Synthesis and protective effect of scutellarein on focal cerebral ischemia/reperfusion in rats

Scutellarein, the main metabolite of scutellarin in vivo, has relatively better solubility, bioavailability and bio-activity than scutellarin. However, compared with scutellarin, it is very difficult to obtain scutellarein from Nature. Therefore, the present study focused on establishing an efficient route for the synthesis of scutellarein by hydrolyzing scutellarin. Neurological deficit score and cerebral infarction volume with the administration of scutellarein were then used to compare its neuroprotective effects on focal cerebral ischemia/reperfusion in rats induced by middle cerebral artery occlusion (MCAO) with those of scutellarin. The results showed that scutellarein had better protective effect on focal cerebral ischemia/reperfusion than scutellarin, which laid the foundation for further research and development of scutellarein as a promising candidate for ischemic cerebro-vascular disease.     

Analysis of amino acid neurotransmitters in hypothalamus of rats during cerebral ischemia-reperfusion by microdialysis and capillary electrophoresis

In this work, focal cerebral ischemia and reperfusion were induced by the model of middle cerebral artery occlusion. The dialysate of extracellular fluid in the hypothalamus of rats were obtained by using brain microdialysis technique. An efficient and sensitive MEKC method for the simultaneous determination of multiple amino acid neurotransmitters in microdialysate was developed by capillary electrophoresis with laser-induced fluorescence detection and 5-(4, 6-dichloro-s-triazin-2-ylamino) fluorescein derivatization. Different parameters that influenced derivatization reaction and CE separation were studied and optimized. This method was used to investigate the dynamic change of fourteen amino acid neurotransmitters in microdialysates during cerebral ischemia/reperfusion period. Our results reveal that MCAO and reperfusion elicited significant increases in the extracellular levels of Arg, Lys, Trp, Phe, Gln, GABA, Asn, Pro, Ser, Ala, Tau, Gly, Glu and Asp. The excitatory/inhibitory neurotransmitter balance was disturbed during ischemia/reperfusion. The dynamic changes and functional status of releasable neurotransmitters during ischemia/reperfusion were discussed.     

Protective effect of Salvia miltiorrhiza extract against renal ischemia-reperfusion-induced injury in rats

The present study investigates the effect of pre-treatment with Salvia miltiorrhiza ethanol extracts (SMEE) on renal function markers, immunity and antioxidant activities in renal ischemia and reperfusion (IR) rats. Wistar rat kidneys were subjected to 60 min of global ischemia at 37 °C followed by 30 min of reperfusion, and were randomly assigned into the sham, IR model and three SMEE-treated groups (n = 8 per group). Results showed that high serum creatinin (Scr), blood urea nitrogen (BUN), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α) and malondialhehyde (MDA) levels, and low antioxidant enzyme activities were observed in IR rats compared to the sham rats. Pre-treatment of Salvia miltiorrhiza ethanol extracts for 20 days prior to IR operation improved renal function, reduced IR induced renal inflammatory and oxidative injury. It is concluded that Salvia miltiorrhiza ethanol extracts could be beneficial in the treatment of renal ischemic injury.     

Ischemic postconditioning protects cardiomyocytes against ischemia/reperfusion injury by inducing MIP2

Cardiomyocytes can resist ischemia/reperfusion (I/R) injury through ischemic postconditioning (IPoC) which is repetitive ischemia induced during the onset of reperfusion. Myocardial ischemic preconditioning up-regulated protein 2 (MIP2) is a member of the WD-40 family proteins, we previously showed that MIP2 was up-regulated during ischemic preconditioning (IPC). As IPC and IPoC engaged similar molecular mechanisms in cardioprotection, this study aimed to elucidate whether MIP2 was up-regulated during IPoC and contributed to IPoC-mediated protection against I/R injury. The experiment was conducted on two models, an in vivo open chest rat coronary artery occlusion model and an in vitro model with H9c2 myogenic cells. In both models, 3 groups were constituted and randomly designated as the sham, I/R and IPoC/hypoxia postconditioning (HPoC) groups. In the IPoC group, after 45 min of ischemia, hearts were allowed three cycles of reperfusion/ischemia phases (each of 30 s duration) followed by reperfusion. In the HPoC group, after 6 h of hypoxia, H9c2 cells were subjected to three cycles of 10 minute reoxygenation and 10 minute hypoxia followed by reoxygenation. IPoC significantly reduced the infarct size, plasma level of Lactate dehydrogenase and creatine kinase MB in rats. 12 h after the reperfusion, MIP2 mRNA levels in the IPoC group were 10 folds that of the sham group and 1.4 folds that of the I/R group. Increased expression of MIP2 mRNA and attenuation of apoptosis were similarly observed in the HPoC group in the in vitro model. These effects were blunted by transfection with MIP2 siRNA in the H9c2 cells. This study demonstrated that IPoC induced protection was associated with increased expression of MIP2. Both MIP2 overexpression and MIP2 suppression can influence the IPoC induced protection.     

Ginkgo biloba extract preserves pyruvate and enhances ascorbate in the cortex of gerbils during focal cerebral ischemia. A microdialysis-liquid chromatography study

The aim of this study was to evaluate dynamic changes in energy-related metabolites in the cortex of gerbils subjected to focal cerebral ischemia after pretreatment with Ginkgo biloba extract. Focal cerebral ischemia was induced by occlusion of the right common carotid artery and the right middle cerebral artery for 60 min in anesthetized gerbils. A microdialysis probe was inserted into the cortex to monitor extracellular lactate. pyruvate and ascorbate during ischemia and reperfusion. The present study demonstrated a dynamic decrease in pyruvate (25% of baseline) and increases in lactate (160% of baseline) and asorbate (300% of baseline) and a 5-fold increase in the lactate:pyruvate (L:P) ratio during cerebral ischemia in the control group. However. pyruvate levels were preserved and ascorbate levels were enhanced with a chronic pretreatment of Ginkgo biloba extract for 8 days (i.p., 100 mg kg(-1) day(-1)). Preservation of pyruvate and enhancement of ascorbate observed in this study may be associated with the neuroprotective effects of Ginkgo biloba extract.     

Protective effect of ischemic postconditioning against ischemia reperfusion-induced myocardium oxidative injury in IR rats

Brief episodes of myocardial ischemia-reperfusion (IR) employed during reperfusion after a prolonged ischemic insult may attenuate the total ischemia-reperfusion injury. This phenomenon has been termed ischemic postconditioning. In the present study, we studied the possible effect of ischemic postconditioning on an ischemic reperfusion (IR)-induced myocardium oxidative injury in rat model. Results showed that ischemic postconditioning could improve arrhythmia cordis, reduce myocardium infarction and serum creatin kinase (CK), lactate dehydrogenase (LDH) and aspartate transaminase (AST) activities in IR rats. In addition, ischemic postconditioning could still decrease myocardium malondialdehyde (MDA) level, and increased myocardium Na+-K+-ATPase, Ca2+-Mg2+-ATPase, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GR) activities. It can be concluded that ischemic postconditioning possesses strong protective effects against ischemia reperfusion-induced myocardium oxidative injury in IR rats.     

Effects of sevoflurane on tight junction protein expression and PKC-α translocation after pulmonary ischemia–reperfusion injury

Pulmonary dysfunction caused by ischemia-reperfusion injury is the leading cause of mortality in lung transplantation. We aimed to investigate the effects of sevoflurane pretreatment on lung permeability, tight junction protein occludin and zona occludens 1 (ZO-1) expression, and translocation of protein kinase C (PKC)-α after ischemia–reperfusion. A lung ischemia-reperfusion injury model was established in 96 male Wistar rats following the modified Eppinger method. The rats were divided into four groups with 24 rats in each group: a control (group C), an ischemia-reperfusion group (IR group), a sevoflurane control group (sev-C group), and a sevoflurane ischemia-reperfusion group (sev–IR group). There were three time points in each group: ischemic occlusion for 45 min, reperfusion for 60 min and reperfusion for 120 min; and there were six rats per time point. For the 120-min reperfusion group, six extra rats underwent bronchoalveolar lavage. Mean arterial pressure (MAP) and pulse oxygen saturation (SpO₂) were recorded at each time point. The wet/dry weight ratio and lung permeability index (LPI) were measured. Quantitative RT-PCR and Western blot were used to measure pulmonary occludin and ZO-1, and Western blot was used to measure cytosolic and membranous PKC-α in the lung. Lung permeability was significantly increased after ischemia–reperfusion. Sevoflurane pretreatment promoted pulmonary expression of occludin and ZO-1 after reperfusion and inhibited the translocation of PKC-α. In conclusion, sevoflurane pretreatment alleviated lung permeability by upregulating occludin and ZO-1 after ischemia–reperfusion. Sevoflurane pretreatment inhibited the translocation and activation of PKC-α, which also contributed to the lung-protective effect of sevoflurane.     

Therapeutic Effect of Icaritin on Cerebral Ischemia-Reperfusion-Induced Senescence and Apoptosis in an Acute Ischemic Stroke Mouse Model

An ischemic stroke is brain damage caused by interruption of blood supply to the brain that can cause death and long-term disability. New medical strategies or therapies are urgently needed for ischemic stroke. Icaritin (ICT) is a metabolite of icariin (ICA), which are two active flavonoid components extracted from Herba epimedii and considered neuroprotective agents in animal models of Alzheimer’s disease and ischemic stroke. The therapeutic effect of ICT on ischemic still remains to be clarified. The aim of this study was to investigate the therapeutic effect of ICT on cerebral ischemia-reperfusion-associated senescence and apoptosis in a middle cerebral artery occlusion (MCAO) mouse model (ischemia for 50 min and reperfusion for 24 h). Administration of ICT after ischemia significantly reduced MCAO-induced neurological damage, infarct volume, and histopathological changes in the brain of acute ischemic stroke mice. ICT treatment could also reduce neuronal apoptosis and senescence and reversed the expression of apoptosis- and senescence-related signaling proteins. These findings suggest that ICT may have therapeutic potential to ameliorate acute ischemic stroke.     

Lack of Neuroprotective Effects of High-Density Lipoprotein Therapy in Stroke under Acute Hyperglycemic Conditions

Introduction: The pleiotropic protective effects of high-density lipoproteins (HDLs) on cerebral ischemia have never been tested under acute hyperglycemic conditions. The aim of this study is to evaluate the potential neuroprotective effect of HDL intracarotid injection in a mouse model of middle cerebral artery occlusion (MCAO) under hyperglycemic conditions. Methods: Forty-two mice were randomized to receive either an intracarotid injection of HDLs or saline. Acute hyperglycemia was induced by an intraperitoneal injection of glucose (2.2 g/kg) 20 min before MCAO. Infarct size (2,3,5-triphenyltetrazolium chloride (TTC)-staining), blood–brain barrier leakage (IgG infiltration), and hemorrhagic changes (hemoglobin assay by ELISA and hemorrhagic transformation score) were analyzed 24 h post-stroke. Brain tissue inflammation (IL-6 by ELISA, neutrophil infiltration and myeloperoxidase by immunohisto-fluorescence) and apoptosis (caspase 3 activation) were also assessed. Results: Intraperitoneal D-glucose injection allowed HDL- and saline-treated groups to reach a blood glucose level of 300 mg/dl in the acute phase of cerebral ischemia. HDL injection did not significantly reduce mortality (19% versus 29% in the saline-injected group) or cerebral infarct size (p = 0.25). Hemorrhagic transformations and inflammation parameters were not different between the two groups. In addition, HDL did not inhibit apoptosis under acute hyperglycemic conditions. Conclusion: We observed a nonsignificant decrease in cerebral infarct size in the HDL group. The deleterious consequences of reperfusion such as hemorrhagic transformation or inflammation were not improved by HDL infusion. In acute hyperglycemia, HDLs are not potent enough to counteract the adverse effects of hyperglycemia. The addition of antioxidants to therapeutic HDLs could improve their neuroprotective capacity.     

Effect of Ginkgo biloba extract 50 on immunity and antioxidant enzyme activities in ischemia reperfusion rats

The aim of the study was to investigate the effect of Ginkgo biloba extract 50 (GBE50), a well-known natural antioxidant, against immunity and antioxidant enzyme activities in ischemia reperfusion (IR) rats. Rats were then divided into six groups fed for 15 days with the same diet: three groups (IV, V, VI) were treated by different doses of GBE50 suspension [20, 40, or 60 mg/kg body weight by oral gavage every day at a fixed time (10.00 a.m.)] (equal to 5, 10 and 20 times, respectively, the maximum recommended human dose), and three groups (I, II, III) were untreated. At the end of the experiment, rats' hearts were subjected to 30 min of ischemia followed by 90 min of reperfusion. Results showed that IR significantly enhanced heart rate, S-T height, myocardium (myeloperoxidase) MPO activity and blood interleukin-8 (IL-8), tumor necrosis factor Alpha (TNF-a), interleukin-1β (IL-1β) levels, blood aspartate transaminase (AST), lactate dehydrogenase (LDH), and creatinine kinase (CK) activities, reduced myocardium sodium-potassium adenosine triphosphatase (Na(+)-K(+)-ATPase), calcium-magnesium adenosine triphosphatase (Ca(2+)-Mg(2+)-ATPase) activities and antioxidant enzyme activities in IR group (III) compared to sham control group (II). Pretreatment of GBE50 markedly significantly reduced heart rate, S-T height, myocardium MPO activity and blood IL-8, TNF-a, IL-1β levels, blood AST, LDH, and CK activities, enhanced myocardium Na(+)-K(+)-ATPase, Ca(2+)-Mg(2+)-ATPase activities and antioxidant enzyme activities in IR group (II) compared to IR group (III). The results suggested that the GBE50 may reduce the oxidative stress in the reperfused myocardium, and increased immunity and antioxidant activities in IR rats.     

Ginsenoside Rb1 attenuates intestinal ischemia reperfusion induced renal injury by activating Nrf2/ARE pathway

Intestinal ischemia reperfusion (IIR) is a serious clinical condition associated with simultaneous multiple organ dysfunction. The aim of this study was to investigate the effects of ginsenoside Rb1 on IIR induced renal injury in mice. An intestinal ischemia reperfusion mouse model was established by superior mesenteric artery (SMA) occlusion for 45 min, followed by reperfusion for 2 h. IIR induced renal injury characterized by increase of BUN, Cr and NGAL in serum, MDA levels and decrease of SOD levels in the renal tissues. Ginsenoside Rb1 (30, 60 mg/kg) given intraperitoneally before reperfusion attennuated renal injury, which was associated with decrease of BUN, Cr and NGAL in serum, MDA levels and increase of SOD levels in the renal tissues. Furthermore, the immunohistochemistry and Western blot data showed that ginsenoside Rb1 dramatically reversed IIR induced renal injury, associated with upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in renal tissues. Our data suggests that ginsenoside Rb1 attenuates acute renal injury induced by intestinal ischemia reperfusion by activating the Nrf2/ARE pathway.     

Lipidomic Profiling of Ipsilateral Brain and Plasma after Celastrol Post-Treatment in Transient Middle Cerebral Artery Occlusion Mice Model

Celastrol, a pentacyclic triterpene isolated from the traditional Chinese medicine Tripterygium wilfordii Hook. F., exhibits effectiveness in protection against multiple central nervous system (CNS) diseases such as cerebral ischemia, but its influence on lipidomics still remains unclear. Therefore, in the present study, the efficacy and potential mechanism of celastrol against cerebral ischemia/reperfusion (I/R) injury were investigated based on lipidomics. Middle cerebral artery occlusion (MCAO) followed by reperfusion was operated in mice to set up a cerebral I/R model. TTC staining and TUNEL staining were used to evaluate the therapeutic effect of celastrol. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS) was employed for lipidomics analysis in ipsilateral hemisphere and plasma. Celastrol remarkably reduced cerebral infarct volume and apoptosis positive cells in tMCAO mice. Furthermore, lipidomics analysis showed that 14 common differentially expressed lipids (DELs) were identified in brain and five common DELs were identified in plasma between the Sham, tMCAO and Celastrol-treated tMCAO groups. Through enrichment analysis, sphingolipid metabolism and glycerophospholipid metabolism were demonstrated to be significantly enriched in all the comparison groups. Among the DELs, celastrol could reverse cerebral I/R injury-induced alteration of phosphatidylcholine, phosphatidylethanolamine and sulfatide, which may be responsible for the neuroprotective effect of celastrol. Our findings suggested the neuroprotection of celastrol on cerebral I/R injury may be partially associated with its regulation of lipid metabolism.     

Neuroprotective effects of sonochemical- synthesized SiO2 nanoparticles in vivo models of ischemic/reperfusion injury in stroke

Cerebral ischemic injury is one of the debilitating diseases that showed inflammation plays an essential role in aggravating ischemic damage. After synthesizing silica nanoparticles (SiO₂ NPs) by sonochemical method, serum parameters in the presence of different concentrations of SiO₂ NPs are measured for toxicity assay. Rats were separated randomly into control, ischemia/reperfusion, and ischemia/reperfusion + SiO₂ NPs groups. Transient forebrain ischemia induced with bilateral occlusion of both common carotid arteries followed by 60minuts of reperfusion. SiO₂ NPs were administered (500 mg/kg/day p.o.) 21 days before ischemia/reperfusion time. Animals sacrificed and frontal cortex and hippocampal tissues used to determine malondialdehyde (MDA) level, nitric oxide (NO), glutathione (GSH) levels, an essential antioxidant, superoxide dismutase (SOD), alterations in the level of cytokines, TNFα, IL-1β, MCP-1, and phosphor Ik-кB. We also revealed the involvement of NF-κB downregulation by using western blotting. We reported on a histological investigation. The results showed that SiO₂ NPs with a diameter of around 50 nm in dose of 500 mg/kg didn't change the level of liver enzyme (including ALT, AST and ALP) and hematological parameters. 500 mg/kg SiO₂ NPs showed significant effects on remission of behavioral impairment. Ischemia/reperfusion oxidative injury in the rat hippocampus demonstrated a significant increase in MDA, TNFα, MCP-1, IL-1β, phosphor Ik-кB, NO levels, and a significant decrease in GSH contents and SOD activities in the hippocampal tissue compared to the control group. Pretreatment of ischemic rats with SiO₂ NPs decreased the elevated levels of MDA, TNFα, MCP-1, IL-1β, phosphor Ik-кB, and NO levels. A significant alteration observed in SOD activities and GSH content results between treated and untreated ischemia/reperfusion brains in rats. Decreased protein level of NF-κB also measured in SiO₂ NPs-treated animals. Untreated ischemia/reperfusion brains had significantly decreased in number of cells in CA1 hippocampus, nevertheless SiO₂ NPs increase the normal cell and decrease the neurodegeneration in hippocampus but it was not significant alteration. SiO₂ NPs reduced the damage caused by cerebral ischemia/reperfusion in rats and its molecular mechanism attributed to the downregulation of NF-κB signaling pathway.     

Simultaneous monitoring of extracellular glucose, pyruvate, lactate and glutamate in gerbil cortex during focal cerebral ischemia by dual probe microdialysis

The aim of this study was to monitor dynamic changes in energy-related metabolites in the cortex of gerbils subjected to cerebral ischemia by a dual probe microdialysis technique. Focal cerebral ischemia was produced in anesthetized gerbils by occlusion of the right common carotid artery and the right middle cerebral artery for 60 min. Two microdialysis probes were inserted into both sides of the cortex to simultaneously monitor extracellular glucose, lactate, pyruvate and glutamate. Dynamic and comparative changes in these analytes, on the ipsilateral and contralateral sides of the brain, were simultaneously monitored by liquid chromatography and a microdialysis analyzer. The present study demonstrated decreases in glucose and pyruvate, increases in lactate and glutamate on the ipsilateral side whereas all analytes remain constant on the contralateral side of cortex during cerebral ischemia. In vitro recovery of each microdialysis probe was performed to ensure the quality of experiments. The detection limits of pyruvate, glutamate, lactate and glucose were 0.2, 1.0, 2.0 and 20 microM, respectively. The intra- and inter-assay correlations were less than 5% in standard mixtures and pooled brain dialysates.     

Monitoring of extracellular pyruvate, lactate, and ascorbic acid during cerebral ischemia: a microdialysis study in awake gerbils

In vivo microdialysis coupled with liquid chromatography was developed for the continuous monitoring of brain neurochemicals during cerebral ischemia in awake, free moving gerbils. The dead volume of the microdialysis system was estimated to be less than 30 μl. The detection limits of the present assay were 0.2 to 2.0 μM for analytes at a signal to noise ratio of five. To validate this assay, a focal cerebral ischemia was produced by occlusion of one common carotid artery for 60 min and then reperfusion for additional 3 h in awake gerbils. A microdialysis probe was inserted into the striatum of the gerbil. Dialysates were autoinjected and analyzed extracellular pyruvate, lactate, and ascorbic acid by liquid chromatography with a UV detector during cerebral ischemia. Significant changes of pyruvate and the lactate/pyruvate ratio were observed. Biphasic and dynamic changes in ascorbic acid and lactate were proposed to correlate a secondary damage. This assay can be used as a tool to study dynamic changes of brain neurochemicals in awake animals.     

The Effect of a New N-hetero Cycle Derivative on Behavior and Inflammation against the Background of Ischemic Stroke

Ischemic stroke triggers a whole cascade of pathological changes in the brain, one of which is postischemic inflammation. Since in such cases thrombolytic therapy is often not possible, methods that modulate inflammation and affect microglia become particularly interesting. We synthesized 3-(2-oxo-4-phenylpyrrolidin-1-yl)propane-1-sulfonate calcium(II) (Compound 4) and studied its anti-inflammatory activity in in vitro and in vivo models of inflammation and ischemia. Macrophage cell line RAW 264.7 was treated with lipopolysaccharides (LPS) and Compound 4 at various dosages to study the cytokine profile using real-time PCR and cytometric bead array (CBA). Stroke in rats was simulated by the middle cerebral artery occlusion method (MCAO). Several tests were performed to characterize the neurological deficit and locomotor activity of the rats, and afterwards, postmortem, the number of astrocytes was counted using immunohistochemistry. Compound 4 in in vitro tests dose-dependently reduced the expression of interleukin-1β (IL1β), and inducible nitric oxide synthase (iNOS) genes in cell culture and increased the concentration of cytokines: interleukin-2, 4, 6 (IL-2, IL-4, and IL-6). In vivo Compound 4 increased the orienting-exploratory behavior, and reduced neurological and motor deficit. The number of astrocytes that promote and support inflammation was lower in the group treated with Compound 4. The stroke volume measured by magnetic resonance imaging (MRI) showed no difference. We have shown that Compound 4 demonstrates anti-inflammatory activity by increasing the synthesis of anti-inflammatory and reducing pro-inflammatory cytokines, and positively affects the neurological deficit in rats. Thus, Compound 4 has a high therapeutic potential in the management of patients after a stroke and requires further study of its neuroprotective properties.     

Delta Sleep-Inducing Peptide Recovers Motor Function in SD Rats after Focal Stroke

Background and Objectives: Mutual effect of the preliminary and therapeutic intranasal treatment of SD rats with DSIP (8 days) on the outcome of focal stroke, induced with intraluminal middle cerebral occlusion (MCAO), was investigated. Materials and Methods: The groups were the following: MCAO + vehicle, MCAO + DSIP, and SHAM-operated. DSIP or vehicle was applied nasally 60 (±15) minutes prior to the occlusion and for 7 days after reperfusion at dose 120 µg/kg. The battery of behavioral tests was performed on 1, 3, 7, 14, and 21 days after MCAO. Motor coordination and balance and bilateral asymmetry were tested. At the end of the study, animals were euthanized, and their brains were perfused, serial cryoslices were made, and infarction volume in them was calculated. Results: Although brain infarction in DSIP-treated animals was smaller than in vehicle-treated animals, the difference was not significant. However, motor performance in the rotarod test significantly recovered in DSIP-treated animals. Conclusions: Intranasal administration of DSIP in the course of 8 days leads to accelerated recovery of motor functions.     

Antioxidant activity of novel indole derivatives and protection of the myocardial damage in rabbits

Novel indole derivatives containing a triazole moiety (1a-d, 2a-c) were synthesized as lead compounds with interesting pharmacological profiles. Their antioxidant activity was investigated on in vitro non-enzymatic rat hepatic microsomal lipid peroxidation. All compounds showed significant effect in the above assay. The effect depended mainly on the attachment position of the triazole moiety on the indole nucleus. The most potent antioxidant derivatives la, 1c and 1d were tested for their protective ability against the oxidative damage of the myocardium after ischemia-reperfusion, in male rabbits which were subjected to 30 min regional ischemia followed by reperfusion. The tested antioxidant compounds 1a, 1c and 1d were continuously infused for 30 min starting at 10th min of ischemia and lasted at 10th min of reperfusion. The concentration of malondialdehyde (MDA, a marker of lipid peroxidation) and hemodynamic parameters (blood pressure and heart rate) were measured in the baseline, at 20th min of the sustained ischemia, 1st and 20th min of reperfusion. It was found that the examined compounds la, 1c and 1d reduced significantly the level of MDA in rabbits under ischemia-reperfusion and proved to be promising substances for further evaluation of anti-ischemic properties.     

High-performance liquid chromatographic detection of hydroxylated benzoic acids as an indirect measure of hydroxyl radical in heart: its possible link with the myocardial reperfusion injury.

The present report describes a method suitable for the indirect assay of hydroxyl radical (OH.), which is likely to be produced during reperfusion of ischemic myocardium. Isolated rat heart perfused by the Langendorff technique was subjected to 30 min of ischemia, followed by 30 min of reperfusion. Salicylic acid (2 mM) was added to the perfusion circuit to trap any OH. radical generated during the experiment. 2,5- and 2,3-dihydroxybenzoic acids (hydroxylated products of salicylic acid) were identified by authentic standards as well as by pure OH.-generating system using high-performance liquid chromatography with electrochemical detection. In addition to serving as a chemical trap for the detection of OH., salicylate attenuated myocardial reperfusion injury as evidenced by reduced formation of creatine kinase, decreased lipid peroxidation, and improved myocardial contractile functions during reperfusion. These results thus provide direct evidence for the presence of OH. in heart and link it to the myocardial reperfusion injury.     

Ischemic preconditioning in the rat hippocampus increases antioxidant activities but does not affect the level of hydroxyl radicals during subsequent severe ischemia

Several studies have demonstrated that ischemic preconditioning increases superoxide dismutase activity, but it is unclear how ischemic preconditioning affects events downstream of hydrogen peroxide production during subsequent severe ischemia and reperfusion in the hippocampus. To answer this question, we investigated whether ischemic preconditioning in the hippocampal CA1 region increases the activities of antioxidant enzymes glutathione peroxidase and catalase, resulting in a decrease in the level of hydroxyl radicals during subsequent severe ischemia-reperfusion. Transient forebrain ischemia was induced by four-vessel occlusion in rats. Ischemic preconditioning for 3 min or a sham operation was performed and a 15-min severe ischemia was induced three days later. Ischemic preconditioning preserved the CA1 hippocampal neurons following severe ischemia. The concentration of 2,3-dihydroxybenzoic acid, an indicator of hydroxyl radical, was measured using in vivo microdialysis technique combined with HPLC. The ischemia-induced increase in the ratio of 2,3-dihydroxybenzoic acid concentration relative to baseline did not differ significantly between preconditioned and control groups. On the other hand, activities of the antioxidant enzymes glutathione peroxidase-1 and catalase were significantly increased at 3 days after ischemic preconditioning in the hippocampus. Our results suggest that, in preconditioned rats, while hydrogen peroxide is generated from severe ischemia, the activity of catalase and glutathione peroxidase-1 is correspondingly increased to eliminate the excessive hydrogen peroxide. However, our results show that the enhanced activity of these antioxidant enzymes in preconditioned rats is not sufficient to decrease hydroxyl radical levels during subsequent severe ischemia-reperfusion.