Intermittent ischemia potentiates intestinal reperfusion injury Elizabeth T. Clark, M D , and Bruce L. Gewertz, M D , Chicago, Ill. We hypothesized that even brief periods of reperfusion interjected between ischemic episodes would increase tissue injury. Studies were performed in a rat small intestine preparation in which metabolic, hemodynamic, and histologic responses to ischemia have been well characterized. Animals were subjected to a total of 30 or 45 minutes of complete intestinal ischemia. Flow interruption was continuous (C, single episode) or intermittent (I, two or three episodes of 15-minute ischemia separated by 5 minutes of reperfusion). In some experiments 5-minute reperfusions were performed with arterial blood depleted of leukocytes (IL). This additional perturbation was included to determine the role of neutrophils that have been strongly implicated in reperfusion injury. In all three protocols histologic sections were obtained after each ischemic insult and after I hour of reperfusion with arterial blood. Villous histology was graded in a blinded fashion with I = normal and 5 = severe injury. No significant differences were found between groups in immediate postischemic histologies before reperfusion. After 1 hour of reperfusion, intermittent episodes of ischemia were associated with significantly worse histologic injury than that seen with comparable durations of continuous ischemia (30 min: I, 4.4 -+ 0.5 vs C, 2.7 - 0.4; 45 min: I, 4.9 -+ 0.2 vs C, 2.8 -+ 0.3). However, if5-minute reperfusions were with leukopenic blood, this effect was markedly reduced (30 min IL, 3.4 --. 0.3; 45 min IL, 3.6 -+ 0.2). Even short periods of reperfusion during an ischemic insult greatly increased mucosal injury. This likely resulted from increased access of leukocytes to ischemic tissues during the brief reperfusion periods, since decreasing leukocyte availability by transiently reperfusing with leukopenic blood attenuated the damage. Maneuvers designed to limit leukocyte adherence to endothelial cells should be considered in the treatment of intestinal ischemia, particularly in patients at risk for repeated episodes of flow interruption. (J VAsc SURe 1991;13:601-6.)
It is now well accepted that much of the tissue injury associated with an ischemic episode is due to reperfusion phenomena that begin only when blood flow is restored.~ The mechanism of injury involves, at least in part, the generation of oxygen-derived free radical species, which destabilize cellular membranes. 2-4 This improved understanding of ischemia and reperfusion in animal models has suggested new strategies to the vascular surgeon treating ischemic syndromes. These have included the use of free radical scavengers and intentional restriction of the initial bolus of oxygenated blood when flow is first restored. Unfortunately, treatment protocols are Supported by American Heart Association grant No. 88-786 (Dr. Gewertz) and National Institutes of Health Grant No. T32H607665-02 (Dr. Clark). Presented at the Fourteenth AnnualMeeting of the Midwestern Vascular Surgical Society,Toledo, Ohio, Sept. 14-15, 1990. Reprint requests: Bruce L. Gewertz,MD, Universityof Chicago, Department of Surgery, 5841 S. Maryland Ave., Box 129, Chicago, IL 60637. 24/6/28207
largely based on experimental preparations subjected to single episodes of ischemia. Such models may not approximate the clinical situation; in practice ischemic insults often recur exposing an organ to multiple cycles of ischemia and reperfusion. We addressed this concern in a series of experiments that more closely parallel the clinical setting. We hypothesized that even brief periods of reperfusion interjected between ischemic episodes would increase tissue injury. Studies were performed in a rat small intestine preparation in which metabolic, hemodynamic, and histologic responses to ischemia have been well characterized. This organ was selected since multiple episodes of mesenteric ischemia are often encountered in clinical practice especially in the nonocclusive variant of the syndrome. Animals were subjected to a total of 30 or 45 minutes of complete intestinal ischemia. Flow interruption was continuous (single episode) or intermittent (two or three episodes of 15 minutes ofischemia separated by 5 minutes of reperfusion). In some 601
Journalof VASCULAR SURGERY
602 Clark and Gewertz
RECOROEH
/
A-vox
I/
~
l
\
-,,,
RESERVOtR
IA
WATER BATt4
Fig. 1. Experiments were performed in denervated autoperfused rat intestinal preparations.
experiments 5-minute reperfusions were performed with arterial blood depleted of leukocytes. This additional intervention was included to determine the role of neutrophils which have been strongly implicated in reperfusion injury. METHODS Animal care and experimentation described below complied with the "Principles of Laboratory Animal Care" and the "Guide for Care and Use of Laboratory Animals" (NIH publication No. 80-23, revised 1985). Experimental model. The intestinal preparation is a modification of that previously described by Anzueto et al. s and Kim and Gewertz6 (Fig. 1). Male Wistar rats weighing 400 to 600 gm (retired breeders, Harlan Sprague-Dawley) were anesthetized by ether inhalation augmented by intraperitoneal injection of sodium pentobarbital (50 mg/ml at 0.1 ml/100 gm). The trachea was intubated with 1.67 mm ID PE 240 tubing (Becton Dickinson Primary Care Diagnostic, Sparks, Md.), and supplemental oxygen was supplied at a rate of 4 L/min. Surgical excision of the duodenum, pancreas, and colon was followed by bilateral ligation of the renal arteries and veins. Periadventitial stripping of the superior me-
senteric artery (SMA) was performed to ensure denervation. The small intestine was wrapped in saline-soaked gauze and cellophane to minimize evaporative losses. Temperature was controlled at 37 ° C with an intermittent thermistor controlled incandescent lamp. Direct cannulation of the right femoral artery allowed continuous monitoring of systemic arterial pressure. After systemic heparinization (100 units heparin/100 gm body weight), an extracorporeal circuit was created to route blood from the right internal carotid artery and superior mesenteric vein (PE190 tubing, 1.19 mm ID) through a continuous A-~rO2 difference analyzer (AVOX Systems, Inc., San Antonio, Texas). Small intestine blood flow was controlled by cannulation of the SMA and use of an infusion pump (Gilson minipuls II; Medical Electronics, Middleton, Wis.) at 6.0 ml/min (calculated to provide 70 to 80 ml/min/100 gm intestine). All extracorporeal blood was collected in a reservoir, pumped through a 37 ° C water bath, and returned to the rat via the right femoral vein. When indicated, SMA occlusion was produced by cessation of pump perfusion. Protocol. Animals (n = 25) were allowed to stabilize for 1 hour after preparation. Sham animals (n -- 3) were observed for 3 hours without ischemic
Volume 13 Number 5 May 1991
Intestinal reperfusion injury potentiated by intermittant ischemia 603
SHAM (N=3)
30 MIN. ISCHEMIA
45 MIN. ISCHEMIA
CONTINUOUS INTERMITTENT INT.& LEUKOPENIC (N=4) (N=4) (N=3)
CONTLNUOUSINTERMITTENT INT.& LEUKOPEN~C (n=4) (n=4) (n=3)
Fig. 2. Animals (n = 25) were subjected to either continuous or intermittent ischemia. Intermittent reperfusion (5 minutes) was performed with either native arterial blood or leukopenic arterial blood.
Grade 1
Grade 2
6racte 3
Gracle 4
6ra~e 5
Fig. 3. Histologic grading system. Epithelial morphologic characteristics of individual intestinal sections were evaluated in a blinded manner by use of a modification of the Chiu system.
insult to establish the stability of the preparation and to document baseline mesenteric vascular resistance (MVR) and oxygen consumption (VO2) (MVR 0.84 + 0.06 at time 0, 0.87 _+ 0.02 at 3 hours; ~rO 2 3.65 _+ 0.73 at time 0, 3.59 _+ 0.42 at 3 hours). In rats with continuous ischemia (C), intestines were subject to either 30 minutes (n = 4) or 45 minutes (n -- 4) of complete flow interruption as a single episode. Fourteen other rats experienced intermittent ischemia presented as two or three episodes of 15 minutes of flow interruption (total ischemia 30 or 45 minutes) separated by 5 minutes of reperfusion. These short intervals of reperfusion were performed with either native arterial blood (I, n - - 8 ) or leukopenic blood (IL, n = 6). Leukopenia (white blood cell cotmt < 5 0 0 / m m 3) was achieved by centtifugation of blood and removal of the buffy coat and confirmed by the absence of white blood cells on peripheral smear. Once the above treatment protocols were completed, the intestines in all animals were reperfused for 1 hour with native arterial blood (Fig. 2).
Histologic sections were obtained after each ischemic insult and after 1 hour of reperfusion. After the experiments rats were killed by exsanguination. Intestinal sections were weighed and fixed in 10% neutral buffered formalin. Histologic evaluation was performed in a blinded manner by use of a modification of the system of Chiu et al.7; this grading system considers both villous height and epithelial morphology. Photomicrographs of the histologic grading system are depicted in Fig. 3. Data analysis. All values are reported as mean _+ SEM. Intestinal blood flow is normalized to 100 gm gut weight stripped of mesentery. Oxygen consumption (VO2) was calculated as the product of A-VO 2 difference and intestinal blood flow. Mesenteric vascular resistance was calculated as the difference between mean arterial pressure and mesenteric venous pressure divided by intestinal blood flow. Statistical analysis was performed with t tests between groups, and significance was assumed for p < 0.05.
604
Journal of VASCULAR SURGERY
Clark and Gewertz
I HR. REPERFUSION
uJ O
< o
u 0 0 0 I.,J
\\x\ \N\\ \\\\ \\\\ x\\\ \xx\
\\\\ \\\\ \\\\
,'x\'~ SHAM
C
I 30 MIN,
IL
C
I 45 MIN,
IL
Fig. 4. Intermittent ischemia (I) significantly increased histologic damage as compared to a continuous (C) insult. This effect was attenuated if 5-minute reperfusions were with leukopenic blood (/L).
Table I. Mesenteric vascular resistance (MVR, mmHg/ml/min • 100 gin) in continuous (C), intermittent (I), and intermittent leukopenic (IL) groups before ischemia and after 1 hour of reperfusion Before ischemia 30 minutes of ischemia Continuous Intermittent Intermittent leukopenic 45 minutes o f ischemia Continuous In termittent In termittent leukopenic
Reperfmion
0.89 -+ 0.13 0.87 + 0.22 0.83 -+ 0.02
0.20 - 0.26 0.89 + 0.10 0.84 -+ 0.04
0.81 + 0.10 0.83 -+ 0.07 0.87 -+ 0.12
0.86 _+ 0 . 0 l 0.86 -+ 0.14 0.88 _+ 0.11
RESULTS In all groups and ischemic intervals, histologic injury was not prominent until after the 1-hour reperfusion period. Immediate postischemic histologies were different from baseline (p < 0.05 all groups), but no differences were found between groups (30 minutes: C, 2.1 _+ 0.2; I, 2.3 + 0.4; IL, 1.9 + 0.1; 45 minutes: C, 2.7 _+ 0.3; I, 3.3 +- 0.4; IL, 2.6 + 0.3). Intermittent episodes of ischemia resulted in significantly worse histologic injury than comparable intervals of continuous ischemia (Fig. 4). For example, 30 minutes of continuous ischemia followed by 1 hour of reperfusion (C) yielded a histologic grade
of 2.7 + 0.4, whereas 30 minutes of intermittent ischemia (two 15-minute periods, I) resulted in a grade of 4.4 + 0.5 (p < 0.05). The eventual injury after intermittent ischemia was attenuated by leukopenic reperfusion during the 5-minute intervals. In the 30-minute flow interruptions, limiting access of leukocytes to the intestine during the 5-minute reperfusions (IL) decreased the eventual injury score to 3.4 + 0.3 (not different from C). Similar results were seen in the 45-minute ischemia groups (C, 2.9 + 0.6; I, 4.9 + 0.2; IL, 3.7 +-- 0.2)~. Since intestinal blood flow was controlled with an infusion pump at 6.0 ml/min, hemodynamic changes are most appropriately monitored by variations in mesenteric vascular resistance. Mean mesenteric vascular resistance in all experimental groups is displayed in Table I. N o statistically significant differences were found in M V R between any groups. VO2 (2.74 - 0.88 ml/min/100 gm at baseline) was predictably depressed after 1 hour of reperfusion in all experimental groups. These values did not vary significantly within total ischemia groups (30 minutes: C, 2.12 + 0.41; I, 2.57-+ 0.32; IL, 2.34 + 0.41; 45 minutes C, 1.91 + 0.54; I, 1.90 -+ 0.23; IL, 2.08 + 0.15), although 45 minutes of total ischemia, regardless of temporal sequence, resulted in more severe depression of VO2 than 30 minutes of total ischemia.
Volume 13 Number 5 May 1991
Intestinal reperfusion injury potentiated by intermittant ischemia 605
DISCUSSION These data demonstrate that multiple episodes of intestinal ischemia interrupted by brief periods of perfusion are associated with increased mucosal injury as compared to comparable continuous ischemic insults. This phenomenon has been previously investigated in myocardial preparations. Geft et al.8 used a canine model and showed that brief ischemic insults that singly would not cause irreversible cellular injury resulted in myocardial necrosis when repeated. Evidence of necrosis was established by increased creatinine kinase release and confirmed by light and electron microscopy. Subramanian et al.9 observed that intermittent global ischemia of the isolated perfused heart (three 20-minute periods of ischemia each followed by 10 minutes ofreperfusion) resulted in increased levels of malondialdehyde (an indicator of free radical formation) as compared to controls. Repeated ischemia-reperfusion injury also adversely affected recovery of cardiac function. Gorlach et al. performed biopsies on 40 patients undergoing coronary artery bypass grafting who were randomized to intermittent ischemia rather than cardioplegic arrest during operation.I° Biopsies of those undergoing intermittent ischemia showed unexpectedly severe myocardial damage with excessive intracellular edema and ultrastructural derangements. Given the role of leukocytes as mediator of the ischemia-reperfusion phenomenon in other organs, we hypothesized that mucosal injury could be attenuated by the use of leukopenic reperfusate between ischemic episodes. Lucchesi et al. 11 showed that leukopenia produced by administration of polymorphonuclear cell (PMN) antisera reduced myocardial infarct size by 43% in a canine model. On histologic examination infarcts in control animals had substantial PMN infiltration, which was virtually absent in leukopenic dogs. Engler et al.12 used leukoplaque filters to produce leukopenia and compared control and leukopenic myocardial preparations after 1 hour of ischemia and 1 hour of reperfusion. Leukocyte depletion prevented excessive tissue edema, ventricular dysrhythmias, and a "no reflow phenomena" as a result of leukocyte "plugging" of capillaries. Korthuis et al.lS examined the role of leukocytes in ischemia-reperfusion injury of skeletal muscle using the well-characterized isolated canine gracilis preparation. Reperfusion with blood depleted of leukocytes prevented the increased microvascular permeability and capillary resistance seen in control preparations. In work more directly related to our experiments,
Hernandez et al. 14 investigated the effects of leukocyte depletion on the generation of ischemiareperfusion injury in a feline model of intestinal ischemia. Systemic treatments included either antineutrophil serum or monoclonal antibody to endothelial receptors responsible for neutrophil adherence. Both neutrophil depletion and prevention of adherence significantly attenuated the increased microvascular permeability associated with 1 hour of ischemia-reperfusion. Our data would strongly support the importance of neutrophils in ischemia-reperfusion. Neutrophil binding to endothelium results in cellular activation and the production of free oxygen radicals and associated toxins. Binding is mediated by the CD 11/CD 18 complex on the neutrophil surface and is stimulated by various chemotactic peptides and lipid mediators. The subsequent release of proteases and the production of free oxygen radicals results in lipid peroxidation, hyaluronic acid degradation, and damage to critical cellular membranes. Our data show that these adverse neutrophil effects are evident even if activated leukocytes are reintroduced to ischemic tissues for relatively brief periods of time (5 minutes) in between periods of total blood flow interruption. The clinical implications of this work extend most directly to (1) the syndrome of "nonocclusive" mesenteric ischemia, where multiple flow interruptions are common, (2) the practice of temporary flow restorations during reconstructive procedures for visceral or extremity ischemia, and (3) low cardiac output states with intermittent left ventricular dysfunction. In such settings thought should be given to decreasing the leukocyte concentration in the initial reperfusate. This could be accomplished with blood filters, PMN antisera, or monoclonal antibodies to endothelial receptor sites. Although not used clinically at this time, these measures should be available in the near future given recent technologic advances. Based on our current understanding of reperfusion phenomena, these extra measures may decrease microvascular injury and improve outcome. The authors thank Mrs. Eileen M. Wayte for manuscript preparation. REFERENCES 1. Parks DA, Granger DN. Contributions of ischemia and reperfusion to mucosal lesion formation. Am J Physiol 1986;250:G749-53. 2. Granger DN, Rutili G, McCord JM. Superoxide radicals in feline intestinal ischemia. Gastroenterology 1981;81: 22-9. 3. Granger DN, Hollwarth ME, Parks DA. Ischemia reperfusion
606
4. 5. 6. 7. 8. 9.
Journal of VASCULAR SURGERY
Clark and Gewertz
injury: role of oxygen-derived free radicals. Acta Physiol Scan Suppl 1986;548:47. Parks DA, Granger DN. Ischemia reperfusion injury: a radical view. Hepatology 1988;3:680-2. Anzueto L, Benoit JN, Granger DN. A rat model for studying the intestinal circulation. Am J Physiol 1984;246: 656-61. Kim EH, Gewertz BL. Chronic digitalis administration alters mesenteric vascular reactivity. J VASC SUV,G 1987;5:382-9. Chiu CJ, McArdle AH, Brown R, et al. Intestinal mucosal lesions in low-flow states. Arch Surg 1970;101:478-83. Geft IL, Fishbein MC, Ninomiya K, et al. Intermittent brief periods of ischemia have a cumulative effect and may cause myocardial necrosis. Circulation 1982;66:1150-3. Subramanian R, Plehn S, Noonan J, et al. Free radical mediated damage during myocardial ischemia and reperfusion and protection by carnitine esters. J Cardiol 1987;76: 41-5.
10. Gorlach G, Sheld HH, Mulch J, et al. Ultrastructure of the human myocardium after intermittent ischemia compared to cardioplegia. Adv Exp Med Biol 1986;194:439-49. 11. Romson J, Hook B, Kunkel S, et al. Reduction of the extent of ischemic myocardial injury by neutrophil depletion in the dog. Circulation 1983;67:1016-23. 12. Engler RL, Daldgren MD, Morris D, et al. Role ofleukocytes in response to acute myocardial ischemia and reflow in dogs. Am J Physiol 1986;251:H314-22. 13. Korthuis RJ, Grisham MB, Granger DN. Leukocyte depletion attenuates vascular injury in postischemic skeletal muscle. Am J Physiol 1988;254:H823-7. 14. Hernandez LA, Grisham MB, Twohig B, et al. Role of neutrophils in ischemia-reperfusion-induced microvascular injury. Am J Physiol 1987;253:H699-703. Submitted Oct. 9, 1990; accepted Jan. 29, 1991.
B O U N D VOLUMES AVAILABLE TO SUBSCRIBERS Bound volumes o f the JOURNAL OF VaSCt3L~ StJR~ERY for 1991 are available to subscribers only. They may be purchased from the publisher at a cost of $59.00 for domestic, $79.13 for Canadian, and $75.00 for international subscribers for Vol. 13 (January to June) and Vol. 14 (July to December). Price includes shipping charges. Each bound volume contains a subject and author index, and all advertising is removed. Copies are shipped within 60 days after publication o f the last issue in the volume. The binding is durable buckram with the journal name, volume number, and year stamped in gold on the spine. Payment must accompany all orders. Contact Subscription Services, Mosby-Year Book, Inc., 11830 Westline Industrial Drive, St. Louis, M O 63146-3318, USA. In the United States call toll free: (800)325-4177, ext. 4351. In Missouri call collect: (314)872-8370, ext. 4351. Subscriptions must be in force to qualify. Bound volumes are not available in place o f a regular JOURNAL subscription.
It is now well accepted that much of the tissue injury associated with an ischemic episode is due to reperfusion phenomena that begin only when blood flow is restored.~ The mechanism of injury involves, at least in part, the generation of oxygen-derived free radical species, which destabilize cellular membranes. 2-4 This improved understanding of ischemia and reperfusion in animal models has suggested new strategies to the vascular surgeon treating ischemic syndromes. These have included the use of free radical scavengers and intentional restriction of the initial bolus of oxygenated blood when flow is first restored. Unfortunately, treatment protocols are Supported by American Heart Association grant No. 88-786 (Dr. Gewertz) and National Institutes of Health Grant No. T32H607665-02 (Dr. Clark). Presented at the Fourteenth AnnualMeeting of the Midwestern Vascular Surgical Society,Toledo, Ohio, Sept. 14-15, 1990. Reprint requests: Bruce L. Gewertz,MD, Universityof Chicago, Department of Surgery, 5841 S. Maryland Ave., Box 129, Chicago, IL 60637. 24/6/28207
largely based on experimental preparations subjected to single episodes of ischemia. Such models may not approximate the clinical situation; in practice ischemic insults often recur exposing an organ to multiple cycles of ischemia and reperfusion. We addressed this concern in a series of experiments that more closely parallel the clinical setting. We hypothesized that even brief periods of reperfusion interjected between ischemic episodes would increase tissue injury. Studies were performed in a rat small intestine preparation in which metabolic, hemodynamic, and histologic responses to ischemia have been well characterized. This organ was selected since multiple episodes of mesenteric ischemia are often encountered in clinical practice especially in the nonocclusive variant of the syndrome. Animals were subjected to a total of 30 or 45 minutes of complete intestinal ischemia. Flow interruption was continuous (single episode) or intermittent (two or three episodes of 15 minutes ofischemia separated by 5 minutes of reperfusion). In some 601
Journalof VASCULAR SURGERY
602 Clark and Gewertz
RECOROEH
/
A-vox
I/
~
l
\
-,,,
RESERVOtR
IA
WATER BATt4
Fig. 1. Experiments were performed in denervated autoperfused rat intestinal preparations.
experiments 5-minute reperfusions were performed with arterial blood depleted of leukocytes. This additional intervention was included to determine the role of neutrophils which have been strongly implicated in reperfusion injury. METHODS Animal care and experimentation described below complied with the "Principles of Laboratory Animal Care" and the "Guide for Care and Use of Laboratory Animals" (NIH publication No. 80-23, revised 1985). Experimental model. The intestinal preparation is a modification of that previously described by Anzueto et al. s and Kim and Gewertz6 (Fig. 1). Male Wistar rats weighing 400 to 600 gm (retired breeders, Harlan Sprague-Dawley) were anesthetized by ether inhalation augmented by intraperitoneal injection of sodium pentobarbital (50 mg/ml at 0.1 ml/100 gm). The trachea was intubated with 1.67 mm ID PE 240 tubing (Becton Dickinson Primary Care Diagnostic, Sparks, Md.), and supplemental oxygen was supplied at a rate of 4 L/min. Surgical excision of the duodenum, pancreas, and colon was followed by bilateral ligation of the renal arteries and veins. Periadventitial stripping of the superior me-
senteric artery (SMA) was performed to ensure denervation. The small intestine was wrapped in saline-soaked gauze and cellophane to minimize evaporative losses. Temperature was controlled at 37 ° C with an intermittent thermistor controlled incandescent lamp. Direct cannulation of the right femoral artery allowed continuous monitoring of systemic arterial pressure. After systemic heparinization (100 units heparin/100 gm body weight), an extracorporeal circuit was created to route blood from the right internal carotid artery and superior mesenteric vein (PE190 tubing, 1.19 mm ID) through a continuous A-~rO2 difference analyzer (AVOX Systems, Inc., San Antonio, Texas). Small intestine blood flow was controlled by cannulation of the SMA and use of an infusion pump (Gilson minipuls II; Medical Electronics, Middleton, Wis.) at 6.0 ml/min (calculated to provide 70 to 80 ml/min/100 gm intestine). All extracorporeal blood was collected in a reservoir, pumped through a 37 ° C water bath, and returned to the rat via the right femoral vein. When indicated, SMA occlusion was produced by cessation of pump perfusion. Protocol. Animals (n = 25) were allowed to stabilize for 1 hour after preparation. Sham animals (n -- 3) were observed for 3 hours without ischemic
Volume 13 Number 5 May 1991
Intestinal reperfusion injury potentiated by intermittant ischemia 603
SHAM (N=3)
30 MIN. ISCHEMIA
45 MIN. ISCHEMIA
CONTINUOUS INTERMITTENT INT.& LEUKOPENIC (N=4) (N=4) (N=3)
CONTLNUOUSINTERMITTENT INT.& LEUKOPEN~C (n=4) (n=4) (n=3)
Fig. 2. Animals (n = 25) were subjected to either continuous or intermittent ischemia. Intermittent reperfusion (5 minutes) was performed with either native arterial blood or leukopenic arterial blood.
Grade 1
Grade 2
6racte 3
Gracle 4
6ra~e 5
Fig. 3. Histologic grading system. Epithelial morphologic characteristics of individual intestinal sections were evaluated in a blinded manner by use of a modification of the Chiu system.
insult to establish the stability of the preparation and to document baseline mesenteric vascular resistance (MVR) and oxygen consumption (VO2) (MVR 0.84 + 0.06 at time 0, 0.87 _+ 0.02 at 3 hours; ~rO 2 3.65 _+ 0.73 at time 0, 3.59 _+ 0.42 at 3 hours). In rats with continuous ischemia (C), intestines were subject to either 30 minutes (n = 4) or 45 minutes (n -- 4) of complete flow interruption as a single episode. Fourteen other rats experienced intermittent ischemia presented as two or three episodes of 15 minutes of flow interruption (total ischemia 30 or 45 minutes) separated by 5 minutes of reperfusion. These short intervals of reperfusion were performed with either native arterial blood (I, n - - 8 ) or leukopenic blood (IL, n = 6). Leukopenia (white blood cell cotmt < 5 0 0 / m m 3) was achieved by centtifugation of blood and removal of the buffy coat and confirmed by the absence of white blood cells on peripheral smear. Once the above treatment protocols were completed, the intestines in all animals were reperfused for 1 hour with native arterial blood (Fig. 2).
Histologic sections were obtained after each ischemic insult and after 1 hour of reperfusion. After the experiments rats were killed by exsanguination. Intestinal sections were weighed and fixed in 10% neutral buffered formalin. Histologic evaluation was performed in a blinded manner by use of a modification of the system of Chiu et al.7; this grading system considers both villous height and epithelial morphology. Photomicrographs of the histologic grading system are depicted in Fig. 3. Data analysis. All values are reported as mean _+ SEM. Intestinal blood flow is normalized to 100 gm gut weight stripped of mesentery. Oxygen consumption (VO2) was calculated as the product of A-VO 2 difference and intestinal blood flow. Mesenteric vascular resistance was calculated as the difference between mean arterial pressure and mesenteric venous pressure divided by intestinal blood flow. Statistical analysis was performed with t tests between groups, and significance was assumed for p < 0.05.
604
Journal of VASCULAR SURGERY
Clark and Gewertz
I HR. REPERFUSION
uJ O
< o
u 0 0 0 I.,J
\\x\ \N\\ \\\\ \\\\ x\\\ \xx\
\\\\ \\\\ \\\\
,'x\'~ SHAM
C
I 30 MIN,
IL
C
I 45 MIN,
IL
Fig. 4. Intermittent ischemia (I) significantly increased histologic damage as compared to a continuous (C) insult. This effect was attenuated if 5-minute reperfusions were with leukopenic blood (/L).
Table I. Mesenteric vascular resistance (MVR, mmHg/ml/min • 100 gin) in continuous (C), intermittent (I), and intermittent leukopenic (IL) groups before ischemia and after 1 hour of reperfusion Before ischemia 30 minutes of ischemia Continuous Intermittent Intermittent leukopenic 45 minutes o f ischemia Continuous In termittent In termittent leukopenic
Reperfmion
0.89 -+ 0.13 0.87 + 0.22 0.83 -+ 0.02
0.20 - 0.26 0.89 + 0.10 0.84 -+ 0.04
0.81 + 0.10 0.83 -+ 0.07 0.87 -+ 0.12
0.86 _+ 0 . 0 l 0.86 -+ 0.14 0.88 _+ 0.11
RESULTS In all groups and ischemic intervals, histologic injury was not prominent until after the 1-hour reperfusion period. Immediate postischemic histologies were different from baseline (p < 0.05 all groups), but no differences were found between groups (30 minutes: C, 2.1 _+ 0.2; I, 2.3 + 0.4; IL, 1.9 + 0.1; 45 minutes: C, 2.7 _+ 0.3; I, 3.3 +- 0.4; IL, 2.6 + 0.3). Intermittent episodes of ischemia resulted in significantly worse histologic injury than comparable intervals of continuous ischemia (Fig. 4). For example, 30 minutes of continuous ischemia followed by 1 hour of reperfusion (C) yielded a histologic grade
of 2.7 + 0.4, whereas 30 minutes of intermittent ischemia (two 15-minute periods, I) resulted in a grade of 4.4 + 0.5 (p < 0.05). The eventual injury after intermittent ischemia was attenuated by leukopenic reperfusion during the 5-minute intervals. In the 30-minute flow interruptions, limiting access of leukocytes to the intestine during the 5-minute reperfusions (IL) decreased the eventual injury score to 3.4 + 0.3 (not different from C). Similar results were seen in the 45-minute ischemia groups (C, 2.9 + 0.6; I, 4.9 + 0.2; IL, 3.7 +-- 0.2)~. Since intestinal blood flow was controlled with an infusion pump at 6.0 ml/min, hemodynamic changes are most appropriately monitored by variations in mesenteric vascular resistance. Mean mesenteric vascular resistance in all experimental groups is displayed in Table I. N o statistically significant differences were found in M V R between any groups. VO2 (2.74 - 0.88 ml/min/100 gm at baseline) was predictably depressed after 1 hour of reperfusion in all experimental groups. These values did not vary significantly within total ischemia groups (30 minutes: C, 2.12 + 0.41; I, 2.57-+ 0.32; IL, 2.34 + 0.41; 45 minutes C, 1.91 + 0.54; I, 1.90 -+ 0.23; IL, 2.08 + 0.15), although 45 minutes of total ischemia, regardless of temporal sequence, resulted in more severe depression of VO2 than 30 minutes of total ischemia.
Volume 13 Number 5 May 1991
Intestinal reperfusion injury potentiated by intermittant ischemia 605
DISCUSSION These data demonstrate that multiple episodes of intestinal ischemia interrupted by brief periods of perfusion are associated with increased mucosal injury as compared to comparable continuous ischemic insults. This phenomenon has been previously investigated in myocardial preparations. Geft et al.8 used a canine model and showed that brief ischemic insults that singly would not cause irreversible cellular injury resulted in myocardial necrosis when repeated. Evidence of necrosis was established by increased creatinine kinase release and confirmed by light and electron microscopy. Subramanian et al.9 observed that intermittent global ischemia of the isolated perfused heart (three 20-minute periods of ischemia each followed by 10 minutes ofreperfusion) resulted in increased levels of malondialdehyde (an indicator of free radical formation) as compared to controls. Repeated ischemia-reperfusion injury also adversely affected recovery of cardiac function. Gorlach et al. performed biopsies on 40 patients undergoing coronary artery bypass grafting who were randomized to intermittent ischemia rather than cardioplegic arrest during operation.I° Biopsies of those undergoing intermittent ischemia showed unexpectedly severe myocardial damage with excessive intracellular edema and ultrastructural derangements. Given the role of leukocytes as mediator of the ischemia-reperfusion phenomenon in other organs, we hypothesized that mucosal injury could be attenuated by the use of leukopenic reperfusate between ischemic episodes. Lucchesi et al. 11 showed that leukopenia produced by administration of polymorphonuclear cell (PMN) antisera reduced myocardial infarct size by 43% in a canine model. On histologic examination infarcts in control animals had substantial PMN infiltration, which was virtually absent in leukopenic dogs. Engler et al.12 used leukoplaque filters to produce leukopenia and compared control and leukopenic myocardial preparations after 1 hour of ischemia and 1 hour of reperfusion. Leukocyte depletion prevented excessive tissue edema, ventricular dysrhythmias, and a "no reflow phenomena" as a result of leukocyte "plugging" of capillaries. Korthuis et al.lS examined the role of leukocytes in ischemia-reperfusion injury of skeletal muscle using the well-characterized isolated canine gracilis preparation. Reperfusion with blood depleted of leukocytes prevented the increased microvascular permeability and capillary resistance seen in control preparations. In work more directly related to our experiments,
Hernandez et al. 14 investigated the effects of leukocyte depletion on the generation of ischemiareperfusion injury in a feline model of intestinal ischemia. Systemic treatments included either antineutrophil serum or monoclonal antibody to endothelial receptors responsible for neutrophil adherence. Both neutrophil depletion and prevention of adherence significantly attenuated the increased microvascular permeability associated with 1 hour of ischemia-reperfusion. Our data would strongly support the importance of neutrophils in ischemia-reperfusion. Neutrophil binding to endothelium results in cellular activation and the production of free oxygen radicals and associated toxins. Binding is mediated by the CD 11/CD 18 complex on the neutrophil surface and is stimulated by various chemotactic peptides and lipid mediators. The subsequent release of proteases and the production of free oxygen radicals results in lipid peroxidation, hyaluronic acid degradation, and damage to critical cellular membranes. Our data show that these adverse neutrophil effects are evident even if activated leukocytes are reintroduced to ischemic tissues for relatively brief periods of time (5 minutes) in between periods of total blood flow interruption. The clinical implications of this work extend most directly to (1) the syndrome of "nonocclusive" mesenteric ischemia, where multiple flow interruptions are common, (2) the practice of temporary flow restorations during reconstructive procedures for visceral or extremity ischemia, and (3) low cardiac output states with intermittent left ventricular dysfunction. In such settings thought should be given to decreasing the leukocyte concentration in the initial reperfusate. This could be accomplished with blood filters, PMN antisera, or monoclonal antibodies to endothelial receptor sites. Although not used clinically at this time, these measures should be available in the near future given recent technologic advances. Based on our current understanding of reperfusion phenomena, these extra measures may decrease microvascular injury and improve outcome. The authors thank Mrs. Eileen M. Wayte for manuscript preparation. REFERENCES 1. Parks DA, Granger DN. Contributions of ischemia and reperfusion to mucosal lesion formation. Am J Physiol 1986;250:G749-53. 2. Granger DN, Rutili G, McCord JM. Superoxide radicals in feline intestinal ischemia. Gastroenterology 1981;81: 22-9. 3. Granger DN, Hollwarth ME, Parks DA. Ischemia reperfusion
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injury: role of oxygen-derived free radicals. Acta Physiol Scan Suppl 1986;548:47. Parks DA, Granger DN. Ischemia reperfusion injury: a radical view. Hepatology 1988;3:680-2. Anzueto L, Benoit JN, Granger DN. A rat model for studying the intestinal circulation. Am J Physiol 1984;246: 656-61. Kim EH, Gewertz BL. Chronic digitalis administration alters mesenteric vascular reactivity. J VASC SUV,G 1987;5:382-9. Chiu CJ, McArdle AH, Brown R, et al. Intestinal mucosal lesions in low-flow states. Arch Surg 1970;101:478-83. Geft IL, Fishbein MC, Ninomiya K, et al. Intermittent brief periods of ischemia have a cumulative effect and may cause myocardial necrosis. Circulation 1982;66:1150-3. Subramanian R, Plehn S, Noonan J, et al. Free radical mediated damage during myocardial ischemia and reperfusion and protection by carnitine esters. J Cardiol 1987;76: 41-5.
10. Gorlach G, Sheld HH, Mulch J, et al. Ultrastructure of the human myocardium after intermittent ischemia compared to cardioplegia. Adv Exp Med Biol 1986;194:439-49. 11. Romson J, Hook B, Kunkel S, et al. Reduction of the extent of ischemic myocardial injury by neutrophil depletion in the dog. Circulation 1983;67:1016-23. 12. Engler RL, Daldgren MD, Morris D, et al. Role ofleukocytes in response to acute myocardial ischemia and reflow in dogs. Am J Physiol 1986;251:H314-22. 13. Korthuis RJ, Grisham MB, Granger DN. Leukocyte depletion attenuates vascular injury in postischemic skeletal muscle. Am J Physiol 1988;254:H823-7. 14. Hernandez LA, Grisham MB, Twohig B, et al. Role of neutrophils in ischemia-reperfusion-induced microvascular injury. Am J Physiol 1987;253:H699-703. Submitted Oct. 9, 1990; accepted Jan. 29, 1991.
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