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1Department of Physiology, University of Maryland School of Medicine, 2Department of Orthopaedics, University of Maryland School of Medicine, 3Department of Diagnostic Radiology, University of Maryland School of Medicine
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An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.
Lovering, R. M., Roche, J. A., Goodall, M. H., Clark, B. B., McMillan, A. An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery. J. Vis. Exp. (51), e2782, doi:10.3791/2782 (2011).
Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.
1. in vivo injury Model and measurement of isometric torque.
2. In situ measurement of whole muscle tension.
3. in vivo MR imaging and/or spectroscopy of rodent skeletal muscles.
All MRI and MRS is performed on a Bruker Biospin (Billerica, MA) 7.0 Tesla MR system equipped with a 12 cm gradient insert (660 mT/m maximum gradient, 4570 T/m/s maximum slew rate) running Paravision 5.0 software.
4. Harvesting and storing muscles.
TAs are harvested after at the end of experiments, weighed, snap frozen in liquid nitrogen, and then stored at -80°C This can be performed at any point in time after the in vivo experiments. Muscles are harvested immediately after the in situ experiments, as this is a terminal procedure. For detailed morphological studies, the animal is fixed with 4% paraformaldehyde via perfusion through the left ventricle.
5. Representative results.
Figure 3 shows representative data from a rat in the in vivo apparatus. The in vivo apparatus is used to obtain maximal torque generated by the dorsiflexor muscles; it is also used to induce injury to these same muscles. Due to the length-tension relationship of muscle, maximal isometric torque typically occurs when the ankle joint is positioned at approximately 20° of plantarflexion (with the foot positioned orthogonal to the tibia considered 0°). After maximal isometric torque is obtained, the foot can then be placed into any position to begin the injury protocol. Figure 3 represents an injury protocol of 30 repetitions with an arc of motion from 0° - 70°. Note the steady decline in torque generated from the isometric phase (filled arrow) and lengthening phase (open arrow) during the contraction-induced injury protocol. Torque is recorded in units of Nmm, but the absolute value depends on the size of the animal and its condition (e.g., injured muscle, fatigued muscle, or muscle lacking a certain protein due to homologous recombination).
Figure 4 shows representative data from a rat in the in situ apparatus. Our in situ apparatus does not involve lengthening contractions; rather, it allows us to isolate, properly align, and measure maximal tension produced by an individual muscle at a known length. Figure 4 shows the gradual loss of force that occurs during a fatigue test in a tibialis anterior muscle of a rat. In this particular example, titanic contractions were performed once every second for 5 minutes. Tension (force) is typically recorded in Newtons (or grams), but like torque, the absolute value depends on the size and condition of the animal. Because muscle weight is obtained immediately after this procedure, the force can be normalized (called "specific force") to muscle cross sectional area.
Figure 5 shows representative data from in vivo imaging of a mouse, such as T1-weighted and T2 parametric mapping (A), 3D tractography from diffusion tensor imaging (B), 1H spectroscopy (C), and 31P spectroscopy. Details are provided in the figure legend.
Figure 1: in vivo apparatus.* To produce the injury, the tibia is stabilized and the foot attached to a motor-driven plate. The ankle dorsiflexors are stimulated via the fibular nerve while the footplate forces the foot into plantar flexion (dotted arrow).
* Lovering & De Deyne, J Biomechanics 2005, used with permission.
Figure 2: In situ apparatus The load cell is mounted to a micromanipulator so that the TA could be adjusted to resting length and aligned properly in the X, Y, and Z directions. The distal tendon of the TA is attached to the load cell and single twitches are induced at different muscle lengths in order to determine L0. A maximal tetanic contraction is obtained to determine maximal contractile activation (P0). Maximal tetanic tension can be performed repeatedly and expressed as percentage of P0, providing an index of fatigue at a desired point in time.
Figure 3: Torque data from in vivo apparatus Representative trace recordings of torque from lengthening contractions in the rat. In this particular example, muscles were stimulated for 200 milliseconds to induce a peak isometric contraction (filled arrow) before lengthening (open arrow) by the footplate through a 70° arc of motion at an angular velocity of 900°/s.
Figure 4: Tension data from in situ apparatus Representative data showing the decline in maximal isometric tetanic tension during repeated stimulation of the tibialis anterior muscle (TA) in a rat. In this example, the TA was isolated, adjusted to optimal length (L0), and then stimulated with a 200 ms tetanic contraction once every second for 5 minutes.
Figure 5: in vivo imaging A: The images show transverse (axial) sections of T1-weighted and T2 parametric mapping from the tibialis anterior muscle (TA). The dotted red box surrounds the TA to show increased increased T2 in the injured (left side) versus uninjured (right side). B: Representative 3D tractography from diffusion tensor imaging (DTI). C: The 1H spectrum of a mouse TA shows several detectable lipid resonances; differentiation between intramyocellular (IMCL) and extramyocellular lipid (EMCL) peaks is obtained using this method. D: The 31P MR spectrum of the rat TA shows phosphocreatine (PCr), inorganic phosphate (Pi), and the three resonances (α, β, γ) of adenosine 5'-triphosphate (ATP).
"Muscle damage" has been defined and measured in many ways. Structural damage is evident in histological findings6,9, but one problem with many of the biological markers used to assess muscle injury, including those used in animal studies, is that they usually do not correlate with the loss of force. Muscle damage is often defined within the context of the assay used to examine it and no one finding can account for the changes in contractility after injury. Since full contractile function can persist despite the presence of injury markers, loss of force may be the most valid measure of injury3, and probably the most relevant.
It is difficult to study muscle injuries in humans, as the incidence is a random event that is difficult to predict and the clinical presentation varies greatly. Therefore much of the data regarding muscle injuries have been ascertained from studies on animals, which provides control over many variables and the ability to study mechanisms of injury and recovery. The in vivo injury apparatus we have described provides a method for assessing contractile function without dissecting the muscle, and thus without the need to euthanize the animal under study. Our custom-designed injury model (patent pending) is based on the same principles used by other to establish contraction-induced injury in animals5,12,15,24. Despite the availability of models in the marketplace, there is little instruction beyond use of the hardware. Our model has specifications in terms of available range of motion and angular velocity that are advantageous17, but our main goal is to share the methods; we have tried to describe procedures from start to finish for producing an injury. Benefits of the in vivo model are that the muscle, anatomy and biomechanics are not altered and that the procedure is not terminal. We use the same location in the tibia for all torque measurements, following sanitary procedures and using a sterile needle for each measurement. The leg can be stabilized without the use of a transosseus pin, but we have found the pin to be superior in terms of reliability and eliminating extraneous movement during the lengthening contractions.
The apparatus used for in vivo torque measurements has several additional advantages. It does not involve any dissection, so there is no need to euthanize the animal under study. The result is that one can measure contractility in the same animal over time, and/or with in vivo imaging such as MRI. Other advantages are that normal anatomy is not altered, the nerve is not bypassed for stimulation (such as for in vitro preparations), and the muscle remains in its normal environment, so the effects of inflammation, hormones, or other factors can be studied. Because it requires the use of fewer animals, whose muscles are subjected to fewer manipulations (e.g., dissection prior to assay of function), we prefer to use the torque measurements whenever possible. The moment arm of the mouse TA is known4 and the muscle can be weighed when the animal is sacrificed. There are some limitations however, compared to isolating the muscle. For example, it is difficult to know the exact length changes that occur during lengthening contractions, and the muscle mass cannot be measured until it is harvested (although it can be estimated based on volume measured via MRI)8.
To determine the "specific force" (force per unit of cross sectional area) of an individual muscle, that muscle needs to be isolated and positioned properly; this also avoids force transmission from nearby muscles10. The in situ apparatus was designed for this purpose. It provides an alternative for measuring contractility of only one muscle with a known length and mass. However this method too has limitations. Although the in situ apparatus provides more experimental control when measuring the force of an individual muscle, the trade-off is that the experiment becomes less physiological. In situ force measurements require a surgical release of the TA muscle, which can alter the anatomy and affect force transmission. The experiment is also terminal, so the muscle cannot be monitored over time.
Diffusion tensor imaging (DTI) is potentially an even more sensitive and earlier marker for muscle damage than standard T2-weighted MRI. The variables obtained with DTI, at least in other tissues such as the brain (1), show a strong and rapid response to damage, whereas the T2 signal can take a prolonged period to change. DTI is based on measurement of the apparent diffusion of water in tissues. The DTI technique has been compared to actual longitudinal sections of the rat TA and it has been shown that DTI directions actually represent local muscle fiber directions in the rat TA muscle19.
MRS provides information on the chemical composition of muscle non-invasively12. Depending on the observed nucleus, MRS allows observation of high-energy phosphates (31P MRS) or lipids (1H MRS). 31P MRS is an ideal tool for the investigation of muscle metabolism because it is non-invasive and can be easily applied to in vivo studies of skeletal muscle. Alternative approaches to the biochemical assay of in situ muscle metabolites, such as needle biopsy, can give significant overestimates of Pi and apparent reductions of PCr1. An animal model provides the obvious benefit of using a controlled injury and comparing in vivo MRS changes to findings in the biochemistry, morphology, and function of the tissue. Changes in high-energy phosphate metabolism are encountered in diseases leading to muscular degeneration2,20. Intracellular pH, as well as the MR signal intensity ratios Pi/PCr (inorganic phosphate [Pi] to phosphocreatine [PCr]), and PDE/PCr (phosphodiester [PDE] to PCr), may provide valuable information regarding the stage and severity of muscle degeneration.
No conflicts of interest declared.
The authors would like to thank Dr., Robert Bloch for his generous donation of laboratory space and facilities and Dr. Rao Gullapalli and Da Shi in the Core for Translational Imaging at Maryland (C-TRIM) and the Magnetic Resonance Research Center (MRRC) for technical support. This work was supported by grants to RML from the National Institutes of Health (K01AR053235 and 1R01AR059179) and from the Muscular Dystrophy Association (#4278), and by a grant to JAR from the Jain Foundation.
|All equipment is the same for mice and rats except for the footplate|
|BUD Value Line Cabinet||Newark Inc||06M4718|
|Multifunction l/O USB-6221M||National Instruments||779808-01|
|Stepper motor controller||Newark Inc||16M4189|
|Stepper Motor||Newark Inc||16M4198|
|Strain Gauge Amplifier||Honeywell||DV-05|
|Foot plate and stabilization device (custom made, patent pending)|
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