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

Summary

This technical report describes a variation of the modified Bergström technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

Abstract

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 Bergström 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.

Introduction

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 Bergström technique^3,4,5, although other techniques are available including the use of a Weil-Blakesley conchotome⁶ or the so called fine-needle^7,8. All these techniques use special needle-like instruments that are designed to pass into muscle and cut a piece of tissue. Specifically, the modified Bergström 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 program^1,9,10,11. Another critical use for muscle biopsies is mechanical experiments such as the measurement of fiber contractile force, muscle fiber stiffness, and history-dependent muscle properties^{12,13,14,15,16}. 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 biopsies^17,18,19. 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 (<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 “open surgeries” 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 “open biopsy” 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 “reinvent the wheel” 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 trauma^1,21. 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’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’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.

Protocol

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 Bergström 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 biopsy collection consult experienced laboratory groups before attempting any human trials.

All studies conducted in this paper were approved by the Ethics Committee of the Faculty of Sport Science at Ruhr University Bochum. Participants gave free written informed consent prior to participating in the study.

1. Experimental preparation

1. Assess exclusion criteria while taking the participant’s detailed medical history during the participant consultation (see below).
   1. Exclude participants if they suffered an injury to the target muscle during the 6 weeks leading up to the biopsy. Ensure participants are generally healthy, aware of no muscle or coagulation disorders, and are not currently on medication that causes blood thinning (e.g., aspirin).
      NOTE: Here, we selected participants who were moderately active and instructed them to refrain from intensive or unaccustomed leg exercises at least 3 days before the biopsy. However, for other research questions, these criteria may change.
2. Adhere to sterilization and aseptic techniques, as regulated by German law and common practice and overseen by the team physician^22,23. This procedure can often be conducted as a “bedside” procedure or in an outpatient surgical suite. Consult the local regulatory body for guidance.
3. Compose the biopsy team. We suggest that the biopsy team includes 4 people. A physician (or trained individual in biopsy collection), one medical assistant working with the physician, one assistant who monitors and interacts with the participant, and one assistant who handles the muscle biopsy immediately after extraction. With these numbers, quick patient care can be administered if a medical emergency occurs during the procedure. If comfortable with the procedure, then the team could be made of only two people: the physician and medical assistant, who would together take on patient care and tissue processing concurrently.
4. Have the participant meet with the project lead/physician to review, discuss, and sign the user consent form. Take a detailed medical history (allergies, injuries or surgeries to the lower limb and TA) and exclude the participant if they meet any of the exclusion criteria. Thoroughly discuss recovery and incision hygiene.
   1. Explain to the participant that they will be sore but able to walk around immediately after the procedure; walking down slopes or stairs is often uncomfortable for the first 48 hours, with full activity usually returning after 72 hours. Finally, explain that, to limit infection and mechanical abrasions, the incision site should remain bandaged for at least 1 week and kept clean.

2. Visualize the Anterior Tibialis with B-mode Ultrasound

1. Instruct the participant to lie down in a comfortable supine position and relax their leg muscles as much as possible. Use a custom-made device (see below) or have the assistant hold the ankle in a slightly dorsiflexed position to mimic that which will be done during the biopsy.
   NOTE: It is important that the participant has a relaxed TA so that it replicates the muscle characteristics during the procedure. During the exam, ask the participant to contract and relax the muscle so that the changes in muscle architecture can be noted.
2. Use an ultrasound probe to visualize the superficial and deep compartments of the TA, to survey the muscle architecture and decide on depth of insertion and needle angle of attack (Figure 2A-B). Indicate landmarks on the skin.
   1. Give particular attention to the selection of a target area that avoids major veins, arteries, or nerves.
   2. Assess the cross section of the muscle, with the goal of identifying the central aponeurosis within TA muscle belly (approximately 1/3 of the leg, distal to the knee, and 2 cm lateral of the tibial crest) (Figure 2B). Record the location and depth of the central aponeurosis (usually 1.5-3 cm) so that care can be taken to not drive the collection (Bergström) needle past this point.
   3. Position the ultrasound probe in the proximal-distal orientation over the target location and visualize the fascicle pennation and muscle thickness (Figure 2A). Use this information to help successfully drive (blindly) the collection needle into the muscle belly. Save images of the target site in both planes for future reference during the surgical procedure.
3. With this information, create a plan for needle movement towards the target area.
   1. Plan to make the incision 1-3 cm distal from the target biopsy area. After the needle is passed into the muscle, rotate the needle to a ~45% angle to the skin along the long axis of the limb, and then driven proximally towards the biopsy area. This strategy limits the chance of driving the needle into the central aponeurosis, if the needle is pushed too hard. Furthermore, the needle can be driven distally or proximally, depending on the handedness of the needle operator.

3. Biopsy procedure

1. Instruct the participant to lay supine on the operating table and relax their leg muscles. Make sure the participant’s line of sight to the biopsy site is blocked by a curtain.
   1. Remove passive tension from the muscle belly by placing the participant’s limb into a device that fixes the ankle into a slightly dorsiflexed position (0-5° from neutral; Figure 3). Ask the patient if they can still relax their muscle, as too much dorsiflexion can potentially make it difficult to relax.
      NOTE: We have found that collecting biopsies from a dorsiflexed foot, no more than 5° of neutral (i.e., the sole of the foot perpendicular to the shank) produces more consistent and larger biopsies than more plantar flexed ankle angles. The device that keeps the ankle dorsiflexed is a custom-made device. However, any number of (cheap) devices can be fabricated that still produce the desired result.
2. Shave, clean, and disinfect the selected incision area, as per standard practices²⁴.
   NOTE: The participant’s “clean” area is about 20 cm proximal-distal and 10 cm medial-lateral of the proposed incision site. However, always consult the institution’s and/or national regulations (if any) on this topic. The disinfection protocol includes scrubbing the skin clean and then disinfecting four times with liberal use of medical-grade disinfection spray. If the participant leaves the table for any reason, the disinfection protocol must be restarted.
3. Administer a suprafascial injection of 1.5 cc of 2% Xylocitin with Epinephrine at the biopsy site, which functions as a local anesthetic and vasoconstrictor. Wait for the allotted affect time of ~20-30 min.
   NOTE: These drugs are myotoxic and thus must never be injected into the muscle, only the subcutaneous tissue. As a reaction to the vasoconstriction, the area of the injection site may turn white (on lighter skin tones) or gray (darker skin tones).
4. Confirm drug effect with skin pitches and gentle pokes with a sterile scalpel.
5. At the previously marked biopsy site, make a 1 cm proximal-distal incision with a sterile scalpel that cuts through the skin and fascia, exposing the muscle belly. Take care to cut the fascia fully because the needle is blunt and will not pass through the fascia.
6. Push the biopsy needle 0.5-1.0 cm into the muscle with an orientation perpendicular to the skin (Figure 2C, 2E).
   NOTE: The operator will feel a change in the tension needed to drive the needle through the different tissue types. The fatty tissue is easy, the fascia is the toughest, and the muscle is in between (but can be variable, based on the participant).
7. Orient the needle to a position of ~45° angle to the skin, along the long axis of the leg (Figure 2D, 2F). Push the needle another 1-2 cm into the muscle until the needle tip is at the target location within the muscle.
   NOTE: The physician should utilize the saved ultrasound images to account for individual variation of muscle dimensions. Because the incision is only large enough to insert the needle, the physician drives the needle blindly through the skin. There is a “feel” that the biopsy operator gains with experience. A novice must learn the skill from a trained biopsy operator (more on this in the discussion).
8. Attach the 100 mL syringe and hose to the biopsy needle (Figure 1G). Apply suction to the Bergström needle by pulling the plunger of the syringe by about 15-20 mL to produce a negative pressure in the needle and sucking the muscle tissue into the needle window. Then, excise the muscle by a quick push(es) of the trocar over the needle window.
   NOTE: Before and during suction, it is sometimes helpful to place light pressure on the skin immediately above the needle window to help push the muscle into the needle.
9. Gently remove the needle from the leg, rotating slowly. There should only be light resistance while extracting the needle. If there is more resistance, this may indicate a partial biopsy cut. It this occurs, return the need to the target location, and reattempt tissue collection.
10. Push the excised tissue towards the needle window using the internal ramrod.
11. Carefully remove the sample from the needle.
    NOTE: Submerging the needle into collection solution (see fiber preparation section) often dislodges the biopsy from the needle. Additionally, the syringe can be used to drive air through the needle and push out the sample. These techniques remove the need to physically touch the biopsy with tweezers and reduces the possibility of damage. If tools, hands (gloved or not) or non-sterile solutions come in contact with the needle, the needle cannot be used furthering during the procedure. Thus, if a second immediate biopsy is needed, then a new sterile needle must be used. This often occurs, so it is a best practice to maintain several sterile needles in reserve.
12. Identify the tissue as muscle and not adipose or connective tissue. Muscle tissue is easily identified from other tissue because of its deep red color (Figure 4A). Sometimes, the collected tissue is not muscle, but fat or connective tissue.
    1. If an adequate amount of muscle tissue is collected, continue the protocol. If there is not enough muscle, attempt the biopsy again.
    2. If a second biopsy is needed, carefully monitor the participant, as a second needle push occasionally makes the participant more uncomfortable than the first.
13. Wash muscle samples immediately in a collection solution and prepare for single fiber experiments (see muscle biopsy handling and storage).
    1. Have an experienced assistant check the sample quality (see below) and assess the need to perform a second biopsy. A separate assistant takes the biopsy for processing, while the rest of the team continues with the participant.
14. Close the incision site.
    1. Close the incision wound with sterile Leukostrip tape. Use one or more pieces to join the edges of the incision site by laying them perpendicular to the long axis of the incision, and then lay further strips in a starshaped pattern to protect against multi-directional loading.
       NOTE: Proper handling of this step will reduce scarring. Suturing the wound can be done but is not necessary. Other options include wound glue.
    2. Place sterile wound dressing (e.g., Leucomed T plus) over the incision site to protect against infection.
    3. Wrap the leg with cohesive elastic bandages (e.g., Unihaft) to limit initial bleeding and protect against external mechanical impact.
    4. Wrap the leg with acrylastic compression bandages to prevent bleeding and protect the deeper bandages from becoming loose or destroyed.

4. Post-biopsy care

1. Ask the participant to walk around immediately after the procedure. There will be localized soreness. Instruct the participant to walk as normally as possible.
2. Instruct the participant to not remove the bandages or let water soak the bandages. They must be kept on for at least: one days for the acrylastic bandage, three days for the cohesive elastic bandage, and seven days for the wound dressing. Inform the participant that they can be rebandaged if needed.
   1. Tailor the post-biopsy care of a participant to the needs of the individual. Have a trained assistant or physician assess the participant and make an appropriate post-biopsy care plan. For this procedure, we suggest that any further in vivo neuromuscular testing of the TA is separated by at least a week from the biopsy.

5. Muscle biopsy handling and storage

1. After tissue extraction, immediately place the tissue into a 5 mL vial containing rigor collection solution (in mM: Tris (50), KCl (2), NaCl (100), MgCl₂ (2), EGTA (1), protease inhibitor tablet (1), pH 7.0) and lightly shake for 4-6 min to wash out blood.
2. Exchange the Rigor solution for fresh rigor, lightly shake for 4-6 min, and then store at 4 °C for 4-6 h to allow the exchange of protease-inhibitor storage solution and blood.
3. Exchange Rigor solution for overnight rigor (in mM: Tris (50), KCl (2), NaCl (100), MgCl₂ (2), EGTA (1), protease inhibitor tablet (1), 50:50 glycerol, pH 7.0), and store at 4 °C for 12-18 h.
4. Exchange overnight rigor for 50:50 collection rigor:glycerol and stored at -20 °C for up to 3 months, or one year in a -80 °C freezer.
   NOTE: This process permeabilizes the fiber membrane which allows for manual addition of calcium into and out of the cell. This process takes time and could be different between different muscles and species.

Results

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 “pressure” sensation in the area around the biopsy needle, with occasional periods of moderate to in...

Discussion

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 biopsies^3,

Materials

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