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Ischemic-Reperfused Rat Skeletal Muscle: The Effect of Vasoactive Intestinal Peptide (VIP) on Contractile Force, Oxygenation and Antioxidant Enzyme Systems
Peptides, Vol. 18, No. 2, pp. 269–275, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/97 $17.00 / .00
PII S0196-9781(96)00289-6
Ischemic-Reperfused Rat Skeletal Muscle: The Effect of Vasoactive Intestinal Peptide (VIP) on Contractile Force, Oxygenation and Antioxidant Enzyme Systems ¨ KSEL ALTIOKKA† NES¸E TUNC ¸ EL,* 1 SERDAR ERDEN,* KUBILAY UZUNER,* GO AND MUZAFFER TUNC ¸ EL† *Department of Physiology, Faculty of Medicine, University of Osmangazi 26040, Eskis¸ehir, Turkey †Department of Analytical Chemistry, Faculty of Pharmacy, University of Anadolu 26480, Eskis¸ehir, Turkey Received 6 August 1996; Accepted 16 October 1996 TUNC ¸ EL, N., S. ERDEN, K. UZUNER, G. ALTIOKKA AND M. TUNC ¸ EL. Ischemic-reperfused rat skeletal muscle: The effect of vasoactive intestinal peptide (VIP) on contractile force, oxygenation and antioxidant enzyme systems. PEPTIDES 18(2) 269– 275, 1997.—The effect of vasoactive intestinal peptide (VIP) on the nerve-stimulated contraction, tissue oxygenation, lipid peroxidation and antioxidant enzymes activities-superoxide dismutase and catalase was investigated in the rat gastrocnemius muscle exposed to 4 h ischemia-4hr reperfusion. Ischemia caused significant decrease in muscle contractile force, oxygenation and superoxide dismutase enzyme activity. Reperfusion of ischemic muscle increased the muscle contractile force and restored the tissue oxygenation to the baseline level. Superoxide dismutase and catalase activities of reperfused muscle increased significantly. However neither ischemia nor reperfusion affected gastrocnemius muscle malondialdehide (MDA) levels. VIP administration at the onset of reperfusion significantly increased skeletal muscle contractile force and tissue oxygenation even higher than baseline and reperfusion values. VIP also normalized the increased superoxide dismutase and catalase activities of reperfused skeletal muscle. In conclusion, VIP, acting as a powerful antioxidant and preserving contractile machinery seems to be a promising endogenous peptide that can salvage the skeletal muscle from severe ischemia-reperfusion injury. q 1997 Elsevier Science Inc. Vasoactive intestinal peptide
Ischemia-reperfusion skeletal muscle
RECENT evidence indicates that vasoactive intestinal peptide (VIP) has modulatory effect on tissue injury (31,36,37). It protects the tissues against injury due to oxidant and other toxins (31). VIP has been found to possess the ability to scavenge oxygen free radicals. VIP can quench in vivo hydroxyl radical formation and can directly scavenge singlet oxygen (17,30). VIP also has a potent protective activity against injury triggerred by xanthine/xanthine oxidase system and inhibits superoxide radical formation by inflammatory cells (6,31,36). In our previous studies, VIP administration in experimental hemorrhagic shock models increased the survival rate and protected the renal tissue from the injury of reperfusion (1,39). In our subsequent studies VIP protected the renal and retinal tissue from ischemia reperfusion injury without any increase in the activity of two endogenous antioxidant enzymes, superoxide dismutase and catalase (40,41). Another study confirming our studies, indicates that VIP protects the heart against reperfusion injury, a common cause of oxidant injury of cardiac muscle (17). In addition to its antioxidant activity, VIP modulates inflamma1
Antioxidant enzymes
Rat
tory cell function and it has an anti-inflammatory activity (31,36). Many studies have implicated that oxygen-derived molecules (free radicals) are the mediators of reperfusion injury in a variety of tissues, including skeletal muscle (7,10,11,14,20,34). Limb ischemia occurs during many forms of vascular insufficiency, including chronic atherosclerotic occlusive disease, arterial embolism, vascular trauma and acute arterial thrombosis. In addition to disease-and-trauma-induced vascular impairment, limb ischemia is often produced electively in surgical procedures which utilize a tourniquet to provide a bloodless operative field (11,20,23,24). After correction of the ischemic event, metabolic factors tend to normalize but, paradoxically, cell damage continues. This persistent decline in cell function is indicative of ongoing muscle destruction during reperfusion. Ischemia with reperfusion may produce tissue edema, compartment syndromes, fibrotic contractures, and contractile dysfunction. However, Faust et al indicate that skeletal muscle exposed to moderately severe total ischemia is not salvaged by the simple
Requests for reprints should be addressed to Nes¸e Tunc¸el Ph.D.; Fax: 222-239 54 00.
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addition of free radical scavengers during reperfusion (10). They suggested that skeletal muscle reperfusion injury is not mediated solely by only oxygen-derived free radicals and that some mechanism/s other than free radical is responsible for the damage of skeletal muscle. In light of the knowledge presented above, VIP, thanks to its multidimensional effects, could be involved in the protection of skeletal muscle against ischemia- reperfusion injury. In the present study, we investigated the effect of VIP on the nerve-stimulated contraction and tissue oxygenation changes of the rat gastrocnemius muscle during ischemia-reperfusion and we also measured lipid peroxidation, antioxidant enzymes activities of the muscle tissue. METHOD
Twenty-four healthy male Wistar rats (200–250 g) were used in the experiments. They were anesthetised with an intraperitoneal injection of urethane (1g.kg.01 ). A heat lamp was used to maintain the body temperature at 37 { .5 7C. The left femoral vein was cannulated (PE 50 tube) for heparinized saline and VIP administration. The right femoral artery was isolated for clamping with an atraumatic microvascular clamp and the right sciatic nerve was isolated from surrounding structures for indirect stimulation (10 V, 7 ms) of the gastrocnemius muscle. A skin incision was made over the anteromedial surface of the right hindlimb, starting at the level of the ankle joint, extending upward to the inguinal ligament. The gastrocnemius muscle was freed from surrounding structures by blunt dissection. And the gastrocnemius muscle was ligated with 2/0 silk suture from its Achilles tendons connected to isometric transducer (Narco Biosystem F 2000 myograph) with a tension that corresponds to maximum contraction of each muscle and the twitch isometric contraction forces were recorded (Narco Biosystem MKIII physiograph). The muscle was covered with a pad and moistened with warmed saline throughout the experiment. Muscles were stimulated via the nerve with a platinum electrode placed under the nerve isolated from the other tissues. After a 30-min stabilization period, the optimum muscle length and the voltage required to produce maximal contraction were established for each muscle individually. Contraction and tissue oxygenation were determined every hour. Only at the time of measurement of muscle contraction and tissue oxygenation pads were removed. Contraction was calculated in terms of mN/g (milinewton/gram) wet tissue. Tissue oxygenation was monitored polarographicaly using a collodion-coated open type gold electrode placed on the muscle (Tacussel PRG-DEL unite Ampero metrique). This system has three electrodes, designated reference (saturated calomel electrode), auxiliary (Pt electrode) and working (collodian-coated open type gold electrode). The gold electrode was implanted in the previously prepared musculus gastrocnemius, without damaging the cells and without obstructing the flow of blood. Reference and auxiliary electrodes were placed subcutaneously. After the electrodes had been placed, the incision was closed with clips to prevent drying, the electrodes were covered with salinesoaked gauzes. The experiments were carried out in four groups.
Group 2: Ischemic Group (n Å 5) The experimental gastrocnemius muscle was subjected to 4 h of ischemia by occlusion of the femoral arter with a microvascular clamp. Following 4 h of ischemic period the rats were killed. Group 3: Ischemic reperfusion group (n Å 5 ) . After 4 h of ischemia the clamp was removed and the muscle was reperfused by the animal’s native circulation. Reperfusion of the muscle was confirmed by measuring the tissue oxygenation of gastrocnemius muscle and the rats were allowed to survive for 4 h. Group 4:Ischemia-Reperfusion / VIP groups ( n Å 6 ) . After 4 h of ischemia VIP was administered ( 25 ng kg 01 ) via bolus iv injection through the left femoral vein catheter just prior to the clamp was removed and the rats were allowed to survive for 4 h. At the end of each experimental protocol just after the right gastrocnemius muscle was dissected the rats were killed by cervical dislocation while still anesthetized. The muscle samples were stored at 0807C for determination of superoxide dismutase, malondialdehyde and catalase enzyme levels. Since the duration of experimental protocol of groups 3 and 4 lasted approximately 9 h, group 1 and 2 protocols were adjusted to last for 9h for the sake of time matching among the groups. Thus, the effect of time on measuring enzyme activity could be eliminated. Preparations of Muscle Samples for Determination of Antioxidant Enzyme Activites Gastrocnemius muscle tissues were homogenized in 10 volume of ice cold 50 mM potassium phosphate buffer pH 7.4. These tissue extracts were then subjected to ultrasonication (15 s) followed by centrifugation (20.000 xg 20 min). Catalase was measured by polarographically, superoxide dismutase, malondialdehyde and protein were measured by spectrophotometrically in the supernatant. Superoxide Dismutase Determination Superoxide dismutase was determined by the modified spectrophotometric method (21). This method depends on quercetin oxidation at pH 9.2. Inhibition of quercetin oxidation was monitored at 406 nm on a Shimadzu 160 A Spectrophotometer. Superoxide dismutase activity was calculated per mg of protein. Catalase Determination Catalase activity was determined by a polarographic method (12). Briefly, a carbon-paste electrode prepared with samples was used as the working electrode of the polarograph (Tacussel model PRG-5) which monitored oxygen produced by the enzymatic disappearance of hydrogen peroxide (42). Catalase activity was calculated in terms of mmol of hydrogen peroxide consumed/mg of protein/min. Malondialdehyde (MDA) Determination Malondialdehyde was determined spectrophotometrically using thiobarbituric acid test, MDA levels were expressed as nmoles/mg muscle tissue (32). Protein was determined by the Lowry method (25).
Group 1: Controls (Sham operated) (n Å 8) The following procedures were applied for groups 2, 3 and 4. After a surgical operation and stabilization period (30 min) muscle contraction and tissue oxygenation were determined in every hour and the rats were let to survive for 8 h.
Data Analysis All data were expressed as the mean { SE. Statistical analysis was performed by using one-way ANOVA and Duncan’s multiple range test.
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Figure 1 shows the effect of ischemia, ischemia- reperfusion and administration of VIP on twitch isometric contractile force of gastrocnemius muscle. Ischemia caused significant decrease in the muscle contractile force within the first hours, F ( 4, 20 ) Å 30.25, p õ 0.01. Reperfusion of muscle increased the muscle contractile force gradually to the baseline levels F ( 8, 36 ) Å 4.48, p õ 0.01. VIP injection on the onset of the reperfusion immediately increased muscle contraction, F ( 8, 45 ) Å 11.05, p õ 0.01. Moreover, muscle contractions are significantly higher than not only the baseline values but also the values of ischemia-reperfusion group, F ( 3, 20 ) Å 7.73, p õ 0.01 for 5.h, F ( 3, 20 ) Å 9.32, p õ 0.01 for 6.h. F ( 3, 20 ) Å 12.39, p õ 0.01 for 7.h. and F ( 3, 20 ) Å 12, p õ 0.01 for 8.h. The effect of ischemia, ischemia-reperfusion and VIP administration on gastrocnemius muscle oxygenation is shown in Fig. 2. Ischemia caused significant decrease in the gastrocnemius muscle oxygenation within the first hour, F(4, 20) Å 133.87, p õ 0.01 and reperfusion restored the oxygenation immediately to the baseline level, F (7, 32) Å 73.88, p õ 0.01. VIP treatment prior to reperfusion increased muscle tissue oxygenation significantly higher than either values of baseline F(7, 32) Å 76.24,
271 p õ 0.01 or reperfusion within first 2 h of reperfusion period, F(3, 19) Å 26.97, p õ 0.01 and F (3, 19) Å 62.25, p õ 0.01 respectively.While gastrocnemius muscle superoxide dismutase activity significantly decreased in ischemic period, superoxide dismutase activity significantly increased during reperfusion (Fig. 3). In VIP administered group, superoxide dismutase activity of tissue muscles is identical to the control levels (Fig. 3), F(3, 16) Å 38.86, p õ 0.01. Catalase activity of skeletal muscle changed at only reperfusion period (Fig.4).Reperfusion of ischemic skeletal muscle caused significant increase in catalase activity of tissue and catalase activity returned to the control level when VIP administered, F (3, 16) Å 9.01, p õ 0.01. Neither ischemia nor reperfusion affected gastrocnemius muscle MDA levels (Fig. 5). DISCUSSION
In the present study, 4 h of complete normothermic ischemia in the rat hind limb appeared not to produce extensive and irreversible damage and it is possible to salvage by reperfusion. This conclusion is based on findings of muscle contractile force, tissue oxygenation and MDA levels.The severity of ischemia produced by 4 h of complete blood flow interruption is shown by the significant reduction of muscle oxygenation from base-
FIG. 1: Twitch isometric contractions of the rat gastrocnemius muscle, F (4, 20) Å 30.25, p õ 0.01 for the ischemic group, F(8, 36) Å 4.48,p õ 0.01 for the reperfusion group and F (8, 45) Å 11.05, p õ 0.01 for the VIP group and F (3, 20) Å 7.73, p õ 0.01 for 5.h, F (3, 20) Å 9.32, p õ 0.01 for 6.h, F(3, 20) Å 12.39,p õ 0.01 for 7.h and F(3, 20) Å 12.0, p õ 0.01 for 8.h of the VIP group.Control n Å 8, Ischemia n Å 5, Ischemiareperfusion n Å 5, Ischemia-reperfusion/VIP n Å 6, Data points: {SE.
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FIG. 2: Oxygenation of gastrocnemius muscle, F (4, 20) Å 133.87, p õ 0.01 for the ischemic group, F(7, 32) Å 73.88, p õ 0.01 the reperfusion group and F (7, 32) Å 76.24, p õ 0.01 for the VIP group, and F(3, 19) Å 26.97, p õ 0.01 and F(3, 19) Å 62.25, p õ 0.01 for within the VIP group. Control n Å 8, Ischemia n Å 5, Ischemia- reperfusion n Å 5, Ischemia-reperfusion/VIP n Å 6, Data points: {SE.
line value of 50 – 60mmHg to 5 – 10 mmHg using muscle pO2 monitor. Restoration of blood supply is confirmed by a rise in the muscle pO2 to near baseline values. Muscle contractile force is also reduced significantly during ischemia but reperfusion can restore contractility of ischemic skeletal muscle gradually. Measurement of muscle contraction provides a functional assessment of the ischemic-reperfused muscle, whereas determination of tissue MDA provides a complementary index of structural oxidative injury of the cell membrane. The increase in MDA suggests increased lipid peroxidation initiated by free radical reactions ( 9 ) . Lipid peroxidation is potentially a very damaging process to the organized structure and function of membranes. In the present study, superoxide dismutase and catalase activity significantly increased during reperfusion period of 4 h ischemic skeletal muscle. It indicates that superoxide anions and hydrogen peroxide increased in the tissue during reperfusion. It is generally known that the enhanced levels of individual reactive oxygen species may lead to increased activities of antioxidant enzymes ( 19 ) . In spite of the increased superoxide anions and hydrogen peroxide, MDA level, as an index of lipid peroxidation, did not change significantly during reperfusion. This can be explained by enhanced superoxide dismutase and catalase activity. It is reasonable to suggest that increased antioxidant enzymes activity were sufficient to quench superoxide anions and hydrogen peroxide. The significance of skeletal muscle reperfusion injury mediated by oxygen-derived free radicals is still controversial (5,29). The role of oxygen free radicals in ischemia and reperfusion injury of skeletal muscle has not been well defined, partly because
of the relative resistance of this tissue to normothermic ischemia (15,33). Belkin et al demonstrated that no additional cellular injury occured during the reperfusion interval of 4 h ischemic skeletal muscle (5). It was concluded that the duration of ischemia rather than reperfusion is the major determinant of skeletal muscle injury after vascular occlusion (5,23). On the other hand, some studies have reported that 3-5 h ischemia followed by reperfusion cause irreversible damage in the skeletal muscles of various species including rats (3,7–11,16,18,22,23,33,34). Those studies indicate that xanthine oxidase-derived oxygen radicals and increased superoxide anions production by activated inflammatory cells may contribute to the injury of ischemia reperfusion.In addition, polymorphonuclear leukocytes associated injury that is mediated by mechanisms other than superoxide anions can ensue during reperfusion. However, our study confirms the others suggestion that rat skeletal muscle can resist under 4 h of complete normothermic ischemia and physiological reperfusion. Our study also indicates that a certain quantities of oxygen free radicals are produced during reperfusion but they are quenched by intracellular free radical scavenging enzymes and thus skeletal muscle tissue can be protected from lipid peroxidation and developing of dysfunction. VIP treatment at the onset of reperfusion significantly increased skeletal muscle contraction force and tissue oxygenation even higher than baseline and reperfusion value. VIP also normalizes the increased superoxide dismutase and catalase activities of reperfused skeletal muscle. As far as we
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FIG. 3: Activity of superoxide dismutase, F(3, 16) Å 38.86, p õ 0.01. Control n Å 8, Ischemia n Å 5, Ischemia-reperfusion n Å 5, Ischemiareperfusion/VIP n Å 6, Data points: {SE.
FIG. 4: Activity of catalase(CAT), F (3, 16) Å 9.01, p õ 0.01. Control n Å 8, Ischemia n Å 5, Ischemia-reperfusion n Å 5, Ischemia-reperfusion/VIP n Å 6, Data points: {SE.
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FIG. 5: Malondialdehyde (MDA) levels of gastrocnemius muscle. Control n Å 8, Ischemia n Å 5, Ischemia-reperfusion n Å 5, Ischemia-reperfusion/VIP n Å 6, Data points: {SE.
know this is the first report presenting data on the effect of VIP on the contractile force, oxygenation and antioxidant enzyme system of ischemic-reperfused skeletal muscle of rat. The effect of VIP on antioxidant enzyme systems suggests that production of superoxide anions and hydrogen peroxide decrease in the reperfused tissue. This can be related to the antioxidant activity of VIP. In our previous studies similar results were observed that VIP protected the rat renal and retinal tissue from ischemia-reperfusion injury without any increase in the activities of two endogenous antioxidant enzymes, superoxide dismutase and catalase ( 40,41 ) . The most important point of this study is that VIP increases the contractile force and oxygenation of skeletal muscle. It has been reported that VIP, stimulating adenylate cyclase activity and cAMP formation increases glycogenolysis in hepatocytes and astrocytes (27,28,35). Increase in cAMP is the stimulation of glycogenolysis via the activation of cascade of phosphorylation leading to the activation of phosphorylase and the inhibition of glycogen synthase in tissues including skeletal muscle (13,27). Thus, it can be to suggested that VIP increasing cAMP in skeletal muscle can induce glycogenolysis. In addition, in the present study VIP increases skeletal muscle tissue oxygenation possibly because of its vasodilator action (35). Therefore, ATP synthesis in the tissue can easily be increased by VIP, which in turn increases skeletal muscle contractile force.Hence, it can be concluded VIP is localized in sciatic nerve VIP may be involved in skeletal muscle energy metabolism homeostasis and contractile force modulation (26,35). It has been reported that VIP has also positive inotropic and vasodilator actions in the isolated ischemic- reperfused rat hearts preparation ( 17 ) . Apart from ischemic-reperfused
isolated heart, other in vivo and in vitro experiments have shown that VIP possesses positive inotropic effect and increases coronary blood flow ( 2,4,35 ) . The mechanism of the positive inotropic effect of VIP has been attributed to its stimulatory effect on adenylate cyclase which concentrate intracellular cAMP that consequently leads to increase in contractile force. In general, in addition to xanthine / xanthine oxidase system, ischemic-reperfused tissue liberate substance capable of activating granulocytes and mast cells ( 3,8,38,40 ) . Mast cells by themselves are the source of the toxic oxygen molecules and other mediators responsible to induce activation of granulocytes. This would explain why allopurinol, as a xanthine oxidase inhibitor, only affords a partial protective effects in ischemic reperfused skeletal muscle, since superoxide radical would be generated in these tissues from sources other than those blocked by allopurinol i.e, activated neutrophils, mast cells and through the cyclooxygenase pathway of prostaglandine synthesis. VIP, preventing mast cell degranulation, inhibiting the release of inflammatory mediators from inflammatory cells and xanthine / xanthine oxidase derived free radical formation, and also directly scavenging hydroxyl radical and singlet oxygen would be more effective to prevent ischemia- reperfusion injury of tissues than other scavengers such as allopurinol, mannitol and desferoxamine ( 9 – 11,14,31,38). In conclusion, our study shows that VIP acting as a powerful antioxidant and preserving contractile machinery seems to be a promising endogenous peptide that can salvage the skeletal muscle from severe ischemia and reperfusion injury. It can decrease the mortality of current techniques of revascularization after prolonged limb ischemia.
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Ischemic-Reperfused Rat Skeletal Muscle: The Effect of Vasoactive Intestinal Peptide (VIP) on Contractile Force, Oxygenation and Antioxidant Enzyme Systems ¨ KSEL ALTIOKKA† NES¸E TUNC ¸ EL,* 1 SERDAR ERDEN,* KUBILAY UZUNER,* GO AND MUZAFFER TUNC ¸ EL† *Department of Physiology, Faculty of Medicine, University of Osmangazi 26040, Eskis¸ehir, Turkey †Department of Analytical Chemistry, Faculty of Pharmacy, University of Anadolu 26480, Eskis¸ehir, Turkey Received 6 August 1996; Accepted 16 October 1996 TUNC ¸ EL, N., S. ERDEN, K. UZUNER, G. ALTIOKKA AND M. TUNC ¸ EL. Ischemic-reperfused rat skeletal muscle: The effect of vasoactive intestinal peptide (VIP) on contractile force, oxygenation and antioxidant enzyme systems. PEPTIDES 18(2) 269– 275, 1997.—The effect of vasoactive intestinal peptide (VIP) on the nerve-stimulated contraction, tissue oxygenation, lipid peroxidation and antioxidant enzymes activities-superoxide dismutase and catalase was investigated in the rat gastrocnemius muscle exposed to 4 h ischemia-4hr reperfusion. Ischemia caused significant decrease in muscle contractile force, oxygenation and superoxide dismutase enzyme activity. Reperfusion of ischemic muscle increased the muscle contractile force and restored the tissue oxygenation to the baseline level. Superoxide dismutase and catalase activities of reperfused muscle increased significantly. However neither ischemia nor reperfusion affected gastrocnemius muscle malondialdehide (MDA) levels. VIP administration at the onset of reperfusion significantly increased skeletal muscle contractile force and tissue oxygenation even higher than baseline and reperfusion values. VIP also normalized the increased superoxide dismutase and catalase activities of reperfused skeletal muscle. In conclusion, VIP, acting as a powerful antioxidant and preserving contractile machinery seems to be a promising endogenous peptide that can salvage the skeletal muscle from severe ischemia-reperfusion injury. q 1997 Elsevier Science Inc. Vasoactive intestinal peptide
Ischemia-reperfusion skeletal muscle
RECENT evidence indicates that vasoactive intestinal peptide (VIP) has modulatory effect on tissue injury (31,36,37). It protects the tissues against injury due to oxidant and other toxins (31). VIP has been found to possess the ability to scavenge oxygen free radicals. VIP can quench in vivo hydroxyl radical formation and can directly scavenge singlet oxygen (17,30). VIP also has a potent protective activity against injury triggerred by xanthine/xanthine oxidase system and inhibits superoxide radical formation by inflammatory cells (6,31,36). In our previous studies, VIP administration in experimental hemorrhagic shock models increased the survival rate and protected the renal tissue from the injury of reperfusion (1,39). In our subsequent studies VIP protected the renal and retinal tissue from ischemia reperfusion injury without any increase in the activity of two endogenous antioxidant enzymes, superoxide dismutase and catalase (40,41). Another study confirming our studies, indicates that VIP protects the heart against reperfusion injury, a common cause of oxidant injury of cardiac muscle (17). In addition to its antioxidant activity, VIP modulates inflamma1
Antioxidant enzymes
Rat
tory cell function and it has an anti-inflammatory activity (31,36). Many studies have implicated that oxygen-derived molecules (free radicals) are the mediators of reperfusion injury in a variety of tissues, including skeletal muscle (7,10,11,14,20,34). Limb ischemia occurs during many forms of vascular insufficiency, including chronic atherosclerotic occlusive disease, arterial embolism, vascular trauma and acute arterial thrombosis. In addition to disease-and-trauma-induced vascular impairment, limb ischemia is often produced electively in surgical procedures which utilize a tourniquet to provide a bloodless operative field (11,20,23,24). After correction of the ischemic event, metabolic factors tend to normalize but, paradoxically, cell damage continues. This persistent decline in cell function is indicative of ongoing muscle destruction during reperfusion. Ischemia with reperfusion may produce tissue edema, compartment syndromes, fibrotic contractures, and contractile dysfunction. However, Faust et al indicate that skeletal muscle exposed to moderately severe total ischemia is not salvaged by the simple
Requests for reprints should be addressed to Nes¸e Tunc¸el Ph.D.; Fax: 222-239 54 00.
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addition of free radical scavengers during reperfusion (10). They suggested that skeletal muscle reperfusion injury is not mediated solely by only oxygen-derived free radicals and that some mechanism/s other than free radical is responsible for the damage of skeletal muscle. In light of the knowledge presented above, VIP, thanks to its multidimensional effects, could be involved in the protection of skeletal muscle against ischemia- reperfusion injury. In the present study, we investigated the effect of VIP on the nerve-stimulated contraction and tissue oxygenation changes of the rat gastrocnemius muscle during ischemia-reperfusion and we also measured lipid peroxidation, antioxidant enzymes activities of the muscle tissue. METHOD
Twenty-four healthy male Wistar rats (200–250 g) were used in the experiments. They were anesthetised with an intraperitoneal injection of urethane (1g.kg.01 ). A heat lamp was used to maintain the body temperature at 37 { .5 7C. The left femoral vein was cannulated (PE 50 tube) for heparinized saline and VIP administration. The right femoral artery was isolated for clamping with an atraumatic microvascular clamp and the right sciatic nerve was isolated from surrounding structures for indirect stimulation (10 V, 7 ms) of the gastrocnemius muscle. A skin incision was made over the anteromedial surface of the right hindlimb, starting at the level of the ankle joint, extending upward to the inguinal ligament. The gastrocnemius muscle was freed from surrounding structures by blunt dissection. And the gastrocnemius muscle was ligated with 2/0 silk suture from its Achilles tendons connected to isometric transducer (Narco Biosystem F 2000 myograph) with a tension that corresponds to maximum contraction of each muscle and the twitch isometric contraction forces were recorded (Narco Biosystem MKIII physiograph). The muscle was covered with a pad and moistened with warmed saline throughout the experiment. Muscles were stimulated via the nerve with a platinum electrode placed under the nerve isolated from the other tissues. After a 30-min stabilization period, the optimum muscle length and the voltage required to produce maximal contraction were established for each muscle individually. Contraction and tissue oxygenation were determined every hour. Only at the time of measurement of muscle contraction and tissue oxygenation pads were removed. Contraction was calculated in terms of mN/g (milinewton/gram) wet tissue. Tissue oxygenation was monitored polarographicaly using a collodion-coated open type gold electrode placed on the muscle (Tacussel PRG-DEL unite Ampero metrique). This system has three electrodes, designated reference (saturated calomel electrode), auxiliary (Pt electrode) and working (collodian-coated open type gold electrode). The gold electrode was implanted in the previously prepared musculus gastrocnemius, without damaging the cells and without obstructing the flow of blood. Reference and auxiliary electrodes were placed subcutaneously. After the electrodes had been placed, the incision was closed with clips to prevent drying, the electrodes were covered with salinesoaked gauzes. The experiments were carried out in four groups.
Group 2: Ischemic Group (n Å 5) The experimental gastrocnemius muscle was subjected to 4 h of ischemia by occlusion of the femoral arter with a microvascular clamp. Following 4 h of ischemic period the rats were killed. Group 3: Ischemic reperfusion group (n Å 5 ) . After 4 h of ischemia the clamp was removed and the muscle was reperfused by the animal’s native circulation. Reperfusion of the muscle was confirmed by measuring the tissue oxygenation of gastrocnemius muscle and the rats were allowed to survive for 4 h. Group 4:Ischemia-Reperfusion / VIP groups ( n Å 6 ) . After 4 h of ischemia VIP was administered ( 25 ng kg 01 ) via bolus iv injection through the left femoral vein catheter just prior to the clamp was removed and the rats were allowed to survive for 4 h. At the end of each experimental protocol just after the right gastrocnemius muscle was dissected the rats were killed by cervical dislocation while still anesthetized. The muscle samples were stored at 0807C for determination of superoxide dismutase, malondialdehyde and catalase enzyme levels. Since the duration of experimental protocol of groups 3 and 4 lasted approximately 9 h, group 1 and 2 protocols were adjusted to last for 9h for the sake of time matching among the groups. Thus, the effect of time on measuring enzyme activity could be eliminated. Preparations of Muscle Samples for Determination of Antioxidant Enzyme Activites Gastrocnemius muscle tissues were homogenized in 10 volume of ice cold 50 mM potassium phosphate buffer pH 7.4. These tissue extracts were then subjected to ultrasonication (15 s) followed by centrifugation (20.000 xg 20 min). Catalase was measured by polarographically, superoxide dismutase, malondialdehyde and protein were measured by spectrophotometrically in the supernatant. Superoxide Dismutase Determination Superoxide dismutase was determined by the modified spectrophotometric method (21). This method depends on quercetin oxidation at pH 9.2. Inhibition of quercetin oxidation was monitored at 406 nm on a Shimadzu 160 A Spectrophotometer. Superoxide dismutase activity was calculated per mg of protein. Catalase Determination Catalase activity was determined by a polarographic method (12). Briefly, a carbon-paste electrode prepared with samples was used as the working electrode of the polarograph (Tacussel model PRG-5) which monitored oxygen produced by the enzymatic disappearance of hydrogen peroxide (42). Catalase activity was calculated in terms of mmol of hydrogen peroxide consumed/mg of protein/min. Malondialdehyde (MDA) Determination Malondialdehyde was determined spectrophotometrically using thiobarbituric acid test, MDA levels were expressed as nmoles/mg muscle tissue (32). Protein was determined by the Lowry method (25).
Group 1: Controls (Sham operated) (n Å 8) The following procedures were applied for groups 2, 3 and 4. After a surgical operation and stabilization period (30 min) muscle contraction and tissue oxygenation were determined in every hour and the rats were let to survive for 8 h.
Data Analysis All data were expressed as the mean { SE. Statistical analysis was performed by using one-way ANOVA and Duncan’s multiple range test.
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Figure 1 shows the effect of ischemia, ischemia- reperfusion and administration of VIP on twitch isometric contractile force of gastrocnemius muscle. Ischemia caused significant decrease in the muscle contractile force within the first hours, F ( 4, 20 ) Å 30.25, p õ 0.01. Reperfusion of muscle increased the muscle contractile force gradually to the baseline levels F ( 8, 36 ) Å 4.48, p õ 0.01. VIP injection on the onset of the reperfusion immediately increased muscle contraction, F ( 8, 45 ) Å 11.05, p õ 0.01. Moreover, muscle contractions are significantly higher than not only the baseline values but also the values of ischemia-reperfusion group, F ( 3, 20 ) Å 7.73, p õ 0.01 for 5.h, F ( 3, 20 ) Å 9.32, p õ 0.01 for 6.h. F ( 3, 20 ) Å 12.39, p õ 0.01 for 7.h. and F ( 3, 20 ) Å 12, p õ 0.01 for 8.h. The effect of ischemia, ischemia-reperfusion and VIP administration on gastrocnemius muscle oxygenation is shown in Fig. 2. Ischemia caused significant decrease in the gastrocnemius muscle oxygenation within the first hour, F(4, 20) Å 133.87, p õ 0.01 and reperfusion restored the oxygenation immediately to the baseline level, F (7, 32) Å 73.88, p õ 0.01. VIP treatment prior to reperfusion increased muscle tissue oxygenation significantly higher than either values of baseline F(7, 32) Å 76.24,
271 p õ 0.01 or reperfusion within first 2 h of reperfusion period, F(3, 19) Å 26.97, p õ 0.01 and F (3, 19) Å 62.25, p õ 0.01 respectively.While gastrocnemius muscle superoxide dismutase activity significantly decreased in ischemic period, superoxide dismutase activity significantly increased during reperfusion (Fig. 3). In VIP administered group, superoxide dismutase activity of tissue muscles is identical to the control levels (Fig. 3), F(3, 16) Å 38.86, p õ 0.01. Catalase activity of skeletal muscle changed at only reperfusion period (Fig.4).Reperfusion of ischemic skeletal muscle caused significant increase in catalase activity of tissue and catalase activity returned to the control level when VIP administered, F (3, 16) Å 9.01, p õ 0.01. Neither ischemia nor reperfusion affected gastrocnemius muscle MDA levels (Fig. 5). DISCUSSION
In the present study, 4 h of complete normothermic ischemia in the rat hind limb appeared not to produce extensive and irreversible damage and it is possible to salvage by reperfusion. This conclusion is based on findings of muscle contractile force, tissue oxygenation and MDA levels.The severity of ischemia produced by 4 h of complete blood flow interruption is shown by the significant reduction of muscle oxygenation from base-
FIG. 1: Twitch isometric contractions of the rat gastrocnemius muscle, F (4, 20) Å 30.25, p õ 0.01 for the ischemic group, F(8, 36) Å 4.48,p õ 0.01 for the reperfusion group and F (8, 45) Å 11.05, p õ 0.01 for the VIP group and F (3, 20) Å 7.73, p õ 0.01 for 5.h, F (3, 20) Å 9.32, p õ 0.01 for 6.h, F(3, 20) Å 12.39,p õ 0.01 for 7.h and F(3, 20) Å 12.0, p õ 0.01 for 8.h of the VIP group.Control n Å 8, Ischemia n Å 5, Ischemiareperfusion n Å 5, Ischemia-reperfusion/VIP n Å 6, Data points: {SE.
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FIG. 2: Oxygenation of gastrocnemius muscle, F (4, 20) Å 133.87, p õ 0.01 for the ischemic group, F(7, 32) Å 73.88, p õ 0.01 the reperfusion group and F (7, 32) Å 76.24, p õ 0.01 for the VIP group, and F(3, 19) Å 26.97, p õ 0.01 and F(3, 19) Å 62.25, p õ 0.01 for within the VIP group. Control n Å 8, Ischemia n Å 5, Ischemia- reperfusion n Å 5, Ischemia-reperfusion/VIP n Å 6, Data points: {SE.
line value of 50 – 60mmHg to 5 – 10 mmHg using muscle pO2 monitor. Restoration of blood supply is confirmed by a rise in the muscle pO2 to near baseline values. Muscle contractile force is also reduced significantly during ischemia but reperfusion can restore contractility of ischemic skeletal muscle gradually. Measurement of muscle contraction provides a functional assessment of the ischemic-reperfused muscle, whereas determination of tissue MDA provides a complementary index of structural oxidative injury of the cell membrane. The increase in MDA suggests increased lipid peroxidation initiated by free radical reactions ( 9 ) . Lipid peroxidation is potentially a very damaging process to the organized structure and function of membranes. In the present study, superoxide dismutase and catalase activity significantly increased during reperfusion period of 4 h ischemic skeletal muscle. It indicates that superoxide anions and hydrogen peroxide increased in the tissue during reperfusion. It is generally known that the enhanced levels of individual reactive oxygen species may lead to increased activities of antioxidant enzymes ( 19 ) . In spite of the increased superoxide anions and hydrogen peroxide, MDA level, as an index of lipid peroxidation, did not change significantly during reperfusion. This can be explained by enhanced superoxide dismutase and catalase activity. It is reasonable to suggest that increased antioxidant enzymes activity were sufficient to quench superoxide anions and hydrogen peroxide. The significance of skeletal muscle reperfusion injury mediated by oxygen-derived free radicals is still controversial (5,29). The role of oxygen free radicals in ischemia and reperfusion injury of skeletal muscle has not been well defined, partly because
of the relative resistance of this tissue to normothermic ischemia (15,33). Belkin et al demonstrated that no additional cellular injury occured during the reperfusion interval of 4 h ischemic skeletal muscle (5). It was concluded that the duration of ischemia rather than reperfusion is the major determinant of skeletal muscle injury after vascular occlusion (5,23). On the other hand, some studies have reported that 3-5 h ischemia followed by reperfusion cause irreversible damage in the skeletal muscles of various species including rats (3,7–11,16,18,22,23,33,34). Those studies indicate that xanthine oxidase-derived oxygen radicals and increased superoxide anions production by activated inflammatory cells may contribute to the injury of ischemia reperfusion.In addition, polymorphonuclear leukocytes associated injury that is mediated by mechanisms other than superoxide anions can ensue during reperfusion. However, our study confirms the others suggestion that rat skeletal muscle can resist under 4 h of complete normothermic ischemia and physiological reperfusion. Our study also indicates that a certain quantities of oxygen free radicals are produced during reperfusion but they are quenched by intracellular free radical scavenging enzymes and thus skeletal muscle tissue can be protected from lipid peroxidation and developing of dysfunction. VIP treatment at the onset of reperfusion significantly increased skeletal muscle contraction force and tissue oxygenation even higher than baseline and reperfusion value. VIP also normalizes the increased superoxide dismutase and catalase activities of reperfused skeletal muscle. As far as we
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FIG. 3: Activity of superoxide dismutase, F(3, 16) Å 38.86, p õ 0.01. Control n Å 8, Ischemia n Å 5, Ischemia-reperfusion n Å 5, Ischemiareperfusion/VIP n Å 6, Data points: {SE.
FIG. 4: Activity of catalase(CAT), F (3, 16) Å 9.01, p õ 0.01. Control n Å 8, Ischemia n Å 5, Ischemia-reperfusion n Å 5, Ischemia-reperfusion/VIP n Å 6, Data points: {SE.
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FIG. 5: Malondialdehyde (MDA) levels of gastrocnemius muscle. Control n Å 8, Ischemia n Å 5, Ischemia-reperfusion n Å 5, Ischemia-reperfusion/VIP n Å 6, Data points: {SE.
know this is the first report presenting data on the effect of VIP on the contractile force, oxygenation and antioxidant enzyme system of ischemic-reperfused skeletal muscle of rat. The effect of VIP on antioxidant enzyme systems suggests that production of superoxide anions and hydrogen peroxide decrease in the reperfused tissue. This can be related to the antioxidant activity of VIP. In our previous studies similar results were observed that VIP protected the rat renal and retinal tissue from ischemia-reperfusion injury without any increase in the activities of two endogenous antioxidant enzymes, superoxide dismutase and catalase ( 40,41 ) . The most important point of this study is that VIP increases the contractile force and oxygenation of skeletal muscle. It has been reported that VIP, stimulating adenylate cyclase activity and cAMP formation increases glycogenolysis in hepatocytes and astrocytes (27,28,35). Increase in cAMP is the stimulation of glycogenolysis via the activation of cascade of phosphorylation leading to the activation of phosphorylase and the inhibition of glycogen synthase in tissues including skeletal muscle (13,27). Thus, it can be to suggested that VIP increasing cAMP in skeletal muscle can induce glycogenolysis. In addition, in the present study VIP increases skeletal muscle tissue oxygenation possibly because of its vasodilator action (35). Therefore, ATP synthesis in the tissue can easily be increased by VIP, which in turn increases skeletal muscle contractile force.Hence, it can be concluded VIP is localized in sciatic nerve VIP may be involved in skeletal muscle energy metabolism homeostasis and contractile force modulation (26,35). It has been reported that VIP has also positive inotropic and vasodilator actions in the isolated ischemic- reperfused rat hearts preparation ( 17 ) . Apart from ischemic-reperfused
isolated heart, other in vivo and in vitro experiments have shown that VIP possesses positive inotropic effect and increases coronary blood flow ( 2,4,35 ) . The mechanism of the positive inotropic effect of VIP has been attributed to its stimulatory effect on adenylate cyclase which concentrate intracellular cAMP that consequently leads to increase in contractile force. In general, in addition to xanthine / xanthine oxidase system, ischemic-reperfused tissue liberate substance capable of activating granulocytes and mast cells ( 3,8,38,40 ) . Mast cells by themselves are the source of the toxic oxygen molecules and other mediators responsible to induce activation of granulocytes. This would explain why allopurinol, as a xanthine oxidase inhibitor, only affords a partial protective effects in ischemic reperfused skeletal muscle, since superoxide radical would be generated in these tissues from sources other than those blocked by allopurinol i.e, activated neutrophils, mast cells and through the cyclooxygenase pathway of prostaglandine synthesis. VIP, preventing mast cell degranulation, inhibiting the release of inflammatory mediators from inflammatory cells and xanthine / xanthine oxidase derived free radical formation, and also directly scavenging hydroxyl radical and singlet oxygen would be more effective to prevent ischemia- reperfusion injury of tissues than other scavengers such as allopurinol, mannitol and desferoxamine ( 9 – 11,14,31,38). In conclusion, our study shows that VIP acting as a powerful antioxidant and preserving contractile machinery seems to be a promising endogenous peptide that can salvage the skeletal muscle from severe ischemia and reperfusion injury. It can decrease the mortality of current techniques of revascularization after prolonged limb ischemia.
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