JOURNAL OF SURGICAL RESEARCH

A Model for Recording the Microcirculatory Changes Associated with Standardized Electrical Injury of Skeletal Muscle JURGEN HUSSMANN, M.D., WILLI A. ZAMBONI, M.D., ROBERT C. RUSSELL, M.D., uC. ROTH, PH.D., JOHN 0. KUCAN, M.D., HANs Suci, B.S., KEVIN BUSH, M.D., TIM BRADLEY, M.D., RIcRD E. BROWN, M.D. Institute for Ptastic and Reconstructive Surgery, Southern Ittinois University, School ofMedicine, Springfietd, Ittinois 62 794-9230 Submitted for publication May 12, 1994

The rate of major limb amputation following high voltage electrical injury remains high despite a decrease in mortality rate. Several theories about the pathophysiology of electrical injury have been discussed in the literature and different clinical regimens have been attempted to decrease the high amputation rate. However, to date, the overall tissue response after electrical injury remains incompletely under stood with nothing new to offer these unfortunate patients. We have deveioped a rat gracilis muscie intravital microscopy preparation in order to better under Q stand the mechanisms of this injury. A standardized 40-V stimulation of 10-sec duration was appiied to the anterior belly of the gracilis muscle which translated into a current bad of 30 mA. The current density was 750 mAJcm2. Sequential intravital assessment ofmicro circuiatory changes before injury, as weil as 5, 15, 30, 60, 120, 180 and 240 min after injury was performed. Consistent findings included initial cessation of biood flow in many capfliary beds, focal flow reversal, venous and arterial vascular spasm. Restitution of microvas cular ftow varied from several minutes to 1 hr and was preceded by vasodilation at 5—15 min folbowing the injury (+16.9 mfrom baseline at 15 mm). Starting at 30 min progressive vasoconstriction was noted (—0.8 pm from baseiine at 30 mm, —31.3 tm from baseline at 4 hr). High resolution observation of neutrophil behavior showed an jncrease in the number of these cells adherent to venuiar endotheiium in areas exhibiting circuiatory disturbances (+11.4 cells at 5 mm, + 15 ceiis at 4 hr). The standardization of this model aiiows a quantitative method of evaluating the microcircuia tory changes associated with eiectrical injury and of studying ways to prevent tissue damage. The microcir culatory changes induced by electrical injury were similar to those reported in ischemia—reperfusion injury of skeietal muscle.

© 1995 Academic Press, Inc.

INTRODUCTION
Industrial electrical injuries have been reported for over 200 years. Electrical current has been used for legal execution purposes in the United States since 1890 [1]. Numerous clinical and experimental investigations on the pathophysiology of electrical injury have been published, and improved fluid resuscitation and intensive care management ofthese patients have lead to a decrease in renal failure and mortality rates. The
pathophysiology of localized microcirculation changes which occur after electrical trauma, however, remains obscure. Several clinical approaches based on different theories of injury development are currently used, but the rate of major limb amputation remains essentially unchanged [2].

“Progressive“ necrosis of electrically injured tissues [3, 4] is believed by some authors to preclude early complete wound debridement and soft tissue coverage since viable tissues may become necrotic over time. Sequential debridement may be necessary to successfully delineate the extent of injury and wound coverage is often delayed several days to weeks after injury.

Other investigators believe that all tissue damage is sustained at the time of the electrical insult [5, 6] and does not develop progressively after injury. Progressive necrosis would then be only a manifestation of a previously sustained injury. Proponents of this theory advocate that early and aggressive debridement with soft tissue coverage is possible if surgeons understand the patterns of electrical injury. The present model was developed to examine themicrocirculatory changes in the rat gracilis muscle that follow exposure to a standardized electrical injury. Specific observations including capillary blood flow, vasospasm, and leukocyte endothelial adherence were made at baseline and up to 4 hr following the injury.

FIG. 1. Preselected arteriole diameter measurements were made for 5 min to 4 hr after injury and were eompared to baseline diameter measurements made before injury. The bar graph represents the average net change in arteriolar diameter from basehne.

MATERIALS AND METHODS
Twenty-flve male Wistar rats weighing between 120 and 220 g were anesthetized intraperitoneally using sodium pentobarbital (42 mg/kg). Additional anesthetic injections of 10 mg/kg were administered over time as needed. All animals were handled in accordance to the
Laboratory of Animal Research Guidelines in place at Southern Illinois University. The gracilis muscle of the right hind limb was dissected through an L-shaped skin incision over the me dial aspect of the groin and thigh region. The gracilis muscle including its anterior and posterior belly was identified and dissected free as first described by Henrich and Hecke [7] and later modified by Zamboni et al. [8] so that only its vascular pedicle remained attached. The proximal and distal ends of two plastic coated platinum—iridium wires, each about 4 cm long
and 0.03 mm in diameter, were carefully stripped of their protective plastic coating over a distance of 1 cm distally. They were placed in a parallel fashion through the proximal and distal ends ofthe previously prepared anterior belly ofthe gracilis muscle about 0.9 cm apart.
The average width of the muscle was 0.4 cm with a thickness of 0.1 cm. Small sutures were used to stretch the muscle to its original in situ length. The rat was positioned on its right side on a microscopic viewing table.

The muscle was protected from desiccation by placing it in a specially constructed chamber and irrigating periodically with phosphate-buffered sahne (PBS) solution. The uninsulated wire ends were attached to an AC regulated power supply (HB4A) with a volt-ohm meter (Beckman 310) connected in parallel. The prepared axial pattern muscle flap was transil luminated and the image projected onto a high resolution video screen allowing visualization ofthe microcir culation. The muscle preparations were allowed to sta bilize for 15—30 min after which time the patterns of vessel branching from the main pedicle were drawn, numbered, and recorded under bw power field to aid in relocation on sequential examinations. This stabilization period was incorporated to minimize initial sys temic effects of Pentobarb anesthesia, which have been reported to affect microvasculatory vasoactivity.

Immediately before the electrical stimulation, the muscle preparation within its chamber was moistened to yield comparable resistance in all preparations. The power suppiy was set up as a voltage source and was utilized to apply a standardized electrical injury of 40 V for 10 sec which translated into a current bad of 30 mA. The current density was 750 mAJcm2 as calculated from the cross-sectional diameter of 0.04 cm2, and the average current of 30 mA measured by ammeter. A volt-ohm meter was used to confirm the voltage debivered. Folbowing the electrical injury the muscle preparation was thoroughly rinsed with PBS. Each preparation was examined using high power resolution at base line immediately before injury, as weil as 5, 15, 30, 60, 120, 180, and 240 min after injury, and findings were recorded on video tape in real time.

The previously Q identified and numbered sites (measure points) were examined for changes in vascular flow patterns, vessel caliber, and leukocyte adherence function. Criteria for our venular measure points included good visibility of 100 segments ofpostcapillary venules (30—70 pm) and clear transillumination through the vessel wall to albow counting of all cells adherent to the venular endothelium over a period of 15 sec. The same areas were ob served before injury and at all observation times after the injury. Arteriolar vessel diameter was measured on the video screen using calipers. Quantification ofrolling and adherent leukocytes was performed in 100 segments of postcapillary venules over a 15-sec time period.

FIG. 2. The numbers of adherent neutrophils in preselected venular segments were counted tor 5 min to 4 hr after injury and were compared to the numbers counted in the same segments before injury. The bar graph represents the average net change in the number of adherent neutrophils from baseline.

FIG. 3. Central portion of anterior belly of gracilis muscle immediately following electrical injury shows focal myofibrilar lysis and contraction band neerosis. (Light microscopy, magnification x500, H.&E.)

PD Dr. med. habil. Jürgen Hussmann

RESULTS
Preliminary studies were performed to develop a standardized electrical injury model. The time of stimulation and voltage were adjusted to produce a consis tent injury which still allowed for observation of  bood circulation. The size (0.9 cm X 0.4 cm) and the thick ness (0.1 cm) of our muscle preparations were constant as evidenced by a consistent amperage of 30 mA delivered by the power supply. Frequent irrigation with PBS solution prevented significant pH changes during the experiments with a mean pH 7.3 before and pH 5.9 directly following the electrical stimulation. PH values returned to preinjury levels within 5—8 min and remained at this level throughout the entire observation period. After establishing this standardized electrical injury, there were reproducible findings of focal cessation or reversal of blood flow, which returned to normal after a period of about 5 min. Those observations were followed by a marked arteriolar vasodilation at 5 to 30 min following the stimulation.

Criteria for inclusion into the study were focal cessation or reversal of blood ftow followed by restitution of flow within 5 to 15 min and good visibility of arteriolar and venular measure points. Twenty-five of the gracilis muscle preparations (80.6%) provided excellent visualization of microcirculatory and cellular changes follow ing the electrical injury. In 10 preparations microcirculatory flow did not return during the 4-hr observation period.

In vivo microscopic observation of the muscle during and following application of the electrical current revealed local bubbling over a short time period indicating electrolysis and suggesting generation of heat at the interfaces between the electrical wire transmitting the current and the gracilis muscle. The pH drop to 5.9 directly after stimulation indicated a direct effect ofelectrolysis on the tissue but this effect lasted for 5 min only. The pH values returned to preinjury levels (mean pH 7.3) within 5—8 min. Our findings suggest a thermal injury dose to the site of these interfaces of approximately 5% of the total muscle area in addition to the electrical field injury of the entire muscle. Immediately
following the electrical injury the blood flow stopped in some vessels or was reversed for a short period of time in the muscle areas not being affected by the severe thermal injury at the interfaces of tissue and wires.

The time to restitution of blood flow varied from several minutes to 1 hr. Artenolar and venular vasospasms were seen in many areas during and following injury. One of the most striking observations after return ofblood ftow was the marked increase in the number of leukocytes adhering to the postcapillary venular endothelium. Vasoconstriction was noted in arterioles adjacent to leukocyte-damaged venules. The increase in neutrophul ad herence from baseline readings before the injury is
shown in Fig. 1; the gradually increasing vasoconstnction after initial vasodilation at 5—15 min is displayed in Fig. 2. Four hours after injury the muscle preparation began to deteriorate and light microscopy findings and video recordings became difficult to interpret.

Histopathological examination of the muscle preparations taken directly following the electrical injury showed only focal myofibrillar lysis and contraction band necrosis in the central portion ofthe anterior belly of the gracilis muscle (Fig. 3). Tissues samples taken 0.2 cm offthe center revealed coagulation necrosis with severe myofibrillar lysis (Fig. 4). Severe coagulation necrosis and complete loss of muscle structure was
fbund immediately adjacent to the ekctncal wire interface ( Fig. 5). Electron microscopical examination of tissue samples taken from the center of the anterior gracilis muscle (Fig. 6) showed the Z-band spaces of the myofibers dose together, suggesting contraction band necrosis.

The thin actin and thick myosin filaments were still visible. There was polymerization and clumping of glycogen. This clumping is a characteristic myofiber change seen in electrical injury. A tissue sample taken 0.2 cm off center revealed two myofibers from this section as shown in Fig. 7. Both show disruption of their ultrastructure, contraction band necrosis, and clumping of the glycogen.

DISCUSSION
The purpose ofthis research was to develop a reliable model that would allow in vivo observation of the deleterious microcirculatory changes which occur in skeletal muscle after electrical injuries. The anatomy of the gracilis muscle was described by Greene [9]. This muscle was ater used by Honig et at. [10] and by Swain and Lalone [11] for hemodynamic and metabolic studies. A preparation of the gracilis muscle for quantitative microcirculatory studies was published by Henrich and Hecke [7] and has been modi fied by Zamboni et at. [8].

Our model was established to allow for evaluation of the microcirculatory changes associated with electrical injury and for the study of means to prevent tissue damage. The 40-V drop over approximately 1 cm length is equivalent to a 4,000-V injury over the length of 1 m which correlates with the distance between a man‘s hand and shoulder. The current density of 750 mAJcm2 compares favorably with the 350 mAJcm2 calculated from the high voltage experiments of Daniel et at. [12] using 4.5 A and assuming an arm diameter of 4 cm for the African Green Monkey used in their study. The application of electrical trauma produces apre dominantly thermal injury dose to the interfaces between the electrical wires and the moist muscle tissue over a distance of 1—2 mm. The intermediate muscle portion ofthe anterior belly shows a pattern ofthermal as weil as electrical feld injury. The central part of the anterior belly of the gracilis muscie over a distance of 4—5 mm shows primarily an electrical feld injury which is where our microscopic observations were made. Thus distinct injury patterns at different locations on the muscle can be studied separately. All injury patterns can be visualized, recorded, and quantitatively anaiyzed by intravital microscopical examination.

FIG. 4. Intermediate portion (0.2 cm off center) of anterior belly of gracilis muscle immediately following electrical injury shows ceagulation necrosis with severe myofibrilar lysis (Light microscopy, magnification x500, H.&E.)

The thermal component of an electrical injury was weil described by Jex-Blake and Oxon in 1913. They reported body temperature elevations of up to 129.5°F within 20 min of an electrocution during which large amounts of electrical energy were applied [1]. We now know that a thermal injury does occur at the entrance and exit areas where current is concentrated and a metal tissue interface exists. Daniel et al. [121 showed
that the pattern oftissue injury is not related to various tissue resistances as was proposed earlier. Laboratory studies by Hunt et cii. [13] and confirmatory mathematical modeling by Lee and Kolodney [141 have shown that the body and, particu]arly, the limbs act as volume
conductors during high voltage electrocution. Robson et al. [151 demonstrated increased levels of prostaglandin derivatives in electrically injured tissue. Increased sur vival of electrically injured tissue treated by a number of prostaglandin inhibitors has also been shown. The
arteriolar vasoconstriction observed in our model may be in part attributed to a level increase in circulating prostaglandins.

Several authors have demonstrated vessei thrombosis after electrical injury [16, 17]. Arteriograms of patients performed between the 2nd and l2th day after electrical injury show arterial pruning of the vascular tree. Jaffe [18] demonstrated a loss ofvessel endothelium and a spectrum of vascular changes ranging in severity from intimal damage to complete necrosis of the whole vessel wall which was also described by Xuewei and Wanrhong [19]. Ponten et at. [20] described massive areas of muscular necrosis in association with a patent vascular tree. Theories of  dehydration [211 and direct cell rupture [22] have both been suggested. Our findings after electrical stimulation of the muscle preparation include initial vasodilation followed by progressive arteriolar vasoconstriction, capillary bed flow disturbances, and increased leukocyte endotheiial adhesion. Previous work on ischemia—reperfusion (1-R) injury by Zamboni et at. [8] showed initial arterial

vasodilation up to 1 hr followed by progressive vasocon striction with increasing numbers of adherent leukocytes to venular endothelium after 4 hr of global ischemia. Following electrotrauma the initial vasodilation lasted up to 30 min and was more severe than the initial vasodilation seen by Zamboni et at. [8] in their I-R model. The findings of leukocyte adherence were almost identical following the two different modes of tissue trauma. Our data suggest that electrical injury mimics some aspects of ischemia—reperfusion injury or that there may be a common pathophysiological pathway.

We found several advantages in the use of our standardized electrical injury model and the study ofmicro circulatory changes and tissue responses after application ofthe electrical injury. During the process ofestablishing the model we found that the anatomy and features of the dominant vascular pedicle were consistent in all rats used and that in smafl rats the muscle was thin enough to allow uninhibited
transillumination and recording of leukocyte—endothelial interactions. The hemodynamically isolated muscle allowed for on-line recording. The femoral vessels could be easily accessed to obtain blood samples derived from the muscle capillary bed. The preparation could also allow
studies ofphysiologic regulatory mechanisms by simultaneous measurements ofpedicle and distant arteriolar blood flow. Our results demonstrated consistent micro circulatory responses to the electrical injury. These in cluded ftow cessationlreversal followed by vasodilation
and progressive vasoconstriction at 30 min postinjury. The other important observation was increased leukocyte endothelial adhesion in venules following electri cal injury.

FIG. 5. Peripheral portion of anterior belly of gracilis muscle immediately following electrical injury shows severe coagulation necrosis and complete loss of muscle structure next to the electrical wire interface. (Light microscopy, magnification x500.)

FIG. 6. Peripheral portion of anterior belly of gracilis muscle immediately following electrical injury shows severe coagulation necrosis
and complete loss of muscle structure next to the electrical wire interface. (Light microscopy, magnification x500.) hick myosin fllaments are still visible. There is polymerization and clumping of glycogen. (Electron microscopy, magnification x 10,000.)

FIG. 7. Intermediate portion (0.2 cm off center) of anterior belly of gracilis muscle immediately following electrical injury shows two myofibers. The myofiber on the lower left hand corner reveals greater disruption ofits ultrastructure compared to the other one. Both show evidence of contraction band necrosis with clumping ofglycogen. (Electron microscopy, magnification x12,000.)

PD Dr. med. habil. Jürgen Hussmann

ACKNOWLEDGMENTS
The authors thank Dr. N. Mody and Jim Vickroy, Department of Pathology, MMC and SIU (Springfield, IL), for the histopathological and electron microscopical preparations and slides.

REFERENCES
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6. Luce, E. A., and Gottlieb, S. E. True high-tension electrical injuries. Ann. Ptast. $urg. 12: 321, 1984.
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9. Greene, E. C. Anatomy ofthe Rat. New York: Hafner, 1955.
10. Honig, C. R., Frierson, J. L., and Patterson, J. L. Comparison of neural controls of resistance and capillary density in resting muscle. Am. J. Physiot. 218: 937, 1970.
11. Swain, D. P., and Lalone, B. J. Rat gracilis muscle preparation for combined macro- and microvascular research. Am. J. Physiot. 242: H713, 1982.
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16. Hunt, J. L., MeManus, W. F., Haney, W. P., et at. Vascular lesions in acute electric injuries. J. Trauma 14: 461, 1974.
17. Koshima, 1., Moriguchi, T., Soeda, S., etat. High-voltage electrical injury: Electron microscopic findings ofinjured vessel, nerve, and muscle. Ann. Ptast. Surg. 26: 587, 1991.
18. Jaffe, R. H. Electropathology: A review of the pathologie changes produced by electric currents. Areh. Pathot. 5: 837, 1928.
19. Xuewei, W., and Wanrhong, Z. Vascular injuries in eleetrical burns—The pathologie basis for mechanism ofinjury. Burns 9: 335, 1983.
20. Ponten, 3., Erikson, U., Johansson, 5. H., et at. New observations on tissue changes along the pathway of the current in an electrical injury. Scand. J. Ptast. Reconstr. Surg. 4: 75, 1970.
21. Torre, C., and Varetto, L. The ultrastructure ofthe electric burn in man: A transmission electron microscopy-scanning eleetron microscopy study. J. Forens. Science 30: 448, 1985.
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