Postconditioning protects against endothelial ischemia-reperfusion injury in the human forearm

Background: Hypoxic cell death follows interruption of blood supply to tissues. Although successful restoration of blood flow is mandatory for salvage of ischemic tissues, reperfusion can paradoxically place tissues at risk of further injury. Brief periods of ischemia applied at the onset of reperfusion have been shown to reduce ischemia-reperfusion (IR) injury, a phenomenon called postconditioning. The aim of this study was to determine whether postconditioning protects against endothelial IR injury in humans, in vivo. Methods and Results: Brachial artery endothelial function was assessed by vascular ultrasound to measure flow-mediated dilation (FMD) in response to forearm reactive hyperemia. FMD was measured before and after IR (20 minutes of arm ischemia followed by 20 minutes of reperfusion) in healthy volunteers. To test the protective effects of postconditioning, 3 cycles of reperfusion followed by ischemia (each lasting 10 or 30 seconds) were applied immediately after 20 minutes of arm ischemia. To determine whether postconditioning needs to be applied at the onset of reperfusion, a 1-minute period of arm reperfusion was allowed before the application of the 10-second postconditioning stimulus. IR caused endothelial dysfunction (FMD 9.1±1.2% pre-IR, 3.6±0.7% post-IR, P<0.001; n=11), which was prevented by postconditioning applied as 10-second cycles of reperfusion/ischemia (FMD 9.9±1.7% pre-IR, 8.3±1.4% post-IR, P=NS; n=11) and 30-second cycles of reperfusion/ischemia (FMD 10.8±1.7% pre-IR, 9.5±1.5% post-IR, P=NS; n=10) immediately at the onset of reperfusion. No protection was observed when the application of the 10-second postconditioning stimulus was delayed for 1 minute after the onset of reperfusion (FMD 9.8±1.2% pre-IR, 4.0±0.9% post-IR, P<0.001; n=8). Conclusions: This study demonstrates for the first time that postconditioning can protect against endothelial IR injury in humans. Postconditioning might reduce tissue injury when applied at the onset of reperfusion by modifying the reperfusion phase of IR

Purine nucleoside phosphorylase: A new marker for free oxygen radical injury to the endothelial cell

The effect of ischemia and reperfusion on purine nucleoside phosphorylase was studied in an isolated perfused rat liver model. This enzyme is localized primarily in the cytoplasm of the endothelial and Kupffer cells; some activity is associated with the parenchymal cells. Levels of this enzyme accurately predicted the extent of ischemia and reperfusion damage to the microvascular endothelial cell of the liver. Livers from Lewis rats were subjected to 30, 45 and 60 min of warm (37° C) no flow ischemia that was followed by a standard reperfusion period lasting 45 min. Purine nucleoside phosphorylase was measured at the end of the no flow ischemia and reperfusion periods as was superoxide generation (O2‐). Bile production was monitored throughout the no flow ischemia and reperfusion periods. Control perfusions were carried out for 120 min. A significant rise in purine nucleoside phosphorylase levels as compared with controls was observed at the end of ischemia in all the three groups. The highest level, 203.5 ± 29.2 mU/ml, was observed after 60 min of ischemia. After the reperfusion period, levels of purine nucleoside phosphorylase decreased in the 30‐ and 45‐min groups 58.17 ± 9.66 mU/ml and 67.5 ± 17.1 mU/ml, respectively. These levels were equal to control perfusions. In contrast, after 60 min of ischemia, levels of purine nucleoside phosphorylase decreased early in the reperfusion period and then rose to 127.8 ± 14.8 mU/ml by the end of reperfusion (p < 0.0001). Superoxide generation at the beginning of reperfusion was higher than in controls with similar values observed at the end of 30, 45 and 60 min of ischemia. During reperfusion, production of superoxide continued. Bile production was significantly lower at the end of 30 min (0.044 ± 0.026 μl/min/gm), 45 min (0.029 ± 0.0022 μ/min/gm) and 60 min of ischemia (0.022 ± 0.008 μ/min/gm) when compared with bile production by control livers during the corresponding time (0.680 ± 0.195, 0.562 ± 0.133 and 0.480 ± 0.100 μ/min/gm respectively; p < 0.001). During reperfusion, rates of bile production were normal after 30 and 45 min of ischemia. In contrast, significantly lower rates of bile production, 0.046 ± 0.36 μ/min/gm (p < 0.001) occurred during reperfusion after 60 min of ischemia. Control livers during the same period produced 0.330 ± 0.056 μl/min/gm of bile. The results indicate that purine nucleoside phosphorylase levels may be a good index of oxidative injury to the liver in ischemia reperfusion and reliably predict the functional state of the organ after reperfusion. Copyright © 1990 American Association for the Study of Liver Disease

Application of a novel method for subsequent evaluation of sinusoids and postsinusoidal venules after ischemia-reperfusion injury of rat liver

Although several intravital fluorescence microscopic studies demonstrated that microcirculatory derangement is induced during liver ischemia-reperfusion, these data were obtained from randomly selected microvascular areas and microvessels, Repeated observation of the identical microvessels has not been performed yet. Using a specially designed cover glass, it is now possible to relocate desired sites of observation repeatedly over the whole reperfusion time, The aim of this study was to determine the impact of reperfusion time on hepatic microvascular perfusion state. Twenty minutes of ischemia induced a significant decrease in sinusoidal perfusion rate (29.1 +/- 10.2%) as compared with baseline values (98.0 +/- 0.3%). At 30, 60, and 120 min of reperfusion, the percentage of perfused sinusoids recovered to 62.8 +/- 6.6, 67.5 +/- 5.7, and 77.2 +/- 5.4%. The number of stagnant leukocytes in the same sinusoids was 6.2 +/- 1.9/lobule at baseline and increased to 22.3 +/- 3.6/lobule at 120 min of reperfusion. The number of leukocytes adhering within postsinusoidal venules was 53.5 +/- 12.5/mm(2) before ischemia and increased to 414.2 +/- 62.5/mm(2) at 120 min of reperfusion. We have demonstrated that during 120 min of reperfusion, there was a steady increase in both sinusoidal and venular leukocyte adhesion along with an attenuation of the initially severely depressed sinusoidal perfusion. a no-reflow phenomenon at an early phase of reperfusion and subsequent reflow were proven

Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling

The "metabolic cocktail" comprising glucose-insulin-potassium administrated at reperfusion reduces infarct size in the in vivo rat heart. We propose that insulin is the major component mediating this protection and acts via Akt prosurvival signaling. This hypothesis was studied in isolated perfused rat hearts (measuring infarct size to area of risk [%]) subjected to 35 minutes regional myocardial ischemia and 2 hours reperfusion. Insulin administered at the onset of reperfusion attenuated infarct size by 45% versus control hearts (P<0.001). Insulin-mediated cardioprotection was found to be independent of the presence of glucose at reperfusion. Moreover, the cell survival benefit of insulin is temporally dependent, in that insulin administration from the onset of reperfusion and maintained for either 15 minutes or for the duration of reperfusion reduced infarct size. In contrast, protection was abrogated if insulin administration was delayed until 15 minutes into reperfusion. Pharmacological inhibition of both upstream and downstream signals in the Akt prosurvival pathway abolished the cardioprotective effects of insulin. Here coadministration of insulin with the tyrosine kinase inhibitor lavendustin A, the phosphatidylinositol3-kinase (PI3-kinase) inhibitor wortmannin, and mTOR/p70s6 kinase inhibitor rapamycin abolished cardioprotection. Steady-state levels of activated/phosphorylated Akt correlated with insulin administration. Finally, downstream prosurvival targets of Akt including p70s6 kinase and BAD were modulated by insulin. In conclusion, insulin administration at reperfusion reduces myocardial infarction, is dependent on early administration during reperfusion, and is mediated via Akt and p70s6 kinase dependent signaling pathway. Moreover, BAD is maintained in its inert phosphorylated state in response to insulin therapy

Reversible Blockade of Complex I or Inhibition of PKCβ Reduces Activation and Mitochondria Translocation of p66\u3csup\u3eShc\u3c/sup\u3e to Preserve Cardiac Function after Ischemia

Aim Excess mitochondrial reactive oxygen species (mROS) play a vital role in cardiac ischemia reperfusion (IR) injury. P66Shc, a splice variant of the ShcA adaptor protein family, enhances mROS production by oxidizing reduced cytochrome c to yield H2O2. Ablation of p66Shc protects against IR injury, but it is unknown if and when p66Shc is activated during cardiac ischemia and/or reperfusion and if attenuating complex I electron transfer or deactivating PKCβ alters p66Shc activation during IR is associated with cardioprotection. Methods Isolated guinea pig hearts were perfused and subjected to increasing periods of ischemia and reperfusion with or without amobarbital, a complex I blocker, or hispidin, a PKCβ inhibitor. Phosphorylation of p66Shc at serine 36 and levels of p66Shc in mitochondria and cytosol were measured. Cardiac functional variables and redox states were monitored online before, during and after ischemia. Infarct size was assessed in some hearts after 120 min reperfusion. Results Phosphorylation of p66Shc and its translocation into mitochondria increased during reperfusion after 20 and 30 min ischemia, but not during ischemia only, or during 5 or 10 min ischemia followed by 20 min reperfusion. Correspondingly, cytosolic p66Shc levels decreased during these ischemia and reperfusion periods. Amobarbital or hispidin reduced phosphorylation of p66Shc and its mitochondrial translocation induced by 30 min ischemia and 20 min reperfusion. Decreased phosphorylation of p66Shc by amobarbital or hispidin led to better functional recovery and less infarction during reperfusion. Conclusion Our results show that IR activates p66Shc and that reversible blockade of electron transfer from complex I, or inhibition of PKCβ activation, decreases p66Shc activation and translocation and reduces IR damage. These observations support a novel potential therapeutic intervention against cardiac IR injury

Tumor Necrosis Factor-α Contributes to Ischemia- and Reperfusion-Induced Endothelial Activation in Isolated Hearts

During myocardial reperfusion, polymorphonuclear neutrophil (PMN) adhesion involving the intercellular adhesion molecule-1 (ICAM-1) may lead to aggravation and prolongation of reperfusion injury. We studied the role of early tumor necrosis factor-α (TNF-α) cleavage and nuclear factor-κB (NF-κB) activation on ICAM-1 expression and venular adhesion of PMN in isolated hearts after ischemia (15 minutes) and reperfusion (30 to 480 minutes). NF-κB activation (electromobility shift assay) was found after 30 minutes of reperfusion and up to 240 minutes. ICAM-1 mRNA, assessed by Northern blot, increased during the same interval. Functional effect of newly synthesized adhesion molecules was found by quantification (in situ fluorescence microscopy) of PMN, given as bolus after ischemia, which became adherent to small coronary venules (10 to 50 mm in diameter). After 480 minutes of reperfusion, ICAM-1–dependent PMN adhesion increased 2.5-fold compared with PMN adhesion obtained during acute reperfusion. To study the influence of NF-κB on PMN adhesion, we inhibited NF-κB activation by transfection of NF-κB decoy oligonucleotides into isolated hearts using HJV-liposomes. Decoy NF-κB but not control oligonucleotides blocked ICAM-1 upregulation and inhibited the subacute increase in PMN adhesion. Similar effects were obtained using BB 1101 (10 μg), an inhibitor of TNF-α cleavage enzyme. These data suggest that ischemia and reperfusion in isolated hearts cause liberation of TNF-α, activation of NF-κB, and upregulation of ICAM-1, an adhesion molecule involved in inflammatory response after ischemia and reperfusion

Postconditioning: a form of "modified reperfusion" protects the myocardium by activating the phosphatidylinositol 3-kinase-akt pathway

Brief intermittent episodes of ischemia and reperfusion, at the onset of reperfusion after a prolonged period of ischemia, confer cardioprotection, a phenomenon termed "ischemic postconditioning" (Postcond). We hypothesized that this phenomenon may just represent a modified form of reperfusion that activates the reperfusion injury salvage kinase (RISK) pathway. Isolated perfused rat hearts were subjected to: (a) 35 minutes of ischemia and 120 minutes of reperfusion, and infarct size was determined by tetrazolium staining; or (b) 35 minutes of ischemia and 7 minutes of reperfusion, and the phosphorylation states of Akt, endothelial NO synthase (eNOS), and p70S6K were determined. Postcond reduced infarct size from 51.2±3.4% to 31.5±4.1% (P<0.01), an effect comparable with ischemic preconditioning (IPC; 27.5±2.3%; P<0.01). Of interest, the combined protective effects of IPC and Postcond were not additive (30.1±4.8% with IPC+Postcond; P=NS). Inhibiting phosphatidylinositol 3-kinase (PI3K) at reperfusion using LY or Wortmannin (Wort) during the first 15 minutes of reperfusion completely abolished Postcond-induced protection (31.5±4.1% with Postcond versus 51.7±4.5% with Postcond+LY, P<0.01; 56.2±10.1% with Postcond+ Wort; P<0.01), suggesting that Postcond protects the heart by activating PI3K-Akt. Western blot analysis demonstrated that Postcond induced a significant increase in phosphorylation of Akt, eNOS, and p70S6K in an LY- and Wort-sensitive manner. In conclusion, we show for the first time that ischemic Postcond protects the myocardium by activating the prosurvival kinases PI3K-Akt, eNOS, and p70S6K in accordance with the RISK pathway

Kinin-B1 receptors in ischaemia-induced pancreatitis: Functional importance and cellular localisation

In this study we compare the role of kininB1 and B2 receptors during ischaemia/reperfusion of rat pancreas. Our investigations were prompted by the observation that infusion of a kininB2 receptor antagonist produced significant improvement in acute experimental pancreatitis. In an acute model with two hours of ischaemia/two hours of reperfusion, application of the kininB1 receptor antagonist (CP-0298) alone, or in combination with kininB2 receptor antagonist (CP-0597), significantly reduced the number of adherent leukocytes in postcapillary venules. In a chronic model with five days of reperfusion, the continuous application of kininB1 receptor antagonist or a combination of kininB1 and B2 receptor antagonists markedly reduced the survival rate. In kininreceptor binding studies kininB1 receptor showed a 22-fold increase in expression during the time of ischaemia/ reperfusion. Carboxypeptidase M activity was upregulated 10-fold following two hours of ischaemia and two hours of reperfusion, provided the appropriate specific ligand, desArg10-kallidin and/or desArg9-bradykinin, was used. The occurrence of kininB1 receptor binding sites on acinar cell membranes was demonstrated by microautoradiography. With a specific antibody, the localisation of kininB1 receptor protein was confirmed at the same sites. In conclusion, we have demonstrated the upregulation of the pancreatic acinar cell kininB1 receptors during ischaemia/reperfusion. The novel functional finding was that antagonism of the kininB1 receptors decreased the survival rate in an experimental model of pancreatitis

Stroke penumbra defined by an MRI-based oxygen challenge technique: 2. Validation based on the consequences of reperfusion

Magnetic resonance imaging (MRI) with oxygen challenge (T2* OC) uses oxygen as a metabolic biotracer to define penumbral tissue based on CMRO2 and oxygen extraction fraction. Penumbra displays a greater T2* signal change during OC than surrounding tissue. Since timely restoration of cerebral blood flow (CBF) should salvage penumbra, T2* OC was tested by examining the consequences of reperfusion on T2* OC-defined penumbra. Transient ischemia (109±20 minutes) was induced in male Sprague-Dawley rats (n=8). Penumbra was identified on T2*-weighted MRI during OC. Ischemia and ischemic injury were identified on CBF and apparent diffusion coefficient maps, respectively. Reperfusion was induced and scans repeated. T2 for final infarct and T2* OC were run on day 7. T2* signal increase to OC was 3.4% in contralateral cortex and caudate nucleus and was unaffected by reperfusion. In OC-defined penumbra, T2* signal increased by 8.4%±4.1% during ischemia and returned to 3.25%±0.8% following reperfusion. Ischemic core T2* signal increase was 0.39%±0.47% during ischemia and 0.84%±1.8% on reperfusion. Penumbral CBF increased from 41.94±13 to 116.5±25 mL per 100 g per minute on reperfusion. On day 7, OC-defined penumbra gave a normal OC response and was located outside the infarct. T2* OC-defined penumbra recovered when CBF was restored, providing further validation of the utility of T2* OC for acute stroke management

The protective effect of M40401, a superoxide dismutase mimetic, on post-ischemic brain damage in Mongolian gerbils

BACKGROUND: Overproduction of free radical species has been shown to occur in brain tissues after ischemia-reperfusion injury. However, most of free radical scavengers known to antagonize oxidative damage (e.g. superoxide dismutase, catalase), are unable to protect against ischemia-reperfusion brain injury when given in vivo, an effect mainly due to their difficulty to gain access to brain tissues. Here we studied the effect of a low molecular weight superoxide dismutase mimetic (M40401) in brain damage subsequent to ischemia-reperfusion injury in Mongolian gerbils. RESULTS: In animals undergoing ischemia-reperfusion injury, neuropathological and ultrastructural changes were monitored for 1–7 days either in the presence or in the absence of M40401 after bilateral common carotid artery occlusion (BCCO). Administration of M40401 (1–40 mg/kg, given i.p. 1 h after BCCO) protected against post-ischemic, ultrastructural and neuropathological changes occurring within the hippocampal CA1 area. The protective effect of M40401 was associated with a significant reduction of the levels of malondialdehyde (MDA; a marker of lipid peroxidation) in ischemic brain tissues after ischemia-reperfusion. CONCLUSION: Taken together, these results demonstrate that M40401 provides protective effects when given early after the induction of ischemia-reperfusion of brain tissues and suggest the possible use of such compounds in the treatment of neurological dysfunction subsequent to cerebral flow disturbances