Quantitative histochemical evaluation of skeletal muscle ischemia and reperfusion injury

JOURNAL

OF SURGICAL

RESEARCH

43, 311-321 (1987)

Quantitative l-tistochemical Evaluation of Skeletal Muscle lschemia and Reperfusion Injuty’32 JOHN BLEBEA, M.D.,*

JOHN C. KERR, B.A.,? JOHN Z. SHUMKO, PH.D.,* RICHARD N. FEINBERG, PH.D.,* AND ROBERT W. HOBSON II, M.D.t,3

Section of Vascular Surgery, Departments of*Surgery New Jersey, Newark, New Jersey, 07103; and tBoston

and $.4natomy, University School

University ofMedicine,

ofMedicine and De&s&y Boston, Massachusetts

of 02118

Submitted for publication July 14, 1986 Acute arterial obstruction to the extremities is associated with significant morbidity and mortality. The evaluation of accompanying skeletal muscle injury has thus far been indirect and imprecise. Triphenyltetrazolium chloride (TX) is an oxidation-reduction indicator which allows for the histochemical quantitation of skeletal muscle injury. In 21 anesthetized nonheparinized adult mongrel dogs, the isolated in vivo gracilis muscle underwent 4, 6, or 8 hr of ischemia with and without reperfusion. The muscles were excised and cut into l-cm segments, representative muscle biopsies for electron microscopy were taken, each segment was stained in 1% TTC, and the total area of staining was measured with computerized planimetry. All control muscles stained completely with a dark red color. After 4, 6, or 8 hr of &hernia, quantitative measurements of muscle staining indicative of normal tissue were present in 98 t l%, 59 f 5%, and 23 +- 9% of the total muscle areas, respectively. Six hours of &hernia followed by reperfusion was associated with only 36 * 9% of the muscle being stained. Segmental TTC staining demonstrated that reperfusion was associated with greater injury, and less TTC staining, in the proximal portion of the gracilis muscle at the site of entry of the major arterial pedicle. The distal muscle did not demonstrate increased damage with reperfusion. It is hypothesized that protection of the distal muscle from reperfusion injury may be due to an absence of reflow farther away from the artery. 0 1987 Academic Press, Inc.

INTRODUCTION

phenyltetrazolium chloride is an oxidationreduction indicator which has been used successfully for the early histochemical diagnosis of myocardial infarcts [4, 51. We have utilized triphenyl tetrazolium chloride (TTC) as a histochemical marker to measure skeletal muscle damage and to distinguish between the ischemic and reperfusion components of this injury.

Acute interruption of arterial blood flow to the extremities is associated with significant morbidity and mortality [ 1, 21. If the period of ischemia is prolonged, the initial injury may be further exacerbated on reperfusion [ 3 1. Evaluation of the degree of skeletal muscle injury has thus far been limited to compartmental pressure measurements and clinical judgement. To study skeletal muscle ischemia and reperfusion injury, we employed a nonheparinized autoperfused in vivo canine gracilis muscle preparation. Tri-

MATERIALS

AND METHODS

Gracilis Muscle Preparation Twenty one adult mongrel dogs of either sex with a mean weight of 23.7 + 0.8 kg (mean f SEM) were used. They were initially anesthetized with intravenous sodium pentobarbital (30 mg/kg body wt) and supplemented thereafter as needed. The animals were intubated and ventilated with room air by a positive pressure ventilator (Harvard Apparatus, Waltham, MA) at a tidal volume

’ Portions of this work were presented at the 28th Annual University Surgical Residents’ Conference, Richmond, VA, February 12, 1986. * Supported in part by a grant from the Veterans’ Administration Research Fund. 3 To whom correspondence and reprint requests should be addressed: Department of Surgery (Dr. Hobson), Boston University Medical Center, Boston, MA 02118. 311

0022-4804/87 $1.50 Copyright 0 1987 by Academic Pxs, Inc. All rights of reproduction in any form reserved.

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of 15 ml/kg and a respiratory rate of 8- 121 min. Arterial blood gas measurements of p02, SO,, and pH were performed during the procedure using a blood gas analyzer (Corning 165, Midland, MI) and ventilatory parameters were adjusted accordingly. Hydration was maintained intravenously with normal saline at a rate of 3-5 ml/kg/hr. Blood pressure was monitored via an indwelling arterial catheter attached to a transducer which was calibrated at the start of each experiment. A thermal blanket and heating lamp were used to maintain the body temperature between 35-39°C. An isolated nonheparinized in vivo gracilis muscle preparation was employed (Fig. l), similar to that described by Duran and Renkin [6] and Kuzon et al. [7]. The gracilis muscles were bilaterally dissected free. All arterial and venous branches of the proximal major vascular pedicle were ligated. The distal minor pedicle was ligated and divided just prior to the beginning of the ischemic period. All other collateral vessels were ligated and sectioned. The gracilis nerve was blocked with 1% lidocaine to prevent muscle spasm and then divided. Both proximal and distal tendons were transected to prevent collateral blood flow. They were then reattached with a continuous suture to maintain original rest-

VOL.

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ing muscle length. A catheter (PE 90, Clay Adams, Parsippany, NJ) was inserted in the femoral artery distal to the major pedicle for blood pressure measurements and sample aspirations. No heparin flushes were used at any time. Gracilis artery blood flows were measured electromagnetically. The transducers of a dual-channel blood flowmeter (Biotronix, Silver Springs, MD) were placed on the femoral artery just proximal to the gracilis artery, while the distal femoral artery was occluded. Gracilis artery blood flows were measured prior to ischemia and at 15-min intervals during reperfusion. One gracilis muscle was randomly chosen to be the experimental muscle and underwent ischemia by the placement of an occluding vascular clamp on the gracilis artery. Eight dogs (15 muscle preparations) underwent 4 (n = 5), 6 (n = 5) or 8 (n = 5) hr of ischemia and were thereafter excised without permitting reperfusion to occur. In 11 dogs, 4(n=2),6(n=7),org(n=2)hrofischemia was followed by 1 hr of reperfusion. Two dogs underwent 6 hr of ischemia with 2 (n = 1) or 4 (n = 1) hr of reperfusion. The contralateral gracilis muscle (n = 13) served as a control in each of these dogs. Following ischemia, with or without re-

Flow Meter

Arterial

//

FIG. 1. The isolated being via the proximal

43, NO.

Calkter

/

gracilis

muscle

preparation

with

all of the vascular

inflow/outflow

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perfusion, the gracilis muscles were excised and transected into l-cm segments using a plastic template. Representative muscle biopsies were taken for electron microscopy from the middle of segments 3, 6, 9, and 12 (numbered proximal to distal) and placed immediately into cold 2% buffered glutaraldehyde, pH 7.4, for fixation.

Electron Microscopic Evaluation Tissue samples were rinsed in 0.1 M cacodylate buffer, postfixed in 1% osmium tetroxide, and dehydrated in a graded series of ethanol and propylene oxide. Samples were then embedded in Epon (Poly/Bed 812, Polysciences, Inc., Warrington, PA). Ultrathin sections were obtained from each block of embedded tissue, stained with uranyl acetate and lead citrate, and examined with a Phillips 300 electron microscope. Representative micrographs were taken of each specimen.

Triphenyltetrazolium Chloride Staining In five animals undergoing 4 (n = l), 6 (n = 2), or 8 (n = 2) hr of ischemia, three repre-

FIG. 2. Two transverse segments of muscle following 6 hr of ischemia and 1 hr of reperfusion. The dark areas indicate the presence of normal dehydrogenase activity and TTC stain uptake. The light pale areas represent damaged and ischemic muscle.

FIG. 3. Gracilis artery blood flow following 6 hr of ischemia and 1 hr of reperfusion. Each curve represents the means + SEM of seven experiments.

sentative segments of both the control and experimental muscles were placed in a 1% TTC solution which included exogenous enzyme substrates. These consisted of Dr-.-malic acid 0.1 M, DL-lactic acid 0.05 M, DL-iSOCk ric acid 0.1 M, and succinic acid 0.1 M (final concentrations). The pH was titrated to 7.9 with NaOH in a 0.2 M Tris buffer solution. All other muscle segments were incubated for 30 min in a 1% TTC solution buffered in 0.2 M Tris buffer, pH 7.9, at 27°C. TTC yields a dark red color at sites of normal dehydrogenase activity. In areas of severely damaged and ischemic muscle cells, which have no dehydrogenase activity, the muscle does not stain with TTC and is pale grey in appearance (Fig. 2). After staining, the muscle surfaces were photographed. The areas of staining in each segment were measured using computerized planimetry (MOP-3, Carl Zeiss, Inc., Oberkochen, West Germany) and presented as the percentage of total surface area of the segment. Muscle lengths are presented as the proportion of the entire length of the muscle. All data are expressed as the means f their standard error (SEM). Statistical comparison between muscle groups was assessed using the Student t test and by analysis of variance with repeated measures using the method of Duncan [8]. Animals were cared for in a licensed animal research facility according to National Institutes of Health guidelines.

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All control muscles stained completely with a dark red color. After 4, 6, or 8 hr of ischemia, quantitative measurements of muscle staining indicative of normal tissue were present in 98 f I%, 59 f 5%, and 23 -+ 9% of the total muscle areas, respectively (Fig. 4). After 4 hr of ischemia, TTC staining was not significantly different from control values. The differences at 6 and 8 hr of ischemia were different from those of control, 4 hr of ischemia, and each other (P < 0.05). FIG. 4. Quantitative TTC staining following various In muscles subjected to ischemia and reperiods of &hernia. With increasing time of ischemia, perfusion, a greater degree of damage was there is progressively less staining of the muscle seg- observed, although this did not reach statistiments. cal significance (P = 0.054). Six hours of ischemia followed by reperfusion was associated with only 36 + 9% of the muscle being RESULTS stained (Table I). Muscles undergoing 6 hr of ischemia and 2 or 4 hr of reperfusion were The baseline preischemic gracilis muscle stained with an amount intermediate beblood flow in those muscles that underwent tween those with and without reperfusion. 6 hr of ischemia and reperfusion was 11.5 The percentage of surface area stained + 0.9 ml/min/ 100 g (n = 14). There was no with TTC within each l-cm muscle segment significant difference between control (10.8 was determined and plotted as a function of + 1.1 ml/min/ 100 g) and experimental the length of the gracilis muscle (Fig. 5). The groups ( 12.8 + 1.9 ml/min/lOO g). A hyper- amount of staining was different between emit response was seen within 15 min of re- those muscles with 4, 6, or 8 hr of ischemia perfusion when experimental muscle blood (P < 0.05). After 6 or 8 hr of ischemia, the flow increased to 204% of the baseline value middle portions of the muscle had the most (Fig. 3). This was different from both base- amount of TTC staining and the distal porline and corresponding control values (P tions the least. Muscles undergoing 6 hr of ischemia and < 0.05). This blood flow rate was sustained throughout the 60-min reperfusion period. reperfusion demonstrated a different pattern All muscle segments, regardless of location of segmental TTC staining compared to or length of ischemia, stained completely those muscles subjected to 6 hr of ischemia when incubated in a TTC solution containalone (Fig. 6). In muscles undergoing reperfusion, staining in muscle segments 4 ing exogenous substrates. TABLE 1 TTC STA~NINCFOLLOWINGVARIOUSPERIODSOFISCHEMIAANDREPERFUSION Time of &hernia

Ischemia only Reperfusion I hr 2 hr 4 hr

4 hr

n

6 hr

n

8 hr

n

98.5 + 1.0%

5

58.7 +- 5.1%

5

23.0 + 8.9%

5

99%

2

36.5 f 8.6% 47.9 43.9

7 1

13.8%

2

1

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ET AL.: ISCHEMIA

I

“OUFS

8

HOUR3

8 HOURS

wmdAL

2

3 1 5 .6 7 .8 .9 AL PROPORTION OF MUSCLE LENGTH

FIG. 5. TTC staining of muscle segments after 4,6, or 8 hr of &hernia plotted as a function of muscle length. Each data point is the mean + SEM of five experiments.

through 7 was significantly decreased, as confirmed by an analysis of variance with repeated measures using the method of Duncan [8]. This trend was not found in muscles undergoing only ischemia without reperfusion. Each group of corresponding segments in the ischemia and ischemia/reperfusion muscles was directly compared. Significant differences were noted in the middle portions of the muscles (Fig. 6). Less injury was noted histochemically in the more proximal and distal portions of the muscles undergoing ischemia and reperfusion. Segmental TTC staining can also be plotted as a function of the distance away from the entry of the gracilis artery into the mus-

I Prlo~

1.1.1.1.1.1.1./.1.1 2 3 .I PROPORTION

.5

.6

.I

OF MUSCLE

8 LENGTH

cle. Figure 7 illustrates that the difference in staining patterns with TTC, and the area of greatest injury defined histochemically on reperfusion, is located at the site of the vascular inflow into the muscle. Electron microscopic evaluation of biopsy specimens demonstrated significant extraand intracellular damage after 6 or more hr of ischemia. Histological changes were more prominent with longer periods of ischemia. Compared to control specimens (Fig, S), those muscles undergoing ischemia and reperfusion showed marked intermyofibrillar edema with myofibrillar separation, mitochondrial swelling, and disorganization of cristae, nuclear chromatin clumping, and sarcolemmal membrane disruption (Fig. 9). These changes were most evident in the middle portions of the muscle. In some specimens, marked capillary endothelial cell swelling was seen. DISCUSSION

Acute arterial obstruction in the extremities, secondary to trauma, embolus, or atherosclerotic disease, is associated with significant morbidity. If revascularization is delayed beyond six hr or if sensory and motor function impairment has already taken place a mortality rate of 40% and limb loss in 27% of patients have been reported [9]. With longer periods of ischemia, the initial skeletal muscle injury may be exacerbated on reper-

.o DA (. _ p < o,oo ) I

6. Segmental TlC staining following 6 hr of ischemia with and without reperfusion. Each curve is the result of five experiments. Significant differences were found between the two groups in the proximal and middle portions of the muscle. FIG.

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-5 PRomu

I.I.I.I.I.I.I.I.I.,.I.I -4 -.1

-2

-.1

0

.I

2

3

.4

PROPORTIONATE MUSCLE LENGTH AWAY FROM GRACILIS ARTERY

.5

.6

DIST*L

FIG. 7. Segmental TTC staining plotted as a function of the distance away from the entry site of the gracilis artery into the muscle.

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fusion, in addition to the deleterious systemic metabolic effects [3]. The evaluation of skeletal muscle ischemic injury has thus far been indirect and limited to compartmental pressure measurements and imprecise clinical judgement. Furthermore, no separate quantitation of the additional damage caused by reperfusion alone has yet been accomplished. The isolated in vivo canine gracilis muscle preparation provides an experimental model in which to measure the ischemic and reperfusion components of muscle injury following acute arterial interruption. Unlike the tourniquet ischemia model, there is no associated venous and lymphatic obstruction or soft tissue injury [lo]. It is technically less demanding then a multiple ligation model which attempts the vascular isolation of an entire limb [S]. The contralateral leg provides an internal control with which to compare the findings in the experimental muscle. Kuzon et al. have evaluated this preparation for use in acute ischemia studies [7]. They found that the two muscles are equivalent metabolically after surgical preparation and isolation on a single vascular pedicle. Blood flow and physiologic measurements are easily accomplished. The response of this model to ischemia and reperfusion is consistent with that of other models. The hyperemic blood flow on reperfusion is similar to that found in whole limb preparations [ 5, 71. Our experience corroborates the conclusion of Kuzon et al. that this preparation is most suitable for the study of skeletal muscle ischemia. Triphenyltetrazolium chloride is a colorless oxidation-reduction indicator which on reduction produces a visible dark red formazan pigment [I]. In the mitochondrial electron transport system the redox potential of TTC is such that electron transfer to it occurs after cytochrome oxidase and just before the final step to oxygen [ 121. TTC can also be reduced by hydrogen from other dehydrogenases and thus allows for a histochemical quantitation of enzyme activity in tissue segments [ 131.

1987

The clinical application of triphenyltetrazolium histochemistry was first demonstrated by Sandritter and Jestadt in 1957 [ 141, and further defined by Nachlas and Schnitka in 1963 [ 131 for the early macroscopic identification of myocardial infarctions. The inability of the ischemic and dehydrogenase deficient myocardium to reduce TTC allowed for planimetric quantitation of infarct size [4, 151. TTC staining has been shown to accurately identify myocardial injury as early as 4 to 6 hr following the acute event and correlates well with findings on electron microscopy and radionuclide studies [ 16- 181. More recently, Chachques et al. have used a related salt, nitroblue tetrazolium chloride, to evaluate skeletal muscle &hernia in rats [ 191. We have applied triphenyltetrazolium chloride histochemical staining for the quantitation of skeletal muscle injury in the canine gracilis muscle. The results of our studies demonstrate that there is a significant and reproducible difference in the amount of TTC staining following various periods of ischemia (Fig. 4). Almost complete staining of the muscle took place after 4 hr of ischemia. Tountas and Bergman similarly found in skeletal muscle that 4 hr of tourniquet ischemia did not produce changes in the histochemical pattern of dehydrogenase activity [20]. As in our study, minimal histologic evidence of tissue injury has been identified by others after 4 hr of total ischemia [lo, 211. Both cellular transmembrane potentials and effective whole muscle function are restored to normal following such short periods of ischemia [22, 231. With increasing length of ischemia, there is a greater amount of muscle damage and a corresponding decrease in quantitative TTC staining. Only 59% of the muscle showed dehydrogenase enzymatic activity following 6 hr of ischemia. Patterson and Klenerman [lo] and others [21] found extensive mitochondrial damage in skeletal muscle following 5 or 6 hr of ischemia, with histologic evidence of injury persisting even a week later. Restitution of cellular function still appears

BLEBEA

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possible after 6 hr of ischemia [24, 251. Functional isometric muscle contraction, however, may be less than 20% of control values 6 days after the ischemic insult [23]. Eight hours of &hernia is associated with only 23% of the muscle showing enzymatic activity by TTC staining. Diffuse histologic changes in the mitochondria, as well as the blood vessels, are evident by light microscopy [26]. Cellular and functional metabolic alterations may now be mostly irreversible [27]. Quantitative histochemical staining thus reflects progressive physiologic and histologic damage seen with increasing periods of skeletal muscle ischemia. The lack of TTC staining indicates a loss of dehydrogenase activity in the corresponding muscle. This could be due to irreversible cellular or enzymatic injury. During shorter periods of ischemia, temporary reversible damage or lack of enzyme substrates may prevent staining of the muscle tissue. We found that adding exogenous substrates to the TTC solution leads to complete muscle staining following 4, 6, or 8 hr of ischemia. Nachlas and Shnitka similarly found that the addition of substrates to the incubation solution decreased the sensitivity of this technique in defining areas of myocardial ischemit injury [ 131. Subsequently, he and others utilizing triphenyltetrazolium chloride staining have not added exogenous substrates during incubation. The differential staining pattern with and without reperfusion and the electron microscopic histologic evaluation nonetheless indicate that there is a direct relationship between TTC staining and skeletal muscle injury. A similar correlation has been documented by others [4, 16-181. Reperfusion following 6 hr of ischemia resulted in a decrease in the amount of muscle TTC staining, from 59 to 37%, compared to 6 hr of ischemia alone (Table 1). An analogous decrease took place in two animals undergoing 8 hr of ischemia and reperfusion. Extrapolation from the results graphed in Fig. 4 indicates that this would be approximately the same amount of muscle staining

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that would be expected if the muscle underwent a total of 7 hr of &hernia. Measured gracilis artery blood flow, the hyperemic increase in flow on reperfusion, and subsequent muscle edema all documented, however, that reperfusion of the muscle did indeed take place. One hour of reperfusion therefore appears to initially injure the muscle to the same degree as an additional equal period of ischemia. The increase in muscle staining seen after 2 and 4 hr of reperfusion, to 48 and 44%, suggests that some reparative processes may take place after longer periods of reperfusion but not enough to decrease the injury to the amount equivalent to 6 hr of ischemia alone. These results confirm the clinical impression of a worsening of the initial ischemic muscle injury following revascularization. Haimovici first characterized the revascularization syndrome as consisting of pain, tenderness, and edema of the muscle associated with systemic metabolic derangements [3]. Presta and Ragnotti have correlated the time course of increases in serum enzyme concentrations and reperfusion of the ischemic extremity to demonstrate that a substantial part of the damage occurs as a consequence of reperfusion, independent of the &hernia itself [25]. Kloner et al. documented that marked intracellular edema and mitochondrial-dense body accumulation took place immediately on reperfusion but was not present with ischemia alone [28]. Our histochemical results indicate a worsening of tissue injury on reperfusion. An analysis of the TTC staining distribution along the length of the muscle indicates a different pattern between those muscles undergoing only ischemia and in those where reperfusion is allowed to take place (Fig. 6). In the latter, the area of least staining and the greatest injury is in the middle portion of the muscle. This corresponds to the entry site of the artery into the gracilis muscle (Fig. 7) and suggests that some component of the reperfusate may mediate the additional injury. One possible mechanism for this reperfusion injury may be the production of oxygen free

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radicals with the introduction of oxygenated blood into the muscle. Such effects would be expected to be most prominent in the area of greatest reflow, nearest the entrance of the artery into the muscle. Similar processes are thought to take place in the heart [29], brain [30], kidney [3 11, and intestine [32]. A recent report by Korthuis et al. indicates that free radical scavengers ameliorate ischemic-induced increased vascular permeability in skeletal muscle [33]. Our finding of greater proximal muscle injury with reperfusion is consistent with this hypothesis. A “no-reflow” phenomenon has been described in the myocardium following temporary coronary ischemia which was associated with endothelial cell swelling and luminal occlusion [ 341. Impaired reperfusion following ischemia has also been documented in skeletal muscle by Strock, who evaluated intravascular distribution of carbon black into vessels following various periods of ischemia [35]. He did not, however, attempt to correlate the severity of tissue injury with the degree of reperfusion. Impaired blood flow to the distal portion of the muscle may spare it some of the deleterious effects of reperfusion. The area nearer to the gracilis artery would therefore be expected to undergo more injury and show less TTC staining. Electron microscopic evaluation confirmed our histochemical findings. Minimal changes were seen following 4 hr of ischemia. This could represent early reversible skeletal muscle injury. With periods of ischemia from 6 to 8 hr, marked histological changes were observed. Disappearance of glycogen granules, irregularity of the Z bands, mitochondrial swelling and disorganization of cristae, swelling of the sarcoplasmic reticulum, and sarcolemmal membrane disruption were found and are similar to the findings noted by others [5, 36, 371. Those muscles undergoing ischemia and reperfusion appeared to develop more significant injury. They also demonstrated more intermyofibrillar edema and separation. Although quantitative morphometric measurements were not done, the greatest area of injury was

1987

apparent in the middle portions of the muscle, compared to the proximal and distal ends. In some segments there was marked capillary endothelial cell swelling occluding the vessel lumen. This may play a role in the previously described no-reflow phenomenon and provide a protective effect from reperfusion injury in the areas distant from the arterial inflow. We have found triphenyltetrazolium chloride to be an effective histochemical marker of skeletal muscle injury. With increasing periods of ischemia, histochemical staining reproducibly documents greater muscle injury. Our experimental model confirms the clinical observation of an accentuation of the initial injury during reperfusion. Such injury was more extensive in the middle portions of the muscle nearest to the entry of the gracilis artery into the muscle. Electron microscopy has confirmed our histochemical findings. The canine gracilis muscle preparation and TTC histochemical staining is a model useful for the evaluation of pharmacologic agents that may ameliorate ischemia and reperfusion injury. REFERENCES 1. Blaisdell, F. W., Steele, M., and Allen, R. E. Management of acute lower extremity arterial &hernia due to embolism and thrombosis. Surgery 84(6): 822, 1978. 2. Tawes, R. L., Harris, E. J., Brown, W. H., Shoor, P. M., et al. Arterial thromboembolism. A twenty year perspective. Arch. Surg. 120: 595, 1985. 3. Haimovici, H. Metabolic complications of acute arterial occlusions. J. Curdiovasc. Surg. 20(4): 349, 1979. 4. Boor, P. J., and Reynolds, E. S. A simple planimetric method for determination of left ventricular mass and necrotic myocardial mass in postmortem hearts. AJCP 68(3): 387, 1977. 5. Padberg, F. T., France, C. D., Kerr, J. C., Lynch, T. G., et al. Alterations in canine hindlimb blood flow with arterial &hernia and reperfusion. Sub mitted for publication. 6. Duran, W. N., and Renkin, E. M. Oxygen consumption and blood flow in resting mammalian skeletal muscle. Amer. J. Physiol. 226( 1): 173, 1974. 7. Kuzon, W., Walker, P., Mickle, D., Harris, K., et al. An isolated skeletal muscle model suitable for acute &hernia studies. J. Surg. Res. 41: 24, 1986.

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8. Snedecor, G. W., and Cochran, W. G. Statistical Methods, 6th ed. Ames: Iowa State Univ. Press, 1968. 9. Kendrick, J., Thompson, B. W., Read, R. C., Campbell, G. S., et al. Arterial embolectomy in the leg. Results in a referral hospital. Amer. J. Surg. 142: 739, 1981. 10. Patterson, S., and Klenerman, L. The effect of pneumatic tourniquets on the ultrastructure of skeletal muscle. J. Bone Joint Surg. 61: 178, 1979. 11. Altman, F. P. A comparison of dehydrogenase activities in tissue homogenates and tissue sections. Biochem. J. 114: 13, 1969. 12. Lippold H. J. Quantitative succinic dehydrogenases histochemistry. Histochemistry 76: 38 1, 1982. 13. Nachlas, M. M., and Shnitka, T. K. Macroscopic identification of early myocardial infarcts by alterations in dehydrogenase activity. Amer. J. Pathol. 42(4): 379, 1963. 14. Sandritter, W., and Jestadt, R. Triphenyltetrazoliumchlorid (TTC) als reduktionsindikator zur makroskopischen diagnose des frischen herzinfarktes. Zentralbl. Allg. Path. 97: 188, 1957. [Abstract] 15. Lie, J. T., Pairolero, P. C. Halley, K. E., and Titus, J. L. Macroscopic enzyme-mapping verification of large, homogenous, experimental myocardial infarcts of predictable size and location in dogs. J. Thorac. Cardiovasc. Stag. 69(4): 599, 1975. 16. Fishbein, M. C., Meerbaum, S., Rit, J., and Lando, U., et al. Early phase acute myocardial infarct size quantification: Validation of the triphenyl tetrazolium chloride tissue enzyme staining technique. Amer. Heart J. lOl(5): 593, 1981. 17. Izquierdo, C., Devous, M. D., Nicod, P., Buja, L. M., et al. A comparison of infarct identification with technetium-99m pyrophosphate and staining with triphenyl tetrazolium chloride. J. Nucl. Med. 24: 492, 1983. 18. Kloner, R. A., Darsee, J. R., DeBoer, L. W. V., and Carlson, N. Early pathologic detection of acute myocardial infarction. Arch. Pathol. Lab. Med. 105: 403, 1981. 19. Chachques, J. C., Fabiani, J. N., Perier, P., Swanson, J., et al. Reversibility of muscular &hernia: A histochemical quantification by the nitroblue tetrazolium (NBT) test. Angiology 36(8): 493, 1985. 20. Tountas, C. P., and Bergman, R. A. Tourniquet &hernia: Ultrastructural and histochemical observations of ischemic human muscle and of monkey muscle and nerve. J Hand. Surg. 2( 1): 3 1, 1977. 2 1. Dahlback, L. O., and Rais, 0. Morphologic changes in striated muscle following &hernia. Acta Chir. Stand.

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22. Enger, E. A., Jennische, E., Medegard, A., and Haljamae, H. Cellular restitution after 3 h of complete tourniquet &hernia. Eur. Surg. Res. 10: 230, 1978. 23. Patterson, S., Klenerman, L. K., Biswas, M., and Rhodes, A. The effect of pneumatic tourniquets on

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skeletal muscle physiology. Ada Orthop. Stand. 52: 171, 1981. Hagemann, H., Schumacher, E., and Hirche, H. Changes of the extracellular K+ activity and K+ balante of the resting skeletal muscle during and after long lasting ischemia. Pfluegers Arch. 373: R60, 1978. Presta, M., and Ragnotti, G. Quantification ofdamage to striated muscle after normothermic or hypothermic ischemia. Clin. Chem. 27(2): 297, 198 1. Scully, R. E., Shannon, J. M., and Dickerson, G. R. Factors involved in recovery from experimental skeletal muscle &hernia produced in dogs. Amer. J. Pathol. 39(6): 721, 1961. Bogdanov, 0. A., Mishnev, 0. D., Kotelnikov, V. M., Goldberg, V. E., et al. Quantitative histochemical characteristics of alterations in red and white fibers of limb skeletal muscle tissue during temporary ischemia and postischemic recirculation. Biull Eksp Biol Med. 98( 11): 530, 1984. [Abstract] Kloner, R. A., Ganote, C. E., Whalen, D. A., and Jennings, R. B. Effect of a transient period of ischemia on myocardial cells. Amer. J. Pathol. 74 399, 1974. Jolly, S. R., Kane, W. J., Baihe, M. B., Abrams, G. D., and Lucchesi, B. R. Canine myocardial reperfusion injury. Its reduction by the combined administration of superoxide dismutase and catalase. Circ Res. 54: 277,

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30. Kontos, HA. Oxygen radicals in cerebral vascular injury. Circ Res. 57(4): 508, 1985. 31. Baker, G. L., Corry, R. J., and Autor, A. P. Oxygen free radical induced damage in kidney subjected to warm ischemia and reperfusion. Ann. Surg. 202(5): 628, 1985. 32. Parks, D. A., Shah, A. K., and Granger, D. N., Oxygen radicals: Effects on intestinal vascular permeability. Amer. J. Physiol. 247: G167, 1984. 33. Korthuis, R. J., Granger, D. N., Townsley, M. I., and Taylor, A. E. The role of oxygen-derived free radicals in ischemia-induced increases in canine skeletal muscle vascular permeability. Circ Res. 57(4): 599, 1985. 34. Kloner, R. A., Ganote, C. E., and Jennings, R. B. The “no-reflow” phenomenon after temporary coronary occlusion in the dog. J. Clin. Invest. 54: 1496, 1974. 35. Strock, P. E., and Majno, G. Vascular responses to experimental tourniquet ischemia. Surg. Gynecol. Obstet.

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36. Sanderson, R. A., Foley, R. K., McIvor, G. W. D., and Kirkaldy-Willis, W. H. Histologic response on skeletal muscle to &hernia. Clin. Orthop. 113: 27, 1975. 37. Santavirta, S., Luoma, A., and Arstila, A. U. Ultrastructural changes in striated muscle after experimental tourniquet ischaemia and short reflow. Eur. Surg. Res. 10: 415, 1978.