We would like to thank Vikram Darbhe and Patrick Franks for their experimental guidance. This work is supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1324585 as well as NIH R01D084009 and F31AR076920. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation or NIH.

Northwestern University&#8217;s Institutional Review Board (IRB) approved the procedures of this study. All participants enrolled in this work gave informed consent prior to beginning the protocol detailed below. 
NOTE: The specific ultrasound system used in this study had EFOV-US capabilities and was adopted because we were able to review details about and validity assessments for the algorithm in the scientific literature22,26; multiple other systems with EFOV-US also exist18,20,30. A linear array transducer 14L5 (frequency bandwidth 5-14 MHz) was utilized. The muscles imaged in this protocol are just a small subset of muscles for which US images have been captured and fascicle lengths measured (e.g., triceps25, extensor carpi ulnaris23, medial gastrocnemius10, vastus lateralis24, biceps femoris8,31). This protocol is intended to provide pointers and describe the necessary standards so that that it may be applied to muscles beyond the two examples we provide.
1. Collecting EFOV-US images of muscles
Preparation
<ol>
	<li>Sonographer Preparation
	<ol>
		<li>Prior to operating the ultrasound system, read through the system&#8217;s manual to become familiar with system safety, care for maintaining the system, system setup and controls, etc. In addition, review the system&#8217;s instructions for obtaining EFOV-US images and be familiar with the method implemented to obtain the EFOV-US images. 
		NOTE: Different ultrasound systems name the EFOV-US mode using different terminology. For example, in the system used here, the EFOV mode is referred to as &#8220;Panoramic Imaging&#8221;. While the technical details of the algorithm implemented in various commercial systems are usually intellectual property and therefore not freely available, from a cursory review, many commercial systems with panoramic ultrasound capabilities describe an approach similar to the one described by Weng et al.22. Evaluating the general validity of measurements acquired from any system, either by obtaining more detailed information directly from the company who manufactures the system, by using an imaging phantom26,32, or by other means (e.g., comparison to animal dissection24) is recommended as an important step before initiating research involving human participants.</li>
		<li>Take time to become familiar with the anatomy of the muscle(s) of interest as well as the surrounding anatomy. It is suggested that the sonographer use an anatomy textbook or preferably an interactive online 3D anatomy model to become familiar with the anatomy of interest.</li>
	</ol>
	</li>
	<li>Participant Preparation
	<ol>
		<li>Explain the protocol of the study to the participant and acquire IRB approved consent prior to beginning the imaging protocol.</li>
		<li>Ask the participant to wear appropriate clothing to enable access to the muscle of interest. For example, if the sonographer plans to image a forearm muscle, the participant should be asked to wear a short-sleeved shirt.</li>
		<li>Seat the participant in an adjustable chair that can be locked in place. Take time to adjust the chair to make the participant as comfortable as possible while still providing access to the muscle of interest. 
		NOTE: If an adjustable chair which can lay completely flat is not available, some study designs may require the use of a table to access the muscle of interest (i.e., hamstrings).</li>
		<li>Place the joint(s) that the muscle of interest spans in a posture that can be controlled and repeated. Use clinical guidance33 for locating anatomical landmarks and implementing goniometry; use ISB standards for defining the joint coordinate system34,35. In general, to measure joint angle, mark anatomical landmarks with skin safe marker (Table of Materials) and then align the center of a handheld goniometer up with the axis of rotation of the joint and the arms of the goniometer up with the joint segments. 
		NOTE: If imaging passive muscle, placing the muscle of interest in a relatively lengthened position is recommended to avoid imaging slack muscle.
		<ol>
			<li>To replicate&#160;the biceps brachii as imaged in this study, seat participants with feet supported, back straight, shoulder at 85&#176; of abduction and 10&#176; of horizontal flexion, elbow at 25&#176; flexion, and forearm, wrist, and fingers at neutral.</li>
			<li>To replicate the tibialis anterior as imaged in this study, seat participants with knee at 60&#176; of flexion and the ankle at 15&#176; of plantar flexion.</li>
		</ol>
		</li>
		<li>Secure the participants limb using cloth straps to minimize movement during the imaging protocol.</li>
	</ol>
	</li>
</ol>
Image Acquisition
<ol start="3">
	<li>Plug in and turn on the ultrasound system. Ensure that the exam is set to Musculoskeletal, the transducer in&#160;use is selected (here we used 14L5), and the transmit frequency is set between 5-17 MHz (here 11MHz was used), a typical frequency range for musculoskeletal imaging. Higher frequencies are generally used for more superficial imaging as they improve resolution but decrease wave penetration.</li>
	<li>Go into the system settings to adjust the footswitch settings. For the purposes of this protocol, we recommend setting the footswitch to start/stop the imaging. If the footswitch in use has multiple pedals, set additional pedals to &#8220;Freeze&#8221; or &#8220;Pause&#8221;, and &#8220;Print&#8221; or &#8220;Store&#8221; the image.</li>
	<li>Apply a generous amount of ultrasound gel to the head of the transducer.</li>
	<li>Place the transducer on the participant&#8217;s skin on the approximate region of interest.</li>
	<li>Move the transducer in the short axis plane of the muscle. Note that the transducer has a small protuberance on one side, called an indicator. The side of the transducer that has the indicator corresponds with the left side of the ultrasound image. When imaging in the short axis, have the sonographer keep the indicator pointed laterally and when the sonographer is in long axis, point the indicator distally.</li>
	<li>Identify the muscle of interest in the short axis plane (perpendicular to muscle fiber direction) and move the transducer distal and proximal to get a full visualization of the muscle path.
	<ol>
		<li>Mark important anatomical landmarks (i.e., the lateral and medial edges of the muscle, the muscle tendon junction, and muscle insertion) using skin safe ink markers (Table of Materials).</li>
	</ol>
	</li>
	<li>Once the location of the muscle has been identified and properly marked, have the sonographer move the ultrasound transducer in the long axis plane (parallel to the muscle fiber direction).</li>
	<li>Beginning at either the distal or proximal end of the muscle, rotate and tilt the transducer to identify the fascicle plane at that point. Make a mark on the skin when the correct transducer position has been established.</li>
	<li>Once the approximate fascicle plane has been established along the entire desired length to be scanned, have the sonographer practice following this path.</li>
	<li>To begin collecting images, put the ultrasound system in EFOV-US mode.</li>
	<li>Starting at one end of the muscle, click the footswitch to start image acquisition and slowly and continuously move the ultrasound transducer in the long axis. Once the end of the muscle has been reached, click the footswitch to end image acquisition.</li>
	<li>Practice and ensure the correct transducer path. This may take several practice images before consistently obtaining &#8220;quality&#8221; EFOV-US images (See section 2 for explanation of quality images).</li>
	<li>To optimize image visibility and clarity, consider adjustments to the following parameters.
	<ol>
		<li>Depth: If image acquisition ends before the desired length of the muscle can be captured, increase the depth of the image (in the system used here, increasing image depth increases the absolute length the scan can be).</li>
		<li>Focus: Place the focus arrow in the lower half of the Image just below the muscle of interest.</li>
		<li>Gain: Ensure the gain is balanced through the depth of the image.</li>
		<li>Speed: Image at the optimum speed as guided by the indicator (in most systems a speed indicator displays on the monitor during panoramic imaging).</li>
	</ol>
	</li>
	<li>Once qualitatively good images have been collected (step 2.1), hit the Print/Store footswitch pedal or a synonymous button on the control panel to save the image.</li>
	<li>Repeat steps 1.13-1.16 until 3 quality EFOV-US images of the muscle are obtained.</li>
	<li>Repeat steps 1.6-1.17 until all muscles of interest are obtained.</li>
	<li>Use a towel to gently wipe the gel from the participant&#8217;s skin. Then have the participant rinse off the area of the skin or use a damp towel to wipe the skin that was exposed to the gel. Dry.</li>
	<li>Wipe gel from the head of the transducer and disinfect.</li>
	<li>Export images as uncompressed DICOM images onto a CD-DVD, flash drive, or through the local network onto a computer.</li>
</ol>
2. Determining &#8220;quality&#8221; of the EFOV-US image
<ol>
	<li>Following step 1.13, have the sonographer identify and evaluate the quality of key anatomical features of the muscle of interest and its surrounding anatomy. This is a qualitative assessment based on the sonographer&#8217;s knowledge of anatomy and musculoskeletal tissue echogenicity (ability of a tissue to reflect ultrasonic waves). For an EFOV-US images to be considered qualitatively &#8220;good&#8221; the following should be met:
	<ol>
		<li>In any long-axis image of a muscle, check that the sonographer can clearly identify the muscle as a hypoechoic (dark) shape with hyperechoic (bright) boundaries which represent the deep and superficial muscle fascia.</li>
		<li>Between the muscle boundaries, check that the sonographer can identify the connective tissue surrounding a muscles fascicle as hyperechoic (bright) lines. 
		NOTE: When imaging multi-pennated muscles, the image should also contain central tendon(s) that show up in the muscle belly, between the deep and superficial muscle fascia, as a hyperechoic (bright) structure.</li>
		<li>Check that the image does not have excessive bending. This is usually indicated by shadows or gaps in the image or a jagged flexible ruler line over the image.</li>
	</ol>
	</li>
	<li>If the image is missing one or more of the tissue structures described in 2.1, deem the image &#8220;qualitatively poor&#8221; and return to live 2D-mode.</li>
</ol>
3. Quanitfying Muscle Fascicle Length
<ol>
	<li>To quantify muscle fascicle length, use ImageJ, an open source image processing platform. ImageJ can be downloaded at https://imagej.net/Downloads. 
	NOTE: Though ImageJ is frequently implemented24,25,31,36,37,38, quantification of muscle fascicle length may be measured using other image processing software8,39 or custom codes40,41.</li>
	<li>Once downloaded, open the ultrasound images as DICOM images in ImageJ by clicking File | Open and selecting the image to analyze.</li>
	<li>To ensure that the DICOM image properties have been preserved, click on the Straight Line tool in the Tools menu and draw a straight line from 0 to 1 cm on the ruler on the side of the ultrasound image. Then go to Analyze | Measure to measure the line made. If the image properties have been preserved, the length of the straight line should be 1 cm.</li>
	<li>To measure fascicle lengths in the image, complete the following.
	<ol>
		<li>Right click on the Straight Line tool.</li>
		<li>Select Segmented Line.</li>
		<li>Move the cursor onto the image and click at one end of the fascicle that has been chosen to be measured. 
		NOTE: Only make measurements on fascicles that the entire fascicle path (i.e., from one aponeurosis to the next aponeurosis or aponeurosis to central tendon) can convincingly be seen.</li>
		<li>Click along the path to ensure curvature in the fascicle path is captured.</li>
		<li>Once the end of the fascicle path is reached, double click to end the line and go to Analyze | Measure to measure the length of the line. 
		NOTE: A new window, &#8220;Results&#8221;, will pop up the first time a measurement is made. What values are displayed can be managed in the Results window by going to Results | Set Measurements.</li>
	</ol>
	</li>
	<li>Repeat steps 3.4.3-3.4.5 until multiple fascicle measures are made in a single image.</li>
	<li>Save fascicle measurements by clicking File | Save on the results tab or the values can be copy and pasted into another document/spreadsheet.</li>
</ol>

The authors have nothing to disclose.

Critical steps in the protocol.
There are a few critical components to obtaining quality EFOV-US images that yield valid and reliable fascicle length measures. First, as indicated in method 1.1.2 it is essential that the sonographer take time to become familiar with the anatomy of the muscle being imaged as well as surrounding muscles, bones, and other soft tissue structures. This will improve the sonographer&#8217;s ability to image the correct muscle and determine if multipl...

Fascicle length is an important parameter of skeletal muscle architecture, which overall is indicative of a muscle&#8217;s ability to produce force1,2. Specifically, a muscle&#8217;s fascicle length provides insight into the absolute range of lengths over which a muscle can generate active force3,4. For example, given two muscles with identical values for all isometric force-generating parameters (i.e., average sarcomere length, pennation angle, physiological cross sectional area, contraction state, etc.) except for fascicle length, the muscle with the longer fascicles would produce its peak force at a longer length and would produce force over a wider range of lengths than the muscle with shorter fascicles3. Quantification of muscle fascicle length is important for understanding both healthy muscle function and changes in a muscle&#8217;s force-generating capacity, which can occur as a result of altered muscle use (e.g., immobilization5,6, exercise intervention7,8,9, high heel wearing10) or a change in the muscle&#8217;s environment (e.g., tendon transfer surgery11, limb distraction12). Measurements of muscle fascicle length were originally obtained through ex vivo cadaveric experiments that allow for direct measurement of dissected fascicles13,14,15,16. The valuable information provided by these ex vivo experiments led to an interest in implementing in vivo methods17,18,19 to address questions that could not be answered in cadavers; in vivo methods allow for quantification of muscle parameters in a native state as well as at different joint postures, different muscle contraction states, different loading or unloading states, and across populations with differing conditions (i.e. healthy/injured, young/old, etc.). Most frequently, ultrasound is the method employed to obtain in vivo muscle fascicle lengths18,19,20; it is quicker, less expensive, and easier to implement than other imaging techniques, such as diffusion tensor imaging (DTI)18,21.
Extended field-of-view ultrasound (EFOV-US) has been demonstrated to be a valid and reliable method for measuring muscle fascicle length in vivo. While commonly implemented, traditional ultrasound (T-US) has a field-of-view which is limited by the ultrasound transducer&#8217;s array length (typically between 4 and 6 cm, although there are probes that extend to 10 cm10)18,20. To overcome this limitation, Weng et al. developed an EFOV-US technology that automatically acquires a composite, two-dimensional &#8220;panoramic&#8221; image (up to 60 cm long) from a dynamic, extended distance scan22. The image is created by fitting together, in real-time, a sequence of traditional, B-mode ultrasound images as the transducer dynamically scans the object of interest. Because sequential T-US images have large overlapping regions, the small differences from one image to the next can be used to calculate the probe motion without the use of external motion sensors. Once the probe motion between two consecutive images is calculated, the &#8220;current&#8221; image is merged successively with the preceding images. The EFOV-US method allows direct measurement of long, curved muscle fascicles and has been demonstrated to be reliable across muscles, trials, and sonographers23,24,25 and valid for both flat and curved surfaces23,26.
Implementing ultrasound to measure muscle fascicle length in vivo is not trivial. Unlike other imaging techniques that involve more automated protocols (i.e., MRI, CT), ultrasound is dependent on sonographer skill and anatomical knowledge27,28. There is concern that probe misalignment with the fascicle plane may cause substantial error in fascicle measures. One study demonstrates little difference (on average &#60; 3 mm) in measures of fascicle length taken using ultrasound and DTI MRI but also shows that measurement precision is low (standard deviation of difference ~12 mm)29. Still, it has been shown that a novice sonographer, with practice and guidance from an experienced sonographer, can obtain valid meaures using EFOV-US23. Thus, efforts should be made to demonstrate appropriate protocols to reduce human error and improve accuracy of measurements obtained using EFOV-US. Ultimately, developing and sharing appropriate protocols may expand the number of experimenters and laboratories that can reproduce fascicle length data from the literature or obtain novel data in muscles which have not yet been studied in vivo.
In this protocol, we demonstrate how to implement the EFOV-US method to obtain high quality musculoskeletal images that can be used to quantify muscle fascicle length. Specifically, we address (a) collecting EFOV-US images of a single upper limb and a single lower limb muscle (b) determining, in real-time, the &#8220;quality&#8221; of the EFOV-US image, and (c) quantifying muscle architecture parameters offline. We provide this detailed guide to encourage the adoption of the EFOV-US method for obtaining muscle fascicle length data in muscles that have gone unstudied in vivo due to their long fascicles.

<table>
	<tbody>
		<tr>
			<td>14L5 linear transducers</td>
			<td>Siemens</td>
			<td>10789396</td>
			<td></td>
		</tr>
		<tr>
			<td>Acuson S2000 Ultrasound System</td>
			<td>Siemens</td>
			<td>10032746</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable chair (Biodex System)</td>
			<td>Biodex Medical Systems</td>
			<td>System Pro 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Skin Marker Medium Tip</td>
			<td>SportSafe</td>
			<td>n/a</td>
			<td>Multi-color 4 Pack recommended</td>
		</tr>
		<tr>
			<td>Ultrasound Gel - Standard 8 Ounce Non-Sterile Fragrance Free Glacial Tint</td>
			<td>MediChoice, Owens &#38;Minor</td>
			<td>M500812</td>
			<td></td>
		</tr>
	</tbody>
</table>

obtaining quality extended field-of-view ultrasound images of skeletal muscle to measure muscle fascicle length

Muscle fascicle length, which is commonly measured in vivo using traditional ultrasound, is an important parameter defining a muscle&#8217;s force generating capacity. However, over 90% of all upper limb muscles and 85% of all lower limb muscles have optimal fascicle lengths longer than the field-of-view of common traditional ultrasound (T-US) probes. A newer, less frequently adopted method called extended field-of-view ultrasound (EFOV-US) can enable direct measurement of fascicles longer than the field-of-view of a single T-US image. This method, which automatically fits together a sequence of T-US images from a dynamic scan, has been demonstrated to be valid and reliable for obtaining muscle fascicle lengths in vivo. Despite the numerous skeletal muscles with long fascicles and the validity of the EFOV-US method for making measurements of such fascicles, few published studies have utilized this method. In this study, we demonstrate both how to implement the EFOV-US method to obtain high quality musculoskeletal images and how to quantify fascicle lengths from those images. We expect that this demonstration will encourage the use of the EFOV-US method to increase the pool of muscles, both in healthy and impaired populations, for which we have in vivo muscle fascicle length data.

Extended field-of-view ultrasound (EFOV-US) was implemented to obtain images from the long head of the biceps brachii and the tibialis anterior in 4 healthy volunteers (Table 1). Figure 1 shows what EFOV-US images of both muscles imaged in this representative imaging session and highlights important aspects of each image such as muscle aponeurosis, central tendon, fascicle path, etc. After the imaging session was over, 3 qualitatively &#8220;good&#8221; images (<strong class...

Watch this Scientific Journal Video about Obtaining Quality Extended Field-of-View Ultrasound Images of Skeletal Muscle to Measure Muscle Fascicle Length at JoVE.com

Obtaining Quality Extended Field-of-View Ultrasound Images of Skeletal Muscle to Measure Muscle Fascicle Length

This study describes how to obtain high quality musculoskeletal images using the extended field-of-view ultrasound (EFOV-US) method for the purpose of making muscle fascicle length measures. We apply this method to muscles with fascicles that extend past the field-of-view of common traditional ultrasound (T-US) probes.

Muscle fascicle length, which is commonly measured in vivo using traditional ultrasound, is an important parameter defining a muscle&#8217;s force ...

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3.5K Views.  Northwestern University. This study describes how to obtain high quality musculoskeletal images using the extended field-of-view ultrasound (EFOV-US) method for the purpose of making muscle fascicle length measures. We apply this method to muscles with fascicles that extend past the field-of-view of common traditional ultrasound (T-US) probes.

Video: Obtaining Quality Extended Field-of-View Ultrasound Images of Skeletal Muscle to Measure Muscle Fascicle Length

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 ...

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 ...

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.

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 ...

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 ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&amp;#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

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 ...

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 ...

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 ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery in humans. Herein we discuss the design of an eccentric exercise protocol to induce damage in the quadriceps muscles, marked by changes in strength, soreness, and plasma creatine kinase levels. This method is simple, ethical, and widely applicable since it is performed in human participants and eliminates the interspecies translation of the results. Subjects perform 300 maximal eccentric contractions of the knee extensor muscles at a speed of 120&#176;/sec on an isokinetic dynamometer. The extent of the damage is measurable using relatively non-invasive isokinetic and isometric measures of strength loss, soreness, and plasma creatine kinase levels over several days following the exercise. Therefore, its application can be directed to specific populations in an attempt to identify mechanisms for muscle adaptation and regeneration.

This article describes a safe and reliable method to induce and quantify exertional skeletal muscle damage in human subjects.

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery ...