Name: collection of skeletal muscle biopsies from the superior compartment of human musculus tibialis anterior for mechanical evaluation
Uploaded: 2020-09-27T14:30:00
Description: Watch this Scientific Journal Video about Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation at JoVE.com

We thank Michaela Rau, Lea-Fedia Rissmann, Michael Marsh, Janina-Sophie Tennler, Kilian Kimmeskamp, and Wolfgang Linke for assisting with the project. Funding for this project was provided by the MERCUR Foundation (ID: An-2016-0050) to DH.

NOTE: Below, we outline a protocol to harvest mechanically undamaged fibers from the TA of volunteers who were enrolled in a separate ongoing study. This protocol is similar to that described by Shanely et al.3, who have described the modified Bergstr&#246;m technique in vastus lateralis. The information presented here has been refined by our research group but may not be ideal for all lab groups or organizational setups. We give only guidelines, and strongly suggests that laboratories new to biop...

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In this report, we described a technique for the biopsy of structurally undamaged muscle tissue from TA. We found that this procedure yields an acceptable content of usable muscle fibers (5-10 fiber bundle preparations per 50 mg of collected tissue) for mechanical testing. Further, we had enough tissue for follow-up mechanical, genetic, and proteomic experiments.
There are several methods typically used for the collection of muscle biopsies3,<sup class="xref"...

The study of human muscle physiology for clinical or research purposes often requires muscle biopsies. For example, a major challenge in human muscle physiology and biomechanics is to distinguish between and understand the various adaptations of muscle performance to exercise. Performance adaptations do not just include structural adaptations (e.g., changes in contractile proteins, muscle architecture) but also include neural adaptations1, which are very hard, if not impossible, to assess separately when testing intact in situ human muscles. Fiber-level experiments remove these higher-order components and allow for a more direct evaluation of muscle contraction and can be collected via biopsy techniques. Muscle biopsies have been collected since at least 18682. Today, the predominant technique to collect muscle biopsies is the modified Bergstr&#246;m technique3,4,5, although other techniques are available including the use of a Weil-Blakesley conchotome6 or the so called fine-needle7,8. All these techniques use special needle-like instruments that are designed to pass into muscle and cut a piece of tissue. Specifically, the modified Bergstr&#246;m technique uses a large modified needle (5 mm needle size here; Figure 1) that has a window close to the needle tip and a smaller internal trocar that moves up and down the needle, cutting the muscle when passing over the needle window. Within this hallow trocar is a ramrod that moves up and down the shaft of the trocar and pushes the biopsy towards the needle window. To pull the muscle into the needle window, a suction hose is attached, which sucks air out of the needle and pulls the muscle into the needle window via negative pressure.
Muscle biopsies are often acquired to study changes in protein content, gene expression, or morphology caused by disease or in a response to an exercise program1,9,10,11. Another critical use for muscle biopsies is mechanical experiments such as the measurement of fiber contractile force, muscle fiber stiffness, and history-dependent muscle properties12,13,14,15,16. Single fiber or fiber bundle mechanics are measured by attaching fibers between a length motor and force transducer on specialized rigs that control fiber length while simultaneously measuring force. By permeabilizing (e.g., skinning) fibers, the sarcolemma membrane becomes permeable to chemicals in the bath solution, allowing for activation control by varying calcium concentration. Furthermore, the effect of contractile properties on chemicals/pharmaceuticals/other proteins can easily be evaluated by adding the reagent in question to the bath solution. However, while this technique is highly used in other animal models, noticeably fewer studies conducted mechanical tests on skinned fibers from human muscle biopsies17,18,19. One reason is that the biopsy tools and protocols are designed to remove as much muscle tissue as possible with less regard for the level of structural damage sustained during tissue extraction. Indeed, a recent biopsy protocol suggests to drive the biopsy needle into the muscle and collect 2-4 chunks of muscle3. The process itself does little damage to the DNA or protein material, but often destroys fiber and sarcomeric structures in such a way that the activation of muscle fibers becomes unstable or impossible. Furthermore, the relative length of fibers within the biopsy are typically short (&#60;2 mm) and not easily handled for mechanical testing. For mechanical testing, ideal fibers are long (3-5 mm) and not structurally damaged.
More advanced tissue extraction techniques can be used to limit fiber damage. For example, one group20 took advantage of previously planned &#8220;open surgeries&#8221; of forearms (e.g., bone fracture repair), where the muscles were fully exposed and a surgeon was able to visualize the muscle structure and carefully dissect relatively large and structurally undamaged samples of muscle tissue (15 mm x 5mm x 5 mm). This &#8220;open biopsy&#8221; technique is favored when participants are undergoing a previously planned procedure, and so limits the pool of potential participants, especially for healthy adults, where no surgeries would otherwise take place. Thus, many biopsies conducted for research purposes are done as an outpatient procedure and the incision site is kept as small as possible to limit infection risk, scarring, and healing time. Therefore, most biopsies are collected blindly (i.e., the operator is unable to see the collection needle as it passes through the fascia into the muscle). This implies that the quality of the biopsy is almost entirely based on the skill and experience of the operator. Every muscle has its own difficulties when collecting tissue, such as risks to violate nerves and blood vessels, selection of an ideal collection depth and location, and deciding on an appropriate body position to keep the muscle as slack as possible. Unfortunately, most of the muscle-specific skillsets are not written down and so each physician must &#8220;reinvent the wheel&#8221; when performing biopsies on muscles new to them. This lack of experience usually leads to several collections with low quality until the physician identifies the best practices for biopsies on that muscle. Novice physicians often learn the skill through conversations with their more experienced colleagues, but relatively few informative and peer-reviewed texts exist on the matter, especially for muscles that are not traditionally used for biopsy collection. If we consider the above information, along with the difficulty of recruiting human volunteers for biopsies, it is clear that more teaching information is needed that maximizes the chances of success for every participant.
Thus, the purpose of this paper was to present a muscle biopsy technique that provides protocols for the successful collection of muscle biopsies with long, undamaged fiber fragments for mechanical tests. Human muscle biopsies are usually carried out on, and the bulk of biopsy training material is on, the musculus vastus lateralis. Its relatively large muscle size and superficial location relative to the skin allows for the collection of adequate muscle tissue, while minimizing patient discomfort and physical trauma1,21. However, there are some limitations to using the vastus lateralis for longitudinal training studies. For example, during experimental protocols that include a training program, participants must refrain from additional training outside of the study for a period that often spans 2-6 months. For athletes, this is often not possible, as the vastus lateralis is usually trained during typical exercises (e.g., squats, jumps), or is generally used for the sport (e.g., running, cycling). These separate training experiences away from the study&#8217;s aim can cause muscular adaptations that alter muscle mechanics, architecture, and physiology in such a way that it is difficult or impossible to know the true effect of the study&#8217;s experimental protocol on muscle properties. For these types of studies, it would be ideal to select a target muscle that is often not the focus of training regiments. The musculus tibialis anterior (TA) is an ideal target muscle that satisfies the requirements above. In addition, training interventions can be targeted towards the TA using controllable approaches, such as with the use of a dynamometer. There is almost no training material pertaining to a TA muscle biopsy. Therefore, we developed a modified protocol to collect relatively undamaged muscle biopsies from the TA.

<table>
	<tbody>
		<tr>
			<td>26 guage subcutaneous needle with 2 ml glass syringe</td>
			<td>B. Braun Melsungen AG 
			Carl-Braun-Stra&#223;e 1 
			34212 Melsungen, Hessen 
			Germany 
			&#160;</td>
			<td>4606027V</td>
			<td>Drug administration</td>
		</tr>
		<tr>
			<td>5mm Berst&#246;m needle</td>
			<td>homemade</td>
			<td>N/A</td>
			<td>Tissue collection. Similar to other Berst&#246;m needles</td>
		</tr>
		<tr>
			<td>Acrylastic</td>
			<td>BSN medical GmbH 
			22771 Hamburg</td>
			<td>269700</td>
			<td>elastic compression bandage</td>
		</tr>
		<tr>
			<td>Complete protease inhibitor cocktail</td>
			<td>Roche Diagnostics, Mannheim, Germany</td>
			<td>11836145001</td>
			<td>Protease inhibitor tabeletes added to all solutions that hold muscle tissue.</td>
		</tr>
		<tr>
			<td>Cutasept</td>
			<td>PAUL HARTMANN AG 
			Paul-Hartmann-Stra&#223;e 12 
			89522 Heidenheim 
			Germany</td>
			<td>9805630</td>
			<td>Disenfectant spray for the skin</td>
		</tr>
		<tr>
			<td>Leucomed T plus</td>
			<td>BSN medical GmbH 
			22771 Hamburg</td>
			<td>7238201</td>
			<td>Transparent wound dressing with wound pad to seal the wound and protect against infection</td>
		</tr>
		<tr>
			<td>Leukostrip</td>
			<td>Smith and Nephew medical Limitied 101 Hessle road, 
			Hull 
			Great Britain</td>
			<td>66002876</td>
			<td>wound closure</td>
		</tr>
		<tr>
			<td>Surgical disposable scalpels</td>
			<td>Aesculap AG 
			Am Aesculap-Platz 
			78532 Tuttlingen 
			Germany</td>
			<td>BA200 series</td>
			<td>Incision</td>
		</tr>
		<tr>
			<td>Unihaft cohesive elastic bandage</td>
			<td>BSN medical GmbH 
			22771 Hamburg</td>
			<td>4589600</td>
			<td>cohesive elastic bandage that protects against mechanical impact</td>
		</tr>
		<tr>
			<td>Xylocitin 2% with Epinephrin</td>
			<td>Milbe GmbH 
			M&#252;nchner Stra&#223;e 15 
			06796 Brehna 
			Germany</td>
			<td>N/A</td>
			<td>Controlled substance anesthesia, vasoconstriction</td>
		</tr>
	</tbody>
</table>

collection of skeletal muscle biopsies from the superior compartment of human musculus tibialis anterior for mechanical evaluation

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

The entire time commitment for a participant was about one hour (10 min consultation, 10 min ultrasound, 20 min surgery preparation and anesthetic administration, 10 min surgery, and 10 min recovery). Often, participants unconsciously activated their TA and needed consistent reminders to keep the muscle as relaxed as possible. When the biopsy needle was inside the muscle, participants usually reported a unique &#8220;pressure&#8221; sensation in the area around the biopsy needle, with occasional periods of moderate to in...

Watch this Scientific Journal Video about Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation at JoVE.com

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...

Welcome to the Ruhr-University Bochum, Faculty of Sports Science. Today, we will demonstrate the collection of skeletal muscle biopsies from the superior compartment of the human musculus tibialis anterior. While biopsy techniques are effective at collecting muscle tissue, the fibers are often structurally damaged in such a way that evaluating fiber mechanics is difficult.We present a variation of a modified Bergstrom technique that limits fiber damage and maximizes fiber length so that the mechanical evaluation of human fibers can be conducted. The demonstration will be conducted by team physician, Dr.Markus de Marees and research scientists, Dr.Daniel Hahn and Anthony Hessel. The participant and physician first meet to review and discuss the procedure.If satisfied, the participant signs the voluntary informed consent contract. Instruct the subject to lay supine and keep their limb as relaxed as possible. Dorsiflex the participant's foot so that the tibialis anterior is slack.Use an ultrasound probe to visualize the transverse plane of the superficial compartment within the tibialis anterior muscle belly, identify a biopsy area with no major arteries or nerves. Finally, record the depth of the central aponeurosis so that the collection needle is never driven past this point. Rotate the ultrasound probe to access the sagittal plane of the identified target area.Save an image of the muscle volume with fascicles visible. The surgery tools are sterilized and prepared before the procedure and are fully described within the written protocol. The Bergstrom needle has a window close to the tip.Within this needle is a smaller hollow trocar, which moves up and down the shaft and cuts the muscle when passing over the needle window. Within the trocar is a smaller ramrod that is used to push the biopsy out of the trocar. To pull the muscle into the needle window, a suction hose is attached to the top of the needle that sucks air out of the needle and pulls the muscle through the needle window by negative pressure.Instruct the participant to lay supine on the procedure table and relax their leg muscles. Replicate the slight ankle dorsiflexion used during ultrasound imaging, and then fix the ankle joint to minimize rotation during the procedure. Shave and disinfect the target incision area.Next administer a superficial injection of 1.5 CCs of 2%xylocitin with epinephrine. This functions as a local anesthetic and vessel constrictor. The affect time is 20 to 30 minutes.Remove the patient's line of sight to the biopsy location with a screen. At the previously marked biopsy site make a one centimeter proximal to distal incision with a sterile scalpel that cuts through the skin and fascia, exposing the underlying muscle tissue. Position the biopsy needle over the incision and orient it perpendicular to the skin.Push the biopsy needle one half to one centimeter into the muscle. There may be some light resistance to the needle movement, but any extra resistance is abnormal and often indicates an inapt incision. Next orient the needle to a distal to proximal position, push the needle another one to two centimeters into the muscle.The exact orientation and travel depth of the needle will be participant specific. And thus based off the ultrasound examination. Attach the syringe hose to the biopsy needle and create negative pressure in the needle.This will draw the muscle tissue through the needle window. Forcefully push the trocar over the needle window to excise the muscle. Remove the needle from the leg and pass to a technician for tissue processing.Use wound closure tape or adhesive to primarily close the wound and apply dressings to protect the wound from infection or mechanical abrasion. Instruct the participant to stand up and walk around. They will be sore, but able to walk around immediately.To remove the tissue from the biopsy window, first expose the tissue, then submerged the needle tip into a vile filled with physiological buffer and vigorously move the needle until the muscle floats out of the needle into the solution. Process muscle tissue for mechanical evaluation using standard methods. We recently collected 22 biopsies from participants using this protocol.The average collection yield was 100 to 200 milligrams of usable muscle tissue. Each biopsy yielded 150 to 300 undamaged fibers of three to five millimeters in length. Fiber bundles were chemically skinned with glycerol and then activated in a high calcium solution at an average sarcomere length of 2.7 microns.Biopsies that are properly stored are viable for mechanical testing for several months. This biopsy procedure takes one hour. Complications from biopsies are very rare, but most likely is an infection of the incision site.A major challenge in human muscle physiology and biomechanics is to distinguish between the various adaptations of muscle performance to exercise and disease. To appropriately identify fiber level adaptations to exercise or disease, biopsies are appropriate technique. However, care must be taken to protect against muscle damage during tissue extraction.The protocol demonstrated here provides a high yield of muscle tissue for these purposes.

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