Limit Ischemia-reperfusion Injury Research Articles

Abstract 2329: Donor Pretreatment with Hypertonic Saline Attenuates Primary Cardiac Allograft Dysfunction

Objective: Hypertonic saline (HTS) has been previously demonstrated to have immune modulatory and vascular protective effects. We assessed the effect of donor pretreatment with HTS on allograft myocardial function in a porcine model of orthotopic heart transplantation. We hypothesized that HTS infusion prior to donor heart arrest and storage would limit ischemia-reperfusion injury and improve myocardial performance following transplantation. Methods: Orthotopic transplants were performed after 6 hours of ischemic allograft storage. Donor pigs were randomly assigned to pretreatment with (n = 7) or without (n = 6) HTS (4.5 ml/kg of 7.5% NaCl) administered 1 hour prior to donor heart arrest. Left ventricular performance was determined after caval occlusion using a Millar micromanometer and conductance catheter. Hemodynamic measurements were obtained using a Swan-Ganz catheter. Results: Administration of hypertonic saline increased serum sodium level from 138 ± 2 mmol/L to 154 ± 4 mmol/L, which normalized to 144 ± 3 mmol/L 1 hour post-infusion. Weaning from CPB and LV performance after transplantation was improved after donor HTS treatment. Successful weaning from CPB was significantly greater in HTS treated hearts (6/7 versus 1/6, p < 0.05). Preload recruitable stroke work post-transplantation was improved compared to control (88 ± 21% versus 35 ± 8% of baseline, p < 0.01). Similarly, maximal elastance was improved compared to control (85 ± 17% versus 42 ± 12% of baseline, p = 0.03). No significant differences in post-transplant central venous, pulmonary artery or pulmonary capillary wedge pressures were observed between groups. However, post-transplant systolic blood pressure was significantly higher in the donor HTS group (60 ± 9 mmHg versus 35 ± 6 mmHg, p = 0.04). Conculsions: Donor HTS pretreatment attenuates post-transplant cardiac allograft myocardial function and improves post-transplant systemic hemodynamic function. HTS may be a novel organ donor intervention to prevent primary graft dysfunction. Further clinical evaluation of this simple and potentially cost-effective intervention is warranted.

Abstract 1905: Restoration of Normoglycemia Re-Establishes the Infarct-Sparing Effect of Postconditioning in Diabetic Mice

Relief of ischemia in a stuttering manner (postconditioning: PostC) reduces infarct size in healthy adult populations, due in part to activation of ERK 1/2 during the early minutes of reflow. However, emerging evidence suggests that PostC fails to limit ischemia-reperfusion injury in the setting of diabetes. Our current aim was to establish whether this loss in efficacy of PostC reflects a permanent diabetes-associated defect in cardioprotective signaling, or whether therapy to restore normoglycemia can re-establish the PostC phenotype. To address this issue, isolated buffer-perfused hearts were obtained from 4 cohorts of C57BL/6J mice: healthy adults; animals injected 2 or 4 wks earlier with streptozotocin (STZ) to induce diabetes; and mice injected 4 wks earlier with STZ that, at 2 wks after being rendered diabetic, received islet cell transplantation (ICT). All hearts underwent 30 min global ischemia and, within each cohort, were assigned to receive either complete, abrupt reperfusion (controls) or PostC with 3 10-sec cycles of stuttered reflow. In hearts from healthy mice: (i) PostC was cardioprotective (infarct size [delineated by tetrazolium staining] was 27±3% vs 50±3% of the LV in PostC hearts vs controls; p<.05); (ii) PostC-induced protection was associated with significant, >2-fold increases in expression of phospho-MEK, phospho-ERK and downstream targets of ERK; and (iii) the benefits of PostC were abrogated by the MEK inhibitor PD98059. At both 2 and 4 weeks post-STZ injection, mice displayed: (i) low plasma insulin levels (0.18±0.08 and 0.07±0.02 vs 1.40±0.20 ng/ml) and hyperglycemia (blood glucose: 556±15 and 558±10 vs 178±7 mg/dl in healthy C57 mice); (ii) no up-regulation of ERK signaling in response to stuttered reflow; and (iii) no infarct-sparing effect of PostC (mean infarct sizes: 62– 63% in STZ-PostC groups vs 58 – 63% in STZ-controls). In contrast, ICT: (i) restored normal insulin and blood glucose levels (1.33±0.09 ng/ml; 189±7 mg/dl) and, most notably (ii) reinstated the up-regulation in ERK signaling and infarct-sparing effect of PostC (infarct size: 31±3% vs 51±5% in STZ±ICT controls; p<0.05). Thus, therapeutic control of insulin and blood glucose re-establishes the protective PostC phenotype in this mouse model of diabetes.

Effects of N-acetylcysteine on myocardial ischemia–reperfusion injury in bypass surgery

Myocardial ischemia-reperfusion injury may complicate coronary artery bypass grafting (CABG) operations. N-Acetylcysteine (NAC) had antioxidant and microcirculatory effects, and inhibits neutrophil aggregation. The aim of this study was to determine the effects of NAC in limiting myocardial ischemia-reperfusion injury in CABG operations. Twenty patients undergoing elective coronary bypass operation with cardiopulmonary bypass were enrolled and randomly assigned to two groups: a control group operated with a routine CABG protocol, and one where NAC was administered intravenously during the operation (NAC group). Blood samples from coronary sinus for tumor necrosis factor-alpha assay, myocardial biopsy specimens for chemiluminescent luminol, and lucigenin measurements of reactive oxygen species were taken. The luminol (specific for (*)OH, H(2)O(2), and HOCl(-) radicals) and lucigenin (specific for O(2) (*-)) levels and the difference ratios after reperfusion were significantly lower in the NAC group. Tumor necrosis factor-alpha levels increased in the control group but, in contrast, a significant decrease was detected in the NAC group (P < 0.01). Creatine kinase-MB levels at 6 and 12 hours were significantly lower in the NAC group (P = 0.02). N-Acetylcysteine has potential effects to limit ischemia reperfusion injury during CABG operations. We believe that its effects on clinical outcome may be more apparent in patients prone to ischemia-reperfusion injury.

Oral therapy with dipyridamole limits ischemia-reperfusion injury in humans

Adenosine receptor stimulation induces several effects that could limit ischemia-reperfusion injury. We hypothesize that treatment with the nucleoside uptake inhibitor dipyridamole increases endogenous adenosine and limits ischemia-reperfusion injury in humans. Ischemia-reperfusion injury was studied in forearm skeletal muscle by technetium Tc 99m-labeled annexin A5 scintigraphy. Ischemia-reperfusion injury was induced by unilateral forearm ischemic exercise. Immediately on reperfusion, annexin A5 labeled with technetium Tc 99m was administered intravenously, and ischemia-reperfusion injury was expressed as the percentage difference in radioactivity between the experimental arm and the control arm 1 and 4 hours after reperfusion. Targeting was quantified in the region of the thenar muscle and forearm flexor muscles. This approach was used in 9 healthy male volunteers after a 1-week treatment with dipyridamole (200 mg, slow release, twice daily) and in 23 control subjects. Dipyridamole treatment significantly reduced annexin A5 targeting in skeletal muscle compared with the control group (thenar region, 13% +/- 7% versus 22% +/- 15% at 1 hour after reperfusion and 9% +/- 6% versus 27% +/- 13% at 4 hours for dipyridamole and control groups, respectively [P = .01]; flexor region, 4% +/- 8% versus 7% +/- 6% at 1 hour after reperfusion and 1% +/- 4% versus 10% +/- 9% at 4 hours for dipyridamole and control groups, respectively [P = .01]). One week of oral treatment with the nucleoside uptake inhibitor dipyridamole (200 mg, slow release, twice daily) significantly limits ischemia-reperfusion injury in humans in vivo, as assessed by technetium Tc 99m-labeled annexin A5 scintigraphy of forearm skeletal muscle.

Small interfering RNA targeting Fas protects mice against renal ischemia-reperfusion injury

Fas-mediated apoptosis has been suggested to contribute to tubular cell death after renal ischemia-reperfusion injury. Here we investigate whether small interfering RNA (siRNA) duplexes targeting Fas protect mice from acute renal failure after clamping of the renal artery. Renal ischemia-reperfusion injury was induced by clamping the renal vein and artery for 15 or 35 min. Mice were treated before or after ischemia with siRNA targeting Fas or a control gene, administered by hydrodynamic injection, low-volume renal vein injection, or both. Treated mice were evaluated for renal Fas protein and mRNA expression, tissue histopathology, and apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining. Blood urea nitrogen and survival were monitored in mice in which the contralateral kidney had been removed. A single hydrodynamic injection of Fas siRNA reduced Fas mRNA and protein expression in the kidney 4-fold. Kidneys from mice that received Fas siRNA two days earlier had substantially less renal tubular apoptosis by TUNEL staining and less tubular atrophy and hyaline damage. Whereas 90% of mice pretreated with saline or GFP siRNA died, only 20% of Fas-siRNA-pretreated animals died. The same survival advantage was provided by a single low-volume Fas siRNA injection into the renal vein. Moreover, postischemic injection through the renal vein protected 38% of mice from death. This study confirms the importance of Fas-mediated apoptosis in renal ischemia-reperfusion injury. Silencing Fas by systemic or local catheterization holds therapeutic promise to limit ischemia-reperfusion injury.

Acute effects of 17beta-estradiol on myocardial pH, Na+, and Ca2+ and ischemia-reperfusion injury.

Evidence suggests that 1) ischemia-reperfusion injury is due largely to cytosolic Ca(2+) accumulation resulting from functional coupling of Na(+)/Ca(2+) exchange (NCE) with stimulated Na(+)/H(+) exchange (NHE1) and 2) 17beta-estradiol (E2) stimulates release of NO, which inhibits NHE1. Thus we tested the hypothesis that acute E2 limits myocardial Na(+) and therefore Ca(2+) accumulation, thereby limiting ischemia-reperfusion injury. NMR was used to measure cytosolic pH (pH(i)), Na(+) (Na(i)(+)), and calcium concentration ([Ca(2+)](i)) in Krebs-Henseleit (KH)-perfused hearts from ovariectomized rats (OVX). Left ventricular developed pressure (LVDP) and lactate dehydrogenase (LDH) release were also measured. Control ischemia-reperfusion was 20 min of baseline perfusion, 40 min of global ischemia, and 40 min of reperfusion. The E2 protocol was identical, except that 1 nM E2 was included in the perfusate before ischemia and during reperfusion. E2 significantly limited the changes in pH(i), Na(i)(+), and [Ca(2+)](i) during ischemia (P < 0.05). In control OVX vs. OVX+E2, pH(i) fell from 6.93 +/- 0.03 to 5.98 +/- 0.04 vs. 6.96 +/- 0.04 to 6.68 +/- 0.07; Na(i)(+) rose from 25 +/- 6 to 109 +/- 14 meq/kg dry wt vs. 25 +/- 1 to 76 +/- 3; [Ca(2+)](i) changed from 365 +/- 69 to 1,248 +/- 180 nM vs. 293 +/- 66 to 202 +/- 64 nM. E2 also improved recovery of LVDP and diminished release of LDH during reperfusion. Effects of E2 were diminished by 1 microM N(omega)-nitro-L-arginine methyl ester. Thus the data are consistent with the hypothesis. However, E2 limitation of increases in [Ca(2+)](i) is greater than can be accounted for by the thermodynamic effect of reduced Na(i)(+) accumulation on NCE.

Suppression of graft coronary artery disease by a brief treatment with a selective epsilonPKC activator and a deltaPKC inhibitor in murine cardiac allografts.

Inhibiting delta protein kinase C (deltaPKC) during reperfusion and activating epsilon PKC (epsilonPKC) before ischemia each limits cardiac ischemic injury. Here, we examined whether limiting ischemia-reperfusion injury inhibits graft coronary artery disease (GCAD) and improves murine cardiac allografting. Hearts of FVB mice (H-2q) were transplanted into C57BL/6 mice (H-2b). epsilonPKC activator (psiepsilonRACK) was injected intraperitoneally (20 nmol) into donor mice 20 minutes before procurement. Hearts were then perfused with psiepsilonRACK (1.5 nmol) through the inferior vena cava (IVC) and subsequently submerged in psiepsilonRACK (0.5 micromol/L) for 20 minutes at 4 degrees C. Before reperfusion, the peritoneal cavity of recipients was irrigated with deltaPKC inhibitor (deltaV1-1, 300 nmol); control animals were treated with normal saline. The total ischemic time to the organ was 50 minutes. Two hours after transplantation, production of inflammatory cytokines and adhesion molecules, cardiomyocyte apoptosis, and caspase-3 and caspase-9 (but not caspase-8) activities were significantly reduced in the PKC regulator-treated group. Fas ligand levels (but not Fas) were also significantly reduced in this group. Importantly, GCAD indices, production of inflammatory cytokines, and adhesion molecules were significantly decreased and cardiac allograft function was significantly better as measured up to 30 days after transplantation. An epsilonPKC activator and a deltaPKC inhibitor together reduced GCAD. Clinically, these PKC isozyme regulators may be useful for organ preservation and prevention of ischemia-reperfusion injury and graft coronary artery disease in cardiac transplantation.

NHE-1 and NBC during pseudo-ischemia/reperfusion in rabbit ventricular myocytes

Despite many studies into the pathophysiology of cardiac ischemia–reperfusion injury, a number of key details are as yet undisclosed. These include the timing and magnitude of the changes in both Na +/H + exchange (NHE-1) and Na +- HCO 3 --cotransport (NBC) transport rates. We fluorimetrically measured H i + fluxes ( J NHE-1 and J NBC) and Na i + fluxes in single contracting rabbit ventricular myocytes subjected to metabolic inhibition, pseudo-ischemia (i.e. metabolic inhibition and extracellular acidosis of 6.4), and pseudo-reperfusion. Metabolic inhibition and pseudo-ischemia inhibited NHE-1 by 43 ± 3.1% and 91 ± 3.6%, and NBC by 66 ± 5.4% and 100%, respectively. Inhibition was due to both an acidic shift of the pH i at which NHE-1 and NBC become quiescent (set-point pH i) and a reduction of the steepness of the pH i - H i + flux profiles. NHE-1 and NBC did not contribute to Na i + loading during metabolic inhibition ( Na i + 18 ± 1.7 mM) or pseudo-ischemia ( Na i + 21 ± 1.7 mM), because pH i acidified less than set-point pH i's. Upon pseudo-reperfusion NBC recovered to 54 ± 7.3% but NHE-1 to 193 ± 11% of aerobic control flux, and set-point pH i's returned to near neutral values. Metabolic inhibition and reperfusion caused an acid load of 18 ± 3.2 mM H + 94% of which were extruded by the hyperactive NHE-1. At pseudo-reperfusion Na i + rose sharply to 31 ± 5.8 mM within 1.5 min and that coincided with hypercontracture. Cariporide not only prevented the Na i + transient, but also inhibited pH i recovery and prevented hypercontracture. Our results are consistent with the view that NHE-1 is active during metabolic inhibition if, like in whole hearts, pH i is driven more acidic than NHE-1 set-point pH i. Furthermore, either an acidic pH i or absence of additional Na i + loading during reperfusion, or both, limit ischemia–reperfusion injury.

Antiatherothrombotic effects of nicotinic acid

Cardiovascular event reduction in hypercholesterolemic subjects appropriately emphasizes the prominent role of statin therapy; however, niacin (nicotinic acid) is also an effective lipid-altering agent that prevents atherosclerosis and reduces cardiovascular events. Niacin has multifarious lipoprotein and anti-atherothrombosis effects that improve endothelial function, reduce inflammation, increase plaque stability, and diminish thrombosis. Niacin reduces the atherogenicity of low-density lipoprotein (LDL) by changing the distribution of small LDL to large LDL subclass, and the susceptibility of LDL to oxidative modification. It is the most effective agent for increasing high-density lipoprotein cholesterol. Moreover, it favorably alters high-density lipoprotein composition, increasing apolipoprotein AI relative to apolipoprotein AII. Niacin reduces blood viscosity through a variety of mechanisms, thus improving blood flow and perfusion through stenotic segments of the vasculature. Finally, niacin has cardioprotective effects that may limit ischemia–reperfusion injury. By preserving glycolysis during periods of ischemia and improving subendocardial blood flow during reperfusion, niacin can improve the functional recovery of the myocardium.

Manganese Superoxide Dismutase

Recent evidence has suggested that ischemia-reperfusion injury is fundamental to the pathogenesis of acute pancreatitis. This study was designed to determine whether acute pancreatitis is associated with elevated serum manganese superoxide dismutase (MnSOD), a key antioxidant enzyme, considered a marker of ischemia-reperfusion injury in myocardial infarction. Thirty-four patients with acute pancreatitis had measurements of MnSOD on days 0, 2, and 5 after recruitment. The patients were recruited within 12 h of admission to hospital and had measurements of MnSOD on days 0, 2, and 5. Patients with severe acute pancreatitis had significantly elevated serum MnSOD concentrations on days 2 and 5 compared with patients with mild acute pancreatitis, but not on the day of recruitment. Elevated serum MnSOD correlated with peripheral plasma markers of lipid peroxidation (malondialdehyde) and neutrophil activation (myeloperoxidase) and was associated with decreased plasma ascorbic acid concentrations. The serial measurement of serum MnSOD may prove useful as a marker of the effectiveness of treatment designed to limit ischemia-reperfusion injury in patients with severe acute pancreatitis.

Ischemia and reperfusion injury: prevention of pulmonary hypertension and leukosequestration following lower limb ischemia

Ischemia is a common clinical event with potentially serious consequences. The major part of tissue damage occurs upon reperfusion and is mediated by activated neutrophils. Ischemia reperfusion injury is manifested by oedema and increased microvascular permeability. This study tested cardiopulmonary functions following 2 h of lower limb ischemia. Anesthetized dogs were randomized into three groups: nonischemic sham dogs ( n = 4), ischemic control dogs ( n = 8) and ischemic dogs pretreated with prostaglandin (PG)E 1 ( n = 8). In control animals, mean pulmonary artery pressure (mPAP) increased 1 min after declamping from 13.37 ± 2.61 mmHg to 16.88 ± 3.68 mm Hg ( P < 0.05). Pulmonary microvascular pressure (Pmv) increased within 1 minute of reperfusion from 7.71 ± 1.87 mm Hg to 10.54 ± 3.71 mm Hg ( P < 0.05). These changes are consistent with increased lung microvascular permeability. White blood cell count fell slightly but not significantly and lung histology showed leukosequestration in alveoli of 171 ± 22 polymorphonuclear leukocyte (PMN) 10 high powered fields (HPF) in the ischemic control group compared with 121 ± 56 PMN 10 HPF in the sham group ( P < 0.05). Systemic arterial pressure, cardiac output, central venous pressure and pulmonary artery wedge pressure were unaffected. In animals pretreated with PGE 1, mPAP and Pmv were unchanged during reperfusion. Lung histologic findings appeared normal and leukosequestration was not observed. PMN counts in alveoli showed 95 ± 26 PMN 10 HPF , lower than in ischemic control animals ( P < 0.05). These data indicate that lower limb ischemia during reperfusion leads to pulmonary hypertension and leukosequestration. PGE 1 infusion is effective in limiting ischemia reperfusion injury.

Inhibition of white blood cell adhesion at reperfusion decreases tissue damage in postischemic striated muscle.

To determine the impact of white blood cell (WBC)-endothelium adhesion on tissue damage in the setting of ischemia-reperfusion injury in striated muscle. The cremaster muscle of four groups of anesthetized Sprague-Dawley rats was subjected to 4 hours of global, warm (37 degrees C) ischemia and 2 hours of reperfusion. At reperfusion two groups of animals received intravenous injections of monoclonal antibodies directed against either CD11b/CD18 (1B6) or ICAM-1 (1A29). The remaining two groups of animals received saline injections (NoRx) or nonreactive IgG1. In vivo light microscopic techniques were used to determine WBC adherence (number of WBCs per 100 microns postcapillary venules) at different intervals of reperfusion. Muscle viability was assessed with computer-assisted image analysis by measuring the optical intensity of transilluminated muscles after incubation with nitroblue tetrazolium. Our results (mean +/- SEM) demonstrate a significant increase in the number of adherent WBCs relative to baseline (8.0 +/- 0.5) after 4 hours of global ischemia in animals receiving NoRx or IgG1. The significant increase occurred at 30 minutes of reperfusion (17.6 +/- 0.6 and 17.4 +/- 0.4 for NoRx or IgG1, respectively) and was sustained for the duration of the experiment. This increase in adherence was attenuated by 1B6 and 1A29 (12.2 +/- 2.2 and 12.4 +/- 0.8, respectively; p < 0.05 compared with NoRx and IgG1). The decrease in WBC adhesion was associated with a decrease in reperfusion injury to the muscle, as indicated by lower optical intensity values for the 1B6 and 1A29 groups (123 +/- 3 and 129 +/- 2) compared with the NoRx and IgG1 groups (151 +/- 2 and 158 +/- 4). Our data support an important role for WBCs in the pathogenesis of ischemia-reperfusion injury. Interfering with the WBC-endothelium interactions by using monoclonal antibodies directed against WBCs and endothelial cell adhesion molecules may help to limit ischemia-reperfusion injury.

Adenosine enhances IL-10 secretion by human monocytes.

Adenosine is a potent endogenous antiinflammatory agent released by cells under metabolically unfavorable conditions. Its effects on the production of IL-10 by human monocytes were presently investigated. Pre-incubation with adenosine dose-dependently enhanced IL-10 release by TNF stimulated human monocytes (+29, +58, and +116% at 1, 10, and 100 muM, respectively.) Adenosine also significantly enhanced IL-10 production after hydrogen peroxide and LPS stimulation and dose-dependently inhibited TNF secretion. Pre-incubation was not mandatory to achieve these effects, since addition of adenosine at the time of or 30 min after the stimulus led to the same results. Blocking IL-10 with anti-IL-10 mAbs partially restored adenosine-induced TNF inhibition. The enhanced IL-10 production was not observed when cells were preincubated with adenosine A1 or A2 receptor agonists (R-phenylisopropyladenosine, 5'-N-ethylcarboxamido-adenosine, and 2-chloroadenosine) and was not affected by pretreatment with theophyllin, an antagonist of both A1 and A2 receptors, or with dipyridamole, an inhibitor of adenosine cellular uptake. In conclusion, adenosine, in the submillimolar concentration range, increases IL-10 secretion by stimulated monocytes. This phenomenon participates in TNF inhibition, a known property of adenosine, but is not mediated through the occupancy of A1 or A2 receptors. This may represent a novel antiinflammatory property of adenosine by which it could modulate inflammation and limit ischemia-reperfusion injury.