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A new quantitative spectrophotometric assay of ischemia-reperfusion injury in skeletal muscle
SCIENTIFIC PAPERS
A New Quantitative Spectrophotometric Assay of Ischemia-Reperfusion Injury in Skeletal Muscle* Michael Belkin, MD, Richard D. Brown, BA, J. Gordon Wright, MO, Wayne W. LaMorte, MD, PhD, Robert W. Hobson II, MD, Boston,Massachusetts
Characterization of ischemia-reperfusion injury of
skeletal muscle remains poorly defined. A new quantitative assay to measure ischemic skeletal muscle injury is described a n d validated in a rat hindlimb model. This biochemical spectrophotometric technique measures triphenyhetrazolium chloride reduction in ischemic muscle: The reduction assay demonstrated significant injury after 3 hours of ischemia (25.4 + 9.7 percent of control activity; p < 0 . 0 5 ) . More severe injury occurred after 4 or more hours (less than 3 percent of control activity; p < 0 . 0 5 ) . This assay is an objective and quantitative method for characterizing ischemia-reperfusion injury.
cute ischemia ofthelower extremity continues to have A significant morbidity and mortality [1-3]. Despite the universal recognition of this problem, characterization and quantification of the ischemia-reperfusion injury of skeletal muscle remains poorly defined [4]. A variety of experimental models have been developed to measure this injury based on indirect measures of skeletal muscle injury, including measurements of membrane potential [5-6], intracellular calcium ion flux [7], and vascular permeability changes [8-10]. Planimetric measurement of infarction in gross muscle segments based on triphenyltetrazolium chloride reduction has been validated as an accurate indicator of injury in both ischemic skeletal and cardiac muscle [11-13]. This technique depends on the ability of the intact mitochondrial reduction-oxidation enzyme systems of muscle to convert the colorless triphenyltetrazolium chloride to a red formazan dye. Since this planimetric method requires recognition of color differences between normal and infarcted muscle, the technique is subjective in its determination of the magnitude of injury. Furthermore, these techniques are not applicable in a small animal model. To overcome these disadvanFrom the Department of Surgical Research, Boston University School of Medicine, Boston, Massachusetts. *This work has been designated the 1988 Peter B. Samuels Prize Essay by a Resident. Dr. Michael Belkin is the recipient of the award. Requests for reprints should be addressed to Michael Belkin, MD, Department of Surgery, Brigham and Women's Hospital, 75 Francis Street, Boston, Massachusetts 02115. Presented at the 16th Annual Meeting of the Society for Clinical Vascular Surgery, Maui, Hawaii, April 6-10, 1988.
tages and obtain a more quantitative method, we developed a biochemical spectrophotometric assay of triphenyltetrazolium chloride reduction and have validated it in a rat hindlimb model of skeletal muscle ischemia. MATERIAL AND METHODS Adult male Wistar rats weighing 250 to 300 g were anesthetized by intraperitoneal injection of pentobarbital sodium (0.055 mg/g) and supplemented as necessary. One hindlimb was randomly selected for ischemia, with the contralateral limb Serving as a control. A no. 18 rubber band (Plymouth Rubber Band, Canton, MA) was wrapped eight times around a plastic cylindrical applicator. The hindlimb was then placed through the cylinder and the rubber band positioned onto the proximal thigh to produce complete ischemia. To validate that the rat hindlimb tourniquet results in complete ischemia and that reperfusion results when the rubber band is removed, perfusion fluorometry studies were undertaken [14]. Five anesthesized rats underwent external jugular vein cannulation with polyethylene-50 tubing. After application of a rubber band tourniquet to one hindlimb, rats were injected with a 6 mg/kg bolus of intravenous fluorescein (Akorn, Abita Springs, LA). The gastrocnemius and soleus muscles were exposed bilaterally and perfusion was measured every 15 minutes with a fiberoptic perfusion fluorometer (Diversatronics, Broomhall, PA). Additional boluses of fluorescein (3 mg/kg) were administered at 2 hours, 45 minutes and 5 hours, 45 minutes of ischemia. After 6 hours of ischemia, the rubber band tourniquets were removed and reperfusion was measured for 1 hour. The triphenyltetrazolium chloride-based assay of skeletal muscle ischemia developed in this study is modified from a previously described assay for succinic dehydrogenase in rat liver [15]. After graded ischemic intervals varying from 1 to 6 hours, the gastrocnemiUs and soleus muscles were excised from both the ischemic and control limbs and were rinsed in ice-cold Ringer's lactate solution. The muscle from the ischemic and control limbs were treated in an identical fashion and all manipulations were performed on ice. The muscle tissue was dissected free of blood vessels, nerves, and fascia. The tissue from each hindlimb was then weighed and homogenized for 90 seconds in 3 ml of 0.25 M sucrose using an Ultra-Turrax | homogenizer (IKA-Works, Cincinnati, OH). Additional sucrose was then added to make a 20 percent homogenate by weight. The homogenate was filtered through a fine stainless steel mesh to remove any remaining fragments of fascia. Protein content of the homogenate was determined by the method of Lowry et al [16]. A 1 ml aliquot of homogenate was then mixed with 1
THE AMERICAN JOURNAL OF SURGERY
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Figure 1. Fluorescein perfusion fluorometry studies of control and ischemic hindlimbs. Mean fluorescence values for five animals are presented. Complete ischemia was demonstrated in the ischemic limbs until the tourniquet was removed at 6 hours, after which reperfusion hyperemia occurred.
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Figure 3. The mean triphenyltetrazolium chloride reduction activities of ischemic limbs 4- 1 standard error are expressed as percentages of control limb activity. Significant injury appeared at 3 hours of ischemia, with further injury at 4, 5, and 6 hours (p <0.05).
ml of 0.15 percent triphenyltetrazolium chloride (Sigma, St. Louis, MO) in 0.033 M phosphate buffer (dibasic sodium phosphate, pH 7.4). Reactions were performed in triplicate. The reaction mixture was incubated at 39 ~C in an agitating water bath for 1 hour. The reaction was then stopped and the colored formazan dye extracted into solution by adding 4 ml of acetone to each reaction tube and vortexing. The reaction tubes were centrifuged for 10 minutes at 1,500 rpm, and the absorbance of the clear red supernatants were measured at 485 nm in a spectrophotometer (Gilford, Oberlin, OH). The absorbances of the reaction triplicates were averaged and unreacted reagent blanks were subtracted to arrive at a final absorption for each muscle sample. Comparison of these absorption values to a standard curve of known reduced triphenyltetrazolium chloride concentrations allowed determination of the micrograms of triphenyltetrazolium chloride reduced per hour per milligram of muscle protein. The activity of each ischemic limb was compared with the contralateral control limb to express the ischemic limb activity as a percentage of the control limb. A series of 42 rats were divided into 7 groups of 6 84
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Figure 2. The mean triphenyltetrazollum chloride ('rrc) reduction activities of the control muscles (4- 1 standard error from each ischemic interval (I) are compared. No significant differences were found among the groups (p = 0.74 by one-way analysis of variance).
animals each. Each group was subjected to an ischemic interval varying from 0 to 6 hours. In those animals subjected to sham ischemia, one limb was randomly selected as experimental and the other as a control for the purpose of comparison. To ensure that the length of the anesthetic period and the various ischemic intervals did not affect the ability of the control muscles to reduce triphenyltetrazolium chloride, the absolute activity of the control muscles (in micrograms of triphenyltetrazolium chloride reduced per hour per mg protein) in each of the seven groups was compared. All comparisons between multiple groups were evaluated by a one-way analysis of variance. Where significant variation was noted, a Duncan's multiple range test (~ = 0.05) was used to identify differences between groups. RESULTS Figure 1 shows the mean measured fluorescence for the five ischemic and control limbs studied. In each control limb, perfusion was sustained throughout the experiment, with increased fluorescence noted with each added bolus of intravenous fluorescein (at 2 hours, 45 minutes and 5 hours, 45 minutes). Conversely, the ischemic limb in each animal demonstrated complete ischemia with no fluorescence until the rubber band tourniquet was removed after 6 hours of ischemia. At that point, reperfusion hyperemia was apparent with fluorescence exceeding that of the control limb. As demonstrated in Figure 2, there was no difference between the control limb activities of the animals subjected to different ischemic intervals (p = 0.74). These results rule out an unrecognized effect of the anesthetic interval on the triphenyltetrazolium chloride reduction activity of muscle. The muscle injury with graded ischemia, as determined by the spectrophotometric triphenyltetrazolium chloride reduction assay, is shown in Figure 3. All ischemic limbs are expressed as the mean percentage of control limb 4- 1 standard error. In animals not subjected to ischemia, experimental limbs had an activity equal to 101 4- 4 percent of the control limbs. A significant variation in triphenyltetrazolium chloride reduction was noted be-
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tween the various ischemic intervals (p <0.0001). Activity was sustained over 1 and 2 hours of ischemia where activities were 95.7 4- 4.8 percent and 84.2 4- 7.3 percent of the control activity, respectively (p >0.05). Significant muscle injury occurred after 3 hours of ischemia, with activity decreasing to 25.4 4- 9.7 percent of the control activity (p <0.05). Beyond 3 hours of ischemia, injury was more severe, with less than 3 percent of control activity demonstrated at 4, 5, and 6 hours of ischemia (p <0.05). COMMENTS The acutely ischemic extremity is one of the most common and serious problems that the vascular surgeon must manage. Depending on the length of the ischemia, significant morbidity, limb loss, and mortality can occur [1-3,17]. Haimovici [18] was the first to describe the acute revascularization syndrome, consisting of pain, edema, tenderness, myoglobinuria, hyperkalemia, and other metabolic derangements that occur when ischemia muscle is revascularized. Prevention of this syndrome and maximal limb salvage depends on an understanding of the various factors contributing to ischemia-reperfusion injury. Recent studies have indirectly implicated oxygen free radicals as important mediators in reperfusion injury of skeletal muscle [6-8,19]. Quantification of the relative importance of ischemic versus reperfusion injury and investigation of factors designed to ameliorate this injury requires an accurate, objective, and reproducible measure of skeletal muscle injury. The spectrophotometric assay of triphenyltetrazolium chloride reduction in the rat hindlimb model of skeletal muscle ischemia meets these criteria. The fluorescein perfusion studies performed in these experiments confirm the consistent and complete ischemia achieved as well as the reactive hyperemia which occurs after tourniquet removal. The use of triphenyltetrazolium chloride reduction is well established as a marker of muscle infarction. Triphenyltetrazolium chloride histochemistry was first employed clinically in 1957 by Sandritter and Jestadt [20]. The reduction-oxidation potential of triphenyltetrazolium chloride is low enough that reduction occurs at the final cytochrome oxidase of the mitochondrial electron transport chain immediately before transfer of electrons to oxygen [21]. The inability to reduce triphenyltetrazolium chloride reflects disruption of mitochondria, the depletion of essential reduction-oxidation coenzymes, or both [22]. This lack of triphenyltetrazolium chloride reduction has been extensively correlated with other measures of skeletal muscle injury. In our previous studies with the isolated canine gracilis muscle preparation [11], electron microscopic studies of muscle that failed to reduce triphenyltetrazolium chloride demonstrated a variety of abnormalities, including mitochondrial swelling, cristae disorganization, swelling of the sarcoplasmic reticulum, and sarcolemmal disruption. These ultrastructural changes identify irreversible injury and confirm that failure of muscle to reduce triphenyltetrazolium chloride is an indicator of cellular infarction. This inability has also shown a close correlation with uptake of techneti-
um 99 pyrophosphate, an additional skeletal muscle injury marker employed in the isolated gracilis preparation
[13,23]. The combination of triphenyltetrazolium chloride reduction with planimetry for macroscopic myocardial infarct mapping and quantification was introduced by Boor and Reynolds [24] in 1977 and has since been used effectively in skeletal muscle studies [9,11,19]. These planimetric techniques are somewhat subjective, however, and are difficult to employ in a small animal model. The allor-none character of tissue staining does not allow a graded measure of cellular injury. The biochemical spectrophotometric assay described in these experiments overcomes these shortcomings and establishes an objective and quantitative technique that takes advantage of the established accuracy of triphenyltetrazolium chloride reduction as a marker of ischemic injury. The spectrophotometric triphenyltetrazolium chloride assay in this rat hindlimb model demonstrates significant skeletal muscle injury first occurring after 3 hours of ischemia. Significantly greater injury occurs at 4, 5, and 6 hours of ischemia. The early appearance of ischemic damage at 3 hours is consistent with previous studies of the ischemic rat hindlimb. Stock and associates [25] studied metabolic changes in rat skeletal muscle in an ischemic hindlimb preparation. These investigators noted a profound decrease in phosphocreatine, adenosine triphosphate, and glycogen, as well as an accumulation of lactate after 3 hours of ischemia. Normalization of the metabolic pattern occurred after several hours of reperfusion. When the ischemic interval was extended to 4 or more hours, however, the abnormal metabolic pattern persisted over 5 to 6 weeks. Similarly, Makitie and Teravainen [26] combined histochemical stains for NADH diaphorase, ATPases, and phosphorylase with histologic studies to evaluate skeletal muscle ischemia in a rat hindlimb model. They reported minimal scattered areas of injury at 2 hours of ischemia, followed by large infarcted areas at 4 hours and total necrosis at 6 hours. Makitie [27] also used electron microscopy and microangiography to investigate ischemic capillary damage in the rat hindlimb. Degeneration of muscle capillaries appeared after 3 hours of ischemia, was extensive after 4 hours, and was complete after 6 hours. Chachques et al [28] used a related tetrazolium salt, nitroblue tetrazolium, in a rat hindlimb ischemia model. They measured the time to gross staining of muscle specimens incubated in a tetrazolium-containing solution. Significant prolongation of the interval to staining occurred after 3 hours of ischemia. Lee et al [7] studied adenosine triphosphate-dependent calcium ion uptake into isolated vesicles of muscle sarcoplasmic reticulum as an index for ischemic injury in a rat hindlimb model. A significant depression of calcium ion uptake was reported after 3 hours of tourniquet ischemia. The model developed and employed in these studies offers considerable advantages for characterizing the injury induced by ischemia and reperfusion. By subjecting ischemic muscle to various periods of reperfusion before assaying for triphenyltetrazolium chloride reduction, an accurate determination of the relative role of reperfusion injury should be possible. Previous studies have only of-
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fered indirect evidence of significant reperfusion injury in skeletal muscle and have not quantified the magnitude of the injury [6-8,19]. The efficacy of controlled reperfusion, hypothermia, free radical scavengers, and other pharmacologic agents in reducing ischemia-reperfusion injury may be systematically evaluated with this model. Furthermore, the possibility of direct clinical application as an indicator of human muscle viability in the setting of acute skeletal muscle ischemia also merits investigation.
REFERENCES 1. Patman R, Thompson J. Fasciotomy in peripheral vascular surgery. Arch Surg 1970; 10h 663-72. 2. Hollier L. Acute arterial occlusion. Cardiovasc Clin 1983; 13: 37-48. 3. Cambria R, Abbott W. Acute arterial thrombosis of the lower extremity. Arch Surg 1984; 119: 784-7. 4. Rutherford RB. Nutrient bed protection during lower extremity arterial reconstruction. J Vasc Surg 1987; 5: 529-34. 5. Perry MO, Shires TS, Albert SA. Cellular changes with graded ischemia and reperfusion. J Vasc Surg 1984; 1: 536-40. 6. Perry MO, Fantini G. Ischemia: profile of an enemy. Reperfusion injury of skeletal muscle. J Vasc Surg 1987; 6: 231-3. 7. Lee KR, Cronenwett JL, Shlafer M, Corpron C, Zelenock GB. Effect of superoxide dismutase plus catalase on Ca 2+ transport in ischemic and reperfused skeletal muscle. J Surg Res 1987; 42: 24-32. 8. Korthuis R J, Granger DN, Townsley MI, Taylor AE. The role of oxygen-derived free radicals in ischemia-induced increases in canine skeletal muscle vascular permeability. Circ Res 1985; 57: 599-609. 9. Wright JG, Kerr JC, Valeri CR, Hobson RW. Endothelial permeability to iodine 125 predicts skeletal muscle injury after ischemia-reperfusion. Curr Surg 1988; 45: 25-7. ! O. Hobson RW, Wright JG, Fox D, Kerr JC. Heparin reduces endothelial permeability and hydrogen ion accumulation in a canine skeletal muscle ischemia-reperfusion model. J Vase Surg 1988; 7: 585-90. 11. Blebea J, Kerr JC, Shumko JZ, Feinberg RN, Hobson RW. Quantitative histochemical evaluation of skeletal muscle ischemia and reperfusion injury. J Surg Res 1987; 43: 311-21. 12. Fishbein MC, Meerbaum S, Rit J, et al. Early phase acute myocardial infarct size quantification: validation of the triphenyl tetrazolium chloride tissue enzyme staining technique. Am Heart J 1981; 101: 593-
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600. 13. Izquierdo C, Devous MD, Nicod P, et al. A comparison of infarct identification with technetium-99m pyrophosphate and staining with triphenyl tetrazolium chloride. J Nucl Med 1983; 24: 492-7. 14. Silverman DG, Cedrone FA, Hurford WE, Bering TG, LaRossa DD. Monitoring tissue elimination of fluorescein with the perfusion fluorometer: a new method to assess capillary blood flow. Surgery 1981; 90: 409-17. 15. Kun E, Abood LG. Colorimetric estimation of succinic dehydrogenase by triphenyltetrazolium chloride. Science 1949; 109: 144-6. 16. Lowry OH, Rosebrough N J, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-75. 17. Kendrick J, Thompson BW, Read RC, et al. Arterial embolectomy in the leg. Results in a referral hospital. Am J Surg 1981; 142: 739-43. 18. Haimovici H. Late arterial embolectomy. Surgery 1959; 46: 77586. 19. Walker PM, Lindsay TF, Labbe R, Mickle DA, Romaschin AD. Salvage of skeletal muscle with free radical scavengers. J Vase Surg 1987; 5: 68-75. 20. Sandritter W, Jestadt R. Triphenyltetrazoliumchlorid (TTC) als reduktionsindikator zur makroskopischen diagnose des frischen herzinfarktes. Zentralbl Allg Path 1957; 97: 97-107. 21. Lippold HJ. Quantitative succinic dehydrogenases histochemistry. Histochemistry 1982; 76: 381-401. 22. Klein H, Puschmann P, Schaper J, Schaper W. The mechanism of the tetrazolium reaction in identifying experimental myocardial infarction. Virchows Arch 1981; 393: 287-91. 23. Labbe R, Lindsay T, Gatley R, et al. Quantitation of post ischemic skeletal muscle necrosis: histochemical and radioisotope techniques. J Surg Res 1988; 44: 45-53. 24. Boor P J, Reynolds ES. A simple planimetric method for determination of left ventricular mass and necrotic muscle mass in postmortem hearts. Am J Clin Pathol 1977; 68: 387-92. 25. Stock W, Bohn H J, Isselhard W. Metabolic changes in rat skeletal muscle after acute arterial occlusion. Vase Surg 1971; 5: 249-55. 26. Makitie J, Teravainen H. Histochemical studies of striated muscle after temporary ischemia in the rat. Acta Neuropathol 1977; 37: 101-9. 27. Makitie J. Microvasculature of rat striated muscle after temporary ischemia. Acta Neuropathol 1977; 37: 247-53. 28. Chachques JC, Fabiani JN, Perier P, Swanson J, Dreyfus G, Carpentier A. Reversibility of muscular isehemia: a histochemical quantification by the nitroblue tetrazolium test. Angiology 1985; 36: 493-9.
VOLUME 156 AUGUST 1988
A New Quantitative Spectrophotometric Assay of Ischemia-Reperfusion Injury in Skeletal Muscle* Michael Belkin, MD, Richard D. Brown, BA, J. Gordon Wright, MO, Wayne W. LaMorte, MD, PhD, Robert W. Hobson II, MD, Boston,Massachusetts
Characterization of ischemia-reperfusion injury of
skeletal muscle remains poorly defined. A new quantitative assay to measure ischemic skeletal muscle injury is described a n d validated in a rat hindlimb model. This biochemical spectrophotometric technique measures triphenyhetrazolium chloride reduction in ischemic muscle: The reduction assay demonstrated significant injury after 3 hours of ischemia (25.4 + 9.7 percent of control activity; p < 0 . 0 5 ) . More severe injury occurred after 4 or more hours (less than 3 percent of control activity; p < 0 . 0 5 ) . This assay is an objective and quantitative method for characterizing ischemia-reperfusion injury.
cute ischemia ofthelower extremity continues to have A significant morbidity and mortality [1-3]. Despite the universal recognition of this problem, characterization and quantification of the ischemia-reperfusion injury of skeletal muscle remains poorly defined [4]. A variety of experimental models have been developed to measure this injury based on indirect measures of skeletal muscle injury, including measurements of membrane potential [5-6], intracellular calcium ion flux [7], and vascular permeability changes [8-10]. Planimetric measurement of infarction in gross muscle segments based on triphenyltetrazolium chloride reduction has been validated as an accurate indicator of injury in both ischemic skeletal and cardiac muscle [11-13]. This technique depends on the ability of the intact mitochondrial reduction-oxidation enzyme systems of muscle to convert the colorless triphenyltetrazolium chloride to a red formazan dye. Since this planimetric method requires recognition of color differences between normal and infarcted muscle, the technique is subjective in its determination of the magnitude of injury. Furthermore, these techniques are not applicable in a small animal model. To overcome these disadvanFrom the Department of Surgical Research, Boston University School of Medicine, Boston, Massachusetts. *This work has been designated the 1988 Peter B. Samuels Prize Essay by a Resident. Dr. Michael Belkin is the recipient of the award. Requests for reprints should be addressed to Michael Belkin, MD, Department of Surgery, Brigham and Women's Hospital, 75 Francis Street, Boston, Massachusetts 02115. Presented at the 16th Annual Meeting of the Society for Clinical Vascular Surgery, Maui, Hawaii, April 6-10, 1988.
tages and obtain a more quantitative method, we developed a biochemical spectrophotometric assay of triphenyltetrazolium chloride reduction and have validated it in a rat hindlimb model of skeletal muscle ischemia. MATERIAL AND METHODS Adult male Wistar rats weighing 250 to 300 g were anesthetized by intraperitoneal injection of pentobarbital sodium (0.055 mg/g) and supplemented as necessary. One hindlimb was randomly selected for ischemia, with the contralateral limb Serving as a control. A no. 18 rubber band (Plymouth Rubber Band, Canton, MA) was wrapped eight times around a plastic cylindrical applicator. The hindlimb was then placed through the cylinder and the rubber band positioned onto the proximal thigh to produce complete ischemia. To validate that the rat hindlimb tourniquet results in complete ischemia and that reperfusion results when the rubber band is removed, perfusion fluorometry studies were undertaken [14]. Five anesthesized rats underwent external jugular vein cannulation with polyethylene-50 tubing. After application of a rubber band tourniquet to one hindlimb, rats were injected with a 6 mg/kg bolus of intravenous fluorescein (Akorn, Abita Springs, LA). The gastrocnemius and soleus muscles were exposed bilaterally and perfusion was measured every 15 minutes with a fiberoptic perfusion fluorometer (Diversatronics, Broomhall, PA). Additional boluses of fluorescein (3 mg/kg) were administered at 2 hours, 45 minutes and 5 hours, 45 minutes of ischemia. After 6 hours of ischemia, the rubber band tourniquets were removed and reperfusion was measured for 1 hour. The triphenyltetrazolium chloride-based assay of skeletal muscle ischemia developed in this study is modified from a previously described assay for succinic dehydrogenase in rat liver [15]. After graded ischemic intervals varying from 1 to 6 hours, the gastrocnemiUs and soleus muscles were excised from both the ischemic and control limbs and were rinsed in ice-cold Ringer's lactate solution. The muscle from the ischemic and control limbs were treated in an identical fashion and all manipulations were performed on ice. The muscle tissue was dissected free of blood vessels, nerves, and fascia. The tissue from each hindlimb was then weighed and homogenized for 90 seconds in 3 ml of 0.25 M sucrose using an Ultra-Turrax | homogenizer (IKA-Works, Cincinnati, OH). Additional sucrose was then added to make a 20 percent homogenate by weight. The homogenate was filtered through a fine stainless steel mesh to remove any remaining fragments of fascia. Protein content of the homogenate was determined by the method of Lowry et al [16]. A 1 ml aliquot of homogenate was then mixed with 1
THE AMERICAN JOURNAL OF SURGERY
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Figure 1. Fluorescein perfusion fluorometry studies of control and ischemic hindlimbs. Mean fluorescence values for five animals are presented. Complete ischemia was demonstrated in the ischemic limbs until the tourniquet was removed at 6 hours, after which reperfusion hyperemia occurred.
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Figure 3. The mean triphenyltetrazolium chloride reduction activities of ischemic limbs 4- 1 standard error are expressed as percentages of control limb activity. Significant injury appeared at 3 hours of ischemia, with further injury at 4, 5, and 6 hours (p <0.05).
ml of 0.15 percent triphenyltetrazolium chloride (Sigma, St. Louis, MO) in 0.033 M phosphate buffer (dibasic sodium phosphate, pH 7.4). Reactions were performed in triplicate. The reaction mixture was incubated at 39 ~C in an agitating water bath for 1 hour. The reaction was then stopped and the colored formazan dye extracted into solution by adding 4 ml of acetone to each reaction tube and vortexing. The reaction tubes were centrifuged for 10 minutes at 1,500 rpm, and the absorbance of the clear red supernatants were measured at 485 nm in a spectrophotometer (Gilford, Oberlin, OH). The absorbances of the reaction triplicates were averaged and unreacted reagent blanks were subtracted to arrive at a final absorption for each muscle sample. Comparison of these absorption values to a standard curve of known reduced triphenyltetrazolium chloride concentrations allowed determination of the micrograms of triphenyltetrazolium chloride reduced per hour per milligram of muscle protein. The activity of each ischemic limb was compared with the contralateral control limb to express the ischemic limb activity as a percentage of the control limb. A series of 42 rats were divided into 7 groups of 6 84
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Figure 2. The mean triphenyltetrazollum chloride ('rrc) reduction activities of the control muscles (4- 1 standard error from each ischemic interval (I) are compared. No significant differences were found among the groups (p = 0.74 by one-way analysis of variance).
animals each. Each group was subjected to an ischemic interval varying from 0 to 6 hours. In those animals subjected to sham ischemia, one limb was randomly selected as experimental and the other as a control for the purpose of comparison. To ensure that the length of the anesthetic period and the various ischemic intervals did not affect the ability of the control muscles to reduce triphenyltetrazolium chloride, the absolute activity of the control muscles (in micrograms of triphenyltetrazolium chloride reduced per hour per mg protein) in each of the seven groups was compared. All comparisons between multiple groups were evaluated by a one-way analysis of variance. Where significant variation was noted, a Duncan's multiple range test (~ = 0.05) was used to identify differences between groups. RESULTS Figure 1 shows the mean measured fluorescence for the five ischemic and control limbs studied. In each control limb, perfusion was sustained throughout the experiment, with increased fluorescence noted with each added bolus of intravenous fluorescein (at 2 hours, 45 minutes and 5 hours, 45 minutes). Conversely, the ischemic limb in each animal demonstrated complete ischemia with no fluorescence until the rubber band tourniquet was removed after 6 hours of ischemia. At that point, reperfusion hyperemia was apparent with fluorescence exceeding that of the control limb. As demonstrated in Figure 2, there was no difference between the control limb activities of the animals subjected to different ischemic intervals (p = 0.74). These results rule out an unrecognized effect of the anesthetic interval on the triphenyltetrazolium chloride reduction activity of muscle. The muscle injury with graded ischemia, as determined by the spectrophotometric triphenyltetrazolium chloride reduction assay, is shown in Figure 3. All ischemic limbs are expressed as the mean percentage of control limb 4- 1 standard error. In animals not subjected to ischemia, experimental limbs had an activity equal to 101 4- 4 percent of the control limbs. A significant variation in triphenyltetrazolium chloride reduction was noted be-
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tween the various ischemic intervals (p <0.0001). Activity was sustained over 1 and 2 hours of ischemia where activities were 95.7 4- 4.8 percent and 84.2 4- 7.3 percent of the control activity, respectively (p >0.05). Significant muscle injury occurred after 3 hours of ischemia, with activity decreasing to 25.4 4- 9.7 percent of the control activity (p <0.05). Beyond 3 hours of ischemia, injury was more severe, with less than 3 percent of control activity demonstrated at 4, 5, and 6 hours of ischemia (p <0.05). COMMENTS The acutely ischemic extremity is one of the most common and serious problems that the vascular surgeon must manage. Depending on the length of the ischemia, significant morbidity, limb loss, and mortality can occur [1-3,17]. Haimovici [18] was the first to describe the acute revascularization syndrome, consisting of pain, edema, tenderness, myoglobinuria, hyperkalemia, and other metabolic derangements that occur when ischemia muscle is revascularized. Prevention of this syndrome and maximal limb salvage depends on an understanding of the various factors contributing to ischemia-reperfusion injury. Recent studies have indirectly implicated oxygen free radicals as important mediators in reperfusion injury of skeletal muscle [6-8,19]. Quantification of the relative importance of ischemic versus reperfusion injury and investigation of factors designed to ameliorate this injury requires an accurate, objective, and reproducible measure of skeletal muscle injury. The spectrophotometric assay of triphenyltetrazolium chloride reduction in the rat hindlimb model of skeletal muscle ischemia meets these criteria. The fluorescein perfusion studies performed in these experiments confirm the consistent and complete ischemia achieved as well as the reactive hyperemia which occurs after tourniquet removal. The use of triphenyltetrazolium chloride reduction is well established as a marker of muscle infarction. Triphenyltetrazolium chloride histochemistry was first employed clinically in 1957 by Sandritter and Jestadt [20]. The reduction-oxidation potential of triphenyltetrazolium chloride is low enough that reduction occurs at the final cytochrome oxidase of the mitochondrial electron transport chain immediately before transfer of electrons to oxygen [21]. The inability to reduce triphenyltetrazolium chloride reflects disruption of mitochondria, the depletion of essential reduction-oxidation coenzymes, or both [22]. This lack of triphenyltetrazolium chloride reduction has been extensively correlated with other measures of skeletal muscle injury. In our previous studies with the isolated canine gracilis muscle preparation [11], electron microscopic studies of muscle that failed to reduce triphenyltetrazolium chloride demonstrated a variety of abnormalities, including mitochondrial swelling, cristae disorganization, swelling of the sarcoplasmic reticulum, and sarcolemmal disruption. These ultrastructural changes identify irreversible injury and confirm that failure of muscle to reduce triphenyltetrazolium chloride is an indicator of cellular infarction. This inability has also shown a close correlation with uptake of techneti-
um 99 pyrophosphate, an additional skeletal muscle injury marker employed in the isolated gracilis preparation
[13,23]. The combination of triphenyltetrazolium chloride reduction with planimetry for macroscopic myocardial infarct mapping and quantification was introduced by Boor and Reynolds [24] in 1977 and has since been used effectively in skeletal muscle studies [9,11,19]. These planimetric techniques are somewhat subjective, however, and are difficult to employ in a small animal model. The allor-none character of tissue staining does not allow a graded measure of cellular injury. The biochemical spectrophotometric assay described in these experiments overcomes these shortcomings and establishes an objective and quantitative technique that takes advantage of the established accuracy of triphenyltetrazolium chloride reduction as a marker of ischemic injury. The spectrophotometric triphenyltetrazolium chloride assay in this rat hindlimb model demonstrates significant skeletal muscle injury first occurring after 3 hours of ischemia. Significantly greater injury occurs at 4, 5, and 6 hours of ischemia. The early appearance of ischemic damage at 3 hours is consistent with previous studies of the ischemic rat hindlimb. Stock and associates [25] studied metabolic changes in rat skeletal muscle in an ischemic hindlimb preparation. These investigators noted a profound decrease in phosphocreatine, adenosine triphosphate, and glycogen, as well as an accumulation of lactate after 3 hours of ischemia. Normalization of the metabolic pattern occurred after several hours of reperfusion. When the ischemic interval was extended to 4 or more hours, however, the abnormal metabolic pattern persisted over 5 to 6 weeks. Similarly, Makitie and Teravainen [26] combined histochemical stains for NADH diaphorase, ATPases, and phosphorylase with histologic studies to evaluate skeletal muscle ischemia in a rat hindlimb model. They reported minimal scattered areas of injury at 2 hours of ischemia, followed by large infarcted areas at 4 hours and total necrosis at 6 hours. Makitie [27] also used electron microscopy and microangiography to investigate ischemic capillary damage in the rat hindlimb. Degeneration of muscle capillaries appeared after 3 hours of ischemia, was extensive after 4 hours, and was complete after 6 hours. Chachques et al [28] used a related tetrazolium salt, nitroblue tetrazolium, in a rat hindlimb ischemia model. They measured the time to gross staining of muscle specimens incubated in a tetrazolium-containing solution. Significant prolongation of the interval to staining occurred after 3 hours of ischemia. Lee et al [7] studied adenosine triphosphate-dependent calcium ion uptake into isolated vesicles of muscle sarcoplasmic reticulum as an index for ischemic injury in a rat hindlimb model. A significant depression of calcium ion uptake was reported after 3 hours of tourniquet ischemia. The model developed and employed in these studies offers considerable advantages for characterizing the injury induced by ischemia and reperfusion. By subjecting ischemic muscle to various periods of reperfusion before assaying for triphenyltetrazolium chloride reduction, an accurate determination of the relative role of reperfusion injury should be possible. Previous studies have only of-
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fered indirect evidence of significant reperfusion injury in skeletal muscle and have not quantified the magnitude of the injury [6-8,19]. The efficacy of controlled reperfusion, hypothermia, free radical scavengers, and other pharmacologic agents in reducing ischemia-reperfusion injury may be systematically evaluated with this model. Furthermore, the possibility of direct clinical application as an indicator of human muscle viability in the setting of acute skeletal muscle ischemia also merits investigation.
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