiorsky, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+human+motion.">Kinetics of human motion.</a> Human Kinetics. , (2002).</li><li>Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ergonomics+and+biology+of+spinal+rotation.">Ergonomics and biology of spinal rotation.</a> Ergonomics. 47, 370-415 (2004).</li><li>Kumar, S., Panjabi, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vivo+axial+rotations+and+neutral+zones+of+the+thoracolumbar+spine.">Vivo axial rotations and neutral zones of the thoracolumbar spine.</a> Journal of spinal. 8, 253-263 (1995).</li><li>Lamoth, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pelvis-thorax+coordination+in+the+transverse+plane+during+walking+in+persons+with+nonspecific+low+back.">Pelvis-thorax coordination in the transverse plane during walking in persons with nonspecific low back.</a> Spine. 27, E92-E99 (2002).</li><li>Gurfinkel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postural+muscle+tone+in+the+body+axis+of+healthy+humans.">Postural muscle tone in the body axis of healthy humans.</a> Journal of Neurophysiology. 96, 2678-2687 (2006).</li><li>Wright, W. G., Gurfinkel, V. S., Nutt, J., Horak, F. B., Cordo, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axial+hypertonicity+in+Parkinson's+disease:+direct+measurements+of+trunk+and+hip+torque.">Axial hypertonicity in Parkinson's disease: direct measurements of trunk and hip torque.</a> Exp. Neurol. 208, 38-46 (2007).</li><li>Cacciatore, T. W., Gurfinkel, V. S., Horak, F. B., Cordo, P. J., Ames, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+dynamic+regulation+of+postural+tone+through+Alexander+Technique+training.">Increased dynamic regulation of postural tone through Alexander Technique training.</a> Human movement science. 30, 74-89 (2011).</li><li>Franzen, E., Gurfinkel, V. S., Wright, W. G., Cordo, P. J., Horak, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haptic+touch+reduces+sway+by+increasing+axial+tone.">Haptic touch reduces sway by increasing axial tone.</a> Neuroscience. 174, 216-223 (2011).</li><li>Franzen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+performance+in+balance,+walking+and+turning+tasks+is+associated+with+increased+neck+tone+in+Parkinson's+disease.">Reduced performance in balance, walking and turning tasks is associated with increased neck tone in Parkinson's disease.</a> Exp. Neurol. 219, 430-438 (2009).</li><li>Wright, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axial+kinesthesia+is+impaired+in+Parkinson's+disease:+Effects+of+levodopa.">Axial kinesthesia is impaired in Parkinson's disease: Effects of levodopa.</a> Exp. Neurol. , (2010).</li><li>Sinkjaer, T., Toft, E., Andreassen, S., Hornemann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stiffness+in+human+ankle+dorsiflexors:+intrinsic+and+reflex+components.">Muscle stiffness in human ankle dorsiflexors: intrinsic and reflex components.</a> J Neurophysiol. 60, 1110-1121 (1988).</li><li>Sherrington, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+plastic+tonus+and+proprioceptive+reflexes.">On plastic tonus and proprioceptive reflexes.</a> Quart. J. Exper. Physiol. 2, 109-156 (1909).</li><li>Wright, W. G., Gurfinkel, V., King, L., Horak, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease+shows+perceptuomotor+asymmetry+unrelated+to+motor+symptoms.">Parkinson's disease shows perceptuomotor asymmetry unrelated to motor symptoms.</a> Neurosci. Lett. 417, 10-15 (2007).</li></ol>

The authors have nothing to disclose.

It is our opinion that Twister can be used address many questions in tonic control. To date, Twister has lead to 7 such publications6-11,14. Probably the most important feature of Twister is that it provides an integrated, kinetic measure of tone. This torque measure of tone is not provided by kinematic, inverse dynamics or electromyographic approaches, and is necessary to answer many questions regarding tone. Also, Twister is unique in not substantially interfering with natural antigravity or postural behavior, and provides a tonic, rather than a phasic perturbation.
One potential use of twister is the quantification of the tonic effects of disease on postural tone. While the intrinsic and reflex stiffness has been well studied for many neurological and musculoskeletal conditions using fast perturbations, the quantitative effect of many diseases on postural tone not well characterized. In particular, Twister can be used to quantify the effects of disorders such as rigidity7,10,14, hypotonia, dystonia, and back and neck pain on the magnitude, distribution and symmetry of postural tone along the body axis. It can also be used to measure axial kinesthesis, eg; perception of body rotation based on muscle proprioceptors and11 perceptuomotor symmetry, eg; representation of straight ahead during axial twisting14. Finally, Twister can be used to study the effect of intervention on these measures of axial postural tone8.
We estimate the cost of hiring an engineering firm to fabricate Twister is approximately $30,000 US. This device can likely be manufactured in-house for a fraction of this cost, as the price of raw materials is low, but significant fabrication is required. Throughout its use, Twister has evolved significantly and continues to do so. There are many basic questions that can be addressed with Twister. We hope that this report will help other investigators build Twisting devices or otherwise stimulate research into this fundamental but poorly understood area.

method to measure tone of axial and proximal muscle

The control of tonic muscular activity remains poorly understood. While abnormal tone is commonly assessed clinically by measuring the passive resistance of relaxed limbs1, no systems are available to study tonic muscle control in a natural, active state of antigravity support. We have developed a device (Twister) to study tonic regulation of axial and proximal muscles during active postural maintenance (i.e. postural tone). Twister rotates axial body regions relative to each other about the vertical axis during stance, so as to twist the neck, trunk or hip regions. This twisting imposes length changes on axial muscles without changing the body's relationship to gravity. Because Twister does not provide postural support, tone must be regulated to counteract gravitational torques. We quantify this tonic regulation by the restive torque to twisting, which reflects the state of all muscles undergoing length changes, as well as by electromyography of relevant muscles. Because tone is characterized by long-lasting low-level muscle activity, tonic control is studied with slow movements that produce "tonic" changes in muscle length, without evoking fast "phasic" responses. Twister can be reconfigured to study various aspects of muscle tone, such as co-contraction, tonic modulation to postural changes, tonic interactions across body segments, as well as perceptual thresholds to slow axial rotation. Twister can also be used to provide a quantitative measurement of the effects of disease on axial and proximal postural tone and assess the efficacy of intervention.

Watch this Scientific Journal Video about Method to Measure Tone of Axial and Proximal Muscle at JoVE.com

Method to Measure Tone of Axial and Proximal Muscle

We have developed a device (Twister) to study the regulation of tonic muscle activity during active postural maintenance. Twister measures torsional resistance and muscular responses in standing subjects during twisting of the body axis. The device can be flexibly configured to study various aspects of tonic control across the neck, trunk, and/or hips.

The control of tonic muscular activity remains poorly understood.  While abnormal tone is commonly assessed clinically by measuring the passive resistance ...

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Medicine

Manual Muscle Testing:  A Method of Measuring Extremity Muscle Strength Applied to Critically Ill Patients

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, and persistent nerve and muscle impairments after hospital discharge.1-6 Using an explicit protocol with a structured approach to training and quality assurance of research staff, manual muscle testing (MMT) is a highly reliable method for assessing strength, using a standardized clinical examination, for patients following ARDS, and can be completed with mechanically ventilated patients who can tolerate sitting upright in bed and are able to follow two-step commands. 7, 8 
This video demonstrates a protocol for MMT, which has been taught to &ge;43 research staff who have performed &gt;800 assessments on &gt;280 ARDS survivors. Modifications for the bedridden patient are included. Each muscle is tested with specific techniques for positioning, stabilization, resistance, and palpation for each score of the 6-point ordinal Medical Research Council scale.7,9-11 Three upper and three lower extremity muscles are graded in this protocol: shoulder abduction, elbow flexion, wrist extension, hip flexion, knee extension, and ankle dorsiflexion. These muscles were chosen based on the standard approach for evaluating patients for ICU-acquired weakness used in prior publications. 1,2.

Survivors of acute respiratory distress syndrome (ARDS) and critical illness frequently develop long-lasting muscle weakness. Manual muscle testing (MMT) is a standardized clinical examination commonly used to measure strength of peripheral skeletal muscle groups. This video demonstrates MMT using the 6-point Medical Research Council scale.

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, ...

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 &#177; 1.1 &#181;m for trueness and 1.6 &#177; 0.6 &#181;m for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 &#177; 2.2 &#181;m, precision: 12.5 &#177; 2.5 &#181;m) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.

Accuracy is a major demand in dental medicine. To verify accuracy, reference scanners are needed. This article presents a new reference scanner with an adjusted scanning method to acquire a broad variety of dental morphologies with high trueness and precision.

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology,&#160;and pathology.

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also underscores the importance of ethical issues associated with animal use in research.

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...