Early Phase Of Ischemia Research Articles

Evaluation of coronary microvascular dysfunction using magnetocardiography: A new application to an old technology

BackgroundIn patients with angina and non-obstructive coronary artery disease (ANOCA), diagnosis of coronary microvascular dysfunction (CMD) remains an unmet need. Magnetocardiography (MCG), is a rest-based, non-invasive scan that can detect weak electrophysiological changes that occur at the early phase of ischemia. ObjectiveThis study assessed the ability of MCG to detect CMD in ANOCA patients as compared to reference standard, invasive coronary flow reserve (CFR). MethodsPatients with ANOCA and invasive coronary physiologic assessment using intracoronary flow measurements with Doppler and thermodilution methods were enrolled. CMD was defined dichotomously as an invasive CFR < 2.0 by Doppler or thermodilution assessment. Noninvasive 36-channel 90-s MCG scan was performed and quantitative assessment of four distinct MCG features was completed. We evaluated the diagnostic performance of 2 or more abnormal MCG features to detect CMD in the overall cohort and performed a subgroup analysis in the subset of patients with Doppler CFR assessment. ResultsAmong 79 ANOCA patients, 25 were CMD positive and 54 patients were CMD negative by CFR. Using invasive CFR as reference, MCG had an ROC AUC of 0.66 with a sensitivity of 68 % and specificity of 65 % for the detection of CMD. In the subgroup with Doppler CFR assessment, MCG had an ROC AUC of 0.76 with a sensitivity of 75 % and specificity of 77 %. ConclusionsIn ANOCA patients, MCG demonstrates the ability to detect CMD using a 90-second non-invasive scan without the need for an intravenous stressor or ionizing radiation. Further investigations are needed to validate an MCG-based diagnostic pathway for CMD.

Prolonged repolarization in the early phase of ischemia is associated with ventricular fibrillation development in a porcine model.

Background: Repolarization prolongation can be the earliest electrophysiological change in ischemia, but its role in arrhythmogenesis is unclear. The aim of the present study was to evaluate the early ischemic action potential duration (APD) prolongation concerning its causes, expression in ECG and association with early ischemic ventricular fibrillation (phase 1A VF). Methods: Coronary occlusion was induced in 18 anesthetized pigs, and standard 12 lead ECG along with epicardial electrograms were recorded. Local activation time (AT), end of repolarization time (RT), and activation-repolarization interval (ARIc) were determined as dV/dt minimum during QRS-complex, dV/dt maximum during T-wave, and rate-corrected RT-AT differences, respectively. Patch-clamp studies were done in enzymatically isolated porcine cardiomyocytes. IK(ATP) activation and Ito1 inhibition were tested as possible causes of the APD change. Results: During the initial period of ischemia, a total of 11 pigs demonstrated maximal ARIc prolongation >10ms at 1 and/or 2.5min of occlusion (8 and 6 cases at 1 and 2.5min, respectively) followed by typical ischemic ARIc shortening. The maximal ARIc across all leads was associated with VF development (OR 1.024 95% CI 1.003-1.046, p = 0.025) and maximal rate-corrected QT interval (QTc) (B 0.562 95% CI 0.346-0.775, p < 0.001) in logistic and linear regression analyses, respectively. Phase 1A VF incidence was associated with maximal QTc at the 2.5min of occlusion in ROC curve analysis (AUC 0.867, p = 0.028) with optimal cut-off 456ms (sensitivity 1.00, specificity 0.778). The pigs having maximal QTc at 2.5min more and less than 450ms significantly differed in phase 1A VF incidence in Kaplan-Meier analysis (log-rank p = 0.007). In the patch-clamp experiments, 4-aminopyridine did not produce any effects on the APD; however, pinacidil activated IK(ATP) and caused a biphasic change in the APD with initial prolongation and subsequent shortening. Conclusion: The transiently prolonged repolarization during the initial period of acute ischemia was expressed in the prolongation of the maximal QTc interval in the body surface ECG and was associated with phase 1A VF. IK(ATP) activation in the isolated cardiomyocytes reproduced the biphasic repolarization dynamics observed in vivo, which suggests the probable role of IK(ATP) in early ischemic arrhythmogenesis.

Spironolactone and eplerenone are cardioprotective during early phase of ischemia in rats submitted to acute coronary occlusion

Introduction: Mineralocorticoid receptor antagonists (MRAs) are effective in reducing left ventricle remodeling and sudden death after acute myocardial infarction (AMI). Objectives: MRAs in vitro display cardioprotective effects, independent of MR; however, it is unknown whether the rapid effects of MRAs are cardioprotective in vivo. This study evaluated the acute effects of spironolactone and eplerenone in the first minutes of AMI. Methods: Wistar Rats, submitted or not to bilateral adrenalectomy, were treated orally with spironolactone (20 mg/kg) or eplerenone (10 mg/kg), and submitted to the left coronary ligation, under anesthesia. Electrocardiogram (ECG) recordings were obtained to evaluate ST-T segment, QT, and QTc intervals. Arterial pressure was also measured before (baseline) and after coronary ligation. Results: Spironolactone or eplerenone given, one hour before coronary ligation, prevented ST-T segment elevation in adrenalectomized and non-adrenalectomized. QT interval analysis showed that MRAs prevented its prolongation after coronary ligation. QT and QTc intervals remained similar to baseline and were smaller than the values displayed by the non-treated group. Animals treated with spironolactone, regardless of adrenalectomy, showed a 3-fold reduced mortality rates compared to the control group. Conclusion: MRAs display acute cardioprotective effects in early phase of AMI, which are independent of aldosterone.

Systematic evaluation during early-phase ischemia predicts outcomes in middle cerebral artery occlusion mice.

Identifying outcome predictors for ischemic stroke is beneficial for choosing correct intervention protocols. Thus, it is necessary to systemically evaluate histological outcome-associated changes such as hemodynamics, behavior, and body weight during the early phase of ischemia. Here, 50 mice were subjected to 45-min middle cerebral artery occlusion (MCAO) using Longa's method. Hemodynamic changes were monitored by Doppler laser probe, and behaviors were evaluated by scales while the tissues were visualized by staining. The results by correlation analysis demonstrated that with a probe located near the posterior boundary zone of MCA territory, the latency of anoxic depolarization, as well as the cerebral blood flow reduction during MCAO were confirmed to be predictors for the infarct volume on day 3 post-ischemia; histology showed that the risk of a space-occupying secondary hemorrhage was significantly correlated with the increase of infarct volume versus the traditional Bederson's neurological deficit scale, a renewed combined behavioral scoring method performed nicely to reflect the severity of tissue lesions. Weight loss was a valuable metric for the enlargement of both infarct volume and secondary hemorrhage. Monitoring changes during early-phase ischemia may benefit the optimization of ischemia models and the discovery of potential intervention targets.See Video Abstract, http:/links.lww.com/WNR/A601).

Abstract 873: Acetate Protects the Heart Against Ischemic Injury by Alternating TCA Cycle-related Metabolites and AMP-activated Protein Kinase

Introduction: Acetate is preferentially taken up in the heart, and converted into acetyl-CoA by acetyl-CoA synthetase2 (AceCS2), which is abundant in the mitochondria of cardiomyocytes, resulting in activation of AMPK through an elevation of AMP/ATP ratio. In this context, we investigated the effects of acetate on myocardial ischemic injury in isolated cardiomyocytes and in vivo mouse hearts. Methods and Results: In isolated cardiomyocytes, 13 C-labelled metabolites were analyzed using capillary electrophoresis-electrospray ionization mass spectroscopy (CE-ESI-MS) in order to determine the fate of exogenously administered 13 C-acetate. The oxygen consumption rate (OCR) was evaluated using a flux analyzer. A variety of 13 C-labelled intermediates in the TCA cycle increased 10 minutes after administration of 13 C-acetate followed by a subsequent increase in OCR. The OCR elevation was sustained more than 2 hours after acetate injection. The acetate-induced OCR elevation was partially blocked by the glycolysis inhibitor 2-Deoxyglucose (2-DG), carnitine palmitoyl transferase1A (CPT-1A) inhibitor (etomoxir), and mitochondrial pyruvate carrier (MPC) inhibitor (UK5099). The acetate-induced OCR elevation was also blocked by AMP-activated protein kinase (AMPK) inhibitor (compound C). These findings indicated that acetate caused an elevation in mitochondrial function through activation of various metabolic pathways, which may be related to activation of the AMPK pathway. Next, left anterior descending artery-ligated hearts were processed with focused microwave and analyzed by CE-MS and matrix-assisted laser desorption/ionization imaging mass spectrometry in order to examine the region-specific metabolite changes at the early phase of ischemia. Nicotinamide adenine dinucleotide (NADH) elevation in the ischemic core region was suppressed by acetate administration. Furthermore, acetate inhibited cardiac remodeling in a protective manner several weeks after myocardial ischemia when viewed by cardiac ultrasound. Conclusion: Acetate caused an increase in OCR via both a significant elevation in TCA cycle metabolites and activation of AMPK pathway, and may have a protective effect on myocardial ischemic injury.

Let-7a regulates expression of β1-adrenoceptors and forms a negative feedback circuit with the β1-adrenoceptor signaling pathway in chronic ischemic heart failure.

BackgroundThe aim of the present study was to investigate the role of microRNA (miRNA) let-7a in down-regulation of β1-adrenoceptors (β1-AR) and elucidate the underlying mechanism of chronic ischemia heart failure (CIHF) in rats.Methods and ResultsCIHF model was established by occlusion of coronary artery for 4 weeks. β1-AR level was obviously down-regulated and let-7a up-regulated in the failing heart 4 weeks after myocardial infarction. Overexpression of let-7a inhibited β1-AR expression in neonatal rat ventricular cells (NRVCs), which was abolished by anti-let-7a antisense inhibitor. The lentivirus vector containing precursor let-7a (len-pre-let-7a) further down-regulated the reduced β1-AR level by CIHF and the effect was reversed by len-AMO-let-7a. Len-negative control did not produce any significant influence on β1-AR expression. Importantly, there exists a negative feedback loop associated with β1-AR regulation through β1-AR/cAMP/PKA/GATA4/let-7a/β1-AR signaling pathway in CIHF. As demonstrated, GATA4 was activated by β1-AR up-regulation through cAMP-PKA signaling pathway in early phase of ischemia, then GATA4 positively regulated let-7a expression which in turn suppressed β1-AR expression.ConclusionsLet-7a regulates β1-AR expression and forms a negative feedback loop with β1-AR signaling pathway in ischemic heart failure. This study provides a new insight into the differential expression of β1-AR in early and later phase of myocardial ischemia.

Memantine mediates neuroprotection via regulating neurovascular unit in a mouse model of focal cerebral ischemia

AimsMemantine is a low-moderate affinity and uncompetitive N-methyl-d-aspartate receptor (NMDAR) antagonist, which is also a potential neuroprotectant in acute ischemic stroke for its particular action profiles. The present study was to reveal the mechanisms involved in the neuroprotection of memantine. Main methodsWe used a mouse model of permanent focal cerebral ischemia via middle cerebral artery occlusion to verify our hypothesis. 2,3,5-Triphenyltetrazolium chloride staining was used to compare infarct size. The amount of astrocytes and the somal volume of the microglia cell body were analyzed by immunohistochemistry and stereological estimates. Western blotting was used to determine the protein expressions. Key findingsMemantine prevented cerebral ischemia-induced brain infarct and neuronal injury, and reduced oxygen-glucose deprivation-induced cortical neuronal apoptosis. Moreover, memantine reduced the amount of the damaged astrocytes and over activated microglia after 24h of ischemia. In the early phase of ischemia, higher production of MMP-9 was observed, and thereby collagen IV was dramatically disrupted. Meanwhile, the post-synaptic density protein 95(PSD-95) was also severely cleavaged. Memantine decreased MMP-9 secretion, prevented the degradation of collagen IV in mouse brain. PSD-95 cleavage was also inhibited by memantine. SignificanceThese results suggested that memantine exerted neuroprotection effects in acute ischemic brain damage, partially via improving the functions of neurovascular unit. Taking all these findings together, we consider that memantine might be a promising protective agent against ischemic stroke.

Early neuroprotective effect with lack of long-term cell replacement effect on experimental stroke after intra-arterial transplantation of adipose-derived mesenchymal stromal cells

Background aimsAdipose-derived mesenchymal stromal cells (AD-MSCs) have high proliferative capacity and ability to secrete trophic factors. Although intra-arterial (IA) transplantation of stem cells induces efficient engraftment to the host brain, it is unclear whether engrafted cells exert their long-term therapeutic effects through a bystander mechanism or a cell replacement mechanism. MethodsAfter induction of ischemia in rats by middle cerebral artery occlusion, we transplanted human AD-MSCs into their carotid arteries with the use of a micro-needle, and we then investigated the therapeutic effects during the early and late phases of ischemia by means of in vivo magnetic resonance imaging, functional and histological analyses. ResultsDuring the early phase of cerebral ischemia, IA transplantation of AD-MSCs attenuated inflammation and enhanced endogenous neurogenesis. Transplanted animals showed a marked improvement in functional tests during the early phase of cerebral ischemia that was less prominent but still significant during the late phase of cerebral ischemia. Although the transplanted cells effectively migrated to the infarct area, only a small number of engrafted cells survived at 8 weeks after transplantation and differentiated into neuronal, glial and endothelial cells. ConclusionsIA transplantation of human AD-MSCs provides an effective therapeutic modality in a rodent model of stroke, of which the main effects are mediated by a bystander mechanism at the early phase of ischemia.

Protective effects of recombinant human erythropoietin on oligodendrocyte after cerebral infarction

To study biological effect of recombinant human erythropoietin (RhEPO) on the expression of oligodendrocyte in the neuron glia antigen 2(NG2), Nogo receptor-interacting protein 1(LINGO-1), myelin basic protein (MBP) and myelin associated glycoprotein (MAG), and to explore the protective mechanism of RhEPO for oligodendrocyte after cerebral infarction. Experimental rats were randomly divided into the treatment group (RhEPO at a dose of 3 000 U/kg) or saline control group. Both groups received intraperitoneal injection of RhEPO after cerebral ischemia in 30 min, 3 h, 6 h, 12 h and 24 h, which was administered daily for 7 days. The modified neurological severity score (mNSS) and histology were analyzed, and immunohistochemistry was used to detect the protein expression of NG2, MAG, MBP and LINGO-1. The overall mNSS of RhEPO treatment group significantly decreased compared with the saline control group on the seventh day after cerebral infarction (P<0.05). Such treatment effect was more obvious in the treatment group at 30 min and 3 h (P<0.01). Compared with the saline control group, the numbers of NG2 positive cells increased in RhEPO treatment group. In contrast, the expression of LINGO-1 protein significantly decreased (P<0.05), with a dramatic decrease observed at 30 min and 3 h (P<0.01). However, the expression of MBP protein decreased more significantly in saline control group, while the level of the MAG protein expression increased. The differences were statistically significant (P<0.05), especially at 30 min (P<0.01). After cerebral ischemia, RhEPO promotes the proliferation of NG2 positive cells, and inhibits the expression of LINGO-1 and MAG proteins. RhEPO improves the proliferation and differentiation of oligodendrocyte precursor cells, which in turn protects neuronal function, particularly at the early phase of ischemia.

Imaging reveals the focal area of spreading depolarizations and a variety of hemodynamic responses in a rat microembolic stroke model.

Spreading depolarizations (SDs) occur in stroke, but the spatial association between SDs and the corresponding hemodynamic changes is incompletely understood. We applied multimodal imaging to visualize the focal area of selected SDs, and hemodynamic responses with SDs propagating over the ischemic cortex. The intracarotid infusion of polyethylene microspheres (d=45 to 53 μm) produced multifocal ischemia in anesthetized rats (n=7). Synchronous image sequences captured through a cranial window above the frontoparietal cortex revealed: Changes in membrane potential (voltage-sensitive (VS) dye method); cerebral blood flow (CBF; laser speckle contrast (LSC) imaging); and hemoglobin (Hb) deoxygenation (red intrinsic optical signal (IOS) at 620 to 640 nm). A total of 31 SD events were identified. The foci of five SDs were seen in the cranial window, originating where CBF was the lowest (56.9±9%), but without evident signs of infarcts. The hyperemic CBF responses to propagating SDs were coupled with three types of Hb saturation kinetics. More accentuated Hb desaturation was related to a larger decrease in CBF shortly after ischemia induction. Microsphere-induced embolization triggers SDs in the rat brain, relevant for small embolic infarcts in patients. The SD occurrence during the early phase of ischemia is not tightly associated with immediate infarct evolution. Various kinetics of Hb saturation may determine the metabolic consequences of individual SDs.

Transferrin Receptor Mediated Brain Uptake During Ischemia and Reperfusion

Drug delivery by transferrin receptor-mediated transport at the blood-brain barrier has shown beneficial effects in animal models of stroke, but it is unclear whether receptor mediated uptake remains functional in the ischemic tissue. The present study addressed that question in a mouse model of brain focal ischemia, permanent or transient middle cerebral artery occlusion (MCAO). Brain accumulation of 125I-labeled 8D3, a mouse-specific transferrin receptor antibody, or of the isotype control UPC-10 used as vascular marker, was measured autoradiographically by phosphorimaging in the core ischemic region on cryostat brain sections up to 24h after ischemia or reperfusion. Cerebral blood flow was quantitatively determined in the same animals after administration of 99mTc-ECD (Neurolite). Apparent volume of distribution obtained with UPC-10 indicated no significant nonspecific leakage of the blood-brain barrier at any time point. Although brain uptake of 8D3 gradually declined compared to healthy tissue under MCAO, VD remained significantly higher than VD of UPC-10 up to 5h. In transient MCAO the brain uptake recovered to levels as in healthy tissue immediately after reperfusion. Transferrin receptor-mediated brain uptake, which is an energy dependent vesicular transport process, is sensitive to reduction in blood supply but remains partially functional for several hours after onset of ischemia. The uptake shows complete recovery after reperfusion. These results support the use of transferrin receptor-mediated brain drug delivery in the early phase of ischemia and in the phase when blood flow is restored. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Effects of xenon and isoflurane on apoptosis and inflammation in a porcine myocardial infarction model

Volatile anaesthetics can reduce the infarction size in myocardial tissue when administered before and during experimentally induced ischaemia. The aim of this study was to investigate whether xenon is beneficial compared to isoflurane in limiting myocardial tissue apoptosis and inflammation induced by experimental ischaemia-reperfusion injury in a porcine right ventricular infarction model. Twenty-one animals used for this study randomly received isoflurane, xenon or thiopental, (n=6-8 per group). Myocardial infarction was induced for 90min, followed by reperfusion for 120min. Tissues from the left and right ventricles were removed from the sites of infarction, reperfusion and remote areas, and processed for immunohistochemistry. Apoptosis (caspase-3 staining) and neutrophilic infiltration (naphthol AS-D chloroacetate-specific esterase) were assessed and evaluated. Statistical analysis was performed using an ANOVA of repeated measures. Density of apoptotic cells were higher in tissues from animals that were anesthetized with xenon. This effect was significant in comparison to isoflurane (p=0.0177). Neutrophilic infiltration was significantly higher in the right compared to the left ventricle (p<0.001), whereas no significant differences in the number of granulocytes based on the anaesthetic regime or the different tissue areas were found. We conclude that xenon, in the early phase of ischaemia and reperfusion, induces a significant increase in apoptosis compared to isoflurane. Therefore, clinical use of this anaesthetic in cardiocompromised patients should be taken with care until more long-term studies have been carried out. The increased neutrophilic infiltration in the right vs. the left ventricle indicates the right ventricle being more susceptible to ischaemia-reperfusion injury.

The role of nitric oxide, reactive oxygen species, and protein kinase C in oxytocin-induced cardioprotection in ischemic rat heart

Ischemia–reperfusion injury is a common complication of heart disease that is the leading cause of death worldwide. Here, we plan to elucidate oxytocin cardioprotection effects against ischemia–reperfusion via nitric oxide (NO), reactive oxygen species (ROS), and protein kinase C (PKC) in anesthetized rat preconditioned myocardium. Forty-eight Sprague-Dawley rats were equally divided into eight groups. All animals were subjected to 25min ischemia and 120min reperfusion. Oxytocin (OT), L-NAME (LNA, a nitric oxide synthase inhibitor), chelerythrine (CHE, a PKC enzyme inhibitor), and N-acetylcysteine (NAC, a ROS scavenger) were used prior to ischemia. Results showed that mean arterial pressure significantly reduced during the first 10min of ischemia and reperfusion in IR, LNA, CHE, and NAC groups (p<0.05). OT prevented mean arterial pressure decline during early phase of ischemia and reperfusion. Cardioprotective effects of OT in infarct size, plasma levels of creatine kinase-MB and lactate dehydrogenase, severity and incidence of ventricular arrhythmias were abolished by L-NAME, chelerythrine, and N-acetylcysteine (p<0.05). The present study showed that OT pretreatment reduces myocardial infarct size and ventricular arrhythmias, and improves mean arterial pressure via NO production, PKC activation, and ROS balance. These findings provide new insight into therapeutic strategies for ischemic heart disease.

Oxytocin protects cardiomyocytes from apoptosis induced by ischemia–reperfusion in rat heart: Role of mitochondrial ATP-dependent potassium channel and permeability transition pore

The current study examines the protective effect of oxytocin (OT) on cardiomyocyte apoptosis modulated by mitochondrial ATP-dependent potassium (mitoKATP) channel and permeability transition pore (mPTP) in the preconditioned myocardium of anesthetized rats. Eighty rats were equally divided into eight groups. The hearts of all animals except for the sham group were subjected to 25min ischemia and 120min reperfusion. Oxytocin, 5-hydroxydeconoate (5-HD), a specific inhibitor of the mitoKATP channel, and atractyloside (ATRC), an mPTP opener, were used prior to ischemia. Hemodynamic parameters were recorded throughout the experiment. Evaluations were made by infarct size, plasma lactate dehydrogenase level (LDH), transmission electron microscopy (TEM) and immunohistochemistry studies. OT prevented mean arterial pressure drop during early phase of ischemia and reperfusion. Treatment with OT before IR induction normalizes cardiomyocytes both in light microscopy and TEM observations. In addition, OT significantly reduced TUNEL- and increased Bcl-2-labeled positive cell number relative to IR (p<0.05). However, 5HD or ATRC inhibited the protective effects of OT on cardiomyocytes damaged by IR (p<0.05). Ultrastructural changes including extensive myofibril loss, sarcolemmal disruption and mitochondrial swelling due to amorphous dens bodies indicate necrosis induction in 5HD and ATRC as well as in IR groups. Restoration of immunohistochemistry parameters and protection against IR-induced ultrastructural changes confirm OT cardioprotective effects via mitoKATP channel and mPTP modulation in apoptosis induced by ischemia–reperfusion.

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Molecular mechanisms underlying neurovascular protection in stroke

Molecular mechanisms underlying neurovascular protection in stroke

Neonatal mouse testis-derived multipotent germline stem cells improve the cardiac function of acute ischemic heart mouse model

Multipotent germline stem (mGS) cells have been established from neonatal mouse testes. We previously reported that undifferentiated mGS cells are phenotypically similar to embryonic stem cells and that fetal liver kinase 1 (Flk1) + mGS cells have a similar potential to differentiate into cardiomyocytes and endothelial cells compared with Flk1 + embryonic stem cells. Here, we transplanted these Flk1 + mGS cells into an ischemic heart failure mouse model to evaluate the improvement in cardiac function. Significant increase in left ventricular wall thickness of the infarct area, left ventricular ejection fraction and left ventricular maximum systolic velocity was observed 4 weeks after when sorted Flk1 + mGS cells were transplanted directly into the hearts of the acute ischemic model mice. Although the number of cardiomyocytes derived from Flk1 + mGS cells were too small to account for the improvement in cardiac function but angiogenesis around ischemic area was enhanced in the Flk1 + mGS cells transplanted group than the control group and senescence was also remarkably diminished in the early phase of ischemia according to β-galactosidase staining assay. In conclusion, Flk1 + mGS cell transplantation can improve the cardiac function of ischemic hearts by promoting angiogenesis and by delaying host cell death via senescence.

Abstract 5137: Visualization of Accumulating Reactive Oxygen Species During Myocardial Ischemia by Real-time Two-photon Imaging of Perfused Rat Hearts

Background: During myocardial ischemia/reperfusion injury, a burst of reactive oxygen species (ROS) is considered to occur at the onset of reperfusion and cause cardiomyocyte death through the loss of mitochondrial membrane potential (ΔΨ m ), but there are evidences against this concept. We aimed to visualize the dynamic changes of ROS during ischemia/reperfusion in intact rat hearts using two-photon laser scanning microscopy. Methods and Results: Langendorff-perfused rat hearts were loaded with 5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl-ester and tetramethylrhodamine ethyl-ester, fluorescent indicators of ROS and ΔΨ m , respectively. Under the two-photon excitation, spatio-temporal changes of ROS and ΔΨ m in response to ischemia/reperfusion were simultaneously monitored at cellular level. As soon as ischemia started, ROS level of each cell began to increase, despite maintained ΔΨ m . Importantly, the rate of ROS accumulation during the early phase of ischemia was variable from cell to cell, and irreversible ΔΨ m loss occurred in an all-or-none manner depending on cellular ROS level, with a clear cut-off value. Upon reperfusion, we were unable to detect a burst of ROS, but there were more cells undergoing similar ΔΨ m loss after the cell-to-cell specific period of latency. Importantly, there was a significant inverse correlation between the level of ROS accumulation during ischemia and the latency until which the cell underwent ΔΨ m loss (r=−0.89). Ischemic preconditioning delayed the increase of ROS during ischemia and protected against ΔΨ m loss. An iron chelator deferoxamine, which reduces the generation of hydroxyl radical through inhibiting the iron-mediated Fenton reaction, neither attenuated the ROS generation nor protected against ΔΨ m loss. A free radical scavenger N-(2-mercaptopropionyl)-glycine attenuated the ROS accumulation, but it did not protect against ΔΨ m loss. Conclusion: In myocardial ischemia/reperfusion, ROS are mainly generated during ischemia, but not after reperfusion, and play a critical role in mitochondrial collapse and cardiomyocyte death. However, reducing the ROS accumulation during ischemia may not suffice to prevent cell death.

Fasudil, a Rho kinase (ROCK) inhibitor, protects against ischemic neuronal damage in vitro and in vivo by acting directly on neurons

Background and purpose Recently, fasudil, a Rho kinase (ROCK) inhibitor, was reported to prevent cerebral ischemia in vivo by increasing cerebral blood flow and inhibiting inflammatory responses. However, it is uncertain whether a ROCK inhibitor can directly protect neurons against ischemic damage. Our purpose was to evaluate both the involvement of ROCK activity in ischemic neuronal damage and any direct neuroprotective effect of fasudil against cerebral infarction. Methods: In vivo, focal cerebral ischemia was induced by permanent middle cerebral artery occlusion in mice, and the resulting infarction was evaluated 24 h later. ROCK expression and activity were assessed using Western blotting and immunohistochemistry. In vitro, the effects of fasudil and hydroxyfasudil (a main metabolite of fasudil) were examined on oxygen–glucose deprivation (OGD)-induced PC12 cell death and on glutamate-induced neurotoxicity in primary cerebral neuronal culture. Results: ROCK expression and activity increased in the striatum, especially in axons, in the early phase of ischemia. Fasudil reduced this ROCK activity and protected against cerebral infarction in vivo. Hydroxyfasudil inhibited OGD-induced PC12 cell death, and fasudil and hydroxyfasudil each attenuated glutamate-induced neurotoxicity in vitro. Conclusions: These findings indicate that ROCK plays a pivotal role in the mechanism underlying ischemic neuronal damage and that a direct effect of fasudil on neurons may be partly responsible for its protective effects against such damage.

Proteomic Analysis of Ischemia-Reperfusion Injury upon Human Liver Transplantation Reveals the Protective Role of IQGAP1

Ischemia-reperfusion injury (IRI) represents a major determinant of liver transplantation. IRI-induced graft dysfunction is related to biliary damage, partly due to a loss of bile canaliculi (BC) integrity associated with a dramatic remodeling of actin cytoskeleton. However, the molecular mechanisms associated with these events remain poorly characterized. Using liver biopsies collected during the early phases of organ procurement (ischemia) and transplantation (reperfusion), we characterized the global patterns of expression and phosphorylation of cytoskeleton-related proteins during hepatic IRI. This targeted functional proteomic approach, which combined protein expression pattern profiling and phosphoprotein enrichment followed by mass spectrometry analysis, allowed us to identify IQGAP1, a Cdc42/Rac1 effector, as a potential regulator of actin cytoskeleton remodeling and maintenance of BC integrity. Cell fractionation and immunohistochemistry revealed that IQGAP1 expression and localization were affected upon IRI and related to actin reorganization. Furthermore using an IRI model in human hepatoma cells, we demonstrated that IQGAP1 silencing decreased the basal level of actin polymerization at BC periphery, reflecting a defect in BC structure coincident with reduced cellular resistance to IRI. In summary, this study uncovered new mechanistic insights into the global regulation of IRI-induced cytoskeleton remodeling and led to the identification of IQGAP1 as a regulator of BC structure. IQGAP1 therefore represents a potential target for the design of new organ preservation strategies to improve transplantation outcome.