Emergency Medicine Research, Carolinas Medical Center, Charlotte, North Carolina 28232-2861
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ABSTRACT |
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This study examines activation of
poly(ADP-ribose) polymerase (PARP) in the ileum during hemorrhage and
resuscitation and determines if inhibition of PARP reduces organ
dysfunction and metabolic acidosis. Awake, nonheparinized rats were
hemorrhaged (40 mmHg, 60 min). Resuscitation used Ringer's solution
(2
multiorgan failure; ileum; gut permeability; 3-aminobenzamide; caspase
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INTRODUCTION |
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WHEN ACTIVATED BY DNA single strand breaks, poly(ADP-ribose) polymerase (PARP) cleaves NAD+ and attaches polymers of ADP-ribose to proteins. PARP normally participates in homeostatic processes (12). In stress conditions, however, excess activation may lead to depletion of NAD+, inhibition of glycolysis, depletion of ATP, and necrotic cell death (2-4, 7, 16, 27-29, 32, 50).
Previous studies of hemorrhagic shock have implicated PARP as a mediator of organ injury in hemorrhage and resuscitation by showing beneficial effects of pharmacological inhibition or by employing mice that are genetically deficient in PARP. Beneficial effects of PARP inhibition in models of hemorrhage and resuscitation include improved mean arterial pressure (MAP) following resuscitation in an underresuscitated model (31, 33), improved vascular contractile responses (30), and reduced organ injury in liver, kidney, and pancreas (21, 22). Knockout mice that lack PARP activity also show a less severe decline in MAP following hemorrhage and resuscitation (19). None of these studies has directly measured the time course of PARP activation, the role of PARP activation in development of increased ileum permeability, or the metabolic effects of PARP activation during the hemorrhage period. Therefore, the purposes of the present study were threefold: first, to characterize the general time course of PARP activation during hemorrhage and resuscitation in the ileum by using a direct enzyme assay technique; second, to determine if inhibition of PARP activity influences the recovery of organ function; and third, to assess the effects of PARP activation on the development of metabolic acidosis during hemorrhage and subsequent resuscitation.
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MATERIALS AND METHODS |
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These experiments were performed in adherence with the National Institutes of Health guidelines on the use of experimental animals. All experiments were performed under protocols approved by the Institutional Animal Care and Use Committee of the Carolinas Medical Center.
Instrumentation
The studies were performed with male Sprague-Dawley rats (300-450 g) that had ad libitum access to Teklad rat chow and water before the experiments. The animals were anesthetized with isofluorane (induction with 3-4% and maintenance with 1-2% isofluorane, balance oxygen) with a small animal anesthesia machine and ventilator. The femoral triangle was prepared with aseptic conditions, and the femoral artery was cannulated with a short segment of PE-50 tubing attached to PE-90 tubing to facilitate blood withdrawal and to monitor blood pressure. A PE-50 catheter was inserted into the femoral vein for resuscitation. Catheters were filled with 101% of their previously determined dead space volume with a sterile solution of 1 U heparin/ml of saline and were routed and exteriorized at the back. After the incisions were sutured, the animals were awakened and regained consciousness for 30 min in a clear chamber made of Plexiglas. The arterial catheter was connected to monitor hemodynamics with a Statham transducer (Statham/Gould Instruments, Millersville, MD) and for digital data acquisition (Acknowledge software, Biopac Systems, Santa Barbara, CA). Values of MAP are presented from the baseline period, the end of the hemorrhage period, and at the end of each phase of the resuscitation period.Hemorrhage and Resuscitation
Hemorrhage was initiated in conscious rats by withdrawing blood from the femoral artery into a syringe containing anticoagulant citrate dextrose (10% vol/vol) at a rate of 1 ml/min with a syringe withdrawal pump (Harvard Apparatus, Holliston, MA). Animals were bled to a MAP of 40 mmHg. Additional blood was withdrawn, or aliquots of Ringer's solution were added as required to maintain animals at 40 mmHg for 60 min. Animals were then resuscitated with 1× the shed blood volume as Ringer solution at a rate of 1 ml/min andThe blood that was shed during hemorrhage was weighed and centrifuged
at 1,500 g for 10 min. The plasma was removed by aspiration, and the PRBC were resuspended in Ringer solution just before
reinfusion. The PRBC solution was gently agitated and temperature
controlled until
PARP Assays
Animals were anesthetized with pentobarbital (15 mg/kg iv), and tissues were removed for the measurement of PARP activity after the sham protocol, following hemorrhage with no resuscitation, following 10 min of resuscitation with the initial Ringer solution, and 60 min after the initiation of resuscitation. Tissues were freeze-clamped in tongs cooled in liquid nitrogen and were stored atWestern Blotting of PARP
Nuclei isolated from the ileum samples as in the PARP enzyme assay or human cells in culture were suspended in lysis buffer [10% (vol/vol) glycerol and 2% (wt/vol) SDS in 83 mM Tris, pH 6.8] and sheared by four passages through a pipette. To part of the homogenate 10%Caspase-3 Assay
Animals were anesthetized with pentobarbital (15 mg/kg iv), ileum samples were removed, and tissues were freeze-clamped in tongs cooled in liquid nitrogen and stored atTreatment Groups for the Study of Organ Dysfunction and Blood Chemistry
The following groups of animals were studied to examine the effects of pharmacological inhibition of PARP on the rise in ileum permeability, release of liver enzymes, and development of metabolic acidosis: 1) pretreatment with PARP inhibitor 3-AB given intravenously at 15 min before the initiation of hemorrhage (20 mg/kg) and at the initiation of resuscitation (10 mg/kg); 2) pretreatment with the structural analog 3-aminobenzoic acid (3-ABA) given intravenously at 15 min before the initiation of hemorrhage (20 mg/kg) and at the initiation of resuscitation (10 mg/kg); 3) posttreatment with PARP inhibitor 3-AB given intravenously as a bolus at 5 min before the initiation of resuscitation (20 mg/kg); and 4) sham-treated animals were time matched, but did not receive hemorrhage or resuscitation procedures.The doses of 3-AB employed in the present studies are comparable with the doses used in previous in vivo studies of hemorrhagic shock (21, 30, 31) and in other models of injury (6, 9, 10, 26, 31, 34, 38, 39, 44, 49). 3-AB also has the capacity to act as an antioxidant at higher concentrations (35). Therefore, one group of animals received the same dose of 3-ABA, which served as an antioxidant control.
Measurements of Organ Dysfunction
Ileum permeability. Measurements of ileum permeability were made between 60 and 90 min of resuscitation. The procedure for assessing ileum permeability was modified from a previously published method (23). Animals were anesthetized with isofluorane (1-2%, balance oxygen), and a laparotomy was performed to allow access to the intestines. Holes were placed at both ends of a 10-cm segment of the ileum proximal to the cecum, and the contents were gently removed by irrigation with 10 ml Ringer solution. One end of the ileum was ligated, and the ileum was filled with 1 ml of FITC-dextran solution (25 mg/ml in 0.1 M PBS, pH 7.2). The other end of the ileum was then ligated so as not to disturb circulation through the major branches of mesenteric circulation to the segment of ileum. The intestines were returned to the abdominal cavity, and the abdomen was sutured. The animals remained on a temperature-controlled heating pad during this measurement. Thirty minutes later, the abdomen was opened and a 1 ml sample of hepatic portal vein blood was removed with an 18-gauge angiocath and syringe. An aliquot (0.1 ml) of whole blood was added to 1.9 ml of fluorescence buffer (50 mM Tris, pH 10.3, containing 150 mM NaCl). The diluted sample was centrifuged (3,000 g for 7 min at 4°C), and the fluorescence of the supernatant was determined with a fluorometer (Perkin-Elmer model L850B, set at 480 nm excitation and 520 nm emission). The original FITC-dextran solution was diluted, and aliquots were added to the fluorescence buffer to obtain a standard curve. This curve was used to quantify the levels of FITC-dextran observed in blood samples.
Blood chemistry and measurement of liver enzymes. Arterial blood samples (0.5 ml) were obtained in a heparinized syringe at the end of the baseline period, at the end of the period of hemorrhagic shock, and at 60 min after the start of resuscitation. These samples were analyzed for blood gases, pH, systemic base excess (SBEc model ABL 520; Radiometer, Westlake, OH), or lactic acid (YSI model 2700 Stat Select analyzer; Yellow Springs Instruments, Yellow Springs, OH). A sample of arterial blood (0.5 ml) was obtained without heparinization 60 min after the start of resuscitation. This sample was centrifuged at 3,000 g (4°C), and the serum was analyzed for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as markers of liver injury with an automated analyzer (VetTest model 8008; IDEXX, Westbrook, ME).
Statistics
Sequential measurements of MAP in the same animals (Fig. 1) were compared with repeated-measures ANOVA on ranks. Individual time points were compared with baseline values by using Dunn's method. Comparisons of PARP activity in ileum isolated at different times (Fig. 2) were made with one-way ANOVA followed by the Student-Newman-Keuls method since these were independent samples. The effect of 3-AB on PARP activity was tested with paired t-tests within each experimental period. Activity of caspase (Fig. 3) in sham-treated and 60-min resuscitated animals was compared by t-test. Treatment effects on ileum permeability (Fig. 4), liver enzymes (Fig. 5), and blood chemistry (Fig. 6) were tested by using one-way ANOVA followed by the Student-Newman-Keuls method. Values are presented as means ± SE with significance accepted at P < 0.05.
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RESULTS |
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Effects of Hemorrhage and Resuscitation
MAP decreased from 124.9 ± 1.8 mmHg at baseline to 39.8 ± 0.8 mmHg at the end of the 60-min hemorrhage period (Fig. 1). The volume of blood shed during the hemorrhage period was 15.49 ± 0.55 ml (3.87 ± 0.10 ml/100 g body wt) in these animals. This shed volume includes the blood sample volume (0.5 ml) taken for blood chemistry measurements at the end of the baseline period. The volume of Ringer solution given toward the end of the hemorrhage period to maintain the hemorrhage pressure near 40 mmHg was 2.38 ± 0.73 ml (0.56 ± 0.16 ml/100g body wt) in these animals. Resuscitation was accomplished with a combination of Ringer solution and PRBC. Blood pressure increased with the infusion of Ringer solution (10-min value), and further increase was observed following infusion of PRBC (30-min value). Blood pressure was maintained well above 100 mmHg through the end of the experimental period, suggesting that there was not severe underresuscitation. Hemorrhagic shock caused significant acidosis (pH 7.09 ± 0.04 vs. 7.43 ± 0.01), accumulation of lactic acid (16.41 ± 0.48 vs. 0.89 ± 0.06 mmol/l), base deficit (Activation of PARP
Ileum samples were isolated from animals at the end of each phase of the protocol. Sham-treated animals exhibited low PARP activity in the ileum (Fig. 2). PARP activity increased significantly (3.6-fold) in ileum samples isolated at the end of the hemorrhage period and was elevated fivefold following the initial 10 min of resuscitation with Ringer solution. Thus PARP activity was stimulated during the hemorrhage period and the initial period of resuscitation in this model. PARP activity was significantly inhibited by the addition of 3-AB in all reactions. PARP activity, expressed per gram wet weight, was 325 pmol · minSimilar measurements of PARP activity were attempted in liver samples.
The activity was not significantly higher in livers isolated after 10 min of resuscitation than in livers isolated from sham-treated animals
(65.5 ± 13.7 vs. 45.0 ± 7.3 pmol · min1 · mg
1 protein,
respectively; P = 0.270). PARP activity measured in the
sham-treated livers was significantly higher than in the sham-treated ileum samples (9.2 ± 2.6 pmol · min
1 · mg
1) but was
similar to the activity observed in the ileum samples isolated after 10 min of resuscitation (45.5 ± 7.6 pmol · min
1 · mg
1). It is
possible that PARP activity may have been stimulated during the
preparation of the tissue extracts, producing high levels of PARP
activity in liver from sham-treated animals. Liver tissue has a
Ca2+/Mg2+ dependent endonuclease
present in the nucleus that actively cleaves DNA (48) and
may have caused nonspecific activation of PARP during the tissue preparation.
Analysis of PARP Cleavage in Ileum
The observation that PARP activity decreased in the ileum after 60 min of resuscitation compared with PARP activity observed after 10 min of resuscitation suggested that PARP may be cleaved by caspase activation. Therefore, we assessed PARP cleavage by Western blotting and directly measured caspase enzyme activity. Western blots of PARP protein in nuclei isolated from ileum samples (Fig. 3A) indicated that there was no disappearance of the 116-kDa active form of PARP and no significant appearance of the 85-kDa breakdown fragment in the sham-treated or the shocked animals. A positive control for the decrease in 116-kDa fragment and appearance of the 85-kDa fragment is also shown to indicate that the technique does detect PARP cleavage when it exists. Human cells were exposed to cyclohexamide (25 µg/ml) for 24 h in culture to induce apoptosis for comparison with control cells. Measurement of caspase-3 activity in the sham-treated and the shocked animals indicated that caspase-3 activity was not elevated at this time of resuscitation (Fig. 3B).Organ Dysfunction
Dextran was infused into the ileum after 60 min of resuscitation, and the hepatic portal blood samples were taken after 90 min of resuscitation to measure ileum permeability (Fig. 4). The level of dextran was elevated 10-fold in the hepatic portal blood of animals pretreated with 3-ABA compared with the level observed in sham-treated animals. Pretreatment with the PARP inhibitor 3-AB resulted in a significant inhibition of the rise in ileum permeability. In contrast with the pretreatment mode of PARP inhibition, the rise in ileum permeability was not prevented with the addition of a bolus of 3-AB given 5 min before resuscitation (Fig. 4).Measurements of liver transferase enzyme activity were made in arterial samples taken at the end of the 60-min resuscitation period. Arterial ALT values increased 6.5-fold in animals subjected to hemorrhage and resuscitation with 3-ABA treatment compared with the sham animals (Fig. 5A). Pretreatment with the PARP inhibitor 3-AB significantly reduced the values of arterial ALT compared with animals receiving 3-ABA pretreatment. There was no significant difference between animals that received 3-ABA pretreatment or 3-AB posttreatment. A similar pattern of liver injury was observed with arterial AST values (Fig. 5B). This suggests that liver injury was reduced by pretreatment with the PARP inhibitor 3-AB but that pretreatment with 3-ABA or posttreatment with a bolus of 3-AB showed evidence of liver injury.
Effects of PARP Inhibition on Acid-Base Status
Sham-treated animals had normal values of pH (Fig. 6A), arterial lactate (Fig. 6B), and arterial base deficit (Fig. 6C). Baseline measurements indicated that blood chemistry was unchanged by pretreatment of the animals with 3-AB or 3-ABA (data not shown). Animals treated with 3-ABA showed significant acidosis (pH = 7.027 ± 0.030), lactic acid accumulation (17.66 ± 1.074 mmol/l), and base deficit ( ![]() |
DISCUSSION |
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This study demonstrates that PARP enzyme activity is elevated in the ileum by hemorrhage alone and during early resuscitation following hemorrhagic shock. Inhibition of PARP activity by pretreatment with 3-AB lessened the rise in permeability of the ileum and reduced the appearance of liver enzymes in the plasma observed after hemorrhage and resuscitation. In addition, the inhibition of PARP slowed metabolic acidosis, lactate accumulation, and base deficit observed during hemorrhagic shock and subsequent resuscitation. In contrast, posttreatment with 3-AB, given as a bolus 5 min before resuscitation, was ineffective in reducing the rise in ileum permeability, the appearance of liver enzymes, and the development of indices of metabolic acidosis in this model. These findings suggest a role of PARP activation in the pathogenesis of organ injury and metabolism during hemorrhagic shock.
Hemorrhagic shock causes mesenteric constriction (40). Intense vasoconstriction induces tissue hypoxia in the ileum that can lead to depletion of tissue ATP levels and enhanced intestinal epithelial permeability (43). Disturbances in the mucosal barrier of the ileum may play a role in the induction of sepsis in trauma and shock via the translocation of bacteria or bacterial products (13). Therefore, it is important to consider the link between excessive PARP activation and intestinal permeability. Increased intestinal permeability associated with ischemia and reperfusion is reduced by PARP inhibition (10, 20) or by PARP knockout in mice (20); however, this relationship has not previously been tested in hemorrhage and resuscitation. The present data indicate that PARP is activated in the ileum as a response to hemorrhage and to hemorrhage with resuscitation and that the inhibition of PARP activity provides protection against the dysfunction of this organ.
PARP activation occurs during hemorrhagic shock in the present studies, suggesting that DNA damage occurs before resuscitation. Severe hemorrhagic shock causes ischemia in the gut. It is clear that reperfusion of ischemic tissue leads to the formation of oxygen radicals (10, 14). In the classic paradigm, resuscitation would be required to stimulate reactive oxygen species to produce DNA damage, which is required for PARP activation. Because PARP activity was stimulated during the hemorrhage period with no resuscitation (Fig. 2), it is important to consider that free radical-mediated DNA injury occurred in the ileum during the period of reduced flow without resuscitation. The respiratory chain is one of the major sources of free radicals in cells (24). Becker and colleagues (1, 41, 42) have recently examined electron leak from the electron transport system during anoxia in cardiomyocytes. These studies indicate that leak of electrons occurs between NADH DH in complex I and the ubisemiquinone site with cytochrome b-c1 segment of complex III when the electron transport system is reduced and some oxygen is present. This occurs even at the low oxygen concentrations observed during ischemia. In addition, low concentrations of arginine cause nitric oxide synthase to form superoxide (45-47) and acidic conditions can cause nonenzymatic formation of nitric oxide (51, 52). This could lead to the formation of peroxynitrite that may damage the DNA. It is unclear whether these conditions contribute to the injury developed in vivo. Recent data show that occlusion of the superior mesenteric artery with no reperfusion produced fragmentation of DNA after 15 min (5% of DNA) and 60 min (12% of DNA) (25). This fragmentation was increased by reperfusion at either time, but the data illustrate that DNA fragmentation can occur in the ileum during low-flow states. Alternatively, there could be intermittent perfusion of the ileum during the hypotensive phase of the experiment, since it is unlikely that there is complete cessation of flow during hemorrhage (40).
The finding that PARP is activated during the hypotensive phase of shock suggests that pretreatment with PARP inhibitors may be required to confer protection. Many studies of PARP inhibitors in hemorrhagic shock have employed models in which the PARP inhibitor was added before the induction of hemorrhagic shock (31, 33). The use of knockout mice also provides a pretreatment absence of PARP-1 activity (19). We have previously observed protection of aortic constrictor responses following the addition of 3-AB at the onset of resuscitation from hemorrhagic shock (30). McDonald et al. (21, 22) also observed a reduction in the appearance of liver, kidney, and pancreatic enzymes in blood when PARP inhibitors were added 5 min before resuscitation and infusion was continued throughout the resuscitation. Two possible explanations may account for the differences in the success of posttreatment with PARP inhibitors following hemorrhagic shock. First, these models employed anesthetized rats and the hemorrhagic shock was probably less severe than the conscious model of hemorrhage used in the present studies (40 mmHg, 3.87 ml blood shed/100 g body wt). Second, the posttreatment in the present study was given as a bolus infusion before resuscitation and was not continued throughout the resuscitation period. Future studies should compare models of different severity and the effects of continual infusion to determine conditions in which posttreatment with PARP inhibitors provides benefits. This approach would further evaluate the clinical application of PARP inhibitors in improving the outcome of resuscitation following hemorrhagic shock.
The increase in PARP activity detected in the present studies of the ileum (5-fold) was similar to the increase observed in organs and isolated cells subjected to a variety of insults (8, 11, 15, 18, 22, 26, 29, 36, 37). A study that specifically measured PARP activity in intestinal mucosal cells found a doubling of PARP activity following splanchnic artery occlusion and reperfusion (10, 20). Technical differences in the preparation of the enzyme for analysis may account for some of the differences in PARP activation of the ileum. For instance, the studies of ischemia and reperfusion of the intestine used permeabilized cells (10), whereas the present studies used nuclei that were permeabilized following their isolation from tissue homogenates.
The dose of 3-AB administered in the present study is comparable with doses used in previous studies of hemorrhagic shock (21, 30, 31) and in other in vivo models of injury (6, 9, 10, 26, 31, 34, 38, 39, 44, 49). At high concentrations, 3-AB also has the capacity to act as an antioxidant (35), which is a possible mode of action that may complicate the interpretation of the data while evaluating the role of PARP in studies employing this agent. The present studies also included a treatment group that received 3-ABA, a compound that has the same benzene ring structure but lacks the amino group and therefore does not inhibit PARP. Therefore, potential antioxidant mechanisms of protection should be observed in this group. Since 3-ABA did not protect against the rise in ileum permeability (Fig. 4), liver ALT, or AST enzymes in blood (Fig. 5), it appears that the protective effects of 3-AB are not due to antioxidant properties. The increase in liver enzymes was attenuated by the addition of 0.03 and 0.3 mg/kg of a water-soluble isoquinolone derivative in another study of hemorrhagic shock in anesthetized rats (22), suggesting that more potent inhibitors may provide beneficial effects at lower doses.
The present studies show that activation of PARP occurs in the ileum at the end of hemorrhagic shock without resuscitation and during the early part of resuscitation. The decline in PARP activity observed following 60 min of resuscitation is not due to cleavage by caspase-3. Inhibition of PARP reduces metabolic acidosis observed during hemorrhage and during resuscitation and reduces the rise in ileum permeability and the release of liver enzymes observed after resuscitation. Thus PARP activation, occurring during hemorrhage and during the early phases of resuscitation, contributes to organ dysfunction. The addition of the PARP inhibitor as a bolus just before resuscitation was ineffective in the present model, suggesting that this approach to therapy may require early treatment or may require subsequent infusion of PARP inhibitor throughout resuscitation.
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ACKNOWLEDGEMENTS |
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We thank Sukhdev S. Brar for his assistance in performing the Western blotting procedures.
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FOOTNOTES |
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This study was supported, in part, by the Charlotte-Mecklenburg Health Services Foundation.
Address for reprint requests and other correspondence: J. A. Watts, Emergency Medicine Research, Carolinas Medical Center, P.O. Box 32861, Charlotte, NC 28232-2861 (E-mail: jwatts{at}carolinas.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 15 September 2000; accepted in final form 23 March 2001.
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