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¹ Nuclear Magnetic Resonance Centre, Department of Medical Biochemistry and Genetics, The Panum Institute² Department of Thoracic Surgery³ Department of Anaesthesia, Rigshospitalet, University of Copenhagen, Denmark

	Abstract

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This study presents a perfused preparation for evaluation of metabolism in pig intercostal muscle in vitro. Preserved vessels and nerves to an intercostal segment including two adjacent ribs allowed for tissue perfusion and electrical stimulation with measurement of contraction force, oxygen consumption and ³¹P-magnetic resonance spectroscopy (³¹P-MRS). When perfused at rest with Krebs–Ringer buffer, the preparation maintained physiological levels of phosphocreatine (PCr), inorganic phosphate (P_i), ATP and pH at a stable oxygen consumption of 0.51 ± 0.01 µmol min^–1 g^–1 for more than 2 h. Tonic stimulation of the nerve caused anaerobic energy consumption as PCr and pH decreased, and both variables recovered after the contraction with half-time values of 7 min. Force increased to 0.040 N g^–1 (range, 0.031–0.103 N g^–1) and it gradually decreased by about 70% during the subsequent 5 min of stimulation. The calculated free ADP concentration increased from 7.4 ± 2.1 nmol g^–1 at rest to 28 ± 12 nmol g^–1 (mean ± S.D.) by the end of the stimulation. Thus anaerobic ATP turnover was zero at rest, 6.1 ± 2 µmol min^–1 g^–1 during the first minute of stimulation and 3.5 ± 0.5 µmol min^–1 g^–1 during the two last minutes, corresponding to the drop in force. When the preparation was left unperfused, anaerobic ATP turnover averaged 0.40 ± 0.15 µmol min^–1 g^–1 for the first 10 min. The preparation can also be applied to human intercostal muscles, as demonstrated in one preliminary experiment. The results demonstrate a stable and functional in vitro preparation of intact perfused intercostal muscles in the pig.

(Received 20 January 2006; accepted after revision 27 April 2006; first published online 28 April 2006)
Corresponding author B. L. Pedersen: NMR Centre, The Panum Institute, Blegdamsvej 3, 2200 Copenhagen N, Denmark. Email: brlp{at}imbg.ku.dk

	Introduction

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Intercostal muscle was introduced as an experimental model by Dillon et al. (1955) for evaluation of the effect of neuromuscular blocking agents on the motor endplate. The muscle samples were obtained from patients undergoing thoracotomy. Subsequently, a similar preparation was used for electrophysiological studies (Creese et al. 1957; Elmqvist et al. 1960), including studies of myasthenia gravis (Elmqvist et al. 1964; Elmqvist & Quastel, 1965; Dahlbäck et al. 1961). In addition, the fibre type composition of the external and internal intercostal muscles from different parts of the thorax, as well as their glycogen and lactate content, has been determined (Mizuno & Secher, 1998). These studies all used relatively small biopsies (15–30 mg) and the experiments were conducted without attempts to perfuse the preparation, induce muscle contraction or otherwise establish conditions similar to the in vivo situation. The intercostal muscles are, however, quite well suited for such studies, since the muscle length is defined by two adjacent ribs and a preparation can be obtained that includes the nerve and the vessels serving the entire section. However, no one has reported attempts to perfuse isolated intercostal muscle by analogy with perfused limb skeletal muscles (Ruderman et al. 1971; Nielsen, 1997) and other organs, such as liver (Bücher et al. 1968; Quistorff, 1985) and kidney (Nishiitsutsuji-Uwo, 1967; Grosse-Siestrup et al. 2002). Since the intercostal muscle is naturally attached to two adjacent ribs, an optimal preparation should include the ribs in order to allow physiological contractions. While this preparation is feasible to acquire from an experimental animal, a similar preparation from humans would need to be obtained by dissection of the muscle and periostium, and subsequent attachment of the latter to some system simulating the ribs. The preparation of the perfused intercostal muscle would be particularly suitable for the study of fibre type-specific metabolism, because of the different fibre type composition of the external and internal intercostal muscles. In humans, the internal intercostal muscles have almost no fast-twitch type-b (FTb) muscle fibres (Mizuno & Secher, 1991), which makes it possible to identify specifically the metabolism of slow-twitch (ST) and fast-twitch type-a (FTa) fibre types and to apply drugs which preferentially block these fibres. Thus depolarizing muscular blocking agents (decamethonium and suxamethonium) inhibit the function of the FT fibres, while non-depolarizing agents inhibit the ST fibres (Mizuno et al. 1994; Mizuno & Secher, 1998). The external intercostal muscles are active predominately during inspiration and the internal predominately during expiration (De Troyer, 2002). The perfused intercostal muscles may be particularly useful for exploring the unique fatigability characteristics of these muscles.

Here we report the development of a preparation of the perfused intercostal muscle from pigs that allows for monitoring of energy turnover by ³¹P-magnetic resonance spectroscopy (³¹P-MRS) and measurement of oxygen consumption at rest and during electrically stimulated contractions. The ³¹P-MRS data indicate that the preparation is stable for several hours when perfused with an oxygenated saline medium and that the energy recovery pattern following electrically induced contractions is similar to what is known for skeletal muscle in vivo.

	Methods

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An intercostal muscle preparation was obtained from nine pigs (Danish Landrace, 70 kg) kept in the laboratory animal facilities within the Panum Institute. The housing conditions complied with FELASA (Federation of European laboratory animal science associations) guidelines. The pigs were anaesthetized with either a single dose of chloralose (1 g (15 kg)^–1) and ventilated with N₂O and isoflorane (30 and 3%, respectively) in oxygen or by I.V. propofol (10 mg ml^–1 kg^–1 h^–1) and fentanyl (250 µg (30 kg)^–1 h^–1). Experiments were performed in accordance with national and local ethical guidelines. Blood coagulation was avoided by the administration of 1 ml heparin (5000 IU/ml, LEO Pharma Nordic, inc, Malmö, Sweden) I.V. prior to surgery. The biopsy (approximately 6 cm x 8 cm) was obtained with the two adjacent ribs from the rear part of the thorax, where the intercostal artery has its largest diameter (Porto da Rocha et al. 2002). The weight of the muscle tissue was 12.9 ± 3.8 g (mean ± S.D.). Surgery lasted 5–10 min, and the biopsy was placed into a plastic bag, submerged in ice-water and transported to the nuclear magnetic resonance (NMR) spectrometer. The biopsy was therefore subjected to 50–70 min of cold ischaemia before perfusion was established. After the biopsy was obtained, the pigs received a lethal dose of pentobarbitone (> 50 mg kg^–1).

The perfusion set-up

The perfusion apparatus was developed to fit a wide-bore, 30 cm diameter, 4.7 T NMR spectrometer (Otsuka Electronics, Magnex Scientific, Oxford, UK) for continuous assessment of muscle energy metabolism by ³¹P-MRS, using a modification of a liver perfusion system (Masson & Quistorff, 1992; Fig. 1). The perfusate was a Krebs–Ringer buffer (mM: 118.5 NaCl, 4.8 KCl, 2.54 CaCl₂.2H₂O, 1.16 KH₂PO₄, 1.18 MgSO₄.7H₂O and 24.9 NaHCO₃) equilibrated with 95% oxygen and 5% carbon dioxide by passing through 2 m of a thin-walled (0.2 mm) siliconized tubing in a 1 l bottle, through which the gas mixture was flushed (Hamilton et al. 1974). Oxygen electrodes, Clark type (probe 5331; Yellow Springs Instruments, OH, USA), were placed immediately before and after the preparation to allow for the calculation of the oxygen consumption, given that the flow is known (Quistorff, 1985). The remaining 3.5 m of the perfusion tubing was gas-proof material (C-FLEX6428; Cole Parmer, in., IL, USA) and was kept at 37°C by a water-jacket. The perfusion model was developed to allow for electrically induced contractions, and force was recorded during the ³¹P-MRS measurements. In order to ovoid interference between the magnetic field and the stimulation electrodes, the electrodes encompassed 1.2 m long agar salt-bridges (1 M NaCl). The force produced by the muscle contractions was measured by a calibrated strain gauge connected to the muscle preparation by a non-magnetic wire.

View larger version (30K): [in this window] [in a new window]   Figure 1.  Schematic view of the perfusion set-up

Perfusion protocol

The intercostal preparation was fastened by plastic screws through the ribs with the pleural side upwards, providing access to the nerve, and the artery and vein were cannulated (INSYTE-W, 0.7 x 19 mm). After administration of 5–10 µl heparin (5000 IU ml^–1) into the artery, a peristaltic pump provided perfusate at 15 ml min^–1, corresponding to 1–2 ml min^–1 g^–1, which was the highest rate attainable without inducing visible oedema and is similar to the rate used by others (Ruderman et al. 1971), i.e. providing about 1 µmol O₂ per gram of muscle per minute (Umbreit et al. 1949). To the Krebs–Ringer perfusate was added 0.5% bovine serum albumin (BSA, fraction V, Sigma no. A-33), 10 mM glucose as substrate, and 20 nM insulin (Actrapid^®, NovoNordic, Inc., Bagsvaerd, Denmark). This high insulin concentration was chosen to counteract the insulin adhesion to the walls of the perfusion line. For calculation of the oxygen consumption, an oxygen solubility of 0.025 ml ml^–1 (10⁵ pascals, 37°C) was assumed (Umbreit et al. 1949).

³¹P-MRS

An Otsuka Electronics Vivospec spectrometer and 4.7 T horizontal superconducting magnet with 31 cm clear bore (Magnex Scientific, Oxford, UK) was used. An oval-shaped surface coil (2.6 cm x 3.6 cm diameter) was fixed 0.5–1 mm under the plastic container holding the preparation. The size and shape of the coil were intended to match the muscles between the ribs. Heterogeneity of the magnetic field was minimized by preshimming on the proton signal followed by fine shimming on the phosphocreatine (PCr) signal. The ³¹P-spectra were obtained at 81.06 MHz by single pulse excitations (150 µs). Data were collected in 2048 data points over 256 ms at 4 s interpulse delay, and 15 or 30 free induction decays (FIDs) were summed in each ³¹P-spectrum, providing for a 60 or 120 s time resolution. Data analysis involved 10 Hz exponential line broadening followed by baseline correction using a semi-manual, cubic-spline procedure (Edwards et al. 1977). The areas of the inorganic phosphate (P_i), PCr and the three ATP peaks were estimated assuming Lorenzian line shape and applying a least-squares fitting routine. Intracellular pH was calculated from the difference in the chemical shift between P_i and PCr (Arnold et al. 1984), with the peak areas corrected for the appropriate saturation factors (Quistorff et al. 1993), and the concentrations of PCr and P_i were calculated in relation to ATP, assumed to be represented by the -ATP peak equal to 5.5 mmol ATP (kg wet wt)^–1 in resting muscle (Bangsbo et al. 1993). A typical resting spectrum is shown in Fig. 2.

View larger version (15K): [in this window] [in a new window]   Figure 2.  31P-MRS spectrum of perfused intercostal muscle from pig The spectrum consists of 60 free induction decays (FIDs) recorded with 5 s interpulse delay. The muscle was perfused in the resting state.

The agar electrodes were placed over the intercostal nerve with an interelectrode distance of 4 cm. In order to determine the metabolic response to contraction, the muscle was stimulated for 5 min at 1 Hz, with 5 ms pulses at a voltage that elicited 50% of the maximal isometric force.

In preliminary experiments, it was found that stimulation for up to 17 min allowed for full recovery.

Calculations

The NMR-measured metabolite variables were normalized to resting values (100%), using the mean of the first seven data points. The PCr degradation and recovery data were fitted to a monoexponential function:

(1)

(2)

allowing for an estimate of t_0.5 as ln(2)/k.

The free ADP concentration was calculated assuming equilibrium of the creatine kinase reaction (Harkema & Meyer, 1997). The maximal aerobic ATP turnover (V_max) was estimated from the time course of the PCr resynthesis (Johansen & Quistorff, 2003). Anaerobic ATP turnover was calculated from the ATP contributions of the creatine kinase and glycogenolysis:

(3)

(4)

as

(5)

where k = 1/(1 + 10^(pH–pKa)) is the stoechiometric coefficient for the H⁺ contribution from eqn (3), pK_a(P_i) = 6.75 and ß = [P_i] + 15 is the buffer capacity (Ratkevicius & Quistorff, 2002).

Statistical analysis

All values are expressed as means ± S.D. One-way ANOVA was used to compare the six intervals in Table 1. A Student's two-tailed t test was used to compare each value following the initial values of PCr, P_i and pH in the perfused and unperfused experiments. In the contraction experiments, the resting baseline was compared with the stimulation and recovery periods using two separate two-tailed t tests.

	Results

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View larger version (11K): [in this window] [in a new window]   Figure 3.  31P-MRS data from perfused intercostal muscle from pigs in the resting state Flow was 15 ml min–1 (approximately 1.5 ml (g muscle)–1). Time resolution was 2.5 min, but only one data point every 5 min is shown. A, PCr () and Pi (); B, pH. Values are means ± S.D., n = 4.

View larger version (15K): [in this window] [in a new window]   Figure 4.  Electrical stimulation of perfused intercostal muscle from a pig, typical experiment A, force measurements during 5 min at 1 Hz of stimulation. In the inset, each line exemplifies a contraction. B, PCr concentration as measured by 31P-MRS.

View larger version (11K): [in this window] [in a new window]   Figure 5.  31P-MRS measurements of unperfused, resting intercostal muscle from pigs Time resolution was 2.5 min, but only one data point every 5 min is shown. A, PCr () and Pi (); B, pH. Values are means ± S.D., n = 5.

	Discussion

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Under aerobic perfused conditions, a physiological metabolic steady state could be maintained for more than 2 h, which is long enough for acute experiments. In preliminary experiments, it was observed that after 2–3 h of perfusion there was a gradual decline in PCr and ATP. One likely explanation is that oedema gradually makes the perfusion less efficient. Another factor may be tissue and vascular injury by formation of reactive oxygen species (ROS) during postischeamic reperfusion (Alper et al. 2002).

Muscle contraction

The drop in muscle force (Fig. 4A) during the 5 min of electrical stimulation, and the change in PCr, P_i and pH, are comparable to data reported on electrically stimulated limb muscle in humans (Boska, 1994; Ratkevicius & Quistorff, 2002; Hartkopp et al. 2003).

The kinetics of the changes of PCr, P_i and H⁺ also resemble those observed in human subjects with a predominance of slow-twitch fibres, which show a relatively small initial decrease in PCr and pH, while subjects with many fast-twitch fibres display a larger anaerobic involvement by the initial contraction. Thus, after graded exercise of the human wrist flexor muscles for 8 min with increasing intensity, Mizuno et al. (1994) reported half-times for PCr and P_i recovery of 1.1 and 0.6 min, respectively, for subjects who had 65% ST fibres. The end-exercise pH was 6.78, and it recovered to the resting level in about 4 min. The half-time for PCr recovery in the present study was about seven times longer than that reported by Mizuno et al. (1994) and Walter et al. (1997); the most likely explanation is oxygen limitation resulting from the limited oxygen supply by the saline perfusion medium (see below). It is not clear why the half-time for P_i recovery in the intercostal muscle was relatively faster than the PCr recovery, resembling the values found for wrist flexor ST fibres (Mizuno et al. 1994). The anaerobic ATP turnover was decreased proportional to force, as found by Boska (1994).

The anaerobic ATP turnover during ischaemia was six- to sevenfold lower compared to values measured by Ratkevicius et al. (1998) in the human gastrocnemius muscle, probably reflecting the fact that the intercostal tissue had been unperfused for 50–70 min prior to the NMR recording and was therefore running out of substrate (PCr and glycogen) for anaerobic ATP production.

The oxygen consumption did not change during stimulation, probably owing to a partial block of perfusion by the high intramuscular pressure of contraction (Berne & Levy, 1998). Thus, flow effectively stops and the oxygen electrodes show unchanged readings. This notion is supported by the ³¹P-MRS data demonstrating anaerobic metabolism with decreasing PCr and pH. The subsequent increase in oxygen consumption during the recovery period is consistent with increased oxygen demand for recovery from the relative anaerobiosis during contraction, and a steady-state resting rate was not reached over the 17 min of observation.

The maximal aerobic ATP turnover may be calculated from the initial rate of PCr resynthesis (Walter et al. 1997), assuming that the major part of the mitochondrial ATP synthesis is used to revert the creatine kinase reaction. The maximal aerobic ATP turnover was 3.63 µmol min^–1 g^–1, corresponding to an oxygen consumption of 0.725 µmol min^–1 g^–1 oxygen, which is about twofold higher than the resting oxygen consumption and 10- to 15-fold lower than the maximum oxygen consumption of the human quadriceps muscle (Rasmussen et al. 2001). The relatively low oxygen consumption is expected, however, since a saline medium, as used in these experiments, contains only some 10% of the oxygen available in blood and therefore the mitochondria will be limited by oxygen availability rather than by ADP, which is normally the major regulator of muscle respiration (Chance et al. 1986). Thus, when the preparation is used for experiments focusing on aerobic metabolism, it would be desirable to use an erythrocyte-containing medium.

Soust et al. (1989) determined blood flow in the intercostal muscle of the newborn lamb to be 0.37 ml min^–1 g^–1 for the external and 0.25 ml min^–1 g^–1 for the internal intercostal muscles. Robertson et al. (1977) reported values for dogs that were much smaller: 0.03 ml min^–1 g^–1 for the external and 0.04 ml min^–1 g^–1 for the internal intercostal muscles, increasing to a total of 0.24 ml min^–1 g^–1 when the respiration rate was increased from 18 to 34 breaths min^–1. Values for humans and pigs have not been reported.

The perfused intercostal muscles may be useful for exploring the unique fatigability characteristics of the intercostal muscles. The ³¹P-MRS recovery kinetics of the intercostals during contraction indicates a slower anaerobic ATP turnover, which might protect against accumulation of P_i and decreasing pH and thereby fatigue or might be a result of a higher aerobic ATP synthesis. Similar conclusions were made by Gandevia et al. (1983). The results of such experiments are of relevance, for example, to reconstructive plastic surgery where an intercostal muscle island flap is used (Kerrigan & Daniel, 1979) and therefore the ischaemia time before reperfusing the muscle is critical for the outcome.

Human intercostal muscle biopsy

To evaluate whether it was possible also to perfuse a human intercostal muscle, one preliminary experiment was performed. A biopsy (2 cm x 3 cm, 5 g) was obtained from a patient undergoing thoracic surgery, with access to the chest cavity by a posterior lateral thoracotomy through the 5th intercostal space with diathermic cutting (Shields et al. 2000). Written informed consent was obtained and the experiment conformed with the Declaration of Helsinki. For the purpose of this study, the intercostal muscles were dissected carefully with a scalpel in order to preserve its nerve and vessels. The muscle was dissected free of the ribs so that the periostium was included. The experiment with the human intercostal muscle biopsy served to evaluate the feasibility of the preparation, and the establishment of perfusion convinced us that a human intercostal preparation in analogy with the pig model is feasible.

Conclusion

We have developed a method for perfusing pig intercostal muscle that allows electrically stimulated contraction and simultaneous ³¹P-MRS. The ³¹P-MRS data indicate that the energy metabolism of the perfused intercostal muscle preparation is stable for at least 2 h when perfused with a simple oxygenated saline medium and that it recovers from the anaerobic metabolic state provoked by electrically induced contractions. Finally, we have established that perfusion of human intercostal muscle is also feasible.

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	Acknowledgements

 
The study was supported by grants from Læge Sofus Carl Emil Friis og hustru Olga Doris Friis's legat and The Danish Research Agency, grant no. 22-03-0113. The authors thank Mr Ib Therkelsen and Mr Palle Koch for expert help with the NMR measurements.

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