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Magnetic Resonance in Medicine 64:262–270 (2010)

A Strictly Noninvasive MR Setup Dedicated to Longitudinal Studies of Mechanical Performance, Bioenergetics, Anatomy, and Muscle Recruitment in Contracting Mouse Skeletal Muscle Benoıˆt Giannesini,* Christophe Vilmen, Yann Le Fur, Christiane Dalmasso, Patrick J. Cozzone, and David Bendahan MR techniques have proven their ability to investigate skeletal muscle function in situ. Their benefit in terms of noninvasiveness is, however, lost in animal research, given that muscle stimulation and force output measurements are usually achieved using invasive surgical procedures, thereby excluding repeated investigations in the same animal. This study describes a new setup allowing strictly noninvasive investigations of mouse gastrocnemius muscle function using 1H-MRI and 31P-MR spectroscopy. Its originality is to integrate noninvasive systems for inducing muscle contraction through transcutaneous stimulation and for measuring mechanical performance with a dedicated ergometer. In order to test the setup, muscle function was investigated using a fatiguing stimulation protocol (6 min of repeated isometric contractions at 1.7 Hz). T2-weighted imaging demonstrated that transcutaneous stimulation mainly activated the gastrocnemius. Moreover, investigations repeated twice with a 7-day interval between bouts did show a high reproducibility in measurements with regard to changes in isometric force and energy metabolism. In conclusion, this setup enables us for the first time to access mechanical performance, energy metabolism, anatomy, and physiology strictly noninvasively in contracting mouse skeletal muscle. The possibility for implementing longitudinal studies opens up new perspectives in many research areas, including ageing, pharmaceutical research, and C gene and cell therapy. Magn Reson Med 64:262–270, 2010. V 2010 Wiley-Liss, Inc. Key words: phosphorus MR spectroscopy; functional magnetic resonance imaging; skeletal muscle fatigue; ergometer; in vivo muscle stimulation; muscle contraction

During the past two decades, transgenic mouse models have been used in order to identify the roles of genes in skeletal muscle development, physiology, and disease, thereby improving our knowledge in biology and medicine. The potential of these experimental models can be enhanced by the utilization of noninvasive techniques

Centre de Re´sonance Magne´tique Biologique et Me´dicale (CRMBM) UMR 6612 CNRS - Universite´ de la Me´diterrane´e, Faculte´ de Me´decine de Marseille, 27, bd Jean Moulin, 13385 Marseille Cedex 05 - France. Grant sponsor: French National Research Agency; Grant number: ANR-07BLAN-0354; Grant sponsors: French Interdisciplinary Small Animal Imaging Program (CEA-CNRS); IBISA Program 2008. *Correspondence to: Benoıˆt Giannesini, PhD, Centre de Re´sonance Magne´tique Biologique et Me´dicale (CRMBM), UMR 6612 CNRS, Universite´ de la Me´diterrane´e, Faculte´ de Me´decine de Marseille, 27, bd Jean Moulin, 13385 Marseille Cedex 05, France. E-mail: benoit.giannesini@ univmed.fr Received 10 February 2009; revised 4 January 2010; accepted 12 January 2010. DOI 10.1002/mrm.22386 Published online in Wiley InterScience (www.interscience.wiley.com). C 2010 Wiley-Liss, Inc. V

such as MR spectroscopy (MRS) and MRI in order to assess muscle function and metabolism in vivo (1–3). These techniques have indeed made possible the transition from in vitro biology to the integrative investigation of skeletal muscle function (4). In that respect, 31P-MRS allows characterization of muscular energy metabolism through the measurement of intracellular pH and the concentration of the major phosphorylated compounds in contracting muscle. These measurements can also be used in order to quantify the relative aerobic and anaerobic contributions to energy production (5,6), as long as force output is recorded simultaneously. Also, 1H-MRI, which distinguishes muscle tissue from fat and bone, provides anatomic information such as muscle volume or cell damage, together with functional information related to muscle activation through the analysis of muscle T2 changes (7,8). However, for metabolic experiments conducted in animal models, muscle has to be stimulated and the corresponding force production must be measured. This is usually achieved through an invasive procedure so that MR techniques no longer have the advantage of being noninvasive. Specifically, muscle contractions are commonly induced via indirect stimulation using an electrode placed on the motor nerve and force output is measured with a transducer attached to the muscle tendon (1,2). These experimental procedures might of course disturb muscle physiology but above all necessitate the sacrifice of the animals at the end of experiments so that repeated investigations in the same animal are prohibited. In that context, the possibility of avoiding any surgical preparation would represent a real advance for the MR investigation of skeletal muscle function in mice. Longitudinal follow-up studies of transgenic mouse models would become feasible, thus opening new perspectives in various research areas, including pharmaceutical research and gene and cell therapies. Although we initially developed a system for rat skeletal muscle (9), the design of a corresponding setup for mice was a real technological challenge, given the order of magnitude difference between rat (300 to 400 g) and mouse weight (25 to 35 g). In the present study, we have designed and built an experimental setup allowing, in the same way as what we initially did for rats, strictly noninvasive MR investigations of mouse gastrocnemius muscle. The corresponding work was devoted to the construction of more sensitive devices for force and MRS-

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A Setup for MR Study of Mouse Muscle Function

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Force Output Measurement

FIG. 1. Schematic representation of the experimental setup. Muscular contractions are induced directly with transcutaneous surface electrodes located at the knee and heel levels. 31P-MR spectra are acquired with an elliptic (8  12mm) surface coil geometrically decoupled within a 20mm-diameter 1H Helmholtz imaging coil. Muscle performance is measured with an ergometer consisting of a foot pedal coupled to a home-built force transducer.

MRI measurements in order to obtain information about mechanical performance, energy metabolism, anatomy, and muscle recruitment in electrically induced muscle contraction. In the second part of the study, we investigated the reproducibility of the corresponding measurements.

Isometric force is measured with a home-built MR-compatible ergometer consisting of a 9  24mm foot pedal coupled to a force transducer. The transducer was constructed by sticking a strain gauge (ref. 1-LY11-6/120A; HBM GmbH, Darmstadt, Germany; 120-ohm internal resistance) according to the manufacturer specifications on a 9  32mm Bakelite slat (0.4mm thickness) in a Wheatstone bridge design (3  120 ohm). The foot pedal rotates freely on an axis that is located at the ankle joint level. The position of the ergometer can be adjusted to modify the angle between foot and lower hind limb. This adjustment enables the gastrocnemius to be stretched passively in order to obtain maximum force production in response to electrical stimulation. Analog electrical signal coming out from the force transducer is amplified with a home-built amplifier (Operational amplifier AD620; Analog Devices, Norwood, MA; gain ¼ 70 dB; bandwidth 0-5 kHz) and converted to a digital signal (PCI-6220; National Instruments, Austin, TX) monitored and recorded on a personal computer using the WinATS software, version 6.5 (Sysma, Aix-en-Provence, France).

Multimodal MR Acquisition MATERIALS AND METHODS

GENERAL OVERVIEW OF THE SETUP The setup is composed of a home-built cylindrical Perspex cradle (100mm diameter, 1450mm length) constructed to be operational in the 4.7-T horizontal magnet of a 47/30 Biospec Avance MR system (Bruker, Karlsruhe, Germany) equipped with a Bruker 120-mm BGA12SL (200 mT/m) gradient insert. The cradle was designed to investigate alternatively the left and the right gastrocnemius muscles of mice weighing 20 to 50 g. It integrates four distinct components allowing muscle stimulation, force output measurement, multimodal MR acquisition, and prolonged anesthesia, with regulation of the animal’s body temperature and breath rate monitoring (Fig. 1). Muscle Contraction Muscle contractions are achieved by transcutaneous electrical stimulation using two rod-shaped 1.5mm-diameter surface electrodes integrated in the cradle and connected to an electrical stimulator (type 215/T; Hugo Sachs Elektronik-Harvard Apparatus GmbH, March-Hugstetten, Germany). One electrode is localized at the foot pedal level (Fig. 1), so that when a mouse is put in the cradle, the electrode is in contact with its heel. The second electrode is inserted in a designed piecework, which is used to immobilize the leg. Thus, when the piecework is placed in the cradle, the integrated-electrode comes up against the animal skin at the knee level. The position of this piece can be accurately adjusted to fit the animal morphology.

MR data are acquired with a home-built spectroscopy/ imaging probe consisting of an elliptic 31P-MRS surface coil (8  12mm) geometrically decoupled inside a 20mm-diameter 1H Helmholtz imaging coil.

Prolonged Anesthesia Anesthesia is maintained by isoflurane (Forene; Abbot France, Rungis, France) inhalation via a home-built facemask connected to an open-circuit gas anesthesia machine (Isotec 3; Ohmeda Medical, Herts, UK). Exhaled and excess gases are removed through a canister filled with activated charcoal (Smiths Industries Medical System, Sheffield, UK) mounted on an electrical pump extractor (Equipement Ve´te´rinaire Minerve, Esternay, France). The animal’s breathing rate is monitored with a small balloon positioned on the abdomen and interfaced to a pressure transducer monitoring kit (ref MX960SCF; Medex Inc., Carlsbad, CA) located outside the magnet. The electrical signal coming from the transducer is amplified (Operational amplifier AD620; Analog Devices; gain ¼ 70 dB; bandwidth 0-5 kHz), analog-to-digital converted and monitored on a personal computer using the ATS software (Sysma). In order to maintain the animal at physiologic temperature during anesthesia, the cradle integrates an electrical heating blanket (PrangþPartner AG, Pfungen, Switzerland) in a feedback loop with a temperature control unit (ref. 507137; Harvard Apparatus) connected to a homebuilt rectal thermometer constructed with a NTC thermistor SMC series (ref: NCP18XW220J03RB-2.2K; Murata, Kyoto, Japan).

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SETUP TESTING Animal Care and Feeding Sixteen 9-week-old C57BL/6 mice (Charles River Laboratory, L’Arbresle, France) weighing 23-28 g were used for these experiments, following the guidelines of the National Research Council Guide for the care and use of laboratory animals, and the French Law on the Protection of Animals. Mice were housed in an environmentally controlled facility (12-12 h light-dark cycle, 22 C) and received water and standard food ad libitum until the time of the experiment.

Giannesini et al.

limb. Multiecho T2-weighted images of these slices (16 echo times equally spaced from 11.79 ms to 188.67 ms; 1010-ms repetition time; two accumulations; 30  30mm field of view; 256  128 matrix size; total acquisition time of 4.3 min) were recorded at rest and immediately after the stimulation protocol. Experiment 2: Mechanical, Anatomic and Metabolic Data Acquisition and Reproducibility Animal preparation, stimulation protocol, and force measurements were done as for experiment 1.

Experimental Protocol

Anatomic Imaging

Experiment 1 (n ¼ 8) was designed to evaluate the pattern of lower hind-limb muscle activation in response to transcutaneous stimulation, using T2 imaging. Experiment 2 (n ¼ 8) was designed to test the ability of the setup to obtain reliable information regarding mechanical performance, anatomy, and energy metabolism and to investigate the reproducibility of the corresponding measurements repeated 1 week later.

Ten consecutive noncontiguous axial scout slices (1mm thickness, spaced 0.5mm), covering the region from the knee to the ankle, were selected across the lower hind limb. RARE images of these slices (eight echoes; effective echo time ¼ 67.9 ms; actual echo time ¼ 16.7 ms; repetition time ¼ 2000 ms; one accumulation; 20  15mm field of view; 256  256 matrix size) were recorded at rest. 31

Experiment 1: Determination of the Pattern of Muscle Activation Animal Preparation Mice were initially anesthetized in an induction chamber (Equipement Ve´te´rinaire Minerve) using 4% isoflurane in 33% O2 and 66% N2O. After shaving the whole left lower hind limb, the anesthetized animal was placed in supine position into the cradle. Electrode cream for electromyography was applied at the knee and heel regions. The head of the mouse was placed in the facemask, which was continuously supplied with 1.75% isoflurane in 33% O2 and 66% N2O throughout the experiment. Corneas were protected from drying by applying ophthalmic cream. The lower hind limb was firmly immobilized in the cradle, and the foot was positioned on the pedal of the ergometer. In this position, the hind limb was centered inside the Helmholtz imaging coil and the belly of the gastrocnemius muscle was located above the 31P-MRS surface coil. The gastrocnemius was passively stretched at rest to give maximum isometric twitch tension in response to submaximal square wave pulses (1-2 mA; 1.5-ms duration). Supramaximal current (2-4 mA) was determined afterward by inducing contractions with increasing current. Stimulation Protocol and Force Measurements The transcutaneous stimulation protocol consisted of 6 min of repeated isometric contractions at a frequency of 1.7 Hz. Contractions were electrically induced with square-wave pulses (2-4 mA; 1.5-ms duration). Isometric force production per twitch (in newtons) was averaged every 30 sec. T2 Imaging Five noncontiguous axial scout slices (1mm thickness, spaced 0.5mm) were selected across the lower hind

P Spectroscopy

Spectra (8-kHz sweep width; 2048 data points) from the gastrocnemius region were continuously acquired throughout the standardized experimental protocol consisting of 6 min of rest, 6 min of stimulation, and 15 min of poststimulation recovery. MR data acquisition was gated to muscle stimulation in order to reduce potential motion artifacts due to contraction. A fully relaxed spectrum (12 scans, 20-sec repetition time) was acquired at rest, followed by fifteen 32-scan and four 64-scan saturated spectra (pulse repetition time ¼ 1.875 sec). The first two of the 32-scan saturated spectra were acquired at rest, the next six during the stimulation period, and the final seven 32-scan and four 64-scan spectra were obtained during the poststimulation recovery period. The increased temporal resolution during the latter stage of the recovery period was necessary in order to clearly assign the inorganic phosphate (Pi) signal. MR Data Processing MR data were processed using a custom-written image analysis program developed on the Interactive Data Language platform (Research Systems, Inc., Boulder, CO). RARE images were used for muscle localization (Fig. 2) and previous MRI studies were used as anatomic references (1,10). T2 Imaging For each slice, zero-filling to a 256  256 matrix and gaussian filtering were performed. T2-weighted images were processed in order to generate T2-maps on a pixel-by-pixel basis by fitting the 1H-MRI data with a single exponential function. Because of imperfect refocusing (hermite refocusing pulse), even echoes were excluded from the analysis. Mean T2 values for tibialis anterior, extensor digitorum longus, flexor, and gastrocnemius regions were measured on T2 maps and averaged for the two slices of largest section by manually outlining regions of interest.

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tive to PCr (13). Time points for the time course of pHi and phosphorylated metabolite concentrations were assigned to the midpoints of the acquisition intervals. The rate constant (krec) of PCr resynthesis during the poststimulation period was determined by fitting the PCr time-dependent changes during this period to a single exponential curve described by the equation: [PCr]t ¼ [PCr]rest  D[PCr] ekrec*t, where [PCr]rest and D[PCr] are, respectively, the concentration of PCr measured at rest and the difference between [PCr]rest and the PCr concentration measured at the end of the stimulation period. The initial rate of PCr resynthesis at the start of the poststimulation period (Virec) was the product of krec and [PCr]cons.

Statistics FIG. 2. Axial RARE image showing different muscle groups of the mouse lower hind limb. Abbreviations are EDL (extensor digitorum longus), F (flexor longus), G (gastrocnemius), and TA (tibialis anterior).

Anatomic Imaging Regions of interest were manually outlined so that the corresponding cross-sectional areas (in square millimeters) could be measured. The gastrocnemius muscle volume (millimeters cubed) was calculated as the sum of the four volumes included between the five consecutive slices. 31

P Spectroscopy

Relative concentrations of phosphocreatine (PCr), Pi, and b-ATP were obtained by a time-domain fitting routine using the AMARES-MRUI Fortran code (11) and appropriate prior knowledge of the ATP multiplets. Signal areas were corrected for magnetic saturation effects using fully relaxed spectra. Absolute concentrations of phosphorylated compounds were expressed relative to a resting b-ATP concentration of 5.43 mM, which was measured by high-performance liquid chromatography in extracts of freeze-clamped gastrocnemius muscle. The high-performance liquid chromatography protocol has been reported previously (12). Intracellular pH (pHi) was calculated from the chemical shift of the Pi signal rela-

FIG. 3. Axial T2-weighted image of left lower mouse hind limb at rest (a), and calculated T2 maps at rest (b) and immediately after 6 min of repeated isometric contractions induced electrically at 1.7 Hz (c).

The effect of bout repetition on variables evolving with respect to time during the rest-stimulation-recovery protocol (isometric force production, metabolite concentrations, and pHi) were tested with two-way repeated-measures analysis of variance (ANOVA) using JMP software (SAS Institute Inc., Cary, NC). Other variables were compared using two-tailed Student’s t test for paired observations or one-way ANOVA followed by Tukey’s test for multiple comparison. Values are means 6 standard error of the mean (SEM). A value of P < 0.05 was taken to indicate statistical significance.

RESULTS Muscle Recruitment T2 maps of the lower hind limb were calculated from multiecho T2-weighted images recorded at rest and immediately after 6 min of repeated isometric contractions (Fig. 3). At rest, T2 values ranged from 21.1 6 0.4 ms in tibialis anterior to 22.9 6 0.3 ms in gastrocnemius muscle (Table 1). As expected, muscle stimulation led to significant T2 increase within each muscle region (Table 1). The largest T2 change was measured in the gastrocnemius (þ29.3 6 1.9%) when compared to other muscles and did not differ among tibialis anterior (þ6.0 6 1.9%), extensor digitorum longus (þ8.0 6 1.4%), and flexor longus (þ10.1 6 1.5%) (Fig. 4).

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Table 1 Muscle T2 (in Milliseconds) Measured in Mouse Lower Hind Limb at Rest and Immediately After 6 Min of Repeated Isometric Contractions Induced at 1.7 Hzy Muscle Tibialis anterior Extensor digitorum longus Flexor longus Gastrocnemius

Rest 21.1 22.9 21.8 22.9

6 6 6 6

Poststimulation 0.4 0.4 0.3 0.3

22.4 24.7 24.0 29.6

6 6 6 6

0.4* 0.4* 0.5* 0.7*

y

Values are shown as means 6 SEM; n ¼ 8. *Significantly different from resting value (P < 0.05).

Gastrocnemius Volume and Mechanical Measurements Gastrocnemius volume, measured with anatomic imaging, averaged 101 6 2mm3 and 100 6 4mm3 at day 0 and day 7, respectively. Typical isometric force signal recorded during the first 10 sec of stimulation inside the MR magnet is shown in Fig. 5a. The changes in isometric force during the stimulation period did not differ significantly (P ¼ 0.76) between both stimulation explorations (Fig. 5b). At the start of the stimulation period, isometric force averaged 379 6 17 mN and 413 6 11 mN at day 0 and day 7, respectively. Force increased transiently in the early stage of the stimulation period to reach a maximal value. Afterward, it decreased progressively until the end of the stimulation period. The final contraction force at the end of the stimulation period did not differ between the two trials, averaging 68.6 6 3.4% and 69.3 6 3.0% of the initial value at day 0 and day 7, respectively.

ments, reaching 3.03 6 0.21 (Table 2), 16.3 6 1.2 mM (Fig. 7a), 1.6 6 0.2 mM (Fig. 7b), and 7.18 6 0.04 (Fig. 7c), respectively, at day 7. During the 6-min stimulation protocols, two-ways repeated-measures ANOVA did not detect any significant difference between time courses obtained at day 0 and day 7 for [PCr], [Pi], pHi, and b-[ATP]. PCr was rapidly consumed at the onset of the stimulation period and reached a steady state after 2 min of stimulation (Fig. 7a). This steady state was maintained until the end of the stimulation periods, when PCr levels were 34.9 6 3.2% and 32.7 6 3.3% of their basal values at day 0 and day 7, respectively. The time course of P exhibited an initial phase of rapid and massive accumulation, followed by a phase of a steady state (Fig. 7b). At the end of the stimulation periods, [Pi] amounted to 13.7 6 0.4 mM at day 0 and 13.3 6 1.3 mM at day 7. During both stimulation periods, pHi decreased progressively to reach 6.69 6 0.04 at day 0 and 6.72 6 0.02 at day 7 at the end of the stimulation period (Fig. 7c). [ATP] decreased slightly during both stimulation protocols (Fig. 7d). At the end of the stimulation periods, it reached 4.6 6 0.6 mM at day 0 and 4.8 6 0.6 mM at day 7.

Energy Metabolism Typical 31P-MRS spectra acquired from a single mouse gastrocnemius muscle are presented in Fig. 6. At day 0 at rest, [PCr]/[b-ATP] ratio was 3.01 6 0.18 (Table 2), [PCr] ¼ 16.4 6 1.0 mM (Fig. 7a), [Pi] ¼ 2.2 6 0.5 mM (Fig. 7b), and pHi ¼ 7.20 6 0.04 (Fig. 7c). These basal values did not change through the repeated measure-

FIG. 4. Changes in T2 of the lower hind-limb muscles after 6 min of repeated isometric contractions induced electrically at 1.7 Hz. Values are shown as means 6 SEM. Abbreviations are EDL (extensor digitorum longus), F (flexor longus), G (gastrocnemius), and TA (tibialis anterior). Statistical difference was tested with one-way ANOVA followed by Tukey’s test for multiple comparisons.

FIG. 5. Isometric force output after electrical stimulation at 1.7 Hz. a: Typical force signal (100 samples/sec) recorded during the first 10 sec of stimulation inside the MR magnet. b: Changes in isometric force averaged every 30 sec during the 6-min stimulation protocol; the P-value is the result of two-way repeated-measures ANOVA used to test the effect of bout repetition. Values are shown as means 6 SEM.

A Setup for MR Study of Mouse Muscle Function

FIG. 6. Typical 31P-MR spectra (32 scans, 1.875-sec repetition time) obtained from a single mouse gastrocnemius muscle at rest, at the end of the stimulation protocol (5-6 min), and in the early stage of the poststimulation recovery period (2-3 min). Abbreviations for signal assignment are Pi, PCr, and g-, a-, and b-phosphates of ATP.

During the poststimulation period, phosphorylated compound levels and pHi progressively reached their prior-stimulation basal value. Both the rate constant (krec) and the rate (Virec) of PCr resynthesis were similar at day 0 and day 7 (Table 2). DISCUSSION This study presents for the first time an experimental setup designed for strictly noninvasive and highly reproducible investigations of mouse gastrocnemius muscle function in vivo, using mechanical performance measurement, 31P-MRS and 1H-MRI. Specific Activation of Gastrocnemius Muscle Our first concern was to determine both location and shape of the transcutaneous electrodes in order to provide a specific activation of the gastrocnemius in response to electrical stimulation. In animal research, MR investigations of skeletal muscle function are commonly performed in this muscle (1,2), which is part of the calf of the lower hind limb and is clearly distinct from the other muscles of the leg. It is directly accessible for MR coils, and its size and thickness are compatible with MR data acquisition. In addition, its contractile activity can be easily induced by electrical stimulation. Sciatic nerve stimulation is widely used for this purpose,

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with a bipolar electrode placed on its gluteal course at the hip level (8,14). Explicit and full activation of the entire innervated muscle mass can be achieved using this particular configuration (8). However, in addition to the risk of disturbing muscle physiology, the surgery required to place the electrode necessitates the sacrifice of the animal after the experiment. Indeed, the nerve must be exposed and carefully cleared of connective tissue in order to prevent electrical resistance to stimulation pulses and must be cut proximally to the electrode attachment to avoid propagation of electrical pulses in the whole animal. Transcutaneous stimulation is an attractive alternative but has been scarcely used. Given that mammalian hind limb is composed of several muscles and that current field distribution between electrodes is difficult to control in living tissue, transcutaneous stimulation of the gastrocnemius might lead to partial activation of antagonist muscles such as the tibialis anterior, flexor longus, or extensor digitorum longus, thereby affecting the net force produced by the gastrocnemius. In that respect, a clear identification of the muscle recruitment pattern is of utmost importance. Accordingly, we used functional muscle T2 MRI in order to distinguish activated from inactivated muscles during exercise as previously described (4,8). This phenomenon, first described in 1965 (15), is actually different from the well-known blood oxygenation level dependent effect underlying brain functional imaging. It would be mainly caused by the osmotically driven fluid shift in response to the intramuscular accumulation of small metabolic osmolites such as Pi and lactic acid during exercise (8,16,17). Using multiecho MRI, we measured T2 in gastrocnemius and in various antagonist muscles, i.e., tibialis anterior, extensor digitorum longus, and flexor longus muscles. At rest, T2 values ranged from 21.1 6 0.4 ms (in tibialis anterior) to 22.9 6 0.3 ms (in gastrocnemius), consistent with data previously obtained in mouse muscle at 4.7 T (18,19). The stimulation protocol induced a significant T2 increase in each muscle, but the magnitude of this increase was the largest in the gastrocnemius, reaching 29.3 6 1.9%. By contrast, it was 6.0 6 1.9% in tibialis anterior, 8.0 6 1.4% in extensor digitorum longus, and 10.1 6 1.5% in flexor longus. It is noteworthy that the activation of gastrocnemius antagonist muscles is not specific to transcutaneous stimulation only. Indeed, this phenomenon occurs during nerve stimulation, possibly as the result of field stimulation of the cut nerve (8). For example in rat, a T2 increase has been previously reported in both Table 2 Energy Metabolism in Mouse Gastrocnemius Muscle*

Basal [PCr]/[b-ATP] Virec (mM min1) krec (min1)

Day 0

Day 7

3.01 6 0.18 4.4 6 0.5 0.47 6 0.06

3.03 6 0.21 4.6 6 0.4 0.50 6 0.04

*Values are shown as means 6 SEM; n ¼ 8. Virec and krec are, respectively, the rate and the rate constant of PCr resynthesis at the start of the poststimulation period. No significant differences were found between data from days 0 and 7.

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FIG. 7. Changes in gastrocnemius [PCr] (a), [Pi] (b), intracellular pH (c), and [bATP] (d) during stimulation (6 min of repeated isometric contractions induced electrically at 1.7 Hz) and poststimulation recovery periods. Time points were assigned to the midpoints of the acquisition intervals. The first time point (t ¼ 0) indicates the resting value. P value is the result of two-way repeated-measures ANOVA used to test the effect of bout repetition on the whole rest-stimulationrecovery protocol. Values are shown as means 6 SEM.

gastrocnemius (30%) and tibialis anterior (11%) in response to 6 min of repeated isometric contractions at 5 Hz induced by sciatic nerve stimulation, despite the fact that the sciatic nerve branch innervating the tibialis anterior was cut, thus theoretically excluding its activation (8). Interestingly, this stimulation protocol at 5 Hz also reduced gastrocnemius isometric twitch force to 56% of the initial value. In the present study, we found that the 6-min stimulation protocol reduced isometric twitch force to 68.6 6 3.4% of the initial value. Thus, for comparable stimulation protocols (duration and intensity),

the pattern of T2 changes in mouse lower hind limb was similar to that elicited via sciatic nerve stimulation in the rat study. These data demonstrate, as previously concluded in rat, a specific activation of the gastrocnemius muscle using our experimental device. Parenthetically, we were unable to perform comparative analyses between transcutaneous and sciatic nerve stimulations because of size limitations. Given the mouse anatomy and the necessary size of the MRI coil, the sciatic nerve electrode would have been positioned inside the MRI coil volume, thereby compromising the

A Setup for MR Study of Mouse Muscle Function

signal-to-noise ratio. In addition, fluid accumulation at this level due to surgery would have strongly altered the T2 measurements. Ergometer The setup of the present study includes an ergometer similar to what has been designed for human experiments (20,21). We actually replaced the direct force measurement at the tendon level, necessitating surgery by an indirect force measurement at the foot level. Similar ergometers suitable for small-animal MR experiments have been scarcely developed, likely because this solution is more technically demanding. As an illustration, an ergometer including an optical force transducer has been constructed to measure force production of rat gastrocnemius upon sciatic nerve stimulation in an MR spectrometer (22). More recently, we have developed an ergometer for the rat hind limb, consisting of a foot pedal mounted on a hydraulic piston connected to a pressure transducer in a hydraulic circuit filled with water (9). In the present setup, a novel ergometer was developed, consisting of a foot pedal connected to a home-built force transducer comprising a strain gauge attached to a Bakelite slat, for which length, width, and thickness were optimized for the range of force measurement, i.e., between 0 and 500 mN. We found that maximal isometric twitch force at the start of repeated contractions averaged 379 6 17 mN at day 0 for 9-week-old mice weighing around 26 g. These values are quantitatively similar to those reported in previous studies in which normal mouse gastrocnemius contractions were elicited by sciatic nerve stimulation and force production was measured by connecting the Achilles tendon to a force transducer with an inextensible thread (23,24). More particularly, maximal isometric twitch force was around 392 mN in mice weighing 24 g (24), and 470 mN in 60-day-old mice (23). Further, the time course of isometric force recorded during the 6-min stimulation period was similar to changes reported in previous studies, with a transient increase in the early stage of the stimulation period, followed by progressive decrease until the end of the stimulation period, illustrating muscle fatigue (8,9,14). Consequently, surgery can be avoided with the present setup for both sciatic nerve stimulation and force measurements, thanks to the combined utilization of transcutaneous stimulation and force measurements using a dedicated ergometer. Energy Metabolism Metabolic variables recorded at rest were strongly similar to those reported in other studies of wild-type mouse hind-limb muscles. At day 0, the PCr/ATP ratio averaged 3.01 6 0.18 and pHi was 7.20 6 0.04, which is consistent with previous MR studies reporting a ratio ranging from 2.7 6 0.1 to 3.2 6 0.1 and a pHi close to 7.18 (1,25–27). Using high-performance liquid chromatography, we found that resting ATP concentration was 5.43 mM. This is very similar to the concentration reported previously (i.e., 5.0 6 0.9 mM) using the same analytical technique (26). In addition, the time courses for PCr,

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ATP, and pHi variations during both stimulation and poststimulation recovery periods were in line with other studies (1,2). Reproducibility of Measurements Given that our device allows for the first time repeated measurements in the same animals, it was critical to investigate the reproducibility of measurements. The stimulation protocol was thus repeated twice, with a 7day interval between bouts. The main energy metabolism variables measured at rest, i.e., [PCr]/[b-ATP] ratio, [PCr], [b-ATP], and pHi, were strictly similar between both explorations. In the same way, stimulation-induced metabolic and force changes were highly reproducible between repeated measurements. As an illustration, both the rate constant (krec) and the rate (Virec) of PCr resynthesis were similar at day 0 and day 7. This high reproducibility clearly demonstrated that the present setup can routinely provide accurate measurements of timedependent changes in metabolite concentrations and contraction force during repeated measurements. Thus, longitudinal effects of therapeutic interventions can now be studied in the same animals. CONCLUSION This setup enables for the first time repeated MR investigations of mouse skeletal muscle function, allowing mechanical performance, energy metabolism, anatomy, and physiology to be assessed strictly noninvasively in contracting gastrocnemius muscle. Longitudinal studies are now possible, thereby opening up numerous perspectives in various research areas, including ageing, pharmaceutical research, and gene and cell therapy. ACKNOWLEDGMENTS We gratefully acknowledge Ferdinand Tagliarini for his expert technical assistance in constructing the force transducer. REFERENCES 1. Cole MA, Rafael JA, Taylor DJ, Lodi R, Davies KE, Styles P. A quantitative study of bioenergetics in skeletal muscle lacking utrophin and dystrophin. Neuromuscul Disord 2002;12:247–257. 2. Goudemant JF, Deconinck N, Tinsley JM, Demeure R, Robert A, Davies KE, Gillis JM. Expression of truncated utrophin improves pH recovery in exercising muscles of dystrophic mdx mice: a 31P NMR study. Neuromuscul Disord 1998;8:371–379. 3. Dunn JF, Frostick S, Brown G, Radda GK. Energy status of cells lacking dystrophin: an in vivo/in vitro study of mdx mouse skeletal muscle. Biochim Biophys Acta 1991;1096:115–120. 4. Giannesini B, Cozzone PJ, Bendahan D. In vivo MR investigation of skeletal muscle function in small animals. MAGMA 2004;17: 210–218. 5. Giannesini B, Izquierdo M, Le Fur Y, Cozzone PJ, Bendahan D. In vivo reduction in ATP cost of contraction is not related to fatigue level in stimulated rat gastrocnemius muscle. J Physiol 2001;536: 905–915. 6. Kemp GJ, Radda GK. Quantitative interpretation of bioenergetic data from 31P and 1H magnetic resonance spectroscopic studies of skeletal muscle: an analytical review. Magn Reson Q 1994;10:43–63. 7. Meyer RA, Prior BM, Siles RI, Wiseman RW. Contraction increases the T(2) of muscle in fresh water but not in marine invertebrates. NMR Biomed 2001;14:199–203.

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