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Superoxide anion release (O2−) after ischemia and reperfusion
JOURNAL
OF SURGICAL
RESEARCH
50,565-568
(1991)
Superoxide Anion Release (0;) after lschemia and Reperfusion JULIE A. FREISCHLAG, M.D., AND DINAH HANNA, M.D. Department Presented
of Surgery,
at the Annual
Meeting
Wadsworth
VA and UCLA Medical
of the Association
for Academic
Center, Los Angeles, California
Surgery, Houston,
90024
Texas, November
14-17, 1990
through preprocedure radiation has shown less tissue damage by some investigators [5]. However, the actual production of superoxide anion by the neutrophil after ischemia and reperfusion has not previously been measured. This study was undertaken to evaluate the production of superoxide anion by neutrophils after 2 hr of ischemia followed by 1 hr of reperfusion (no clinical reperfusion injury seen) and after 3 hr of ischemia followed by 1 hr of reperfusion (significant reperfusion injury seen) and to correlate release of superoxide anion to clinical reperfusion injury.
Neutrophils have been implicated as mediators of the reperfusion injury following ischemia. In order to measure neutrophil activation, 0, was determined after 2 hr of ischemia followed by 1 hr of reperfusion (no clinical reperfusion injury) and 3 hr of ischemia followed by 1 hr of reperfusion (significant clinical reperfusion injury). Using New Zealand white rabbits, baseline blood samples were drawn from an ear artery. The right iliac and femoral arteries were exposed and clamped. Just prior to clamp release, blood was obtained from the right iliac vein (ischemia). After 1 hr of reperfusion, blood was again taken from the right iliac vein (reperfusion). Neutrophils were isolated from the blood samples. 0, was determined by the reduction of cytochrome c using a spectrophotometer. In the 2-hr group, results (expressed as pmole O,/min/2 X lo* cells) were: baseline, 0.337 + 0.025; ischemia, 0.512 + 0.039;* and reperfusion, 0.634 f 0.064*. (*P < .05 as compared to baseline). In the 3-hr group, results were: baseline, 0.391 + 0.038; ischemia, 0.413 + 0.051; and reperfusion, 0.258 -t 0.043** (**P < 0.05 as compared to 2 hr reperfusion). A significant increase in 0, was seen after 2 hr of ischemia followed by 1 hr of reperfusion. However, little 0, increase was seen after 3 hr of ischemia and a significant 0, decrease was seen after 1 hr of reperfusion. We conclude: (1) Neutrophil 0, is stimulated early in ischemia followed by reperfusion; (2) after reperfusion injury occurs (3 hr), neutrophils have been activated and 0, can no longer be stimulated; and (3) 0, in this model may be involved in the clinical re0 1991 Academic Press. 1~. perfusion injury seen.
MATERIALS
AND METHODS
Male New Zealand white rabbits (2-5 kg) were purchased from Universal Rabbitry, California. All rabbits underwent procedures approved by the animal research committee whose guidelines were followed. The rabbits were anesthetized using acepromazine and ketamine administered intramuscularly. Baseline blood samples of 10 ml each were then taken from an ear artery. A midline laparotomy and right groin incision were then performed in order to expose the aorta and both iliac and right femoral arteries. All collateral branches were ligated from the aorta to the right common femoral artery. Clips were placed on both internal iliac arteries and the right profunda femoris artery. Vascular clamps were then placed on the right common iliac and common femoral arteries for either 2 or 3 hr. Ten rabbits were used in both the 2- and 3-hr ischemia group. Disappearance of the right anterior tibialis pulse was confirmed by Doppler. All rabbits were monitored by an arterial line which had been placed in the right carotid artery. Body temperature was maintained by heating pads and warming lights. An intravenous line was placed in an ear vein and normal saline was given at 5 ml/kg/hr. After the ischemic period had elapsed, a 20-gauge angiocatheter (Critikon) was placed in the right common iliac vein. A blood sample of 10 ml was taken from the right common iliac vein just prior to clamp release. After the clamp was released, return of the anterior tibialis pulse was confirmed by Doppler. After 1 hr of reperfusion, a similar blood sample was taken again from the
INTRODUCTION The presence of the neutrophil has been identified in many tissues which have been rendered ischemic and then are subsequently reperfused [ 1,2]. In skeletal muscle, neutrophils have been held responsible for the production of the majority of the oxygen free radicals involved in the reperfusion injury due to the fact that skeletal muscle possesses small amounts of xanthine oxidase which is present only in the vascular endothelium [3,4]. Neutropenia achieved experimentally by filters or 565
0022.4804/91
$1.50
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
566
JOURNAL
OF SURGICAL
RESEARCH:
right iliac vein. The rabbit was then sacrificed after clinical examination of the experimental and control limbs. All three blood samples (baseline, ischemia, and reperfusion) were immediately combined with phosphatebuffered saline with glucose and gelatin (PBSG-G) in a 1:l ratio. This solution was combined and thoroughly mixed with an equal volume of 6% dextran (Sigma Chemical Co.) and the erythrocytes were allowed to sediment for 45 min. The supernatant was removed and spun at 1200 rpm for 15 min. The pellet was resuspended in 8 ml of PBSG-G and layered over a 3 ml of Histopaque 1077 (Sigma). This was centrifuged at 1500 rpm for 30 min to separate the lymphocytes and monocytes from the neutrophils. The pellet, consisting of the neutrophils and any remaining erythrocytes, was resuspended in PBSG-G, lysed with distilled water, and 3.5% saline was added to restore the osmolarity. The solution was spun at 1200 rpm for 15 min and the purified neutrophils were resuspended, counted, and diluted to a cell population of 2 X lo6 cells/ml. Differential slides were made to determine purity and evaluation by the trypan blue exclusion test was used to determine neutrophil viability. The production of superoxide anion was determined using a modified method of the difference spectra technique developed by Cohen and Chovaniec [6, 71. Thirty microliters of cytochrome c (Sigma) in a 1 mmole concentration was added to 820 ~1 of preheated Hank’s solution (37’C) in a 1 ml cuvette. Neutrophils in a concentration of 2 X lo6 cells/ml in a volume of 50 ~1 were then added to the cuvette. The neutrophils were then stimulated by PMA (4-/3-phorbol-12+myristate-13-a-acetate; Sigma). At an OD of 550, the sample was read over 10 min for the change in absorbance which reflected the reduction of cytochrome c by the superoxide anion release. This was recorded on graph paper. Samples were run in duplicate. The same reaction was run in the presence of superoxide dismutase (Sigma) which served as a control. The rate of superoxide anion production was taken from the linear portion of the curve between 2 and 6 min. Using the extinction coefficient for cytochrome c, data were expressed as pmole O;/min/B X lo6 cells [8]. Statistical analysis was performed using ANOVA. RESULTS
Differential counts revealed neutrophil purity to be 90-100%. Cell viability as determined by trypan blue was 95-97%. The results of superoxide anion production are shown in Table 1 and Fig. 1. A significant increase in 0; was seen after 2 hr of ischemia which remained elevated after 1 hr of reperfusion. Clinically, no change was seen in the experimental limb as compared to the control limb. However, no significant increase in 0, was seen after 3 hr of ischemia and a significant decrease was then seen after 1 hr of reperfusion. Clinically, after 3 hr of ischemia, a significant ischemit injury was seen which worsened during reperfusion. The experimental limb was stiff and swollen de-
VOL.
50, NO. 6, JUNE
1991
spite an excellent pulse obtained by Doppler in the anterior tibialis artery after both the ischemic and reperfusion period. DISCUSSION
The release of oxygen free radicals has been implicated in reperfusion to be the mechanism which causes tissue damage [g-11]. Oxygen free radicals, which include superoxide anion, the hydroxyl radical, and hydrogen peroxide, cause tissue damage by altering the function of specific proteins and glycosaminoglycans through membrane crosslinking. Through the process of lipid peroxidation, further tissue damage occurs [ 121. The source of the oxygen free radical in reperfusion is somewhat controversial. In experimental models of intestinal ischemia in the cat, the source is primarily xanthine dehydrogenase which is then converted to xanthine oxidase in the intestinal endothelial cells upon the restoration of oxygen [ 131. There is little xanthine oxidase in skeletal muscle as it is only present in the endothelial cells of the vascular structures. Therefore, the generation of the oxygen free radicals must originate elsewhere. Neutrophils have been identified microscopically in skeletal muscle during reperfusion after ischemia. Tissue edema, microcirculation thrombosis, and neutrophil obstruction of small capillaries have been observed [ 141. These neutrophils obstruct flow in the capillaries despite the proximal larger vessels being patent and this has been called the “no reflow” phenomenon. Neutrophils also have been shown to migrate into infarcted areas of the myocardium after ischemia and reperfusion. Elimination of neutrophils by the use of filters prior to ischemia in canine myocardial experiments has demonstrated less tissue destruction and ischemia-induced arrhythmias as shown by Engler and associates [2]. The production of xanthine oxidase and oxygen free radicals initially by skeletal muscle capillary endothelial cells may serve as the chemoattractant for the neutrophils through the activation of complement and histamine [ 15, 161. Neutrophils are then recruited to the site of ischemia and reperfusion and are activated by substances released by the endothelial cells. The neutrophils then produce more superoxide anion through their cell surface NADPH oxidase system which attracts more neutrophils as well as causes more extensive tissue damage [17]. Our study demonstrates that neutrophils are stimulated to produce superoxide anion early in ischemia (2 hr) and remain stimulated after reperfusion even though a clinical reperfusion injury is not yet seen. This finding supports the theory previously described which proposes that neutrophil activation occurs secondary to some stimulation related to ischemia which may be the release of xanthine oxidase from the vascular endothelial cells. Oxygen, which becomes available after reperfusion, can further stimulate the production of superoxide anion.
FREISCHLAG
AND HANNA:
SUPEROXIDE
TABLE Superoxide
2 hr of ischemia: 3 hr of ischemia:
1 hr of reperfusion 1 hr of reperfusion
Note. Data are expressed as pmole O;/min/2 * P < 0.05 as compared to baseline. ** P c 0.05 as compared to 2 hr.
Baseline
Ischemia
Reperfusion
10 10
0.337 +- 0.025 0.391 +- 0.038
0.512 * 0.039* 0.413 f 0.051
0.634 f 0.064* 0.258 -t 0.043**
X lo6 cells.
0.6 -
0.5 -
?I (0 0
0.4 -
b
. ‘N 0 0 0 E a.
b
1
n
0.7 -
L? . 2c
1 IT
0.3 -
oxide anion, and can no longer be stimulated in our assay. In our assay, PMA activates the release of superoxide anion and will not do so if the neutrophil has already been stimulated prior to the test. Therefore, superoxide anion release from neutrophils in our study correlated well with the clinical appearance of the experimental limb. Methods aimed at preventing ischemia-induced reperfusion injury in skeletal muscle therefore must deactivate the neutrophil prior to reperfusion. In skeletal muscle ischemia, unlike in transplantation where the organ can be pretreated prior to ischemia, there is rarely an opportunity to treat prior to ischemic interval. Therefore, pharmacological agents need to be investigated which can block the neutrophil’s activation after ischemia and prior to reperfusion. In summary, our study evaluated the 0; of neutrophils after 2 hr of ischemia followed by 1 hr of reperfusion (no clinical reperfusion injury seen) and after 3 hr of ischemia followed by 1 hr of reperfusion (significant reperfusion injury seen). A significant increase in 0; in the 2-hr group was seen after both ischemia and reperfusion. Further studies should include evaluation of the neutrophils obtained from other sites, such as the contralateral limb, after the ischemia and reperfusion interval to determine if this effect is a systemic one as well as later in the reperfusion period to determine how long this effect is manifested. No such increase in 0; was seen after 3 hr of ischemia with an actual decrease seen after reperfusion. These findings demonstrate that neutrophil activation correlates with reperfusion injury in this model. Prevention of such an injury will rely on methods which can deactivate the neutrophil prior to reperfusion.
0.2 -
ACKNOWLEDGMENT
--+-
0.1:
567
Anion Production
After 3 hr of ischemia, however, the neutrophils were not able to be stimulated as seen after 2 hr of ischemia. This finding was now associated with a severe clinical ischemic injury with a swollen and stiff extremity. After reperfusion in the 3-hr group, the superoxide anion release was decreased, even below baseline values. Substances released after reperfusion may be more potent in their ability to stimulate the neutrophil. The clinical reperfusion injury in the experimental limb also was worse. This decrease reflects the fact that the neutrophils have already been activated, released their super-
01 =
ANION RELEASE
.
‘
2 hrs tiemia.1
hr rep&&ion
3 hrs ischemm.
1 hr reperfwon
.
,
.
,
b = baseline i = ischemia r = reperfusion
.
,
,
The authors thank Jeffrey with the statistical analysis.
A. Gorbein,
Dr. PH, for his assistance
,
REFERENCES Time
cells
sampled
1. FIG. 1. Graph demonstrating neutrophil lease after 2 hr of ischemia, 1 hr of reperfusion hr of reperfusion.
superoxide anion reand 3 hr of ischemia, 1
Romson, J. L., Schork, A., and emit myocardial lation 67: 1016,
Hook, B. G., Kunkel, S. L., Abrams, G. D., Lucchesi, B. R. Reduction of the extent of ischinjury by neutrophil depletion in the dog. Circu1983.
568
JOURNAL
2.
Engler, R. L., Dahlgren, M. D., Morris, D. D., Peterson, M. A., and Schmid-Schonbein, G. W. Role of leukocytes in response to acute myocardial ischemia and reflow in dogs. Am. J. Physiol.
OF SURGICAL
RESEARCH:
251:H314,1986. 3.
4.
5.
6.
7.
8.
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: 599, 1985. Belkin, M., LaMorte W. L., Wright, J. G., and Hobson, R. W., III. The role of leukocytes in the pathophysiology of skeletal muscle ischemic injury. J. Vast. Surg. 10: 14, 1989. Korthuis, R. J., Grisham, M. B., and Granger, D. N. Leukocyte depletion attenuates vascular injury in postischemic skeletal muscle. Am. J. Physiol. 254: H823, 1988. Cohen, H. J., and Chovaniec, M. E. Superoxide generation by digitonin-stimulated guinea pig granulocytes-A basis for a continuous assay for monitoring superoxide production and for study of the activation of the generating system. J. Clin. Inuest. 61: 1081,1978. Cohen, H. J., and Chovaniec, M. E. Superoxide generation by digitonin-stimulated guinea pig granulocytes-The effects of Nethyl maleimide, divalent cations, and glycolytic and mitochondrial inhibitors on the activation of the superoxide generating system. J. Cl&. Invest. 61: 1088, 1978. Freischlag, J. A., Backstrom, B., Kelly, D., Keehn, G., and Busuttil, R. W. Comparison of blood and peritoneal neutrophil activity in rabbits with and without peritonitis. J. Surg. Res. 40: 145, 1986.
VOL.
50, NO. 6, JUNE
1991
9.
Parks, D. A., Bulkley, G. B., and Granger, D. N. Role of oxygen free radicals in shock, ischemia and organ preservation. Surgery 94: 428,1983.
10.
Gardner, T. J., Stewart, J. R., Casale, A. S., Downey, J. M., and Chambers, D. E. Reduction of myocardial ischemic injury with oxygen-derived free radical scavengers. Surgery 94: 423, 1983.
11.
McCord, J. M. Oxygen-derived free radicals in postischemic sue injury. NEJM 312: 159, 1985.
tis-
12.
McCord, J. M. The superoxide free radical: Its biochemistry pathophysiology. Surgery 94: 412,1983.
and
13.
Parks, D. A., and Granger, D. N. Ischemia-induced vascular changes: Role of xanthine oxidase and hydroxyl radicals. Am. J. Phystil. 245: G285,1983.
14.
Engler, R. L., Schmid-Schonbein, G. W., and Pavelic, R. S. Leukocyte capillary plugging in myocardial ischemia and reperfusion in the dog. Am. J. Pathol. 111: 98,1983.
15.
Sacks T., Moldow, C. F., Craddock, P. R., Bowers, T. K., and Jacob, H. S. Oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes-An in vitro model of immune vascular damage. J. Clin. Inuest. 61: 1161, 1978.
16.
Ward, P. A., Till, G. O., Kunkel, R., and Beauchamp, C. Evidence for role of hydroxyl radical in complement and neutrophil dependent tissue injury. J. Clin. Invest. 72: 189, 1983.
17.
Petrone, W. F., English, D. K., Wong, K., and McCord, J. M. Free radicals and inflammation: Superoxide-dependent activation of a neutrophil chemotactic factor in plasma. Proc. Natl. Acad. Sci. 77: 1159,198O.
OF SURGICAL
RESEARCH
50,565-568
(1991)
Superoxide Anion Release (0;) after lschemia and Reperfusion JULIE A. FREISCHLAG, M.D., AND DINAH HANNA, M.D. Department Presented
of Surgery,
at the Annual
Meeting
Wadsworth
VA and UCLA Medical
of the Association
for Academic
Center, Los Angeles, California
Surgery, Houston,
90024
Texas, November
14-17, 1990
through preprocedure radiation has shown less tissue damage by some investigators [5]. However, the actual production of superoxide anion by the neutrophil after ischemia and reperfusion has not previously been measured. This study was undertaken to evaluate the production of superoxide anion by neutrophils after 2 hr of ischemia followed by 1 hr of reperfusion (no clinical reperfusion injury seen) and after 3 hr of ischemia followed by 1 hr of reperfusion (significant reperfusion injury seen) and to correlate release of superoxide anion to clinical reperfusion injury.
Neutrophils have been implicated as mediators of the reperfusion injury following ischemia. In order to measure neutrophil activation, 0, was determined after 2 hr of ischemia followed by 1 hr of reperfusion (no clinical reperfusion injury) and 3 hr of ischemia followed by 1 hr of reperfusion (significant clinical reperfusion injury). Using New Zealand white rabbits, baseline blood samples were drawn from an ear artery. The right iliac and femoral arteries were exposed and clamped. Just prior to clamp release, blood was obtained from the right iliac vein (ischemia). After 1 hr of reperfusion, blood was again taken from the right iliac vein (reperfusion). Neutrophils were isolated from the blood samples. 0, was determined by the reduction of cytochrome c using a spectrophotometer. In the 2-hr group, results (expressed as pmole O,/min/2 X lo* cells) were: baseline, 0.337 + 0.025; ischemia, 0.512 + 0.039;* and reperfusion, 0.634 f 0.064*. (*P < .05 as compared to baseline). In the 3-hr group, results were: baseline, 0.391 + 0.038; ischemia, 0.413 + 0.051; and reperfusion, 0.258 -t 0.043** (**P < 0.05 as compared to 2 hr reperfusion). A significant increase in 0, was seen after 2 hr of ischemia followed by 1 hr of reperfusion. However, little 0, increase was seen after 3 hr of ischemia and a significant 0, decrease was seen after 1 hr of reperfusion. We conclude: (1) Neutrophil 0, is stimulated early in ischemia followed by reperfusion; (2) after reperfusion injury occurs (3 hr), neutrophils have been activated and 0, can no longer be stimulated; and (3) 0, in this model may be involved in the clinical re0 1991 Academic Press. 1~. perfusion injury seen.
MATERIALS
AND METHODS
Male New Zealand white rabbits (2-5 kg) were purchased from Universal Rabbitry, California. All rabbits underwent procedures approved by the animal research committee whose guidelines were followed. The rabbits were anesthetized using acepromazine and ketamine administered intramuscularly. Baseline blood samples of 10 ml each were then taken from an ear artery. A midline laparotomy and right groin incision were then performed in order to expose the aorta and both iliac and right femoral arteries. All collateral branches were ligated from the aorta to the right common femoral artery. Clips were placed on both internal iliac arteries and the right profunda femoris artery. Vascular clamps were then placed on the right common iliac and common femoral arteries for either 2 or 3 hr. Ten rabbits were used in both the 2- and 3-hr ischemia group. Disappearance of the right anterior tibialis pulse was confirmed by Doppler. All rabbits were monitored by an arterial line which had been placed in the right carotid artery. Body temperature was maintained by heating pads and warming lights. An intravenous line was placed in an ear vein and normal saline was given at 5 ml/kg/hr. After the ischemic period had elapsed, a 20-gauge angiocatheter (Critikon) was placed in the right common iliac vein. A blood sample of 10 ml was taken from the right common iliac vein just prior to clamp release. After the clamp was released, return of the anterior tibialis pulse was confirmed by Doppler. After 1 hr of reperfusion, a similar blood sample was taken again from the
INTRODUCTION The presence of the neutrophil has been identified in many tissues which have been rendered ischemic and then are subsequently reperfused [ 1,2]. In skeletal muscle, neutrophils have been held responsible for the production of the majority of the oxygen free radicals involved in the reperfusion injury due to the fact that skeletal muscle possesses small amounts of xanthine oxidase which is present only in the vascular endothelium [3,4]. Neutropenia achieved experimentally by filters or 565
0022.4804/91
$1.50
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
566
JOURNAL
OF SURGICAL
RESEARCH:
right iliac vein. The rabbit was then sacrificed after clinical examination of the experimental and control limbs. All three blood samples (baseline, ischemia, and reperfusion) were immediately combined with phosphatebuffered saline with glucose and gelatin (PBSG-G) in a 1:l ratio. This solution was combined and thoroughly mixed with an equal volume of 6% dextran (Sigma Chemical Co.) and the erythrocytes were allowed to sediment for 45 min. The supernatant was removed and spun at 1200 rpm for 15 min. The pellet was resuspended in 8 ml of PBSG-G and layered over a 3 ml of Histopaque 1077 (Sigma). This was centrifuged at 1500 rpm for 30 min to separate the lymphocytes and monocytes from the neutrophils. The pellet, consisting of the neutrophils and any remaining erythrocytes, was resuspended in PBSG-G, lysed with distilled water, and 3.5% saline was added to restore the osmolarity. The solution was spun at 1200 rpm for 15 min and the purified neutrophils were resuspended, counted, and diluted to a cell population of 2 X lo6 cells/ml. Differential slides were made to determine purity and evaluation by the trypan blue exclusion test was used to determine neutrophil viability. The production of superoxide anion was determined using a modified method of the difference spectra technique developed by Cohen and Chovaniec [6, 71. Thirty microliters of cytochrome c (Sigma) in a 1 mmole concentration was added to 820 ~1 of preheated Hank’s solution (37’C) in a 1 ml cuvette. Neutrophils in a concentration of 2 X lo6 cells/ml in a volume of 50 ~1 were then added to the cuvette. The neutrophils were then stimulated by PMA (4-/3-phorbol-12+myristate-13-a-acetate; Sigma). At an OD of 550, the sample was read over 10 min for the change in absorbance which reflected the reduction of cytochrome c by the superoxide anion release. This was recorded on graph paper. Samples were run in duplicate. The same reaction was run in the presence of superoxide dismutase (Sigma) which served as a control. The rate of superoxide anion production was taken from the linear portion of the curve between 2 and 6 min. Using the extinction coefficient for cytochrome c, data were expressed as pmole O;/min/B X lo6 cells [8]. Statistical analysis was performed using ANOVA. RESULTS
Differential counts revealed neutrophil purity to be 90-100%. Cell viability as determined by trypan blue was 95-97%. The results of superoxide anion production are shown in Table 1 and Fig. 1. A significant increase in 0; was seen after 2 hr of ischemia which remained elevated after 1 hr of reperfusion. Clinically, no change was seen in the experimental limb as compared to the control limb. However, no significant increase in 0, was seen after 3 hr of ischemia and a significant decrease was then seen after 1 hr of reperfusion. Clinically, after 3 hr of ischemia, a significant ischemit injury was seen which worsened during reperfusion. The experimental limb was stiff and swollen de-
VOL.
50, NO. 6, JUNE
1991
spite an excellent pulse obtained by Doppler in the anterior tibialis artery after both the ischemic and reperfusion period. DISCUSSION
The release of oxygen free radicals has been implicated in reperfusion to be the mechanism which causes tissue damage [g-11]. Oxygen free radicals, which include superoxide anion, the hydroxyl radical, and hydrogen peroxide, cause tissue damage by altering the function of specific proteins and glycosaminoglycans through membrane crosslinking. Through the process of lipid peroxidation, further tissue damage occurs [ 121. The source of the oxygen free radical in reperfusion is somewhat controversial. In experimental models of intestinal ischemia in the cat, the source is primarily xanthine dehydrogenase which is then converted to xanthine oxidase in the intestinal endothelial cells upon the restoration of oxygen [ 131. There is little xanthine oxidase in skeletal muscle as it is only present in the endothelial cells of the vascular structures. Therefore, the generation of the oxygen free radicals must originate elsewhere. Neutrophils have been identified microscopically in skeletal muscle during reperfusion after ischemia. Tissue edema, microcirculation thrombosis, and neutrophil obstruction of small capillaries have been observed [ 141. These neutrophils obstruct flow in the capillaries despite the proximal larger vessels being patent and this has been called the “no reflow” phenomenon. Neutrophils also have been shown to migrate into infarcted areas of the myocardium after ischemia and reperfusion. Elimination of neutrophils by the use of filters prior to ischemia in canine myocardial experiments has demonstrated less tissue destruction and ischemia-induced arrhythmias as shown by Engler and associates [2]. The production of xanthine oxidase and oxygen free radicals initially by skeletal muscle capillary endothelial cells may serve as the chemoattractant for the neutrophils through the activation of complement and histamine [ 15, 161. Neutrophils are then recruited to the site of ischemia and reperfusion and are activated by substances released by the endothelial cells. The neutrophils then produce more superoxide anion through their cell surface NADPH oxidase system which attracts more neutrophils as well as causes more extensive tissue damage [17]. Our study demonstrates that neutrophils are stimulated to produce superoxide anion early in ischemia (2 hr) and remain stimulated after reperfusion even though a clinical reperfusion injury is not yet seen. This finding supports the theory previously described which proposes that neutrophil activation occurs secondary to some stimulation related to ischemia which may be the release of xanthine oxidase from the vascular endothelial cells. Oxygen, which becomes available after reperfusion, can further stimulate the production of superoxide anion.
FREISCHLAG
AND HANNA:
SUPEROXIDE
TABLE Superoxide
2 hr of ischemia: 3 hr of ischemia:
1 hr of reperfusion 1 hr of reperfusion
Note. Data are expressed as pmole O;/min/2 * P < 0.05 as compared to baseline. ** P c 0.05 as compared to 2 hr.
Baseline
Ischemia
Reperfusion
10 10
0.337 +- 0.025 0.391 +- 0.038
0.512 * 0.039* 0.413 f 0.051
0.634 f 0.064* 0.258 -t 0.043**
X lo6 cells.
0.6 -
0.5 -
?I (0 0
0.4 -
b
. ‘N 0 0 0 E a.
b
1
n
0.7 -
L? . 2c
1 IT
0.3 -
oxide anion, and can no longer be stimulated in our assay. In our assay, PMA activates the release of superoxide anion and will not do so if the neutrophil has already been stimulated prior to the test. Therefore, superoxide anion release from neutrophils in our study correlated well with the clinical appearance of the experimental limb. Methods aimed at preventing ischemia-induced reperfusion injury in skeletal muscle therefore must deactivate the neutrophil prior to reperfusion. In skeletal muscle ischemia, unlike in transplantation where the organ can be pretreated prior to ischemia, there is rarely an opportunity to treat prior to ischemic interval. Therefore, pharmacological agents need to be investigated which can block the neutrophil’s activation after ischemia and prior to reperfusion. In summary, our study evaluated the 0; of neutrophils after 2 hr of ischemia followed by 1 hr of reperfusion (no clinical reperfusion injury seen) and after 3 hr of ischemia followed by 1 hr of reperfusion (significant reperfusion injury seen). A significant increase in 0; in the 2-hr group was seen after both ischemia and reperfusion. Further studies should include evaluation of the neutrophils obtained from other sites, such as the contralateral limb, after the ischemia and reperfusion interval to determine if this effect is a systemic one as well as later in the reperfusion period to determine how long this effect is manifested. No such increase in 0; was seen after 3 hr of ischemia with an actual decrease seen after reperfusion. These findings demonstrate that neutrophil activation correlates with reperfusion injury in this model. Prevention of such an injury will rely on methods which can deactivate the neutrophil prior to reperfusion.
0.2 -
ACKNOWLEDGMENT
--+-
0.1:
567
Anion Production
After 3 hr of ischemia, however, the neutrophils were not able to be stimulated as seen after 2 hr of ischemia. This finding was now associated with a severe clinical ischemic injury with a swollen and stiff extremity. After reperfusion in the 3-hr group, the superoxide anion release was decreased, even below baseline values. Substances released after reperfusion may be more potent in their ability to stimulate the neutrophil. The clinical reperfusion injury in the experimental limb also was worse. This decrease reflects the fact that the neutrophils have already been activated, released their super-
01 =
ANION RELEASE
.
‘
2 hrs tiemia.1
hr rep&&ion
3 hrs ischemm.
1 hr reperfwon
.
,
.
,
b = baseline i = ischemia r = reperfusion
.
,
,
The authors thank Jeffrey with the statistical analysis.
A. Gorbein,
Dr. PH, for his assistance
,
REFERENCES Time
cells
sampled
1. FIG. 1. Graph demonstrating neutrophil lease after 2 hr of ischemia, 1 hr of reperfusion hr of reperfusion.
superoxide anion reand 3 hr of ischemia, 1
Romson, J. L., Schork, A., and emit myocardial lation 67: 1016,
Hook, B. G., Kunkel, S. L., Abrams, G. D., Lucchesi, B. R. Reduction of the extent of ischinjury by neutrophil depletion in the dog. Circu1983.
568
JOURNAL
2.
Engler, R. L., Dahlgren, M. D., Morris, D. D., Peterson, M. A., and Schmid-Schonbein, G. W. Role of leukocytes in response to acute myocardial ischemia and reflow in dogs. Am. J. Physiol.
OF SURGICAL
RESEARCH:
251:H314,1986. 3.
4.
5.
6.
7.
8.
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: 599, 1985. Belkin, M., LaMorte W. L., Wright, J. G., and Hobson, R. W., III. The role of leukocytes in the pathophysiology of skeletal muscle ischemic injury. J. Vast. Surg. 10: 14, 1989. Korthuis, R. J., Grisham, M. B., and Granger, D. N. Leukocyte depletion attenuates vascular injury in postischemic skeletal muscle. Am. J. Physiol. 254: H823, 1988. Cohen, H. J., and Chovaniec, M. E. Superoxide generation by digitonin-stimulated guinea pig granulocytes-A basis for a continuous assay for monitoring superoxide production and for study of the activation of the generating system. J. Clin. Inuest. 61: 1081,1978. Cohen, H. J., and Chovaniec, M. E. Superoxide generation by digitonin-stimulated guinea pig granulocytes-The effects of Nethyl maleimide, divalent cations, and glycolytic and mitochondrial inhibitors on the activation of the superoxide generating system. J. Cl&. Invest. 61: 1088, 1978. Freischlag, J. A., Backstrom, B., Kelly, D., Keehn, G., and Busuttil, R. W. Comparison of blood and peritoneal neutrophil activity in rabbits with and without peritonitis. J. Surg. Res. 40: 145, 1986.
VOL.
50, NO. 6, JUNE
1991
9.
Parks, D. A., Bulkley, G. B., and Granger, D. N. Role of oxygen free radicals in shock, ischemia and organ preservation. Surgery 94: 428,1983.
10.
Gardner, T. J., Stewart, J. R., Casale, A. S., Downey, J. M., and Chambers, D. E. Reduction of myocardial ischemic injury with oxygen-derived free radical scavengers. Surgery 94: 423, 1983.
11.
McCord, J. M. Oxygen-derived free radicals in postischemic sue injury. NEJM 312: 159, 1985.
tis-
12.
McCord, J. M. The superoxide free radical: Its biochemistry pathophysiology. Surgery 94: 412,1983.
and
13.
Parks, D. A., and Granger, D. N. Ischemia-induced vascular changes: Role of xanthine oxidase and hydroxyl radicals. Am. J. Phystil. 245: G285,1983.
14.
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