Cardiac contractile JURETA Department
injury
W. HORTON
AND
of Surgery,
University
after intestinal D. JEAN
WHITE
of Texas Southwestern
HORTON, JURETA W., AND D. JEAN WHITE. CU&ZC COW tractile injury after intestinal ischemia-reperfusion. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H1164-Hl170, 1991.Experimental and clinical data suggest that even a brief period of intestinal ischemia followed by reperfusion initiates a sequence of events that include release of inflammatory mediators and multiorgan failure. In this study, 41 rats were subjected to occlusion of the superior mesenteric artery (SMA) for 20 min and collateral arcade ligation. Twelve rats were sham operated and served as controls (group I). Groups of rats with SMA occlusion were killed at several time intervals after reperfusion (group 2, 2-3 h; group 3, 4-5 h; group 4, 12-16 h). In group 5, rats were pretreated with enterally administered allopurinol (10 mg* kg-’ *day-‘) for 4 days before the intestinal ischemia episode and were studied 2-3 h after reperfusion. In vivo studies confirmed that 20 min of intestinal ischemia produced a transient bradycardia (P < 0.05) and no change in systemic blood pressure, acid-base balance, or hematocrit. In vitro studies showed marked cardiac contractile depression as early as 2 h after ischemia-reperfusion as indicated by a fall in left ventricular pressure (LVP; from 77 t 3 to 63 t 4 mmHg, P = 0.01) (from 1,827 + 59 to 1,557 t 99 mmHg/s, P < and +dP/dt,,, 0.02) and -dP/dt,,, (from 1,267 t 57 to 953 t 67 mmHg/s, P = 0.02), a rightward shift in LV function curves, and a decreased responsiveness to perfusate Ca*+. Allopurinol pretreatment prevented ischemia-reperfusion-mediated deficits in cardiac contraction and relaxation. We concluded that 1) intestinal ischemia-reperfusion produces significant cardiac contractile dysfunction that persists for several hours and 2) the cardioprotective effects of allopurinol treatment before ischemia-reperfusion indicate that oxygen-derived free radicals contribute, in part, to the cardiac defects. myocardial depression; mesenteric occlusion; rats; reperfusion injury
ischemia;
mesenteric
artery
PREVIOUS STUDIES have shown that even a brief period
of intestinal ischemia followed by reperfusion produces direct injury to the intestine (4, 11, 26) and triggers the release of numerous inflammatory mediators, including oxygen-derived free radicals, arachidonic acid products, cytokines, prostaglandins, and endotoxin (9, 10, 21, 24, 25, 28). Both clinical and experimental studies have suggested that altered capillary permeability and a loss of intestinal barrier function in ischemia potentiate multiorgan system failure (1, 3, 5, 17, 23, 27, 29, 34). In this regard, Schmeling et al. (31) showed that intestinal ischemia-reperfusion produced acute lung injury characterized by increased lung permeability, capillary endothelial cell injury, and reduced lung tissue high-energy phosphate stores. Although many animal models have been developed to study ischemic injury to the intestine, a wide range of superior mesenteric artery (SMA) occlusion times (4-100 min) and mortality rates (8-80%) have H1164
0363-6135/91
$1.50
ischemia-reperfusion
Medical Center, Dallas, Texas 75235-9031
suggested that SMA occlusion does not produce consistent ischemic injury (11, 23, 24, 27, 29, 32). We recently developed a model of intestinal ischemia that included occlusion of the SMA immediately distal to the right colic artery plus occlusion of the collateral arcades from the right colic and jejunal arteries (18). Studies from our laboratory showed that SMA occlusion with interruption of collateral flow for 20 min produced a reproducible and consistent mortality and suggested that the primary mechanism by which intestinal ischemia-reperfusion mediated multiorgan failure was the generation of oxygen-derived free radicals (18, 19). This study was designed to examine the possibility that cardiac dysfunction occurs after experimental intestinal ischemia-reperfusion. In addition, allopurinol was administered enterally in clinically acceptable doses to examine the contribution of oxygen-derived free radicals to cardiac contractile defects after intestinal ischemia and reperfusion. METHODS
A total of 78 pathogen-free adult male Sprague-Dawley rats (weighing 300-400 g) were purchased from Sasco, Omaha, NE. All rats were acclimatized to their surroundings for 3-4 days before experimental intervention. The study was divided into three phases: 1) to examine the time sequence for the development of cardiac dysfunction after intestinal ischemia-reperfusion, 2) to provide quantitative cardiac contraction and relaxation data after untreated SMA occlusion plus collateral interruption, and 3) to determine if allopurinol treatment before SMA occlusion prevents ischemia-reperfusion-induced cardiac dysfunction. General anesthesia was achieved with methoxyflurane and was augmented by intramuscular injection of 20 mg/kg ketamine. The research protocol was executed in accordance with the guidelines of the Institutional Review Board for Animal Research at the University of Texas Southwestern Medical Center. In the first phase of this study, 41 male SpragueDawley rats were studied at various time intervals to characterize the development of cardiac dysfunction after ischemia-reperfusion. After achieving a suitable level of anesthesia, the rats underwent midline laparotomy and evisceration. Collateral arcades were ligated, and the SMA was occluded with an atraumatic microvascular clip for 20 min. Use of this technique renders the cecum and -75% of the small intestine ischemic. Twelve animals designated as sham ischemic animals underwent an identical preparation consisting of midline laparotomy and evisceration (group 1). In those animals, the lateral arcades were dissected and the SMA was
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CARDIAC
EFFECTS
OF
freed, but there was no occlusion of the arterial supply. After 20 min of mesenteric ischemia (or sham ischemia for the control group), the occluding clamp on the SMA was removed, and the intestine was returned to the peritoneal cavity; the abdomen was closed in two layers with 3-O chromic, and the skin was closed with skin staples. All animals were allowed to recover and to take food and water ad libitum. Ten rats subjected to intestinal ischemia (group 2) were killed between 2 and 3 h after reperfusion, and the hearts were harvested for in vitro assessment of cardiac function. In group 3, 10 rats were killed 4-5 h after 20 min of ischemia followed by reperfusion, and in group 4, rats were killed 12-16 h after ischemia-reperfusion (n = 11). At each time period (2-3, 4-5, or 12-16 h), four sham ischemia controls were killed, and in vitro contraction and relaxation indexes were studied. Because there was no significant difference in the control hearts harvested at any time after sham ischemia-reperfusion, these groups were combined and reported as one control group. In the second phase of this study, allopurinol (Sigma Chemical, St. Louis, MO) was administered intragastritally by gavage for 4 days before the ischemic insult was suspended in water (group 5, n = 10). Allopurinol and administered at a dose of 10 mg. kg-’ day? In seven rats, 1 ml of water was administered intragastrically each day, and these rats served as vehicle-treated controls. Allopurinol-treated rats and vehicle-treated controls underwent midline laparotomy and evisceration after anesthesia as described for groups l-4. On the basis of the studies in the first phase, we determined that cardiac dysfunction occurred as early as 2 h after ischemiareperfusion and persisted up to 16 h after reperfusion. For these reasons the animals pretreated with allopurinol were killed, and the cardioprotective effects were studied at a time when cardiac depression was maximal (2-3 h after ischemia-reperfusion). In the third phase of this study, hemodynamic function (systemic blood pressure and heart rate) and acid-base balance were studied at baseline, after 20 min of intestinal ischemia, and 2 and 24 h after reperfusion. In two groups of animals (sham ischemia, n = 8, and ischemiareperfused, n = lo), anesthesia was achieved as described above; a catheter (PE-50) was placed in the carotid artery, tunneled to the nape of the neck, filled with heparin, and occluded. Blood pressure was measured with a Gould-Statham transducer (Oxnard, CA), and tracings were recorded on a physiological recorder (model 81, Siemans). Blood samples (0.20 ml) were collected anaerobically from the carotid artery catheter for measurement of arterial pH, blood gases, and hematocrit. Blood collected for assessment of acid-base status and hematocrit was replaced with warm lactated Ringer solution. After completing ischemia-reperfusion studies on the experimental day, the abdomen was closed with 3-O chromic and skin staples; animals were returned to individual cages and were allowed food and water at will. Twenty-four hours after the ischemia-reperfusion insult, systemic blood pressure and heart rate were measured and a blood sample was collected to assess metabolic function. Isolated coronary perfused hearts. At the designated time period (2-3, 4-5, or 12-16 h after 20-min ischemia l
INTESTINAL
ISCHEMIA
H1163
followed by reperfusion), the rats were anticoagulated with sodium heparin (1,000 U) and decapitated with a guillotine. Hearts were rapidly excised and placed in icecold Krebs-Henseleit buffer to remove the blood. The composition of this buffer was (in mM) 24.0 NaHC03, 118.0 NaCl, 4.7 KCl, 1.2 KH2P04, 1.2 MgS04, 1.25 CaC12, and 11.1 glucose. A small stab incision was made in the ventricular apex, and a collapsed latex balloon connected to a short polyethylene tube (-12 cm) was placed into the left ventricle. The apical stab incision allowed for free drainage of any thesbian flow entering the left ventricle. The hearts were then perfused in a retrograde manner through a catheter placed in the aorta with prefiltered and oxygenated (95% 02-5% CO*) KrebsHenseleit buffer at a pH of 7.4, a temperature of 37°C and a coronary flow rate of 5 ml/min. Previous studies in our laboratory have shown that this flow rate produces maximal ventricular performance with the longest stability over time (unpublished data). The perfused heart was enclosed in a water-jacketed chamber to maintain humidity and temperature. The balloon was filled with bubble-free distilled water, and the polyethylene tubing was connected to a Statham transducer. All balloons were individually calibrated to assure that each balloon was used on the flat portion of its compliance curve. Volume was then injected into the balloon to achieve a desired left ventricular end-diastolic volume and pressure. In a few hearts from each experimental group, myocardial temperature was monitored using a 27-gauge thermistor needle inserted superficially into the left ventricular muscle; the temperature was stable over time for all hearts (37.1 t 0.2”C). After instrumentation, hearts were allowed to stabilize for 30 min before any experiments were performed. Isolated hearts were excluded from further study if they failed to achieve a stable developed pressure or if they developed persistent arrhythmias. Less than 10% of the isolated hearts were excluded for these reasons. After stabilization, left ventricular pressure and the rate of left ventricular rise (+dP/dt,,,) and fall (-dP/dt,,,), heart rate, and coronary perfusion pressure were measured simultaneously on a multichannel polygraph (Grass Instruments, Quincy, MA). In add’ti ion, left ventricular compliance, the time to maximal left ventricular pressure, the time to 90% relaxation, and the time to maximal &dP/dt were calculated as previously described ( 13). Statistical analysis. All values are expressed as means t SE. Statistical comparison of groups was performed with an analysis of variance and a multiple-comparison procedure where appropriate (Student-Newman-Keuls). Differences between experimental groups were considered significant at P < 0.05. RESULTS
In vivo hemodynamic variables obtained during baseline period, 20 min of sham ischemia, and at 20, 40, and 60 min after sham reperfusion were not significantly different from control values, confirming the stability of this preparation (Table 1). Twenty minutes of intestinal ischemia was associated with no change in mean arterial blood pressure, a significant bradycardia, and no change in arterial pH, blood gases, serum bicarbonate, or he-
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H1166 TABLE
CARDIAC
EFFECTS
OF
INTESTINAL
ISCHEMIA
1. Hemodynamic response to intestinal &hernia-reperfusion
Mean arterial blood pressure, Sham Ischemic Heart rate, beats/min Sham Ischemic
Postreperfusion
End of 20 min Ischemia
Baseline
2h
24 h
mmHg 96t6 108*6
87k6 94t6
104t6 98k7
109*5 lOOt8
252k13 231t9
201*7* 160t8*
221t13 179t13
26024 233t9
7.43kO.01 7.38kO.08
7.40t0.02 7.4OkO.02
7.4OkO.01 7.38t0.01
42tl 46t3
36&Z 42t2
34*1 33k2
43t2 46tl
38*1 41*1
40&l 40&l
39t1 4221
34*1* 32*3*
PH
Sham Ischemic Pco~, mmHg Sham Ischemic Hematocrit, Sham Ischemic Values
are means
7.41t0.01 7.36kO.01
t
%
* SE. * Significant
change
from
baseline
values,
P < 0.05. t Significant
matocrit ratio. Reperfusion resulted in a transient hypotension (mean arterial blood pressure fell from 108 t 6 to 88 t 7 mmHg, P < 0.05) but no change in heart rate or acid base balance. Two hours after reperfusion of the ischemic bowel, all indexes of hemodynamic function returned to baseline values and remained stable for 24 h after reperfusion (Table 1). Table 2 summarizes the in vitro cardiac perfusion data from animals subjected to SMA occlusion and killed at varying times after reperfusion of the ischemic bowel. Significant cardiac dysfunction was documented in hearts from animals subjected to 20 min of intestinal ischemia and killed at either 2-3 or 4-5 h after reperfusion. Cardiac dysfunction, indicated by the significant reduction in left ventricular pressure and in rates of left ventricular pressure rise and fall, as well as a decrease in time to peak pressure, was further confirmed by a shift of left ventricular function curves calculated for these groups downward and to the right of those curves calculated for the sham ischemia group (Fig. 1). In addition, hearts harvested from rats after ischemia and reperfusion TABLE
difference
between
groups
at
P < 0.05.
had a significantly reduced responsiveness to increases in exogenous calcium as well as a reduced ventricular response to increased coronary flow compared with control hearts. As seen in Table 3, allopurinol pretreatment for 4 days before ischemia-reperfusion produced cardiac contraction and relaxation indexes that were not statistically different from those measured in control hearts. In addition, left ventricular function curves calculated for this group were similar to those calculated for sham ischemic controls and were significantly better than those generated by untreated ischemic reperfused hearts (Fig. 2). Hearts from rats pretreated with allopurinol generated dose-response curves to exogenous Ca2’ nearly identical to those generated by control hearts and significantly better than those generated by hearts from untreated rats killed 2-3 h after ischemia-reperfusion (Fig. 3). Incremental increases in coronary flow rate (from 3 to 12 ml/min) failed to overcome ischemia-reperfusion-induced cardiac dysfunction, as indicated by the significantly lower left ventricular pressure and +dP/dt,,, at
2. In . vitro cardiac performance Group 1 Sham Ischemia
LVP, mmHg +dP/dt, mmHg/s -dP/dt, mmHg/s Differential ratio dP/dt (DP 40), mmHg CPP, mmHg CVR, mmHg ml-’ min HR, beats/min TTP, ms R&O, ms Time to +dP/dt,,,, ms Time to -dP/dt,,,, ms Serum electrolytes, meq/l Na’ K+ Cll
l
Group 2 Ischemia Plus 2-3 h Reperfusion
Group 3 Ischemia Plus 4-5 h Reperfusion
Group 4 Ischemia Plus 12-16 h Reperfusion
77*3 1,827+59 1,267+57 1.45t0.05 1,738*49 62.2t4.4 12.4k0.8 25Ok13 81&Z 80&l 41t1 49&l
63&4* 1,557+99* 953t67* 1.65&0.07* 1,488+90* 53k7 10.7k1.5 252t13 76&l* 79t2 4Okl 47t1
67t4* 1,596+81* 1,078+70 1.51t0.06* 1,561+65* 50t5 10.0~1.0 223&11 81t2 81t2 42tl 50t1
66&Z* 1,647+69* 1,059+53* 1.5720.05 1,581+60* 53t3 10.7kO.7 226kll 82kl 8021 45t0.8* 52tl
142tl 6.1t0.3 107.4t0.8
139kl 5.5kO.2 105.1t0.9
138t2 6.1k0.2 105.6kl.O
142tl 6.6kO.2 106.7t0.6
Values are means & SE. LVP, left ventricular pressure; kdP/dt, maximal rate of left ventricular pressure rise (+) and fall (-); dP/dt (DP 40), rate of LVP rise at a developed pressure of 40 mmHg; CPP, coronary perfusion pressure; CVR, coronary vascular resistance; TPP, time to peak isovolumic pressure; RTSo, time to 90% relaxation. * Significant difference among groups at P 5 0.05.
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CARDIAC
LV Pressure (mmHg)
EFFECTS
OF
INTESTINAL
A
FIG. 1. Left ventricular (LV) performance after 20 min of intestinal ischemia followed by reperfusion. Experimental groups were killed at either 2-3 or 4-5 h after removing clip on superior mesenteric artery. All values are means & SE. &dP/ dt, rate of LV pressure rise (+) or fall (-). * Significant difference between groups at P < 0.05.
Sham Ischemia/2-3 hrs reperfusion Ischemia/4-5 hrs reperfusion
Ol
H1167
-dP/dt (mmHg/sec)
+dP/dt (mmHg/sec)
1000
n l
ISCHEMIA
5000
-10
0
20
10
-10
0
10
20
Left Ventricular End Diastolic Pressure
mmw
3. Effects of allopurinol on in vitro cardiac performance TABLE
Sham Ischemia
LVP, mmHg +dP/dt, mmHg/s -dP/dt,,,, mmHg/s dP/dt (DP 40), mmHg/s CPP, mmHg CVR, mmHg. ml-’ min HR, beats/min TTP, ms R%o, ms Time to +dP/dt,,,, ms Time to -dP/dt,,,, ms l
Values
are means
Allopurinol-Treated Ischemia
77k3 1,827+59 1,267rt57 1,738*49 62k4 12.4k0.8
7322 1,696+60 1,231+46 1,622*56 72k5 14.5t1.1
250t13 81&Z 80tl 41&l 49t1
269t12 84k2 78k2 43k2 45&l
t SE. See Table
2 for definitions
of abbreviations.
each level of flow in the untreated ischemia-reperfusion group. In contrast, allopurinol-treated hearts harvested at an identical time after reperfusion of the ischemic bowel (2-3 h) had ventricular performance-coronary flow relationships that were identical to those generated by control hearts (Fig. 4). During 30 min of in vitro perfusion with Krebs buffer, there was a nonsignificant increase in left ventricular pressure and in dP/dt,,, in all animals, attesting to the stability of the Langendorff heart preparation. Left ventricular compliance was identical in all experimental groups over the experimental period, and allopurinol pretreatment did not alter this relationship (Fig. 5). DISCUSSION
The rat has been commonly used as a model to evaluate mesenteric ischemia, and SMA occlusion has been most commonly used to produce ischemic injury to the intestine. Mesenteric collateral circulation in rats is analogous to that in humans. Vascular arcades that connect the
jejunal to the ileal arteries and the ileal colic artery to the right colic artery are quite small, with an external diameter of CO.5 mm. However, the efficiency with which these arteries transmit blood has been previously demonstrated in our laboratory, as documented by the failure of SMA occlusion alone to alter blood flow (measured with radioactive microspheres) to the middle segment of the ischemic bowel compared with blood flow measured in the proximal and distal segments (18). In the present study, occlusion of the SMA with collateral interruption produced intestinal ischemia that was grossly visible during the procedure. The immediate pallor of the ischemic segment and a sharp line of demarcation between perfused and hypoperfused sections confirmed adequate bowel ischemia using this technique. This model of intestinal ischemia and reperfusion injury is reproducible, simple, and clinically relevant. As previously demonstrated by our laboratory, this model produces a profound, but reversible, decrease in intestinal blood flow and in hemodynamic stability and a significant bowel edema after reperfusion, as well as reproducible histological injury to the intestinal mucosa (18, 19). The data presented here extend our previous studies, confirming that intestinal ischemia-reperfusion injury produces significant cardiac dysfunction. Cardiac injury was characterized by a decreased ability of the ventricle to develop pressure, alterations in the rate of left ventricular pressure rise and fall, and a shift in the left ventricular function curves downward and to the right of those calculated for control hearts, suggesting ventricular failure. These cardiac contraction and relaxation deficits could not be overcome by increases in perfusate calcium or by maximal increases in preload. These data suggest that 1) the release or activation of toxic factors from the ischemic bowel target the heart during intestinal reperfusion, and 2) profound cardiac contractile dysfunction after a brief episode of intestinal ischemia reduces cardiac output, contributing, in part, to periph-
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H1168
CARDIAC
LV Pressure (mmHg)
EFFECTS
OF
INTESTINAL
+dPMt (mmHg/sec)
ISCHEMIA
dP/dt (mmHg/sec)
* FIG. 2. Effects of allopurinol on left ventricular performance after intestinal ischemia and 2-3 h of reperfusion. All values are means k SE. * Significant difference between groups at P < 0.05.
loo0
n Sham 0
lschemia,Untfeated
5WLeft Ventricular End Diastolic Pressure (mmHg)
era1 perfusion deficits and eventual multiorgan failure. However, it must be emphasized that in our study contractile performance was evaluated in a non-blood-perfused, nonworking heart that was almost entirely neutrophi1 free. It is likely that in vivo activation, sequestration, and adherence of leukocytes target particular organs such as the heart; in addition, activation of numerous plasma factors, such as cytokines, complement products, and myocardial depressant factors, produced at the site of LV Pressure (mmHg) loo-
+dP/dt (mmHg/sec)
-dP/dt (mmHg/sec)
2500
2500
2000
2000 -
.
80 -
ischemic injury likely impair cardiac function after systemic distribution. Therefore it is apparent that oxygenderived free radical-induced cardiac injury is only one aspect of a complex sequence of pathological events that follows a brief period of intestinal ischemia. Several previous studies have suggested that the intestinal ischemic injury previously described in our model and by others was likely related to the effects of interrupted aerobic metabolism, the accumulation of by-prod-
1 .
1500
60 -
1500 .
1000
1000 .
n Sham l IschemiaJntreated 0
500
FIG. 3. Calcium dose-response curves generated by untreated ischemia-reperfusion, allopurinol-treated ischemia-reperfusion, and control experimental groups. All values are means k SE. * Significant difference between groups at P < 0.05.
500 -
lschemia + Allopurinol
\ 0
.
-1.1’
00
Perfusate Calcium (mM) Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.155) on October 17, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
CARDIAC
LV Pressure (mmHg)
EFFECTS
OF
INTESTINAL
+dP/dt (mmHg/sec)
H1169
ISCHEMIA
-dP/dt (mmHg/sec)
2500
2500
60 FIG. 4. Ventricular performance coronary flow relationships. All values are means t SE. * Significant difference between groups at P < 0.05.
2
n
Sham
0 0
Ischemia,Untreated lschemia + Allopurind
6
10
500
14
2
10
6
14
2
6
10
14
Coronary Flow Rate (ml/min) n l A A
Sham Ischemia,2-3 lschemia&5 Ischemia,l2-16
hrs repeffusion hrs reperfusion hrs reperfusion
0
Ischemia,2-3
hrs reperfusion
0.00
0.03 Left Ventriarlar
FIG.
5. Left ventricular
compliance
0.06
0.09
Volume
+ Allopurinol
0.12
(ml)
in all experimental
groups.
ucts of ATP degradation, and the generation of toxic oxygen metabolites generated via xanthine oxidase-dependent mechanisms (9, 24, 28). The overproduction of toxic oxygen metabolites appears to play a central role in the production of multiorgan system failure, and the heart may be a particularly susceptible organ. We have previously shown that the cardiac dysfunction that is characteristic of several types of trauma with shock, such as burn injury (13, 15) and hemorrhagic shock (14), was related to the generation of oxygen-derived free radicals. Whether the generation of oxygen-derived free radicals
is due to generation of xanthine oxidase-dependent or phagocyte-derived sources remains unclear. Several studies have suggested that sequestration of neutrophils in the ischemic tissue during hypoperfusion and the activation of neutrophils during reperfusion damage peripheral organs directly (2,12,22,31). In this regard, previous studies in other models of ischemic injury have shown that neutrophil depletion significantly improved peripheral organ function after reperfusion of ischemic injury (30, 31). The protective effects of allopurinol pretreatment suggest that blocking xanthine oxidase prevents formation of the toxic free radicals, enhancing cardiac performance at a time when intestinal ischemia-mediated cardiac dysfunction was maximal. An alternate mechanism by which allopurinol may protect the heart during an episode of intestinal ischemia-reperfusion was suggested by studies by Das et al. (7) and Garcia et al. (a), who demonstrated that allopurinol and oxypurinol, its metabolite, function as free radical scavengers independent of their inhibitory effects on xanthine oxidase. The idea that allopurinol pretreatment can prevent reperfusion injury after ischemia is not a new concept. As early as 1969 the cardioprotective effects of allopurino1 were confirmed in hemorrhagic shock (6). More recently, allopurinol pretreatment has been shown to improve survival of acute skin flaps (16), limit infarct size after ischemia-reperfusion (33), and improve survival after mesenteric ischemia-reperfusion (19, 20). A significant finding of the present study is that the protective effect of allopurinol was achieved with enteral doses that would not cause intolerable side effects. If a patient population that is at risk for an ischemic event can be identified, allopurinol can be studied for its prophylactic effects, since it is innocuous, easily administered enterally once a day, and has been used extensively in the past.
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H1170
CARDIAC
EFFECTS
OF
In summary, cardiac contraction and relaxation defects occurred as early as 2 h and persisted for 12-16 h after mesenteric ischemia-reperfusion. Although numerous studies have implicated oxygen-derived free radicals as primary mediators in the pathogenesis of ischemiareperfusion injury, it is likely that a number of factors such as cytokines, complement products, and myocardial depressant factors are produced at the site of tissue injury and subsequently impair cardiac function after systemic distribution. In our study, allopurinol, given enterally for 4 days before the ischemic insult, prevented mesenteric ischemia-reperfusion-induced cardiac dysfunction, confirming that oxygen-derived free radicals contribute, in part, to the cardiac deficits.
INTESTINAL
14.
15.
16.
17.
18.
19. The authors thank Nancy Bain for superb secretarial assistance. Address for reprints requests: J. W. Horton, Dept. of Surgery, Univ. of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235-9031. Received
22 January
1991; accepted
in final
form
22 May
20.
1991. 21.
REFERENCES 1. BOORSTEIN, J. M., L. J. DACEY, AND J. L. CRONENWETT. Pharmacologic treatment of occlusive mesenteric ischemia in rats. J. Surg. Res. 44: 555-560, 1988. 2. BROWN, M. F., A. J. Ross III, J. DASHER, D. L. TURLEY, M. M. ZIEGLER, AND J. A. O’NEILL, JR. The role of leukocytes in mediating mucosal injury of intestinal ischemia/reperfusion. J. Pediatr. Surg. 25: 214-217,199O. 3. CARRICO, C. J., J. L. MEAKINS, J. C. MARSHALL, D. FRY, AND R. V. MAIER. Multiple-organ failure syndrome. Arch. Surg. 121: 196208,1986. 4. CHIU, C. J., A. MCARDLE II, R. BROWN, H. J. SCOTT, AND F. N. GURD. Intestinal mucosal lesions in low-flow states. I. A morphological, hemodynamic and metabolic reappraisal. Arch. Surg. 101: 478-483,197O. 5. CRISSINGER, K. D., AND D. N. GRANGER. Mucosal injury induced by ischemia and reperfusion in the piglet intestine: influences of age and feeding. Gastroenterology 97: 920-926, 1989. 6. CROWELL, J. W., C. E. JONES, AND E. E. SMITH. Effect of allopurinol in hemorrhagic shock. Am. J. Physiol. 216: 744-748, 1969. 7. DAS, D. K., R. M. ENGELMAN, R. CLEMENT, H. OTANI, M. R. PRASAD, AND P. S. RAO. Role of xanthine oxidase inhibitor as free radical scavenger: a novel mechanism of action of allopurinol and oxypurinol in myocardial salvage. Biochem. Biophys. Res. Commun. 148: 314-319,1987. 8. GARCIA, J. G., C. M. ROLLAN, M. A. R. ENRINQUEZ, M. H. MADRUGA, E. M. HERNANDEZ, J. F. M. NUNEZ, AND A. G. ALONSO. Improved survival in intestinal ischemia by allopurinol not related to xanthine-oxidase inhibition. J. Surg. Res. 48: l44146, 1990. 9. GRANGER, D. N., D. A. PARKS, AND M. HOLLWARTH. Role of oxygen radicals in ischemic bowel disorders. Pediatr. Surg. Int. 1: 15-20,1986. 10. GRANGER, D. N., G. RUTILI, AND J. M. MCCORD. Superoxide radicals in feline intestinal ischemia. Gastroenterology 81: 22-29, 1981. 11. HAGLUND, U., T. ABE, C. AHREN, I. BRAIDE, AND 0. LUNDGREN. The intestinal mucosal lesions in shock. I. Studies on the pathogenesis. Eur. Surg. Res. 8: 435-447, 1976. 12. HERNANDEZ, L. A., M. B. GRISHAM, B. TWOHIG, K. ARFORS, J. M. HARLAN, AND D. N. GRANGER. Role of neutrophils in ischemiareperfusion-induced microvascular injury. Am. J. Physiol. 253 (Heart Circ. Physiol. 22): H699-H703, 1987. 13. HORTON, J. W., C. R. BAXTER, AND D. J. WHITE. Differences in
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
ISCHEMIA
cardiac responses to resuscitation from burn shock. Surg. Gynecol. Obstet. 168: 201-213, 1989. HORTON, J. W., AND K. R. BORMAN. Possible role of oxygenderived, free radicals in cardiocirculatory shock. Surg. Gynecol. Obstet. 165: 293-300, 1987. HORTON, J. W., D. J. WHITE, AND C. R. BAXTER. The role of oxygen-derived free radicals in burn-induced myocardial contractile depression. J. Burn Care Rehab. 9: 589-598, 1988. IM, M. J., P. N. MANSON, G. B. BULKEY, AND J. F. HOOPES. Effects of SOD and allopurinol on the survival of acute island skin flaps. Ann. Surg. 201: 357, 1986. MATUSCHAK, G. M., AND J. E. FINALDO. Organ interactions in the adult respiratory distress syndrome during sepsis. Chest 94: 400406,1988. MEGISON, S. M., J. W. HORTON, H. CHAO, AND P. B. WALKER. A new model for intestinal ischemia in the rat. J. Surg. Res. 49: l68173,199o. MEGISON, S. M., J. W. HORTON, H. CHAO, AND P. B. WALKER. Prolonged survival and decreased mucosal injury after low-dose enteral allopurinol prophylaxis in mesenteric ischemia. J. Pediatr. Surg. 25: 917-921,199O. MEGISON, S. M., J. W. HORTON, H. CHAO, AND P. B. WALKER. High dose versus low dose enteral allopurinol for prophylaxis in mesenteric ischemia. Circ. Shock 30: 323-329, 1990. MOZES, T., E. WIESZT, AND T. ZAHAJSZKY. Role of PGFz, in the superior mesenteric artery-induced shock. J. Surg. Res. 47: 476481,1989. OTAMIRI, T. Oxygen radicals, lipid peroxidation, and neutrophil infiltration after small-intestinal ischemia and reperfusion. Surgery St. Louis 105: 593-597,1989. OTAMIRI, T., R. SJODAHL, AND C. TAGESSON. An experimental model for studying reversible intestinal ischemia. Actu Chir. Scund. 153: 51-56,1987. PARKS, D. A., G. B. BULKLEY, D. N. GRANGER, S. R. HAMILTON, AND J. M. MCCORD. Ischemic injury in the cat small intestine: role of superoxide radicals. Gustroenterology 82: 9-15, 1982. PARKS, D. A., AND D. N. GRANGER. Ischemia-induced vascular changes: role of xanthine oxidase and hydroxyl radicals. Am. J. Physiol. 245 (Gastrointest. Liver Physiol. 8): G285-G289, 1983. PARKS, D. A., AND D. N. GRANGER. Contributions of ischemia and reperfusion to mucosal lesion formation. Am. J. Physiol. 250 (Gustrointest. Liver Physiol. 13): G749-G753, 1986. PARKS, D. A., B. GROGAARD, AND D. N. GRANGER. Comparison of partial and complete arterial occlusion models for studying intestinal ischemia. Surgery St. Louis 92: 896-901, 1982. PERRY, M. A., S. WADHWA, D. A. PARKS, W. PICKARD, AND D. N. GRANGER. Role of oxygen radicals in ischemia-induced lesions in the cat stomach. Gustroenterology 90: 362-367, 1986. PRIEBE, C. In discussion of Bailey, R. W., G. B. Bulkley, S. R. Hamilton, J. B. Morris, and U. H. Haglund. Protection of the small intestine from nonocclusive mesenteric ischemic injury due to cardiogenic shock. Am. J. Surg. 153: 108, 1987. ROMSON, J. L., B. G. HOOK, S. L. KUNKEL, G. D. ABRAMS, M. A. SCHORK, AND B. R. LUCCHESI. Reduction of the extent of ischemic myocardial injury by neutrophil depletion in the dog. Circulation 67: 1016-1023,1983. SCHMELING, D. J., M. G. CATY, K. T. OLDHAM, K. S. GUICE, AND D. B. HINSHAW. Evidence for neutrophil-related acute lung injury after intestinal ischemia-reperfusion. Surgery St. Louis 106: 195202,1989. TALBERT, B. J., AND D. W. TUGGLE. The effects of maturity and timed ischemia on survival rates in necrotizing enterocolitis. Curr. Surg. 45: 307-309,1988. WERNS, S. W., M. J. SHEA, S. E. MITSOS, R. C. DYSKO, J. C. FANTONE, M. A. SCHORK, G. D. ABRAMS, B. PITT, AND B. R. LUCCHESI. Reduction of the size of infarction by allopurinol in the ischemic-reperfused canine heart. Circulation 73: 518-524, 1986. WILMORE, D. W., R. J. SMITH, S. T. O’DWYER, D. 0. JACOBS, T. R. ZIEGLER, AND X. D. WANG. The gut a central organ after surgical stress. Surgery St. Louis 104: 917-927, 1988.
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injury
W. HORTON
AND
of Surgery,
University
after intestinal D. JEAN
WHITE
of Texas Southwestern
HORTON, JURETA W., AND D. JEAN WHITE. CU&ZC COW tractile injury after intestinal ischemia-reperfusion. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H1164-Hl170, 1991.Experimental and clinical data suggest that even a brief period of intestinal ischemia followed by reperfusion initiates a sequence of events that include release of inflammatory mediators and multiorgan failure. In this study, 41 rats were subjected to occlusion of the superior mesenteric artery (SMA) for 20 min and collateral arcade ligation. Twelve rats were sham operated and served as controls (group I). Groups of rats with SMA occlusion were killed at several time intervals after reperfusion (group 2, 2-3 h; group 3, 4-5 h; group 4, 12-16 h). In group 5, rats were pretreated with enterally administered allopurinol (10 mg* kg-’ *day-‘) for 4 days before the intestinal ischemia episode and were studied 2-3 h after reperfusion. In vivo studies confirmed that 20 min of intestinal ischemia produced a transient bradycardia (P < 0.05) and no change in systemic blood pressure, acid-base balance, or hematocrit. In vitro studies showed marked cardiac contractile depression as early as 2 h after ischemia-reperfusion as indicated by a fall in left ventricular pressure (LVP; from 77 t 3 to 63 t 4 mmHg, P = 0.01) (from 1,827 + 59 to 1,557 t 99 mmHg/s, P < and +dP/dt,,, 0.02) and -dP/dt,,, (from 1,267 t 57 to 953 t 67 mmHg/s, P = 0.02), a rightward shift in LV function curves, and a decreased responsiveness to perfusate Ca*+. Allopurinol pretreatment prevented ischemia-reperfusion-mediated deficits in cardiac contraction and relaxation. We concluded that 1) intestinal ischemia-reperfusion produces significant cardiac contractile dysfunction that persists for several hours and 2) the cardioprotective effects of allopurinol treatment before ischemia-reperfusion indicate that oxygen-derived free radicals contribute, in part, to the cardiac defects. myocardial depression; mesenteric occlusion; rats; reperfusion injury
ischemia;
mesenteric
artery
PREVIOUS STUDIES have shown that even a brief period
of intestinal ischemia followed by reperfusion produces direct injury to the intestine (4, 11, 26) and triggers the release of numerous inflammatory mediators, including oxygen-derived free radicals, arachidonic acid products, cytokines, prostaglandins, and endotoxin (9, 10, 21, 24, 25, 28). Both clinical and experimental studies have suggested that altered capillary permeability and a loss of intestinal barrier function in ischemia potentiate multiorgan system failure (1, 3, 5, 17, 23, 27, 29, 34). In this regard, Schmeling et al. (31) showed that intestinal ischemia-reperfusion produced acute lung injury characterized by increased lung permeability, capillary endothelial cell injury, and reduced lung tissue high-energy phosphate stores. Although many animal models have been developed to study ischemic injury to the intestine, a wide range of superior mesenteric artery (SMA) occlusion times (4-100 min) and mortality rates (8-80%) have H1164
0363-6135/91
$1.50
ischemia-reperfusion
Medical Center, Dallas, Texas 75235-9031
suggested that SMA occlusion does not produce consistent ischemic injury (11, 23, 24, 27, 29, 32). We recently developed a model of intestinal ischemia that included occlusion of the SMA immediately distal to the right colic artery plus occlusion of the collateral arcades from the right colic and jejunal arteries (18). Studies from our laboratory showed that SMA occlusion with interruption of collateral flow for 20 min produced a reproducible and consistent mortality and suggested that the primary mechanism by which intestinal ischemia-reperfusion mediated multiorgan failure was the generation of oxygen-derived free radicals (18, 19). This study was designed to examine the possibility that cardiac dysfunction occurs after experimental intestinal ischemia-reperfusion. In addition, allopurinol was administered enterally in clinically acceptable doses to examine the contribution of oxygen-derived free radicals to cardiac contractile defects after intestinal ischemia and reperfusion. METHODS
A total of 78 pathogen-free adult male Sprague-Dawley rats (weighing 300-400 g) were purchased from Sasco, Omaha, NE. All rats were acclimatized to their surroundings for 3-4 days before experimental intervention. The study was divided into three phases: 1) to examine the time sequence for the development of cardiac dysfunction after intestinal ischemia-reperfusion, 2) to provide quantitative cardiac contraction and relaxation data after untreated SMA occlusion plus collateral interruption, and 3) to determine if allopurinol treatment before SMA occlusion prevents ischemia-reperfusion-induced cardiac dysfunction. General anesthesia was achieved with methoxyflurane and was augmented by intramuscular injection of 20 mg/kg ketamine. The research protocol was executed in accordance with the guidelines of the Institutional Review Board for Animal Research at the University of Texas Southwestern Medical Center. In the first phase of this study, 41 male SpragueDawley rats were studied at various time intervals to characterize the development of cardiac dysfunction after ischemia-reperfusion. After achieving a suitable level of anesthesia, the rats underwent midline laparotomy and evisceration. Collateral arcades were ligated, and the SMA was occluded with an atraumatic microvascular clip for 20 min. Use of this technique renders the cecum and -75% of the small intestine ischemic. Twelve animals designated as sham ischemic animals underwent an identical preparation consisting of midline laparotomy and evisceration (group 1). In those animals, the lateral arcades were dissected and the SMA was
Copyright @ 1991 the American Physiological Society
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CARDIAC
EFFECTS
OF
freed, but there was no occlusion of the arterial supply. After 20 min of mesenteric ischemia (or sham ischemia for the control group), the occluding clamp on the SMA was removed, and the intestine was returned to the peritoneal cavity; the abdomen was closed in two layers with 3-O chromic, and the skin was closed with skin staples. All animals were allowed to recover and to take food and water ad libitum. Ten rats subjected to intestinal ischemia (group 2) were killed between 2 and 3 h after reperfusion, and the hearts were harvested for in vitro assessment of cardiac function. In group 3, 10 rats were killed 4-5 h after 20 min of ischemia followed by reperfusion, and in group 4, rats were killed 12-16 h after ischemia-reperfusion (n = 11). At each time period (2-3, 4-5, or 12-16 h), four sham ischemia controls were killed, and in vitro contraction and relaxation indexes were studied. Because there was no significant difference in the control hearts harvested at any time after sham ischemia-reperfusion, these groups were combined and reported as one control group. In the second phase of this study, allopurinol (Sigma Chemical, St. Louis, MO) was administered intragastritally by gavage for 4 days before the ischemic insult was suspended in water (group 5, n = 10). Allopurinol and administered at a dose of 10 mg. kg-’ day? In seven rats, 1 ml of water was administered intragastrically each day, and these rats served as vehicle-treated controls. Allopurinol-treated rats and vehicle-treated controls underwent midline laparotomy and evisceration after anesthesia as described for groups l-4. On the basis of the studies in the first phase, we determined that cardiac dysfunction occurred as early as 2 h after ischemiareperfusion and persisted up to 16 h after reperfusion. For these reasons the animals pretreated with allopurinol were killed, and the cardioprotective effects were studied at a time when cardiac depression was maximal (2-3 h after ischemia-reperfusion). In the third phase of this study, hemodynamic function (systemic blood pressure and heart rate) and acid-base balance were studied at baseline, after 20 min of intestinal ischemia, and 2 and 24 h after reperfusion. In two groups of animals (sham ischemia, n = 8, and ischemiareperfused, n = lo), anesthesia was achieved as described above; a catheter (PE-50) was placed in the carotid artery, tunneled to the nape of the neck, filled with heparin, and occluded. Blood pressure was measured with a Gould-Statham transducer (Oxnard, CA), and tracings were recorded on a physiological recorder (model 81, Siemans). Blood samples (0.20 ml) were collected anaerobically from the carotid artery catheter for measurement of arterial pH, blood gases, and hematocrit. Blood collected for assessment of acid-base status and hematocrit was replaced with warm lactated Ringer solution. After completing ischemia-reperfusion studies on the experimental day, the abdomen was closed with 3-O chromic and skin staples; animals were returned to individual cages and were allowed food and water at will. Twenty-four hours after the ischemia-reperfusion insult, systemic blood pressure and heart rate were measured and a blood sample was collected to assess metabolic function. Isolated coronary perfused hearts. At the designated time period (2-3, 4-5, or 12-16 h after 20-min ischemia l
INTESTINAL
ISCHEMIA
H1163
followed by reperfusion), the rats were anticoagulated with sodium heparin (1,000 U) and decapitated with a guillotine. Hearts were rapidly excised and placed in icecold Krebs-Henseleit buffer to remove the blood. The composition of this buffer was (in mM) 24.0 NaHC03, 118.0 NaCl, 4.7 KCl, 1.2 KH2P04, 1.2 MgS04, 1.25 CaC12, and 11.1 glucose. A small stab incision was made in the ventricular apex, and a collapsed latex balloon connected to a short polyethylene tube (-12 cm) was placed into the left ventricle. The apical stab incision allowed for free drainage of any thesbian flow entering the left ventricle. The hearts were then perfused in a retrograde manner through a catheter placed in the aorta with prefiltered and oxygenated (95% 02-5% CO*) KrebsHenseleit buffer at a pH of 7.4, a temperature of 37°C and a coronary flow rate of 5 ml/min. Previous studies in our laboratory have shown that this flow rate produces maximal ventricular performance with the longest stability over time (unpublished data). The perfused heart was enclosed in a water-jacketed chamber to maintain humidity and temperature. The balloon was filled with bubble-free distilled water, and the polyethylene tubing was connected to a Statham transducer. All balloons were individually calibrated to assure that each balloon was used on the flat portion of its compliance curve. Volume was then injected into the balloon to achieve a desired left ventricular end-diastolic volume and pressure. In a few hearts from each experimental group, myocardial temperature was monitored using a 27-gauge thermistor needle inserted superficially into the left ventricular muscle; the temperature was stable over time for all hearts (37.1 t 0.2”C). After instrumentation, hearts were allowed to stabilize for 30 min before any experiments were performed. Isolated hearts were excluded from further study if they failed to achieve a stable developed pressure or if they developed persistent arrhythmias. Less than 10% of the isolated hearts were excluded for these reasons. After stabilization, left ventricular pressure and the rate of left ventricular rise (+dP/dt,,,) and fall (-dP/dt,,,), heart rate, and coronary perfusion pressure were measured simultaneously on a multichannel polygraph (Grass Instruments, Quincy, MA). In add’ti ion, left ventricular compliance, the time to maximal left ventricular pressure, the time to 90% relaxation, and the time to maximal &dP/dt were calculated as previously described ( 13). Statistical analysis. All values are expressed as means t SE. Statistical comparison of groups was performed with an analysis of variance and a multiple-comparison procedure where appropriate (Student-Newman-Keuls). Differences between experimental groups were considered significant at P < 0.05. RESULTS
In vivo hemodynamic variables obtained during baseline period, 20 min of sham ischemia, and at 20, 40, and 60 min after sham reperfusion were not significantly different from control values, confirming the stability of this preparation (Table 1). Twenty minutes of intestinal ischemia was associated with no change in mean arterial blood pressure, a significant bradycardia, and no change in arterial pH, blood gases, serum bicarbonate, or he-
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H1166 TABLE
CARDIAC
EFFECTS
OF
INTESTINAL
ISCHEMIA
1. Hemodynamic response to intestinal &hernia-reperfusion
Mean arterial blood pressure, Sham Ischemic Heart rate, beats/min Sham Ischemic
Postreperfusion
End of 20 min Ischemia
Baseline
2h
24 h
mmHg 96t6 108*6
87k6 94t6
104t6 98k7
109*5 lOOt8
252k13 231t9
201*7* 160t8*
221t13 179t13
26024 233t9
7.43kO.01 7.38kO.08
7.40t0.02 7.4OkO.02
7.4OkO.01 7.38t0.01
42tl 46t3
36&Z 42t2
34*1 33k2
43t2 46tl
38*1 41*1
40&l 40&l
39t1 4221
34*1* 32*3*
PH
Sham Ischemic Pco~, mmHg Sham Ischemic Hematocrit, Sham Ischemic Values
are means
7.41t0.01 7.36kO.01
t
%
* SE. * Significant
change
from
baseline
values,
P < 0.05. t Significant
matocrit ratio. Reperfusion resulted in a transient hypotension (mean arterial blood pressure fell from 108 t 6 to 88 t 7 mmHg, P < 0.05) but no change in heart rate or acid base balance. Two hours after reperfusion of the ischemic bowel, all indexes of hemodynamic function returned to baseline values and remained stable for 24 h after reperfusion (Table 1). Table 2 summarizes the in vitro cardiac perfusion data from animals subjected to SMA occlusion and killed at varying times after reperfusion of the ischemic bowel. Significant cardiac dysfunction was documented in hearts from animals subjected to 20 min of intestinal ischemia and killed at either 2-3 or 4-5 h after reperfusion. Cardiac dysfunction, indicated by the significant reduction in left ventricular pressure and in rates of left ventricular pressure rise and fall, as well as a decrease in time to peak pressure, was further confirmed by a shift of left ventricular function curves calculated for these groups downward and to the right of those curves calculated for the sham ischemia group (Fig. 1). In addition, hearts harvested from rats after ischemia and reperfusion TABLE
difference
between
groups
at
P < 0.05.
had a significantly reduced responsiveness to increases in exogenous calcium as well as a reduced ventricular response to increased coronary flow compared with control hearts. As seen in Table 3, allopurinol pretreatment for 4 days before ischemia-reperfusion produced cardiac contraction and relaxation indexes that were not statistically different from those measured in control hearts. In addition, left ventricular function curves calculated for this group were similar to those calculated for sham ischemic controls and were significantly better than those generated by untreated ischemic reperfused hearts (Fig. 2). Hearts from rats pretreated with allopurinol generated dose-response curves to exogenous Ca2’ nearly identical to those generated by control hearts and significantly better than those generated by hearts from untreated rats killed 2-3 h after ischemia-reperfusion (Fig. 3). Incremental increases in coronary flow rate (from 3 to 12 ml/min) failed to overcome ischemia-reperfusion-induced cardiac dysfunction, as indicated by the significantly lower left ventricular pressure and +dP/dt,,, at
2. In . vitro cardiac performance Group 1 Sham Ischemia
LVP, mmHg +dP/dt, mmHg/s -dP/dt, mmHg/s Differential ratio dP/dt (DP 40), mmHg CPP, mmHg CVR, mmHg ml-’ min HR, beats/min TTP, ms R&O, ms Time to +dP/dt,,,, ms Time to -dP/dt,,,, ms Serum electrolytes, meq/l Na’ K+ Cll
l
Group 2 Ischemia Plus 2-3 h Reperfusion
Group 3 Ischemia Plus 4-5 h Reperfusion
Group 4 Ischemia Plus 12-16 h Reperfusion
77*3 1,827+59 1,267+57 1.45t0.05 1,738*49 62.2t4.4 12.4k0.8 25Ok13 81&Z 80&l 41t1 49&l
63&4* 1,557+99* 953t67* 1.65&0.07* 1,488+90* 53k7 10.7k1.5 252t13 76&l* 79t2 4Okl 47t1
67t4* 1,596+81* 1,078+70 1.51t0.06* 1,561+65* 50t5 10.0~1.0 223&11 81t2 81t2 42tl 50t1
66&Z* 1,647+69* 1,059+53* 1.5720.05 1,581+60* 53t3 10.7kO.7 226kll 82kl 8021 45t0.8* 52tl
142tl 6.1t0.3 107.4t0.8
139kl 5.5kO.2 105.1t0.9
138t2 6.1k0.2 105.6kl.O
142tl 6.6kO.2 106.7t0.6
Values are means & SE. LVP, left ventricular pressure; kdP/dt, maximal rate of left ventricular pressure rise (+) and fall (-); dP/dt (DP 40), rate of LVP rise at a developed pressure of 40 mmHg; CPP, coronary perfusion pressure; CVR, coronary vascular resistance; TPP, time to peak isovolumic pressure; RTSo, time to 90% relaxation. * Significant difference among groups at P 5 0.05.
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CARDIAC
LV Pressure (mmHg)
EFFECTS
OF
INTESTINAL
A
FIG. 1. Left ventricular (LV) performance after 20 min of intestinal ischemia followed by reperfusion. Experimental groups were killed at either 2-3 or 4-5 h after removing clip on superior mesenteric artery. All values are means & SE. &dP/ dt, rate of LV pressure rise (+) or fall (-). * Significant difference between groups at P < 0.05.
Sham Ischemia/2-3 hrs reperfusion Ischemia/4-5 hrs reperfusion
Ol
H1167
-dP/dt (mmHg/sec)
+dP/dt (mmHg/sec)
1000
n l
ISCHEMIA
5000
-10
0
20
10
-10
0
10
20
Left Ventricular End Diastolic Pressure
mmw
3. Effects of allopurinol on in vitro cardiac performance TABLE
Sham Ischemia
LVP, mmHg +dP/dt, mmHg/s -dP/dt,,,, mmHg/s dP/dt (DP 40), mmHg/s CPP, mmHg CVR, mmHg. ml-’ min HR, beats/min TTP, ms R%o, ms Time to +dP/dt,,,, ms Time to -dP/dt,,,, ms l
Values
are means
Allopurinol-Treated Ischemia
77k3 1,827+59 1,267rt57 1,738*49 62k4 12.4k0.8
7322 1,696+60 1,231+46 1,622*56 72k5 14.5t1.1
250t13 81&Z 80tl 41&l 49t1
269t12 84k2 78k2 43k2 45&l
t SE. See Table
2 for definitions
of abbreviations.
each level of flow in the untreated ischemia-reperfusion group. In contrast, allopurinol-treated hearts harvested at an identical time after reperfusion of the ischemic bowel (2-3 h) had ventricular performance-coronary flow relationships that were identical to those generated by control hearts (Fig. 4). During 30 min of in vitro perfusion with Krebs buffer, there was a nonsignificant increase in left ventricular pressure and in dP/dt,,, in all animals, attesting to the stability of the Langendorff heart preparation. Left ventricular compliance was identical in all experimental groups over the experimental period, and allopurinol pretreatment did not alter this relationship (Fig. 5). DISCUSSION
The rat has been commonly used as a model to evaluate mesenteric ischemia, and SMA occlusion has been most commonly used to produce ischemic injury to the intestine. Mesenteric collateral circulation in rats is analogous to that in humans. Vascular arcades that connect the
jejunal to the ileal arteries and the ileal colic artery to the right colic artery are quite small, with an external diameter of CO.5 mm. However, the efficiency with which these arteries transmit blood has been previously demonstrated in our laboratory, as documented by the failure of SMA occlusion alone to alter blood flow (measured with radioactive microspheres) to the middle segment of the ischemic bowel compared with blood flow measured in the proximal and distal segments (18). In the present study, occlusion of the SMA with collateral interruption produced intestinal ischemia that was grossly visible during the procedure. The immediate pallor of the ischemic segment and a sharp line of demarcation between perfused and hypoperfused sections confirmed adequate bowel ischemia using this technique. This model of intestinal ischemia and reperfusion injury is reproducible, simple, and clinically relevant. As previously demonstrated by our laboratory, this model produces a profound, but reversible, decrease in intestinal blood flow and in hemodynamic stability and a significant bowel edema after reperfusion, as well as reproducible histological injury to the intestinal mucosa (18, 19). The data presented here extend our previous studies, confirming that intestinal ischemia-reperfusion injury produces significant cardiac dysfunction. Cardiac injury was characterized by a decreased ability of the ventricle to develop pressure, alterations in the rate of left ventricular pressure rise and fall, and a shift in the left ventricular function curves downward and to the right of those calculated for control hearts, suggesting ventricular failure. These cardiac contraction and relaxation deficits could not be overcome by increases in perfusate calcium or by maximal increases in preload. These data suggest that 1) the release or activation of toxic factors from the ischemic bowel target the heart during intestinal reperfusion, and 2) profound cardiac contractile dysfunction after a brief episode of intestinal ischemia reduces cardiac output, contributing, in part, to periph-
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H1168
CARDIAC
LV Pressure (mmHg)
EFFECTS
OF
INTESTINAL
+dPMt (mmHg/sec)
ISCHEMIA
dP/dt (mmHg/sec)
* FIG. 2. Effects of allopurinol on left ventricular performance after intestinal ischemia and 2-3 h of reperfusion. All values are means k SE. * Significant difference between groups at P < 0.05.
loo0
n Sham 0
lschemia,Untfeated
5WLeft Ventricular End Diastolic Pressure (mmHg)
era1 perfusion deficits and eventual multiorgan failure. However, it must be emphasized that in our study contractile performance was evaluated in a non-blood-perfused, nonworking heart that was almost entirely neutrophi1 free. It is likely that in vivo activation, sequestration, and adherence of leukocytes target particular organs such as the heart; in addition, activation of numerous plasma factors, such as cytokines, complement products, and myocardial depressant factors, produced at the site of LV Pressure (mmHg) loo-
+dP/dt (mmHg/sec)
-dP/dt (mmHg/sec)
2500
2500
2000
2000 -
.
80 -
ischemic injury likely impair cardiac function after systemic distribution. Therefore it is apparent that oxygenderived free radical-induced cardiac injury is only one aspect of a complex sequence of pathological events that follows a brief period of intestinal ischemia. Several previous studies have suggested that the intestinal ischemic injury previously described in our model and by others was likely related to the effects of interrupted aerobic metabolism, the accumulation of by-prod-
1 .
1500
60 -
1500 .
1000
1000 .
n Sham l IschemiaJntreated 0
500
FIG. 3. Calcium dose-response curves generated by untreated ischemia-reperfusion, allopurinol-treated ischemia-reperfusion, and control experimental groups. All values are means k SE. * Significant difference between groups at P < 0.05.
500 -
lschemia + Allopurinol
\ 0
.
-1.1’
00
Perfusate Calcium (mM) Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.155) on October 17, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
CARDIAC
LV Pressure (mmHg)
EFFECTS
OF
INTESTINAL
+dP/dt (mmHg/sec)
H1169
ISCHEMIA
-dP/dt (mmHg/sec)
2500
2500
60 FIG. 4. Ventricular performance coronary flow relationships. All values are means t SE. * Significant difference between groups at P < 0.05.
2
n
Sham
0 0
Ischemia,Untreated lschemia + Allopurind
6
10
500
14
2
10
6
14
2
6
10
14
Coronary Flow Rate (ml/min) n l A A
Sham Ischemia,2-3 lschemia&5 Ischemia,l2-16
hrs repeffusion hrs reperfusion hrs reperfusion
0
Ischemia,2-3
hrs reperfusion
0.00
0.03 Left Ventriarlar
FIG.
5. Left ventricular
compliance
0.06
0.09
Volume
+ Allopurinol
0.12
(ml)
in all experimental
groups.
ucts of ATP degradation, and the generation of toxic oxygen metabolites generated via xanthine oxidase-dependent mechanisms (9, 24, 28). The overproduction of toxic oxygen metabolites appears to play a central role in the production of multiorgan system failure, and the heart may be a particularly susceptible organ. We have previously shown that the cardiac dysfunction that is characteristic of several types of trauma with shock, such as burn injury (13, 15) and hemorrhagic shock (14), was related to the generation of oxygen-derived free radicals. Whether the generation of oxygen-derived free radicals
is due to generation of xanthine oxidase-dependent or phagocyte-derived sources remains unclear. Several studies have suggested that sequestration of neutrophils in the ischemic tissue during hypoperfusion and the activation of neutrophils during reperfusion damage peripheral organs directly (2,12,22,31). In this regard, previous studies in other models of ischemic injury have shown that neutrophil depletion significantly improved peripheral organ function after reperfusion of ischemic injury (30, 31). The protective effects of allopurinol pretreatment suggest that blocking xanthine oxidase prevents formation of the toxic free radicals, enhancing cardiac performance at a time when intestinal ischemia-mediated cardiac dysfunction was maximal. An alternate mechanism by which allopurinol may protect the heart during an episode of intestinal ischemia-reperfusion was suggested by studies by Das et al. (7) and Garcia et al. (a), who demonstrated that allopurinol and oxypurinol, its metabolite, function as free radical scavengers independent of their inhibitory effects on xanthine oxidase. The idea that allopurinol pretreatment can prevent reperfusion injury after ischemia is not a new concept. As early as 1969 the cardioprotective effects of allopurino1 were confirmed in hemorrhagic shock (6). More recently, allopurinol pretreatment has been shown to improve survival of acute skin flaps (16), limit infarct size after ischemia-reperfusion (33), and improve survival after mesenteric ischemia-reperfusion (19, 20). A significant finding of the present study is that the protective effect of allopurinol was achieved with enteral doses that would not cause intolerable side effects. If a patient population that is at risk for an ischemic event can be identified, allopurinol can be studied for its prophylactic effects, since it is innocuous, easily administered enterally once a day, and has been used extensively in the past.
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H1170
CARDIAC
EFFECTS
OF
In summary, cardiac contraction and relaxation defects occurred as early as 2 h and persisted for 12-16 h after mesenteric ischemia-reperfusion. Although numerous studies have implicated oxygen-derived free radicals as primary mediators in the pathogenesis of ischemiareperfusion injury, it is likely that a number of factors such as cytokines, complement products, and myocardial depressant factors are produced at the site of tissue injury and subsequently impair cardiac function after systemic distribution. In our study, allopurinol, given enterally for 4 days before the ischemic insult, prevented mesenteric ischemia-reperfusion-induced cardiac dysfunction, confirming that oxygen-derived free radicals contribute, in part, to the cardiac deficits.
INTESTINAL
14.
15.
16.
17.
18.
19. The authors thank Nancy Bain for superb secretarial assistance. Address for reprints requests: J. W. Horton, Dept. of Surgery, Univ. of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235-9031. Received
22 January
1991; accepted
in final
form
22 May
20.
1991. 21.
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