Author for correspondence: Martin Anetseder, Department of Anesthesiology, University of Würzburg, Oberdürrbacher Straße 6, D-97080 Würzburg, Germany (fax +49 931 201 30009, e-mail Anetseder_M@klinik.uni-wuerzburg.de).
Abstract: Microdialysis is a valuable tool to measure tissue responses. We hypothesized that skeletal muscle metabolism can be modulated by microdialysis applied drugs which alter cytosolic calcium concentration. With approval of the local animal care committee, the hind limbs of sacrificed male Sprague Dawley rats were perfused either with Ringer's solution or with dantrolene 1 μM at 30 ml hr−1 and 21 °. Microdialysis probes in both hind limbs were perfused at 1 μl min−1 either with sorbitol 80 mM, calcium 20 mM, 40 mM, 80 mM, caffeine 40 mM, 80 mM, and halothane 10 vol% respectively, and at the contralateral adductor muscle with Ringer as control. Lactate was measured spectrophotometrically in the dialysate at 15 min. intervals. Lactate levels as measured by intramuscular microdialysis were not influenced by intramuscular application of sorbitol 80 mM compared to control measurements with Ringer's solution. Local application of calcium 20 mM, 40 mM, 80 mM, caffeine 40 mM, 80 mM, and halothane 10 vol% via microdialysis increased lactate concentrations, while organ perfusion by dantrolene 1 μM reduced the caffeine-induced lactate increase. Modulation of intramuscular lactate metabolism by exogenous compounds via microdialysis probes generates new insights in skeletal muscle metabolism.
For excitation-contraction coupling, vertebrate skeletal muscle has generated a unique control mechanism for intracellular calcium regulation (Berchtold et al. 2000; Stokes & Wagenknecht 2000). Several drugs are known to modify cytosolic calcium release and reuptake. While caffeine and halothane increase myoplasmic calcium via different mechanisms (Franzini-Armstrong & Protaso 1997), the hydantoine derivative dantrolene lowers cytosolic calcium by reducing calcium release from the sarcoplasmic reticulum (Zhao et al. 2001). Microdialysis is a suitable technique for measuring dialyzable interstitial compounds like lactate near physiologic conditions. Furthermore, based on diffusion kinetics, microdialysis allows delivery of exogenous drugs into the interstitial space modulating local tissue concentration of compounds like lactate. Clinical research focuses on extracellular fluid concentrations of endogenous agents while little is known about local metabolic reactions induced by drug application via microdialysis probes (Müller 2002).
As a step in the development of a metabolic test to diagnose susceptibility to malignant hyperthermia, we hypothesized that intramuscular lactate concentrations as an indirect parameter of skeletal muscle metabolism can be modulated by drugs which alter cytosolic calcium concentration and which are applied via microdialysis.
Materials and Methods
Animals and experimental protocol. With approval of the local animal care committee, healthy male Sprague Dawley rats weighing 250–300 mg were anaesthetized with isoflurane (Abbott, Germany) while breathing spontaneously. After laparotomy and intravenous injection of 500 IU of heparin (Roche, Germany), the animals were sacrificed. The abdominal aorta was ligated and cannulated anterogradely to allow isolated perfusion of the hind limbs by a roller pump (VP/1000 Infusomat; Fresenius, Germany) with carboxygenated (95% CO2/5% O2) perfusate containing either plain Ringer's solution or Ringer/dantrolene 1 μM (Procter & Gamble, Germany) at 30 ml hr−1 and 21 °.
Microdialysis probes were placed bilaterally in the medial region of the thigh, which consists of the gracilis, adductor longus, magnus and brevis muscle, and the pectineus muscle by help of an introducer cannula. The contralateral probe served as intraindividual control perfused solely by Ringer's solution. After measurement of baseline lactate levels, the unilateral microdialysis probe was perfused for 120 min. by a modified Ringer solution containing either sorbitol 80 mM (Sigma Chemicals, Germany), calcium 20 mM, calcium 40 mM, calcium 80 mM, caffeine 40 mM or caffeine 80 mM (all Merck, Germany), respectively. In a further series of experiments, lipophilic halothane (Eurim-Pharm, Germany) was dissolved in soy bean oil at 10 vol% and applied to the tissue via a second microdialysis probe with the membrane in parallel and next to a Ringer perfused microdialysis probe. At the end of each experiment, a single bolus of methylene blue (Neopharma, Germany) was injected through the aortic cannula to assure homogenous perfusion of the hind limbs. If reperfusion differed macroscopically the experiment was excluded.
Microdialysis set-up. Microdialysis probes with a polycarbonate membrane, a molecular cut-off at 20,000 Dalton, a membrane length of 10 mm and a diameter of 0.5 mm were used (CMA/20, CMA, Sweden). Prior to the first experiment, the probes were perfused with alcohol 70% followed by aqua destillata to eliminate glycerol from the membrane. Diffusion properties of the membrane were determined before and after every 5th experiment by measurement of the in vitro recovery for lactate at 1 μl min.−1 flow with Ringer's solution while mounted in Eppendorf tubes containing lactate 40 mM. The in vitro recovery was calculated by the ratio of recovered lactate in the dialysate and the lactate concentration outside the probe. To evaluate the recovery for caffeine respectively calcium the probes were placed in Ringer's solution and perfused with caffeine 80 mM respectively calcium 80 mM at 1 μl min.−1 perfusion flow. For this setting recovery was defined as the ratio of the drug concentrations in the solution surrounding the microdialysis probe and the perfusate. Probes with an in vitro recovery for lactate <70%, caffeine <65% and calcium <55% were discarded.
For microdialysis of lactate in the isolated perfused skeletal muscle preparation, the inlet tubings of the microdialysis probes were connected via a syringe selector (CMA/110, CMA, Sweden) to syringes in a micro pump (CMA/100, CMA, Sweden) and perfused with Ringer's solution or modified Ringer's solution at 1 μl min.−1. Once the probe was implanted into the tissue, unbound molecules <20 kD in the extracellular fluid were able to diffuse through the semipermeable membrane into the perfusate of the probe, while drugs in the perfusate were delivered into the interstitial fluid. The dialysate in the outlet tubing was collected and analyzed at 15 min intervals. Lactate was measured in the dialysate indirectly by a spectrophotometer. In detail, a test kit containing lactate oxidase 400 U l−1, peroxidase 2400 U l−1, and buffer at a pH of 7.2 (Sigma Chemicals, Germany) converts a chromogene dye in linear proportion to the concentration of lactate. The absorption of the dye is measured by a spectrophotometer at λ=540 nm (HP 8453-UV-Visible spectrophotometer; Hewlett Packard, Germany). Before sample analysis, a standard curve for lactate was established by using lactate 20 mM, 40 mM and 80 mM (Sigma Chemicals, Germany). Reproducibility was described with a coefficient of variation below 3%, correlation coefficient to the Sigma lactic dehydrogenase procedure was 0.999, and recovery was >97%.
Statistics. Data are shown as median and quartiles. In vitro results with isolated skeletal muscle perfusion were statistically evaluated for drug effects in comparison to the corresponding intraindividual control measurement in the contralateral limb using the Mann-Whitney-U-test, Bonferroni correction was used for multiple comparisons. To detect a dose-dependent effect after calcium and caffeine application, the relative increase in lactate, i.e. the difference between lactate increase and contralateral control measurement, at different concentrations of calcium and caffeine was statistically analysed by the Kruskal-Wallis- and Mann-Whitney-U-test. P<0.05 was considered significant.
The in vitro recovery of the microdialysis membranes at 1 μl min.−1 was 82 (74–84)% for lactate (n=12), 79 (69–83)% for caffeine (n=10) and 59 (58–61)% for calcium (n=4).
After equilibration of the microdialysis probes in the adductor muscles, plain Ringer perfusion increased local lactate concentrations in the isolated perfused hind limb of the rat similarly in the right and in the left limb (n=6 per group). Sorbitol 80 mM had no significant effect on local lactate concentrations compared to control measurements (n=6 per group).
Lactate concentrations were significantly increased after application of calcium 20 mM, 40 mM and 80 mM compared to the contralateral limb (n=6 per group). Similarly, caffeine 40 mM and 80 mM in the microdialysis perfusate raised initial lactate levels in the verum limb significantly higher than in the control limb (n=6 per group). Halothane 10 vol% dissolved in soy bean oil increased local lactate concentrations significantly different from the control measurements (n=6 per group). The lactate increase after caffeine 40 mM was reduced by isolated organ perfusion with dantrolene 1 μM compared to dantrolene-free Ringer's solution (n=6 per group) (table 1).
Table 1. Modulation of intramuscular lactate by application of drugs that increase or decrease cytosolic calcium concentrations in skeletal muscle. Baseline lactate levels [mM] and relative modulation of intramuscular lactate [%].
The group “Caffeine 40 mM (RL)” served as control group for comparison with caffeine 40 mM/dantrolene 1 μM; median and quartiles; n=6 per group;
*P<0.05 for right (RL) versus left hind limb (LL).
Compared to calcium 20 mM, application of calcium 40 mM and 80 mM increased local lactate concentrations dose-dependently, while at higher concentrations no dose-response effect was detectable (table 2). Furthermore, a dose-response relationship of the lactate increase was demonstrated after application of caffeine 40 mM and 80 mM (table 3).
Table 2. Percentage increase in lactate (lactate minus contralateral control in %) after calcium 20 mM; calcium 40 mM and calcium 80 mM.
Median and quartiles; n=6; *P<0.05 for Calcium 20 mM vs. Calcium 40 mM; +P<0.05 for Calcium 20 mM vs. Calcium 80 mM; #P<0.05 for Calcium 40 mM versus calcium 80 mM.
Histological evaluation of three muscle biopsies after isolated organ perfusion and caffeine application revealed minimal interstitial oedema, but no intramuscular necrosis (fig. 1).
The present study demonstrates that microdialysis is an excellent tool to modulate interstitial lactate levels by exogenous application of agents known to increase or decrease intracellular calcium concentration. For the development of a metabolic test to diagnose susceptibility to malignant hyperthermia, a hypermetabolic skeletal muscle syndrome, modulation of intramuscular metabolism is the underlying principle. The local application of a small dose of a calcium-releasing drug like caffeine or halothane was shown to increase local lactate concentrations dose-dependently and reliably.
The preserved integrity of the tissue allows analysis of dialyzable compounds from the interstitial fluid. Hereby, microdialysis is dependent on a perfusate similar to the extracellular fluid (Ungerstedt 1991). In addition, dialyzable exogenous compounds like caffeine may be applied to the tissue via the microdialysis membrane while its effects at the target site can be measured at the same time (Benveniste & Huttemeier 1990).
With regard to methodological aspects, the time-dependent increase of lactate in the present study is attributed to the artificial perfusion by Ringer's solution with a far lower oxygen transport capacity than blood. Therefore, an intraindividual control measurement in the contralateral limb served to differentiate an intrinsic increase of lactate from extrinsic drug effects. Osmotic effects of the microdialysis perfusate on lactate levels were excluded by measurement of similar lactate levels with and without sorbitol 80 mM in the perfusate. Furthermore, systemic aortic injection of methylene blue at the end of each experiment excluded lactate modulation by altered perfusion i.e. vascular occlusions caused by an embolus. Since the placement of a microdialysis probe itself may increase local lactate due to tissue injury, an equilibration time of at least 15 min. is necessary for stable baseline lactate levels in muscle tissue (Korth et al. 2000). Histological examination of three muscle biopsies after isolated organ perfusion and caffeine application did not reveal significant signs of cell oedema or necrosis.
For in vivo calibration of microdialysis several methods were established. The recovery of substances from the extracellular fluid depends on membrane length, perfusion flow, diffusion properties of the semipermeable microdialysis membrane and of the surrounding tissue. Due to different diffusion coefficients between water and muscle tissue, e.g. influenced by local blood flow, in vitro and in vivo recovery are not the same (De Lange et al. 1997). In vitro experiments demonstrated a huge variability of substance recovery, e.g. 19% for adenosine compared to 45% for pyruvate at equal conditions (Eliasson 1991). In the present study, microdialysis probes were calibrated exclusively in vitro. Initial lactate concentrations before drug application were similar between ipsilateral and contralateral limb. Only the relative change in lactate were of interest in our investigation.
In skeletal muscle metabolism, calcium represents a major intracellular second messenger that stimulates glycolysis, glyocogenolysis as well as mitochondrial energy turnover and muscle contraction. Consecutively, lactate increases with enhanced aerobic and anaerobic glycolysis (Stryer 1994). Calcium application via microdialysis increases intramuscular calcium concentration directly while application of caffeine increases cytosolic calcium levels by lowering the threshold of calcium release via the ryanodine receptor (Stokes & Wagenknecht 2000). The present study clearly demonstrates that calcium and caffeine applied via microdialysis increases local lactate concentration dose-dependently. The lactate plateau reached after perfusion with calcium 40 mM and 80 mM is most likely a result of maximal stimulation. In contrast, lactate levels were reduced by dantrolene. Therefore, lactate modulation in this study is mainly mediated via a pharmacological effect rather than by toxic effects to the tissue. This finding is supported by non-significant histologic alterations in selected muscle probes. It is of note that the high drug concentrations used, e.g. 80 mM of caffeine and 10 vol% halothane, are the concentrations in the perfusate while the tissue concentrations remain unknown. Reducing perfusion flow rate (0.1–0.5 μl min.−1) increases recovery nearly to 100% by enhanced diffusion via the semipermeable membrane from the perfusate into the tissue and from the interstitial fluid into the dialysate respectively. However, the small dialysate volume prolongs collection periods and rapid interstitial changes are masked (Ungerstedt 1991).
It is likely that the substances used influence local tissue perfusion. While halothane is a strong vasodilator, caffeine and calcium induce vasoconstriction (Boyle & Maher 1995). Depending on local flow rates, lactate levels are reported to be either increased or decreased. In our study, the dose-dependent modulation of lactate is clearly a drug effect of caffeine and calcium. Based on the experimental set-up, we assume that vasodilating and vasoconstricting actions are probably small.
The in vivo recovery for caffeine is estimated to be 20 to 40% and this points towards a much lower tissue concentration compared to the perfusate. In previous experimental models, a steep decline of drug concentrations around the semipermeable membrane of the microdialysis probe was described (Armberg & Lindefors 1989; Chen et al. 2002). We assume that due to a relatively low in vivo recovery and the dilution of the exogenous drug in the interstitial space, a toxic drug effect resulting in the measured lactate increase is unlikely.
Similarly to caffeine, the volatile anaesthetic, halothane enhances calcium release in skeletal muscle fibers (Endo 1983) and increases local lactate concentrations (Rosenberg et al. 1977). While plain halothane may act as a strong detergent and may induce severe cell damage after pure intravenous injection (Berman & Tattersall 1982), the same drug allows intravenous anaesthesia, when dissolved in a lipophilic carrier like soy bean oil, comparable to the inhalative route and without associated organ failure or cell damage (Musser et al. 1999). We perfused halothane dissolved in soy bean oil through an intramuscular microdialysis probe allowing only halothane to diffuse through the membrane. The increase of intramuscular lactate levels is likely triggered by halothane while soy bean oil itself did not increase lactate in pigs (Schuster et al. 2006).
The hydantoin derivate dantrolene reduces cytosolic calcium concentration by inhibition of sarcoplasmic calcium release without influencing calcium reuptake (Zhao et al. 2001). This is supported by our finding that caffeine-induced lactate increase was impaired by systemic perfusion of dantrolene confirming its antagonistic effect on sarcoplasmic calcium release.
The results of this study indicate, that (a) isolated organ perfusion is a suitable model for investigation of skeletal muscle metabolism by microdialysis, (b) lactate levels via microdialysis are not influenced by varying osmotic pressures, (c) calcium, caffeine and halothane applied via microdialysis increase local intramuscular lactate concentrations, and (d) systemic perfusion with dantrolene reduces caffeine-induced lactate increase. Microdialysis may be a valuable tool to study intramuscular lactate metabolism and to enable extrinsic modulation of local skeletal metabolism through different drugs. This minimally invasive technique may generate new insights into neuromuscular diseases like glycogenosis, mitochondrial myopathies or malignant hyperthermia.