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reperfusion injury to skeletal muscle
Free Radical Biology & Medicine, Vol. 30, No. 9, pp. 979 –985, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter
PII S0891-5849(01)00485-3
Original Contribution MEASUREMENT OF FREE RADICAL PRODUCTION BY IN VIVO MICRODIALYSIS DURING ISCHEMIA/REPERFUSION INJURY TO SKELETAL MUSCLE DAVID PATTWELL, ANNE MCARDLE, RICHARD D. GRIFFITHS,
and
MALCOLM J. JACKSON
Department of Medicine, University of Liverpool, Liverpool, UK (Received 25 October 2000; Revised 16 January 2001; Accepted 25 January 2000)
Abstract—Microdialysis techniques have been used to detect hydroxyl radical and superoxide release into the interstitial space of anaesthetized rat anterior tibialis muscles during a period of prolonged (4 h) limb ischemia and subsequent reperfusion. Data indicate that reperfusion of the ischemic skeletal muscle was associated with a large increase in hydroxyl radical activity in the interstitial space, which may contribute to the significant oxidation of muscle glutathione, protein thiols, and lipids also seen in this model. No evidence for release of superoxide into the interstitial space was found during reperfusion, although this was observed during electrically stimulated contractile activity of the rat limb muscle. These data imply that therapeutic approaches aimed at reduction of hydroxyl radical generation in the interstitial fluid are more likely to be beneficial in reduction of skeletal muscle reperfusion injury than approaches designed to scavenge superoxide radicals. © 2001 Elsevier Science Inc. Keywords—Hydroxyl radical, Superoxide, Salicylate, Cytochrome c, Anterior tibialis muscle, Free radicals
INTRODUCTION
skeletal muscle, and we have confirmed that this protection is apparent with ascorbate, but not some other potential scavengers [6]. Work with a variety of other tissues indicates that oxygen radicals play a key role in complex mechanisms of damage following reperfusion. It has been claimed that superoxide, produced from xanthine oxidase in the reperfused vascular endothelium, induces upregulation of adhesion molecules on the luminal surface of the endothelial cell. These molecules then react with complementary ligands on circulating neutrophils, which are consequently arrested and activated, releasing damaging proteases and further oxidants [7]. Much of these data implicating oxidants in the damaging process following reperfusion have involved measurement of indirect indicators of free radical activity or use of nonspecific scavengers to reduce oxidant damage. Clear understanding of the processes involved will require identification and quantification of the primary radical species generated although currently, appropriate techniques for this in complex biological materials are lacking. One technique that may overcome this problem involves the use of microdialysis. This technique has been used to study the metabolism of brain, adipose, and more recently skeletal muscle [8]. It is minimally inva-
There is increasing evidence that oxidizing free radical species play an important role in the damage to skeletal muscle that occurs in various situations, such as following excessive or unaccustomed exercise or in certain disease states (see [1] for a review). Skeletal muscle is recognized to be relatively resistant to injury due to ischemia, but there are important clinical examples of where this damage does occur, such as following prolonged use of a tourniquet in orthopedic surgery or following surgery to correct arterial occlusion [2,3]. A major portion of the damage that occurs following ischemia is sustained at the time of the reperfusion and the mechanisms underlying this process have been the subject of considerable study. The role of reactive oxygen or nitrogen species in skeletal muscle reperfusion injury is currently unclear. Several investigators (e.g., Oredsson et al. [4] and Perler et al. [5]) have reported that administration of scavengers of free radical species reduced reperfusion injury to Address correspondence to: Prof. Malcolm J. Jackson, Department of Medicine, University of Liverpool, Liverpool L69 3GA, UK; Tel: ⫹44 (151)706-4072; Fax: ⫹44 (151)706-5802; E-Mail: [email protected]. 979
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sive and enables continuous monitoring of the extracellular environment of the tissue under study and produces very little interference with the normal physiological state. Previous studies have used microdialysis in conjunction with salicylate (2-hydroxybenzoic acid) to look for evidence of hydroxyl radical generation in tissue extracellular fluid [9] and we have also recently presented evidence that microdialysis techniques can be used to detect superoxide release into the extracellular space of skeletal muscle [10]. The aims of this study were therefore to examine the effect of prolonged ischemia and reperfusion of skeletal muscle on the release of superoxide and hydroxyl radicals into the extracellular space. In order to more clearly interpret the data obtained, we have also examined the effect of a superimposed period of contractile activity on this release. We hypothesized that reperfusion of skeletal muscle after a prolonged period of ischemia, or a short period of ischemia with superimposed contractile activity, would lead to release of superoxide and/or hydroxyl radicals into the extracellular fluid that would be detectable with microdialysis techniques. MATERIALS AND METHODS
Ischemia and reperfusion of the rat limb The technique of ischemia-reperfusion injury to the rat hind limb muscles described previously by McArdle et al. [8] was used. Adult female Wistar rats (approximately 200 g) were anaesthetized with sodium pentobarbitone (65 mg/100 IP) throughout the procedure. MAB 3.8.10 microdialysis probes (10 mm membrane, 0.5 mm wide, Metalant AB, Stockholm, Sweden) with a molecular weight cut-off of 35,000 daltons were placed into the anterior tibialis muscles of both legs. The probe was placed by initially inserting a 22G needle in a splittubing introducer into the muscle, the needle was removed and the microdialysis probe inserted prior to removal of the introducer. The femoral artery of one leg was exposed. The probes were perfused with either 0.5 mM salicylic acid (Sigma Chemical Co., Poole Dorset, UK) or 50 M cytochrome c (Sigma Chemical Co.) in isotonic saline at a flow rate of 1 l/min or 4 l/min, respectively. They were allowed to stabilize for 30 min and a baseline sample collected for 30 min from both legs. The femoral artery of one leg was then occluded using a microvessel clip for 4 h. Within this period samples were collected from both probes over sequential 30 min periods. Following collection of the sample immediately preceding reperfusion, the microvessel clip was removed and the ischemic limb was allowed to reperfuse. Samples were collected every 30 min from both limbs for a further 60 min. At the end of the
experimental protocol, rats were euthanized with an overdose of anesthesia. Anterior tibialis muscles were removed and frozen rapidly in liquid nitrogen and all samples were stored at ⫺70°C prior to analysis. Contractile activity of the ischemic rat hind limb Animals were prepared as above and additionally surface electrodes were placed around the upper limb and ankle of the ischemic limb. Immediately after occlusion of the femoral artery, the limb was subjected to 30 min of repetitive isometric contractions at a frequency of 100 Hz. with a pulse width of 0.1m s for half a second every 5 s. After the period of contractile activity the limb remained ischemic for 30 min. Microdialysis samples were collected from both limbs for 30 min periods. Control microdialysis studies Microdialysis samples were also obtained from anterior tibialis muscles of control animals under anesthesia, but not subjected to any ischemia and reperfusion or to contractile activity. Samples were collected for 30 min periods over 5 h. Anterior tibialis muscles were also obtained from freshly killed nonmanipulated animals as control tissue Analysis of microdialysates 2,3-Dihydroxybenzoic acid (2,3-DHB) and 2,5-dihydroxybenzoic acid (2,5-DHB) generated from the salicyate in the microdialysis fluids were measured as an index of reaction with hydroxyl radicals [11]. 2,3-DHB and 2,5-DHB were measured by HPLC with electrochemical detection as described by Halliwell et al., [12]. The HPLC system comprised a Rheodyne injector valve, HPLC pump (Gilson Model 303), Spherisorb 5 ODS column (HPLC Technology): 25 ⫻ 4.6 mm with guard column and C-8 cartridge (BDH) and an electrochemical detector (Gilson Model 141). The HPLC eluant consisted of 34 mM sodium citrate, 27.7 mM acetate buffer (pH 4.75) mixed with methanol 97.2:2.8, v/v. Standard solutions of 2,3-DHB, 2,5-DHB were prepared in HPLCgrade water. These solutions were stable at 4°C for a period of one week. Twenty l samples or standards were eluted at a rate of 0.9 ml/min, and monitored at ⫹65 V with the electrochemical detector. Reduction of cytochrome c in the microdialysis fluids was used to detect superoxide radicals as described by McArdle et al. [10] based on the approach followed by Reid et al. [13] and Green and Hill [14]. Samples were analyzed using scanning visible spectrometry. The superoxide content was calculated from the absorbance at
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Table 1. Glutathione, Protein Thiol, and MDA Content of Rat AT Muscles
Control muscle Test animal: contralateral control muscles Test animal: reperfused muscles
Total glutathione content (mol/g protein)
Oxidized glutathione content (percentage of total)
Protein thiol content (mol/g protein)
MDA content (mol/g protein)
16.6 ⫾ 1.2 15.2 ⫾ 3.9 9.1 ⫾ 2.3*
5.0 ⫾ 0.3 4.0 ⫾ 1.0 4.5 ⫾ 1.5
369 ⫾ 15 343 ⫾ 15 182 ⫾ 13*
10.3 ⫾ 0.7 16.5 ⫾ 3.8 31.10 ⫾ 4.7*
*p ⬍ .05 compared with both the AT muscle of control animals and contralateral AT muscles of test animals.
550 nm in comparison with the isobestic wavelengths at 542 and 560 nm. A molar extinction coefficient for reduced cytochrome c of 21,000 was used for calculation of the superoxide anion concentration.
significant changes were seen over the time course of the study. In the test animals values rose rapidly and within 30 min of occlusion of the femoral artery the content of 2,3 DHB was elevated in the ischemic limb compared with
Biochemical analyses The total and oxidized glutathione content of anterior tibialis muscles were analyzed by the enzymatic recycling assay described by Anderson [15]. Muscle protein thiol content was analyzed by titration of muscle proteins with 5,5-dithiolbis-2-nitro-benzoic acid [16] and the extet of lipid peroxidation in muscle samples was determined by HPLC measurements of the malonaldehyde (MDA) content [17].
Statistical analyses Data are presented as mean ⫾ SEM of values from 4 – 6 animals for each experiment. Data were initially analyzed by analysis of variance followed by Student’s t-test where appropriate. RESULTS
The effect of the sustained period of ischemia followed by 1 h reperfusion on biochemical indices of oxidation within the anterior tibialis muscle is shown in Table 1. Muscle MDA content was grossly elevated in comparison with both the contralateral control muscle and fresh control tissue. This was accompanied by loss of both protein thiols and glutathione from the muscles, although there was no evidence for an increase in the tissue oxidized glutathione content. Detectable levels of the salicylate oxidation products, 2,3 and 2,5 dihydroxybenzoate (DHB) were found in all microdialysates from the anterior tibialis muscles (Fig. 1). In microdialysates from the anterior tibialis muscle of anesthetized control animals not subjected to ischemia, these values remained unchanged throughout the 5 h of study. Values varied between 70 and 120 pmol 2,3 DHB/30 min and 55 and 100 pmol 2,5 DHB/30 min. No
Fig. 1. (A) 2,3-dihydroxybenzoic acid content of microdialysis fluid from the AT muscle of a limb exposed to 4 h ischemia and 1 h reperfusion (■) and the contralateral control muscle (●). Samples were collected every 30 min. *p ⬍ .05 compared with nonischemic contralateral muscles. (B) 2,5-dihydroxybenzoic acid content of microdialysis fluid from the AT muscle of a limb exposed to 4 h ischemia and 1 h reperfusion (■) and the contralateral control muscle (●). Samples were collected every 30 min. *p ⬍ .05 compared with nonischemic contralateral muscles.
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Fig. 2. Superoxide, detected as reduction of cytochrome c, in microdialysis fluid from the AT muscle of limbs exposed to 4 h ischemia and 1 h reperfusion (■) and the contralateral control muscle (●). Samples were collected every 30 min.
control, although this significance was not maintained until after 210 min of ischemia (Fig. 1A). Mean values for 2,3 DHB from both the ischemic and contralateral control limbs were grossly elevated compared with those from the anesthetized control animals. On reperfusion the 2,3 DHB content of microdialysis fluid from the previously ischemic limb showed large rises in comparison with the contralateral limb, remaining elevated at 1 h postreperfusion. Similar data were obtained for the 2,5 DHB content of the microdialysates with levels during the ischemic period being approximately 10-fold elevated compared with that from the anterior tibialis muscle of the anesthetized control animals. At the end of the ischemic period and during reperfusion the levels of 2,5 DHB from the ischemic and reperfused muscle was also significantly elevated compared with the values from the contralateral muscle (Fig. 1B). No significant differences in the reduction of cytochrome c within the microdialysates were seen between the ischemic/reperfused limb and the contralateral limb (Fig. 2). The overall level of reduction of cytochrome c fell over the first 90 min following insertion of the probe,
but did not appear to be influenced by the ischemia or reperfusion. When the ischemic muscle was stimulated to contract via surface electrodes, a significant rise in the reduction of cytochrome c was observed (Table 2); the values remaining elevated in comparison with the 30 min period following stimulation. DISCUSSION
The model of ischemia/reperfusion to the hind limb of the anesthetized rat that was used in this study has previously been shown to produce significant amounts of damage to skeletal muscle, in particular to the type II fibers [18] and to lead to substantial loss of adenine nucleotides from the muscle tissue [8]. The data presented here demonstrate that this model is also associated with substantial peroxidation of muscle lipids and loss of protein thiols and glutathione (Table 1). Our previous analyses of indicators of oxidation in reperfused muscle have been inconsistent [6,19], but other investigators have reported increased evidence for oxidation of skeletal muscle following reperfusion injury. Saez et al. [20]
Table 2. Superoxide Detected in Microdialysates from Ischemic and Contracting Rat AT Muscle Pre-ischemia (nmoles/30 min)
0–30 Min ischemia (nmoles/30 min)
30–60 Min ischemia (nmoles/30 min)
7.9 ⫾ 1.7 7.5 ⫾ 1.3
5.2 ⫾ 0.7 12.0 ⫾ 1.6*
3.9 ⫾ 0.6 7.3 ⫾ 2.6
Ischemia alone Ischemic muscle stimulated to contract at 0–30 min *p ⬍ .05 compared with ischemic muscle at the sam time point.
Measurement of free radical production
reported an increased content of MDA in blood following hind-limb bilateral ischemia in the rats, and Fantini and Yoshioka reported increased lipid peroxidation in ischemic/reperfused rat skeletal muscle that was prevented by prior treatment with deferoxamine [21]. Use of the microdialysis technique has revealed that this increased oxidation of cellular molecules is associated with an increased generation of 2,3 DHB and 2,5 DHB from salicylate in the microdialysate in comparison with that from the muscle of the contralateral limb (Fig. 1). Previous data have shown that both of these products can be produced by reaction of salicylate with hydroxyl radical, but 2,5 DHB may also be produced by an enzymatic pathway [22,23]. The implication of these data is therefore that ischemia to skeletal muscle is associated with some increase in hydroxyl radical generation in the interstitial fluid and that during reperfusion a further substantial increase in hydroxyl radical activity occurs leading to oxidation of key molecules. Several groups have previously used production of 2,3 DHB from salicylate in microdialysis fluids as evidence that hydroxyl radical activity had increased in a variety of tissues [9,24,25]. The absolute amount of 2,3 DHB produced in the microdialysis probe prior to ischemia was approximately 70 pmol/30 min, which is equivalent to 0.5% of the salicylate in the microdialysate. This value was essentially unchanged in the limb of the control rats, but increased substantially following the onset of ischemia in both ischemic and contralateral control limbs. The extent of this was that during the ischemic period the amount of 2,3 DHB in the microdialysates from both ischemic and contralateral limbs was approximately 500 pmol/30min, equivalent to 3.3% of the salicylate. It seems unlikely that a rise of this magnitude (i.e., approximately 7-fold) would have occurred in hydroxyl radical activity in both the ischemic and contralateral limbs, although no previous data appear to have been presented on this point. An alternative explanation for these changes may relate to the compromised blood flow in the limb. Salicylic acid has a low molecular weight (138 daltons) and some must be lost from the probe to the interstitial space. In fully perfused animals this will be rapidly cleared to the circulation, but in the ischemic limb it is likely to diffuse relatively slowly away from the probe providing a potential source of salicylate that may re-enter the probe. Furthermore, this salicylate would be increasingly oxidized within the interstitial space. This potential explanation for the large rise in 2,3 and 2,5 DHB content during ischemia does not immediately explain the rise from the contralateral control muscle, but some evidence suggests that this model may be associated with compensatory changes in blood flow in the contralateral control muscle [8] that may have contributed to the data
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observed. It is also possible that hydroxyl radical generation in the contralateral limb may have increased due to systemic responses triggered by the ischemic limb. Limb ischemia is known to induce the release of a number of angiogenic factors [26] and to activate monocytes leading to cytokine release [27], substances that may influence free radical production at sites distant to the ischemic limb. Surprisingly there was no evidence for an increase in superoxide release during the ischemic or reperfusion periods. Our previous data have demonstrated a significant release of hypoxanthine from rat skeletal muscle following 3 h of ischemia and during reperfusion [8]. We hypothesized that this would lead to generation of superoxide radicals via endothelial xanthine oxidase, but no evidence for this was seen. In order to demonstrate that this lack of a detectable rise in superoxide was not a technical artifact, the effect of contraction of skeletal muscle was examined. Previous workers have shown that superoxide is released from skeletal muscle during contractions [13] and we have detected this rise as an increased reduction of cytochrome c in microdialysis fluids from contracting mouse AT muscle [10]. Contraction of the rat AT muscle at the beginning of the ischemic period was found to induce a significant rise in the reduction of microdialysate cytochrome c (Table 2) confirming that the technique had sufficient sensitivity to detect a rise in superoxide in the rat extracellular fluid. These data therefore indicate that any rise in interstitial superoxide during ischemia and reperfusion must have been relatively small in comparison with that seen following contraction, and below the sensitivity of the microdialysis detection technique. The relative release of superoxide by skeletal muscle cells on contraction is large [10] and must be sufficient to permit detection by the microdialysis technique in the presence of extracellular superoxide dismutase, which acts to maintain low extracellular levels of superoxide by generation of hydrogen peroxide. Our previous data indicate that a significant proportion of the basal reduction of cytochrome c detected in muscle microdialysates is either due to superoxide derived from nonskeletal muscle cells or due to alternative low molecular weight substances found in interstitial fluid that are capable of reducing cytochrome c [10]. This may also imply that changes in the release of superoxide by muscle must be substantial to be detectable by these techniques. We therefore conclude that, in contrast to contractile activity of skeletal muscle, reperfusion of the ischemic AT muscle was not associated with a large rise in extracellular superoxide content, although an increased hydroxyl radical activity was detected. Current theories of the pathogenesis of ischemic/reperfusion damage implicate an increased superoxide production from endothelial xanthine oxidase [7,28] in initiating
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up-regulation of adhesion molecules on the luminal surface of endothelial cells and/or neutrophils leading to their arrest and activation releasing damaging proteases and further oxidants. Our data do not support the possibility that significant amounts of superoxide are present in the extracellular fluid to play such a role, although such changes may also be initiated by extracellular hydroxyl radical. This has clear implications for design of agents to prevent the action of free radical species in ischemic reperfusion injury. Thus, for instance, these data imply that application of extracellular superoxide dismutase would have little effect, whereas strategies to reduce hydroxyl radical production (e.g., by chelation of iron) would be predicted to be beneficial. Published data are contradictory concerning this point. There are a significant number of publications claiming beneficial effects of extracellular superoxide dismutase against skeletal muscle reperfusion injury [29 –32], but a number also report no effect of superoxide dismutase in comparison with beneficial effects of iron chelation [21,33, 34]. Although microdialysis offers a potentially powerful approach to monitoring reactive oxygen species within tissues in vivo, the data can only be considered semiquantitiative unless specific information relating to the recovery of the analyte is available [8,35]. In the case of superoxide and hydroxyl radicals, such data are not available because of the instability and reactivity of the molecules and the lack of appropriate generation and detection systems. Thus, for instance, both Forman and Azzi [36] and Reid and co-workers [13] comment that the apparent rates of production and diffusion of superoxide are influenced by the presence of trapping agents/ scavengers, precluding their use in the type of experiments required to define recoveries of these molecules across the microdialysis probe. A further limitation of the microdialysis approach is that, although the probe is inserted into the bulk of the muscle tissue, the microdialysate reflects interstitial fluid content. Thus our inability to detect a rise in microdialysate superoxide does not exclude a role for this molecule within the endothelial or muscle cells. Nevertheless, many of the major scavenging agents used in this area are membrane impermeable (e.g., superoxide dismutase, deferoxamine) and would not have access to the cells. Currently, little is known about the ability of these agents to rapidly enter the interstitial space, but a knowledge of the radical species present in the extracellular fluids is potentially highly relevant to our understanding of the pathophysiology of the ischemia/reperfusion process and its manipulation. Acknowledgements — The authors would like to thank the Mersey Kidney Research Fund and the Wellcome Trust for financial support and Drs. F. McArdle and A. Crowe for helpful scientific discussions.
REFERENCES [1] Reznick, A. Z.; Packer, L.; Sen, C. K.; Holloszy, J. O.; Jackson, M. J., eds. Oxidative stress in skeletal muscle. Basel, Switzerland: Birkhauser Verlag; 1998. [2] Klenerman, L.; Biswas, M.; Hulands, G. H.; Rhodes, A. M. Systemic and local effects of the application of a tourniquet. J. Bone Joint Surg. Br. 62:385–388; 1980. [3] Patterson, S.; Klenerman, L. The effects of pneumatic tourniquets on the ultrastructure of skeletal muscle. J. Bone Joint Surg. Br. 61:178 –183; 1979. [4] Oredsson, S.; Plate, G.; Quarford, P. Allopurinol—free radical scavenger—reduces reperfusion injury in skeletal muscle. Eur. J. Vasc. Surg. 5:47–52; 1991. [5] Perler, B. A.; Tohmed, A. G.; Bulkley, G. B. Inhibition of the compartment syndrome by the ablation of free radical-mediated reperfusion injury. Surgery 108:40 – 47; 1990. [6] Bushell, A.; Klenerman, L.; Davis, H.; Grierson, I.; Jackson, M. J. Ischemia-reperfusion-induced muscle damage: protective effect of corticosteroids and antioxidants in rabbits. Acta Orthop. Scand. 67:393–398; 1996. [7] Bulkley, G. B. Reactive oxygen metabolites and reperfusion injury: aberrant triggering of reticuloendothelial function. Lancet 334:934 –936; 1994. [8] McArdle, A.; Khera, G.; Edwards, R. H. T.; Jackson, M. J. In vivo microdialysis—A novel technique for analysis of chemical activators of muscle pain. Muscle Nerve 22:1047–1052; 1999. [9] Obata, T. Use of microdialysis for in vivo monitoring of hydroxyl radical generation in the rat. J. Pharm. Pharmacol. 49:724 –730; 1997. [10] McArdle, A.; Pattwell, D.; Vasilaki, A.; Griffiths, R. D.; Jackson, M. J. Contractile activity-induced oxidative stress: cellular origin and adaptive responses. Am. J. Physiol. Cell. 280:C621–C627; 2001. [11] Richmond, R.; Halliwell, B.; Chauhan, J.; Darbe, A. Superoxidedependent formation of hydroxyl radicals: detection of hydroxyl radicals by the hydroxylation of aromatic compounds. Anal. Biochem. 118:328 –335; 1981. [12] Halliwell, B.; Grootveld, M.; Gutteridge, J. M. C. Methods for the measurement of hydroxyl radicals in biochemical systems: deoxyribose degradation and aromatic hydroxylation. Methods Biochem. Anal. 33:59 –90; 1988. [13] Reid, M. B.; Haack, K. E.; Franchek, K. M.; Valberg, P. A.; Kobzik, L.; West, S. Reactive oxygen in skeletal muscle. II. Extracellular release of free radicals. J. Appl. Physiol. 73:1805– 1809; 1992. [14] Green, M. J.; Hill, A. O. Chemistry of dioxygen. Methods Enzymol. 105:13–17; 1984. [15] Anderson, M. Determination of glutathione and glutathione disulphide. Methods Enzymol. 113:548 –555; 1985. [16] DiMonte, D.; Rose, D.; Bellomo, G.; Eklow, L.; Orrenius, S. Alterations in intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes. Arch. Biochem. Biophys. 235:334 –342; 1984. [17] Chirico, S. High Performance Liquid Chromatography-based thiobabituric acid tests. Methods Enzymol. 233:314 –318; 1990. [18] Bushell, A. J.; Klenerman, L.; Davies, H. M.; Grierson, I.; Jackson, M. J. Damage to skeletal muscle induced by prolonged ischemia and reperfusion. Transplant Proc. 27:2834 –2835; 1995. [19] Klenerman, L.; Lowe, N. M.; Miller, I.; Fryer, P. R.; Green, C. J.; Jackson, M. J. Dantrolene sodium protects against experimental ischemia and reperfusion damage to skeletal muscle. Acta Orthop. Scand. 66:352–358; 1995. [20] Saez, J. C.; Cifuentes, F.; Ward, P. H.; Gunther, B.; Vivaldi, E. Tourniquet shock in rats: effects of allopurinol on biochemical changes of the gastrocnemius muscle subjected to ischemia followed by reperfusion. Biochem. Med. Metab. Biol. 35:199 –209; 1986. [21] Fantini, G.; Yoshioka, T. Deferoxamine prevents lipid peroxidation and attenuates reoxygenation injury in postischemic skeletal muscle. Am. J. Physiol. 264:H1953–H1959; 1993. [22] Rumble, R. H.; Roberts, M. S.; Wanwimolruk, S. Determination
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[30] Bowler, D. J.; McLaughlin, R.; Kelly, C. J.; O’Farrell, D. A.; Moran, K.; Bouchier-Hayes, D. Recombinant human manganese superoxide dismutase attenuates early but not delayed skeletal muscle dysfunction following reperfusion injury. Eur. J. Vasc. Endovasc. Surg. 18:216 –221; 1999. [31] O’Farrell, D.; Chen, L. E.; Seaber, A. V.; Murrell, G. A.; Urbaniak, J. R. Efficacy of recombinant human manganese superoxide dismutase compared to allopurinol in protection of ischemic skeletal muscle against “no-reflow”. J. Reconst. Microsurg. 11:207– 214; 1995. [32] Deune, E. G.; Koopman, R.; Smith, M. E.; Hong, S. P.; Ozbek, M. R.; Khouri, R. K. Prevention of ischemia-reperfusion injury with a synthetic metalloprotein superoxide dismutase mimic, SC52608. Plast. Reconstr. Surg. 98:711–718; 1996. [33] Potter, R. F.; Ellis, C. J.; Tyml, K.; Groom, A. C. Effect of superoxide dismutase and 21-aminosteroids (lazaroids) on microvascular perfusion following ischemia-reperfusion in skeletal muscle. Int. J. Microcirc. Clin. Exp. 14:313–318; 1994. [34] Oredsson, S; Plate, G.; Qvarfordt, P. Experimental evaluation of oxygen free radical scavengers in the prevention of reperfusion injury in skeletal muscle. Eur. J. Surg. 160:97–103; 1994. [35] Bienveniste, H.; Huttemeier, P. C. Microdialysis-theory and applications. Prog. Neurobiol. 35:195–215; 1990. [36] Forman, H. J.; Azzi, A. On the virtual existence of superoxide anions in mitochondria: thoughts regarding its role in pathophysiology. FASEB J. 11:374 –375; 1997.
PII S0891-5849(01)00485-3
Original Contribution MEASUREMENT OF FREE RADICAL PRODUCTION BY IN VIVO MICRODIALYSIS DURING ISCHEMIA/REPERFUSION INJURY TO SKELETAL MUSCLE DAVID PATTWELL, ANNE MCARDLE, RICHARD D. GRIFFITHS,
and
MALCOLM J. JACKSON
Department of Medicine, University of Liverpool, Liverpool, UK (Received 25 October 2000; Revised 16 January 2001; Accepted 25 January 2000)
Abstract—Microdialysis techniques have been used to detect hydroxyl radical and superoxide release into the interstitial space of anaesthetized rat anterior tibialis muscles during a period of prolonged (4 h) limb ischemia and subsequent reperfusion. Data indicate that reperfusion of the ischemic skeletal muscle was associated with a large increase in hydroxyl radical activity in the interstitial space, which may contribute to the significant oxidation of muscle glutathione, protein thiols, and lipids also seen in this model. No evidence for release of superoxide into the interstitial space was found during reperfusion, although this was observed during electrically stimulated contractile activity of the rat limb muscle. These data imply that therapeutic approaches aimed at reduction of hydroxyl radical generation in the interstitial fluid are more likely to be beneficial in reduction of skeletal muscle reperfusion injury than approaches designed to scavenge superoxide radicals. © 2001 Elsevier Science Inc. Keywords—Hydroxyl radical, Superoxide, Salicylate, Cytochrome c, Anterior tibialis muscle, Free radicals
INTRODUCTION
skeletal muscle, and we have confirmed that this protection is apparent with ascorbate, but not some other potential scavengers [6]. Work with a variety of other tissues indicates that oxygen radicals play a key role in complex mechanisms of damage following reperfusion. It has been claimed that superoxide, produced from xanthine oxidase in the reperfused vascular endothelium, induces upregulation of adhesion molecules on the luminal surface of the endothelial cell. These molecules then react with complementary ligands on circulating neutrophils, which are consequently arrested and activated, releasing damaging proteases and further oxidants [7]. Much of these data implicating oxidants in the damaging process following reperfusion have involved measurement of indirect indicators of free radical activity or use of nonspecific scavengers to reduce oxidant damage. Clear understanding of the processes involved will require identification and quantification of the primary radical species generated although currently, appropriate techniques for this in complex biological materials are lacking. One technique that may overcome this problem involves the use of microdialysis. This technique has been used to study the metabolism of brain, adipose, and more recently skeletal muscle [8]. It is minimally inva-
There is increasing evidence that oxidizing free radical species play an important role in the damage to skeletal muscle that occurs in various situations, such as following excessive or unaccustomed exercise or in certain disease states (see [1] for a review). Skeletal muscle is recognized to be relatively resistant to injury due to ischemia, but there are important clinical examples of where this damage does occur, such as following prolonged use of a tourniquet in orthopedic surgery or following surgery to correct arterial occlusion [2,3]. A major portion of the damage that occurs following ischemia is sustained at the time of the reperfusion and the mechanisms underlying this process have been the subject of considerable study. The role of reactive oxygen or nitrogen species in skeletal muscle reperfusion injury is currently unclear. Several investigators (e.g., Oredsson et al. [4] and Perler et al. [5]) have reported that administration of scavengers of free radical species reduced reperfusion injury to Address correspondence to: Prof. Malcolm J. Jackson, Department of Medicine, University of Liverpool, Liverpool L69 3GA, UK; Tel: ⫹44 (151)706-4072; Fax: ⫹44 (151)706-5802; E-Mail: [email protected]. 979
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sive and enables continuous monitoring of the extracellular environment of the tissue under study and produces very little interference with the normal physiological state. Previous studies have used microdialysis in conjunction with salicylate (2-hydroxybenzoic acid) to look for evidence of hydroxyl radical generation in tissue extracellular fluid [9] and we have also recently presented evidence that microdialysis techniques can be used to detect superoxide release into the extracellular space of skeletal muscle [10]. The aims of this study were therefore to examine the effect of prolonged ischemia and reperfusion of skeletal muscle on the release of superoxide and hydroxyl radicals into the extracellular space. In order to more clearly interpret the data obtained, we have also examined the effect of a superimposed period of contractile activity on this release. We hypothesized that reperfusion of skeletal muscle after a prolonged period of ischemia, or a short period of ischemia with superimposed contractile activity, would lead to release of superoxide and/or hydroxyl radicals into the extracellular fluid that would be detectable with microdialysis techniques. MATERIALS AND METHODS
Ischemia and reperfusion of the rat limb The technique of ischemia-reperfusion injury to the rat hind limb muscles described previously by McArdle et al. [8] was used. Adult female Wistar rats (approximately 200 g) were anaesthetized with sodium pentobarbitone (65 mg/100 IP) throughout the procedure. MAB 3.8.10 microdialysis probes (10 mm membrane, 0.5 mm wide, Metalant AB, Stockholm, Sweden) with a molecular weight cut-off of 35,000 daltons were placed into the anterior tibialis muscles of both legs. The probe was placed by initially inserting a 22G needle in a splittubing introducer into the muscle, the needle was removed and the microdialysis probe inserted prior to removal of the introducer. The femoral artery of one leg was exposed. The probes were perfused with either 0.5 mM salicylic acid (Sigma Chemical Co., Poole Dorset, UK) or 50 M cytochrome c (Sigma Chemical Co.) in isotonic saline at a flow rate of 1 l/min or 4 l/min, respectively. They were allowed to stabilize for 30 min and a baseline sample collected for 30 min from both legs. The femoral artery of one leg was then occluded using a microvessel clip for 4 h. Within this period samples were collected from both probes over sequential 30 min periods. Following collection of the sample immediately preceding reperfusion, the microvessel clip was removed and the ischemic limb was allowed to reperfuse. Samples were collected every 30 min from both limbs for a further 60 min. At the end of the
experimental protocol, rats were euthanized with an overdose of anesthesia. Anterior tibialis muscles were removed and frozen rapidly in liquid nitrogen and all samples were stored at ⫺70°C prior to analysis. Contractile activity of the ischemic rat hind limb Animals were prepared as above and additionally surface electrodes were placed around the upper limb and ankle of the ischemic limb. Immediately after occlusion of the femoral artery, the limb was subjected to 30 min of repetitive isometric contractions at a frequency of 100 Hz. with a pulse width of 0.1m s for half a second every 5 s. After the period of contractile activity the limb remained ischemic for 30 min. Microdialysis samples were collected from both limbs for 30 min periods. Control microdialysis studies Microdialysis samples were also obtained from anterior tibialis muscles of control animals under anesthesia, but not subjected to any ischemia and reperfusion or to contractile activity. Samples were collected for 30 min periods over 5 h. Anterior tibialis muscles were also obtained from freshly killed nonmanipulated animals as control tissue Analysis of microdialysates 2,3-Dihydroxybenzoic acid (2,3-DHB) and 2,5-dihydroxybenzoic acid (2,5-DHB) generated from the salicyate in the microdialysis fluids were measured as an index of reaction with hydroxyl radicals [11]. 2,3-DHB and 2,5-DHB were measured by HPLC with electrochemical detection as described by Halliwell et al., [12]. The HPLC system comprised a Rheodyne injector valve, HPLC pump (Gilson Model 303), Spherisorb 5 ODS column (HPLC Technology): 25 ⫻ 4.6 mm with guard column and C-8 cartridge (BDH) and an electrochemical detector (Gilson Model 141). The HPLC eluant consisted of 34 mM sodium citrate, 27.7 mM acetate buffer (pH 4.75) mixed with methanol 97.2:2.8, v/v. Standard solutions of 2,3-DHB, 2,5-DHB were prepared in HPLCgrade water. These solutions were stable at 4°C for a period of one week. Twenty l samples or standards were eluted at a rate of 0.9 ml/min, and monitored at ⫹65 V with the electrochemical detector. Reduction of cytochrome c in the microdialysis fluids was used to detect superoxide radicals as described by McArdle et al. [10] based on the approach followed by Reid et al. [13] and Green and Hill [14]. Samples were analyzed using scanning visible spectrometry. The superoxide content was calculated from the absorbance at
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Table 1. Glutathione, Protein Thiol, and MDA Content of Rat AT Muscles
Control muscle Test animal: contralateral control muscles Test animal: reperfused muscles
Total glutathione content (mol/g protein)
Oxidized glutathione content (percentage of total)
Protein thiol content (mol/g protein)
MDA content (mol/g protein)
16.6 ⫾ 1.2 15.2 ⫾ 3.9 9.1 ⫾ 2.3*
5.0 ⫾ 0.3 4.0 ⫾ 1.0 4.5 ⫾ 1.5
369 ⫾ 15 343 ⫾ 15 182 ⫾ 13*
10.3 ⫾ 0.7 16.5 ⫾ 3.8 31.10 ⫾ 4.7*
*p ⬍ .05 compared with both the AT muscle of control animals and contralateral AT muscles of test animals.
550 nm in comparison with the isobestic wavelengths at 542 and 560 nm. A molar extinction coefficient for reduced cytochrome c of 21,000 was used for calculation of the superoxide anion concentration.
significant changes were seen over the time course of the study. In the test animals values rose rapidly and within 30 min of occlusion of the femoral artery the content of 2,3 DHB was elevated in the ischemic limb compared with
Biochemical analyses The total and oxidized glutathione content of anterior tibialis muscles were analyzed by the enzymatic recycling assay described by Anderson [15]. Muscle protein thiol content was analyzed by titration of muscle proteins with 5,5-dithiolbis-2-nitro-benzoic acid [16] and the extet of lipid peroxidation in muscle samples was determined by HPLC measurements of the malonaldehyde (MDA) content [17].
Statistical analyses Data are presented as mean ⫾ SEM of values from 4 – 6 animals for each experiment. Data were initially analyzed by analysis of variance followed by Student’s t-test where appropriate. RESULTS
The effect of the sustained period of ischemia followed by 1 h reperfusion on biochemical indices of oxidation within the anterior tibialis muscle is shown in Table 1. Muscle MDA content was grossly elevated in comparison with both the contralateral control muscle and fresh control tissue. This was accompanied by loss of both protein thiols and glutathione from the muscles, although there was no evidence for an increase in the tissue oxidized glutathione content. Detectable levels of the salicylate oxidation products, 2,3 and 2,5 dihydroxybenzoate (DHB) were found in all microdialysates from the anterior tibialis muscles (Fig. 1). In microdialysates from the anterior tibialis muscle of anesthetized control animals not subjected to ischemia, these values remained unchanged throughout the 5 h of study. Values varied between 70 and 120 pmol 2,3 DHB/30 min and 55 and 100 pmol 2,5 DHB/30 min. No
Fig. 1. (A) 2,3-dihydroxybenzoic acid content of microdialysis fluid from the AT muscle of a limb exposed to 4 h ischemia and 1 h reperfusion (■) and the contralateral control muscle (●). Samples were collected every 30 min. *p ⬍ .05 compared with nonischemic contralateral muscles. (B) 2,5-dihydroxybenzoic acid content of microdialysis fluid from the AT muscle of a limb exposed to 4 h ischemia and 1 h reperfusion (■) and the contralateral control muscle (●). Samples were collected every 30 min. *p ⬍ .05 compared with nonischemic contralateral muscles.
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Fig. 2. Superoxide, detected as reduction of cytochrome c, in microdialysis fluid from the AT muscle of limbs exposed to 4 h ischemia and 1 h reperfusion (■) and the contralateral control muscle (●). Samples were collected every 30 min.
control, although this significance was not maintained until after 210 min of ischemia (Fig. 1A). Mean values for 2,3 DHB from both the ischemic and contralateral control limbs were grossly elevated compared with those from the anesthetized control animals. On reperfusion the 2,3 DHB content of microdialysis fluid from the previously ischemic limb showed large rises in comparison with the contralateral limb, remaining elevated at 1 h postreperfusion. Similar data were obtained for the 2,5 DHB content of the microdialysates with levels during the ischemic period being approximately 10-fold elevated compared with that from the anterior tibialis muscle of the anesthetized control animals. At the end of the ischemic period and during reperfusion the levels of 2,5 DHB from the ischemic and reperfused muscle was also significantly elevated compared with the values from the contralateral muscle (Fig. 1B). No significant differences in the reduction of cytochrome c within the microdialysates were seen between the ischemic/reperfused limb and the contralateral limb (Fig. 2). The overall level of reduction of cytochrome c fell over the first 90 min following insertion of the probe,
but did not appear to be influenced by the ischemia or reperfusion. When the ischemic muscle was stimulated to contract via surface electrodes, a significant rise in the reduction of cytochrome c was observed (Table 2); the values remaining elevated in comparison with the 30 min period following stimulation. DISCUSSION
The model of ischemia/reperfusion to the hind limb of the anesthetized rat that was used in this study has previously been shown to produce significant amounts of damage to skeletal muscle, in particular to the type II fibers [18] and to lead to substantial loss of adenine nucleotides from the muscle tissue [8]. The data presented here demonstrate that this model is also associated with substantial peroxidation of muscle lipids and loss of protein thiols and glutathione (Table 1). Our previous analyses of indicators of oxidation in reperfused muscle have been inconsistent [6,19], but other investigators have reported increased evidence for oxidation of skeletal muscle following reperfusion injury. Saez et al. [20]
Table 2. Superoxide Detected in Microdialysates from Ischemic and Contracting Rat AT Muscle Pre-ischemia (nmoles/30 min)
0–30 Min ischemia (nmoles/30 min)
30–60 Min ischemia (nmoles/30 min)
7.9 ⫾ 1.7 7.5 ⫾ 1.3
5.2 ⫾ 0.7 12.0 ⫾ 1.6*
3.9 ⫾ 0.6 7.3 ⫾ 2.6
Ischemia alone Ischemic muscle stimulated to contract at 0–30 min *p ⬍ .05 compared with ischemic muscle at the sam time point.
Measurement of free radical production
reported an increased content of MDA in blood following hind-limb bilateral ischemia in the rats, and Fantini and Yoshioka reported increased lipid peroxidation in ischemic/reperfused rat skeletal muscle that was prevented by prior treatment with deferoxamine [21]. Use of the microdialysis technique has revealed that this increased oxidation of cellular molecules is associated with an increased generation of 2,3 DHB and 2,5 DHB from salicylate in the microdialysate in comparison with that from the muscle of the contralateral limb (Fig. 1). Previous data have shown that both of these products can be produced by reaction of salicylate with hydroxyl radical, but 2,5 DHB may also be produced by an enzymatic pathway [22,23]. The implication of these data is therefore that ischemia to skeletal muscle is associated with some increase in hydroxyl radical generation in the interstitial fluid and that during reperfusion a further substantial increase in hydroxyl radical activity occurs leading to oxidation of key molecules. Several groups have previously used production of 2,3 DHB from salicylate in microdialysis fluids as evidence that hydroxyl radical activity had increased in a variety of tissues [9,24,25]. The absolute amount of 2,3 DHB produced in the microdialysis probe prior to ischemia was approximately 70 pmol/30 min, which is equivalent to 0.5% of the salicylate in the microdialysate. This value was essentially unchanged in the limb of the control rats, but increased substantially following the onset of ischemia in both ischemic and contralateral control limbs. The extent of this was that during the ischemic period the amount of 2,3 DHB in the microdialysates from both ischemic and contralateral limbs was approximately 500 pmol/30min, equivalent to 3.3% of the salicylate. It seems unlikely that a rise of this magnitude (i.e., approximately 7-fold) would have occurred in hydroxyl radical activity in both the ischemic and contralateral limbs, although no previous data appear to have been presented on this point. An alternative explanation for these changes may relate to the compromised blood flow in the limb. Salicylic acid has a low molecular weight (138 daltons) and some must be lost from the probe to the interstitial space. In fully perfused animals this will be rapidly cleared to the circulation, but in the ischemic limb it is likely to diffuse relatively slowly away from the probe providing a potential source of salicylate that may re-enter the probe. Furthermore, this salicylate would be increasingly oxidized within the interstitial space. This potential explanation for the large rise in 2,3 and 2,5 DHB content during ischemia does not immediately explain the rise from the contralateral control muscle, but some evidence suggests that this model may be associated with compensatory changes in blood flow in the contralateral control muscle [8] that may have contributed to the data
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observed. It is also possible that hydroxyl radical generation in the contralateral limb may have increased due to systemic responses triggered by the ischemic limb. Limb ischemia is known to induce the release of a number of angiogenic factors [26] and to activate monocytes leading to cytokine release [27], substances that may influence free radical production at sites distant to the ischemic limb. Surprisingly there was no evidence for an increase in superoxide release during the ischemic or reperfusion periods. Our previous data have demonstrated a significant release of hypoxanthine from rat skeletal muscle following 3 h of ischemia and during reperfusion [8]. We hypothesized that this would lead to generation of superoxide radicals via endothelial xanthine oxidase, but no evidence for this was seen. In order to demonstrate that this lack of a detectable rise in superoxide was not a technical artifact, the effect of contraction of skeletal muscle was examined. Previous workers have shown that superoxide is released from skeletal muscle during contractions [13] and we have detected this rise as an increased reduction of cytochrome c in microdialysis fluids from contracting mouse AT muscle [10]. Contraction of the rat AT muscle at the beginning of the ischemic period was found to induce a significant rise in the reduction of microdialysate cytochrome c (Table 2) confirming that the technique had sufficient sensitivity to detect a rise in superoxide in the rat extracellular fluid. These data therefore indicate that any rise in interstitial superoxide during ischemia and reperfusion must have been relatively small in comparison with that seen following contraction, and below the sensitivity of the microdialysis detection technique. The relative release of superoxide by skeletal muscle cells on contraction is large [10] and must be sufficient to permit detection by the microdialysis technique in the presence of extracellular superoxide dismutase, which acts to maintain low extracellular levels of superoxide by generation of hydrogen peroxide. Our previous data indicate that a significant proportion of the basal reduction of cytochrome c detected in muscle microdialysates is either due to superoxide derived from nonskeletal muscle cells or due to alternative low molecular weight substances found in interstitial fluid that are capable of reducing cytochrome c [10]. This may also imply that changes in the release of superoxide by muscle must be substantial to be detectable by these techniques. We therefore conclude that, in contrast to contractile activity of skeletal muscle, reperfusion of the ischemic AT muscle was not associated with a large rise in extracellular superoxide content, although an increased hydroxyl radical activity was detected. Current theories of the pathogenesis of ischemic/reperfusion damage implicate an increased superoxide production from endothelial xanthine oxidase [7,28] in initiating
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up-regulation of adhesion molecules on the luminal surface of endothelial cells and/or neutrophils leading to their arrest and activation releasing damaging proteases and further oxidants. Our data do not support the possibility that significant amounts of superoxide are present in the extracellular fluid to play such a role, although such changes may also be initiated by extracellular hydroxyl radical. This has clear implications for design of agents to prevent the action of free radical species in ischemic reperfusion injury. Thus, for instance, these data imply that application of extracellular superoxide dismutase would have little effect, whereas strategies to reduce hydroxyl radical production (e.g., by chelation of iron) would be predicted to be beneficial. Published data are contradictory concerning this point. There are a significant number of publications claiming beneficial effects of extracellular superoxide dismutase against skeletal muscle reperfusion injury [29 –32], but a number also report no effect of superoxide dismutase in comparison with beneficial effects of iron chelation [21,33, 34]. Although microdialysis offers a potentially powerful approach to monitoring reactive oxygen species within tissues in vivo, the data can only be considered semiquantitiative unless specific information relating to the recovery of the analyte is available [8,35]. In the case of superoxide and hydroxyl radicals, such data are not available because of the instability and reactivity of the molecules and the lack of appropriate generation and detection systems. Thus, for instance, both Forman and Azzi [36] and Reid and co-workers [13] comment that the apparent rates of production and diffusion of superoxide are influenced by the presence of trapping agents/ scavengers, precluding their use in the type of experiments required to define recoveries of these molecules across the microdialysis probe. A further limitation of the microdialysis approach is that, although the probe is inserted into the bulk of the muscle tissue, the microdialysate reflects interstitial fluid content. Thus our inability to detect a rise in microdialysate superoxide does not exclude a role for this molecule within the endothelial or muscle cells. Nevertheless, many of the major scavenging agents used in this area are membrane impermeable (e.g., superoxide dismutase, deferoxamine) and would not have access to the cells. Currently, little is known about the ability of these agents to rapidly enter the interstitial space, but a knowledge of the radical species present in the extracellular fluids is potentially highly relevant to our understanding of the pathophysiology of the ischemia/reperfusion process and its manipulation. Acknowledgements — The authors would like to thank the Mersey Kidney Research Fund and the Wellcome Trust for financial support and Drs. F. McArdle and A. Crowe for helpful scientific discussions.
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