Identification of nonviable muscle in electric burns with nitroblue tetrazolium

JOURNAL

OF SURGICAL

RESEARCH

37, 369-375 (1984)

Identification

of Nonviable Muscle in Electric Burns with Nitroblue Tetrazolium

JOHN L. HUNT, M.D., AND ELLEN L. HECK, B.S., M.T. Department of Surgery, University of Texas Health Science Center, 5323 Harry Hines, Dallas, Texas 75235 Submitted for publication June 13, 1983 Both experimental and clinical evaluation of nitroblue tetrazolium (NBT) as a method of identifying ischemic and necrotic muscle is described. Colorless NBT was reduced to a blue formazin by viable muscle but remained colorless in necrotic muscle. Muscle samples were rated for viability on a scale of O-10096 based on the relative amount of muscle sample that reduced NBT. There was good correlation between gross appearance, distribution of staining, and histologic findings in all nonviable experimental and clinical muscle specimens. All tissue that reacted with NBT proved to be viable histologically. Metabolically active or viable muscle fibers are rich in respiratory enzymes (dehydrogenases).The loss of dehydrogenase activity from ischemic or necrotic muscle can be detected by NBT reduction capacity. NBT technique identified nonviable tissue, clearly showed the even distribution of muscle damage characteristic of electric burns, was useful in dehning ischemic muscle prior to obvious necrosis, and was a rapid, simple, and reliable semiquantitative test that can be used intraoperatively. 0 I984 Academic PI-W, Inc.

shaved and a 5 X 5 X 2-mm metal electrode was taped to the inner aspect of the skin of Acute electric burns are commonly asso- each thigh. The animals were shocked with ciated with underlying muscle damage. A 250 V for 10 sec. Animals were resuscitated major clinical problem in the care of this with intraperitoneal Ringer’s lactate. Groups injury is the early and accurate intraoperative of four animals were sacrificed at 2, 24, 48, recognition of injured muscle. Because early and 72 hr postinjury, respectively. Muscle gross assessmentof ischemic, yet not grossly slices measuring l/2 X l/2 cm were removed necrotic muscle is often difficult, a conser- from directly under, adjacent to, and remote vative approach with regards to debridement from the contact site. All fat and facia were is most often followed. For this reason mul- trimmed away and each specimen was washed tiple operative procedures are often necessary. in normal saline to remove any blood. Each In addition, a heterogeneous or mixed distri- piece of tissue was cut in half. One half was bution of muscle injury, viable muscle adja- sent for microscopic evaluation and the other cent to nonviable muscle both grossly and half incubated at 37°C for 10 min in NBT. microscopically, complicates early clinical The top of each specimen was covered with identification. This report describes both ex- a minimum of 2 cc of NBT. perimental and preliminary clinical evaluaSixteen patients with an acute electric burn tion of nitroblue tetrazolium (NBT) reduction (average TBSA 18%, range 4-50%) and asto identify ischemic and/or skeletal muscle. sociated deep muscle damage had intraoperative muscle slices taken for both clinical and microscopic assessmentof viability and MATERIALS AND METHODS NBT reduction. The average day of surgery A reproducible electric injury rat model postinjury was 4 with a range of 3-8 days. was used [4]. Sixteen Sprague-Dawley rats Specimens were obtained by the operating weighing between 250 and 350 g were anes- surgeon from areaswith muscle that appeared thetized with an intraperitoneal injection of grossly viable, nonviable, and of questionable sodium phenobarbital. Each hind leg was viability. INTRODUCTION

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NBT was mixed by combining 50 mg of NBT powder with one part phosphate buffer, ph 7.4, and 8 parts of saline. This was heated to 37°C. The solution was kept refrigerated and the container wrapped in tinfoil. Histologic features of viability included the following: (1) skeletal muscle cells packed with parallel myofibrils, (2) muscle cells that contained numerous oval nucleoli along each edge of the myocyte, and (3) cross striations which were prominent in the bundle of parallel myofibrils. Histologic criteria indicating nonviable tissue included the following: (1) disrupted and fragmented myocytes, (2) amorphous eosinophilic or hypereosinophilic muscle fibers, (3) narrow nucleoli along edges of myocytes, and (4) absence of distinct parallel myofibrils with normal intact nucleoli. Two pathologists reviewed all the tissue sections and both agreedon the above criteria. A few precautions should be followed to avoid false negative or false positive results with NBT. An inferior batch of NBT may be obtained from the commercial supply. To detect this, a fresh muscle sample should be used whenever an experimental or clinical evaluation is planned. Next, prompt placement of the specimen in NBT is imperative to diminish the postmortem biochemical changes secondary to autolysis. Next, incubation with at least 2 cc of NBT over the upper edge of the specimen is imperative, otherwise uneven staining from the top to the bottom may occur. Finally, incubation of the specimen longer than 20 min results in not only darker staining but staining of tissue which previously had been unstained at 10 min. This presumably occurs because of diffusion of reduced NBT into nonviable tissue or NBT with time will eventually react with even the smallest amount of available substrates. The patient served as his own control each time a test was performed. A piece of obviously viable muscle (pink and contracted with electrical stimulation) and nonviable muscle (pale, no bleeding, and no contraction with electrical stimulation) were placed in NBT. These were then compared with muscle

of unknown viability that was placed in NBT solution. The intense blue formazin reaction which occurred in the viable muscle was tissue bound. Attempts to elute the formazin were unsuccessful in a variety of solutions including alcohol, toluene, and triten. Since the color could not be satisfactorily extracted, spectrophotometric measurements were not performed. RESULTS

In this animal model the extent of tissue damage both surrounding and deep to the contact point was the same at 24, 48, and 72 hr postinjury. Skin and muscle directly under the contact site had full-thickness damage. There was less muscle damage the more centrifugal the muscle was from the point of contact. At 2 hr postinjury demarcation between viable and nonviable muscle was not clearly evident. At 24 hr though there was a distinct visual demarcation between the viable and nonviable muscle. Clinical assessmentof viability was grossly determined by the color of the muscle, presence or absence of bleeding, and presence or absence of muscle contraction to electrical stimulation or pinching with forceps. Very vigorous contraction was consistent with normal viable muscle whereas no contraction represented nonviable muscle. “Sluggish” or weak contraction was associated with a mixture of viable and nonviable muscle. Conclusive histologic identification of nonviable tissue was impossible when muscle sections were taken at 2 and 24 hr postinjury. Early microscopic changes consistent with nonviable tissue were evident though at 48 hr. At 72 hr histologic criteria of necrosis were obvious. NBT is a colorless solution. It is reduced to a purple formazin precipitate in viable muscle (Fig. 1). Irreversibly ischemic and/or necrotic skeletal muscle remains colorless in NBT. Muscle samples were grossly rated for viability on a scale of from 0 to 100% based

HUNT AND HECK: NONVIABLE

MUSCLE

FIG. 1. Muscle biopsies in NBT for 10 min reveal: Left specimen shows dark staining tissue at bottom; middle section shows several dark areas surrounded by unstained nonviable tissue; upper section is without stain and is not viable. Right specimen. Arrow at junction to viable (dark) and nonviable (light) and muscle.

on the relative amount of muscle sample that reduced NBT. Muscle samples which reduced NBT 100% (all the sample turned dark blue) represented viable tissue. If the sample remained completely colorless in NBT, all tissue was nonviable. In the animal model there was very good correlation between clinical assessmentof viability and the presence or absence of NBT reduction. Incomplete reduction of a sample represented a mixture of viable and nonviable muscle. Once it was established that viability could be determined accurately in the animal model, a comparison of viability by gross clinical criteria, pathologic analysis, and NBT reduction was performed on muscle biopsies in acute electric injuries. Sixty specimens were obtained from 14 patients during their

multiple operative procedures. Fifteen specimens were interpreted clinically to be 100% viable. One hundred percent tissue reduction of NBT occurred in each specimen and pathologic interpretation revealed viable tissue in each instance. Twenty tissue samples were clinically determined to be 100% nonviable, all failed to reduce NBT, and microscopic evaluation revealed nonviable tissue in each instance. Twenty-five tissue sections were clinically deemed of mixed viability. NBT reduction in each slice revealed an irregular or mixed precipitation of the formazin. Estimation of NBT reduction varied between 20 and 80%. Histologic examination of these tissue slices revealed a mixed component of viable and nonviable muscle fibers (Fig. 2). Gross and

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FIG. 2. Sections from muscle biopsy on left in Fig 1. (A) Area of total necrosis. Muscle cells composed of amorphous eosinophilic material (light stain). (B) Area of mixed viability contains amorphous cellular material adjacent to distinct appearing myofibrils with cross striations and normal oval nucleoli. (C) Area of normal muscle (H & E X35).

microscopic assessmentof tissue viability was tissue). When the clinical observer assessed very accurate and correlated closely when tissue reduction by NBT to be between 20 NBT reduction was between 0 and 20% and 80%, both gross and microscopic assess(indicating a majority of nonviable tissue) or ment of viability was at best only semiquan80 and 100% (indicating a majority of viable titative. The gross assessmentof tissue via-

HUNT

AND

HECK:

bility again varied from one observer to another even among the most experienced surgeons. The ratio of viable to nonviable tissue commonly varied from one microscopic field to another in each section. In addition, different observers interpreted the ratio differently. DISCUSSION

Although various intraoperative methods have been employed to identify necrotic and/ or irreversibly “ischemic” skeletal muscle in acute electric injuries, all have met with only limited success.Early gross identification of necrotic muscle is often very difficult. Bleeding does not always signify viable muscle. Characteristically the small intramuscular vessels,those not grossly visible to the naked eye, are thrombosed while the larger muscular branches are patent and bleed readily during debridement [5]. Therefore, during debridement bleeding frequently occurs in and about areas of nonviable muscle. This in turn can stain or clot on muscle thereby further confusing the identification of viable from nonviable tissue. Contractility to electrical stimulation may vary markedly [3]. Very active muscle contraction is a good sign of viability. The greater the amount of nonviable muscle in an area, the less overall contractility to electrical stimulation. Significant amounts of nonviable muscle can be intermixed with viable muscle and muscle still will contract. The use of intraoperative frozen sections has been advocated to identify viable muscle, but unequivocal histologic verification of viability early postinjury is difficult even in experienced hands when utilizing permanent tissue sections [12, 11. Multiple anatomic areas often require surgical debridement in the patient with an electrical injury and the use of intraoperative frozen sections would be time consuming and very costly. Most surgeons are very conservative when debriding muscle injured by electricity. It is far preferable to wait until muscle is obviously

NONVIABLE

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necrotic, unfortunately this often requires multiple operative procedures. Although local sepsis is often associated with small areas of incompletely debrided muscle of questionable viability, this poses no threat to life or limb. On the other hand, lethal systemic sepsis is an ever present threat if initial debridement is either delayed or inadequate when gross nonviable muscle is evident. This is one instance where anaerobic infection can complicate a bum injury. The early detection of nonviable muscle has eluded physicians, specifically pathologists, for many years. In no area has this been more true than in the early gross identification of myocardial infarctions [lo]. Unequivocal gross identification of an acute myocardial infarction does not become apparent before 24-48 hr following occlusion of a major coronary artery in man and between 7 and 24 hr in experimental animals [ 1l]. It is well known that the serum level of certain enzymes becomes elevated alter an acute myocardial infarction. Along with this there has been noted a decrease in certain enzymatic reactions in the infarcted myocardium itself. With this in mind, multiple investigators have evaluated a variety of oxidation-reduction indicators for the morphological identification of early myocardial infarctions 113, 61. Metabolically active or viable muscle fibers are rich in respiratory enzymes, particularly dehydrogenases. The loss of dehydrogenase activity in ischemic and necrotic muscle can be detected by lack of NBT reduction capacity. Nachlas first reported the use of nitroblue tetrazolium (NBT as an enzyme-mapping technique [ 111. McVie [9] and Lie [8] have demonstrated that the macroscopic enzymemapping technique is an accurate method of identifying and locating myocardial infarctions in the experimental animal. Feldman, using coronary perfusion with nitroblue tetrazolium, has grossly detected zones of myocardial infarction of about 24 hr duration [2]. NBT is a sensitive tetrazolium salt with good chromogenic properties thereby making

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it ideal to grossly identify the topography in myocardial infarctions. NBT acts as a hydrogen ion acceptor. NBT reduction reflects the activities of dehydrogenasesoxidizing endogenous substratesand transferring the liberated electrons either directly or through coenzymes to the tetrazolium salt. The loss of nonspecific dehydrogenase activity from ischemic and necrotic muscle fibers is the basis for this oxidation-reduction identification of nonviable tissue by NBT [7]. Normal muscle cells stain blue with NBT and those that are damaged, i.e., ischemic and/or necrotic by infarction, do not stain. What do the biochemical changes represented by the nonreduction of NBT in ischemit and frankly necrotic muscle fibers represent? Areas of tissue not stained with NBT could either represent depletion of endogenous substrates or the loss of respiratory enzymes. The addition of substrate, such as succinate or DPN, with NBT has been shown to lead to the disappearance of differential staining with formazin in early lesions but not in late or old infarcted tissue. This is taken to indicate that substrate deficiency was primarily responsible for the lack of NBT reduction in ischemic muscle while enzyme loss was the cause in frankly necrotic muscle. Macroscopic enzyme marking of ischemic or nonviable myocardium has been reported positive as early as 3% hr aider an experimental myocardial infarction. With the experimental electric injury model used in this study, identification of nonviable tissue between 2 and 8 hr with NBT was unclear, but NBT identification of viable and nonviable muscle at 24 hr was found to be very reliable. As in most clinical circumstances when dealing with an acute electric injury, tissue is generally not debrided until 24 hr after injury and most often not until 48-72 hr. Tissue directly under or adjacent to the contact site was most severely injured and NBT reduction was 0. The further away the tissue was from the contact site, the less dense and more

diffuse the injured tissue and the greater the ratio of viable to nonviable muscle. Because interpretation of NBT reduction is based on visual assessment,it represents a semiquantitative test and consequently its clinical usefulness as a guide to surgical debridement of tissue must be approached cautiously, specifically when tissue reduction is estimated to be between 20 and 80%. While this test is more sensitive than the naked eye in the early identification of nonviable muscle, other clinical parameters such as contractility, bleeding, and color must obviously be considered when making the ultimate decision of if and how much tissue debridement is required. In conclusion, macroscopic enzymatic identification of necrotic skeletal muscle in acute electric injuries with NBT provides the surgeon with a reliable test to aid in the intraoperative identification of nonviable tissue. The technique clearly shows the uneven distribution of muscle damage characteristic of an acute electric burn. It is a rapid, simple, and reliable semiquantitative test. REFERENCES 1. Derias, N. W., and Adams, C. W. M. Nitroblue tetrazolium test: Early gross detection of human myocardiaf infarcts. Brit. .I. Exp. Pathol. 59: 254, 1978. 2. Feldman, S., Glagov, S., Wissler, R. W., and Hughes, R. H. Postmortem delineation of infarcted myocardium. Coronary perfusion with nitro blue tetrazolium. Arch. Pathol. Lab. Med. 100: 55, 1976. 3. Harman, J. W., and Gwinn, R. P. The recovery of skeletal muscle fibers from acute ischemia as determined by histologic and chemical methods. Amer. J. Pathol. 25: 741, 1949. 4. Hunt, J. L., Mason, A. D., Jr., Masterson, T. S., and Pruitt, B. A., Jr. The pathophysiology of acute electic injuries. J. Trauma 16: 335, 1976. 5. Hunt, J. L., McManus, W. F., Haney, W. P., and Pruitt, B. A., Jr. Vascular lesions in acute electric injuries. J. Trauma 14: 461, 1974. 6. Jesmdt, R., and Sandritter, W. Erfahrungen mit der TTC (Triphenyltetrazoliumchlorid) Reaktion fur die pathologisch-anatomische Diagnose des frischen Hetzinfarktes, Ztschr. Kreislaufirsch. 48: 802, 1959.

HUNT AND HECK: NONVIABLE 7. Knight, B. Early myocardial infarction. Practical methods for its post-mortem demonstration. J. Foren. Med. 14: 101, 1967. 8. Lie, J. T., Pairolero, P. C., Halley, K. E., and Titus, J. L. Macroscopic enzyme-mapping verification of large, homogeneous,experimental myocardial infarcts of predictable size and location in dogs. J. Thoruc Cardiovasc. Surg. 69: 599, 1975.

9. McVie, J. G. Postmortem detection of inapparent myocardial infarction. J. Clin. Pathol. 23: 203, 1970. 10. Morales, A. R., and Fine, G. Early human myocardial infarction. Arch. Pathol. 82: 9, 1966.

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Nachlas, M. M., and Shnitka, T. K. Macroscopic identification of early myocardial infarcts by aherations in dehydrogenaseactivity. Amer. J. Pathol. 42: 379, 1963. 12. Quinby, W. C., Jr., Burke, J. F., T&tad, R. L., and Caulfield, J. The use of microscopy as a guide to primary excision of high-tension electrical bums. II.

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13. Sandritter, W., and Jestiidt, R. Triphenyhetrazohumchlorid (TTC) als Reduktionsindikator zur Makroskopischen Diagnose des Frischen Herzinfarktes. (Abstract) Zentrulbl. AlZg. Path. 97: 188, 1957.