Minocycline inhibits 5-lipoxygenase expression and accelerates functional recovery in chronic phase of focal cerebral ischemia in rats

AimsWe previously reported that minocycline attenuates acute brain injury and inflammation after focal cerebral ischemia, and this is partly mediated by inhibition of 5-lipoxygenase (5-LOX) expression. Here, we determined the protective effect of minocycline on chronic ischemic brain injury and its relation with the inhibition of 5-LOX expression after focal cerebral ischemia.Main methodsFocal cerebral ischemia was induced by 90 min of middle cerebral artery occlusion followed by reperfusion for 36 days. Minocycline (45 mg/kg) was administered intraperitoneally 2 h and 12 h after ischemia and then every 12 h for 5 days. Sensorimotor function was evaluated 1–28 days after ischemia and cognitive function was determined 30–35 days after ischemia. Thereafter, infarct volume, neuron density, astrogliosis, and 5-LOX expression in the brain were determined.Key findingsMinocycline accelerated the recovery of sensorimotor and cognitive functions, attenuated the loss of neuron density, and inhibited astrogliosis in the boundary zone around the ischemic core, but did not affect infarct volume. Minocycline significantly inhibited the increased 5-LOX expression in the proliferated astrocytes in the boundary zone, and in the macrophages/microglia in the ischemic core.SignificanceMinocycline accelerates functional recovery in the chronic phase of focal cerebral ischemia, which may be partly associated with the reduction of 5-LOX expression.     

Antioxidative effects of Panax notoginseng saponins in brain cells

Oxidative stress resulting from accumulation of reactive oxygen species (ROS) is involved in cell death associated with neurological disorders such as stroke, Alzheimer's disease and traumatic brain injury. Antioxidant compounds that improve endogenous antioxidant defenses have been proposed for neural protection. The purpose of this study was to investigate the potential protective effects of total saponin in leaves of Panax notoginseng (LPNS) on oxidative stress and cell death in brain cells in vitro. Lactate dehydrogenase (LDH) assay indicated that LPNS (5 μg/ml) reduced H₂O₂-induced cell death in primary rat cortical astrocytes (23 ± 8% reduction in LDH release vs. control). Similar protection was found in oxygen and glucose deprivation/reoxygenation induced SH-SY5Y (a human neuroblastoma cell line) cell damage (78 ± 7% reduction vs. control). The protective effects of LPNS in astrocytes were associated with attenuation of reactive oxygen species (ROS) accumulation. These effects involved activation of Nrf2 (nuclear translocation) and upregulation of downstream antioxidant systems including heme oxygenase-1 (HO-1) and glutathione S-transferase pi 1 (GSTP1). These results demonstrate for the first time that LPNS has antioxidative effects which may be neuroprotective in neurological disorders.     

Mrp-8 and -14 mediate CNS injury in focal cerebral ischemia

Several reports have recently demonstrated a detrimental role of Toll-like receptors (TLR) in cerebral ischemia, while there is little information about the endogenous ligands which activate TLR-signaling. The myeloid related proteins-8 and-14 (Mrp8/S100A8; Mrp14/S100A9) have recently been characterized as endogenous TLR4-agonists, and thus may mediate TLR-activation in cerebral ischemia. Interestingly, not only TLR-mRNAs, but also Mrp8 and Mrp14 mRNA were found to be induced in mouse brain between 3 and 48 h after transient 1 h focal cerebral ischemia/reperfusion. Mrp-protein was expressed in the ischemic hemisphere, and co-labeled with CD11b-positive cells. To test the hypothesis that Mrp-signaling contributes to the postischemic brain damage, we subjected Mrp14-deficient mice, which also lack Mrp8 protein expression, to focal cerebral ischemia. Mrp14-deficient mice had significantly smaller lesion volumes when compared to wild-type littermates (130 ± 16 mm³ vs. 105 ± 28 mm³) at 2 days after transient focal cerebral ischemia (1 h), less brain swelling, and a reduced macrophage/microglia cell count in the ischemic hemisphere. We conclude that upregulation and signaling of Mrp-8 and-14 contribute to neuroinflammation and the progression of ischemic damage.     

Intervention action of total flavonoids from root of Ilex pubescens in cerebral ischemic tolerance with blood stasis

The aim of this study was to explore the action characters of total flavonoids from MDQ on cerebral ischemic tolerance with blood stasis. Fully understanding the mechanism of action of total flavonoids from MDQ is helpful for the development of new drugs and the utilization of resources. Male Wistar rat model of blood stasis was established by injecting dexamethasone into the intramuscular side of the thigh. Then they were given related drugs via an intragastric administration for a successive 10 days. After 7 days, the following occurred: firstly, the method of blocking the bilateral common carotid artery (CCA) was used for 10 min, followed by a restoration of perfusion. After 72 h, we performed a temporary occlusion of the rat’s middle cerebral artery for 2 h with an intraluminal thread method. This was followed by reperfusion for 24 h, respectively, to establish the rat model of cerebral ischemic tolerance with blood stasis. Viscosity of the whole blood was measured after the last administration was given blood. Brain was removed, and then the activity of ATP enzyme and T-SOD was determined. To observe the pathological changes of the hippocampus area by HE staining, and the expression of Bcl-2 and Bax were observed by immunohistochemical method. The rat model of cerebral ischemic tolerance with blood stasis was copied successfully. The whole blood viscosity, the activity of NOS, the content of Gluin in the ischemic brain in the IPC model group and the ischemia–reperfusion group were increased significantly. The activity of ATPase was decreased significantly. Compared with the ischemia–reperfusion model group, the activity of ATPase and the whole blood viscosity in the ischemic preconditioning (IPC) group were increased significantly. The activity of NOS and the content of Gluin were decreased significantly. The degree of pathological injury of the brain tissue was also relieved significantly. Total flavonoids of MDQ were used, improving blood circulation, improving energy metabolism, activating endogenous anti-oxidative capability, enhancing the antiapoptotic effect, and relieving the injury of the nerve cell. Hence, the use of MDQ flavonoids improves the tolerance ability of cerebral ischemia.     

Effect of total flavonoids of Radix Ilicis pubescentis on cerebral ischemia reperfusion model

This paper aims to observe the effects of total flavonoids of Radix Ilicis pubescentis on mouse model of cerebral ischemia reperfusion. Mice were orally given different doses of total flavonoids of Radix Ilicis pubescentis 10 d, and were administered once daily. On the tenth day after the administration of 1 h in mice after anesthesia, we used needle to hook the bilateral common carotid artery (CCA) for 10 min, with 10 min ischemia reperfusion, 10 min ischemia. Then we restored their blood supply, copy the model of cerebral ischemia reperfusion; We then had all mice reperfused for 24 h, and then took their orbital blood samples and measured blood rheology. We quickly removed the brain, with half of the brain having sagittal incision. Then we fixed the brains and sectioned them to observe the pathological changes of brain cells in the hippocampus and cortex. We also measured the other half sample which was made of brain homogenate of NO, NOS, Na⁺-K⁺-, ATP enzyme Mg²⁺-ATPase and Ca²⁺-ATPase. Acupuncture needle hook occlusion of bilateral common carotid arteries can successfully establish the model of cerebral ischemia reperfusion. After comparing with the model mice, we concluded that Ilex pubescens flavonoids not only reduce damage to the brain nerve cells in the hippocampus and cortex, but also significantly reduce the content of NO in brain homogenate, the activity of nitric oxide synthase (NOS) and increases ATP enzyme activity (P < 0.05, P < 0.01). In this way, cerebral ischemia reperfusion injury is improved. Different dosages of Ilex pubescens flavonoids on mouse cerebral ischemia reperfusion model have good effects.     

Hyperthermic preconditioning protects astrocytes from ischemia/reperfusion injury by up-regulation of HIF-1 alpha expression and binding activity

It has been demonstrated that hypoxia-inducible factor-1 alpha (HIF-1 alpha) mediates ischemic tolerance induced by hypoxia/ischemia or pharmacological preconditioning. In addition, preconditioning stimuli can be cross-tolerant, safeguarding against other types of injury. We therefore hypothesized HIF-1 alpha might also be associated with ischemic tolerance induced by hyperthermic preconditioning. In the present study, we demonstrated for the first time that 6 h of hyperthermia (38 °C or 40 °C) could induce a characteristic “reactive” morphology and a significant increase in the expression of bystin in astrocytes. We also showed that pre-treatment with 6 h of hyperthermia resulted in a significant increase in cell viability and a remarkable decrease in lactate dehydrogenase (LDH) release and apoptosis development in the astrocytes that were exposed to 24 h of ischemia and a subsequent 24 h of reperfusion. Analysis of mechanisms showed that hyperthermia could lead to a significant increase in HIF-1 alpha expression and also the HIF-1 binding activity in the ischemia/reperfusion astrocytes. The data provide evidence to our hypothesis that the up-regulation of HIF-1 alpha is associated with the protective effects of hyperthermic preconditioning on astrocytes against ischemia/reperfusion injury.     

Iron overload,measured as serum ferritin,increases brain damage induced by focal ischemia and early reperfusion

High levels of iron, measured as serum ferritin, are associated to a worse outcome after stroke. However, it is not known whether ischemic damage might increase ferritin levels as an acute phase protein or whether iron overload affects stroke outcome. The objectives are to study the effect of stroke on serum ferritin and the contribution of iron overload to ischemic damage.Swiss mice were fed with a standard diet or with a diet supplemented with 2.5% carbonyl iron to produce iron overload. Mice were submitted to permanent (by ligature and by in situ thromboembolic models) or transient focal ischemia (by ligature for 1 or 3 h).Treatment with iron diet produced an increase in the basal levels of ferritin in all the groups. However, serum ferritin did not change after ischemia. Animals submitted to permanent ischemia had the same infarct volume in the groups studied. However, in mice submitted to transient ischemia followed by early (1 h) but not late reperfusion (3 h), iron overload increased ischemic damage and haemorrhagic transformation.Iron worsens ischemic damage induced by transient ischemia and early reperfusion. In addition, ferritin is a good indicator of body iron levels but not an acute phase protein after ischemia.     

3-Bromo-7-nitroindazole attenuates brain ischemic injury in diabetic stroke via inhibition of endoplasmic reticulum stress pathway involving CHOP

AimsThe role of nitric oxide (NO) and endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of cerebral ischemic/reperfusion (I/R) injury and diabetes. The aim of the study was to investigate the neuroprotective potential of 3-bromo-7-nitroindazole (3-BNI), a potent and selective neuronal nitric oxide synthase (nNOS) inhibitor against ER stress and focal cerebral I/R injury associated with comorbid type 2 diabetes in-vivo.Main methodsType 2 diabetes was induced by feeding high-fat diet and streptozotocin (35 mg/kg) treatment in rats. Focal cerebral ischemia was induced by 2 h middle cerebral artery occlusion (MCAO) followed by 22 h of reperfusion. Immunohistochemistry and western blotting methods were employed for the detection and expression of ER stress/apoptosis markers [78 kDa glucose regulated protein (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP)]. TUNEL assay for DNA fragmentation was also performed.Key findingsThe diabetic rats subjected to cerebral I/R had prominent neurological damage and functional deficits compared with sham-operated rats. Massive DNA fragmentation was observed in ischemic penumbral region of diabetic brains. Concomitantly, the enhanced immunoreactivity and expression of ER stress/apoptosis markers were noticed. 3-BNI (30 mg/kg, i.p.) treatment significantly inhibited the cerebral infarct, edema volume and improved functional recovery of neurological deficits. The neuroprotection was further evident by lesser DNA fragmentation with a concomitant reduction of GRP78 and CHOP.SignificanceThe study demonstrates the neuroprotective potential of 3-BNI in diabetic stroke model which may be partly due to inhibition of ER stress pathway involving CHOP.     

Inhibition of nNOS reduces ischemic cell death through down-regulating calpain and caspase-3 after experimental stroke

In vitro nitric oxide (NO) regulates calpain and caspase-3 activation, and in vivo neuronal nitric oxide synthase (nNOS), calpain and caspase-3 participate in the ischemic brain injury. Our objective was to investigate whether nNOS was involved in the ischemic brain injury through activating calpain and caspase-3 during experimental stroke. Rats received 1-h ischemia by intraluminant filament, and then reperfused for 23 h (R 23 h). nNOS inhibitor 7-nitroindozale (7-NI, 50 mg/kg) was administrated intraperitoneally 5 min before ischemia. Our data showed that treatment with 7-NI markedly reduced neurological deficits, the brain swelling, and the infarct volume at R 23 h. Enzyme studies revealed significant suppression of the activities of m-calpain and caspase-3 in penumbra and core, and the activities of μ-calpain in penumbra, but not in core, in 7-NI-treated rats versus vehicle-treated rats. Western blot analysis demonstrated that 7-NI markedly increased the levels of MAP-2 and spectrin in penumbra and core compared with vehicle-treated rats. Histopathological studies displayed that 7-NI significantly reduced the necrotic cell death in penumbra and core, and apoptotic cell death in penumbra, but not in core. These data demonstrate the involvement of NO produced by nNOS in the ischemic neuronal injury through affecting the activation of calpain and caspase-3 in penumbra and core after experimental stroke, which provides a new perspective on possible mechanisms of action of nNOS inhibition in cerebral ischemia.     

Ginsenoside Rg1 provides neuroprotection against blood brain barrier disruption and neurological injury in a rat model of cerebral ischemia/reperfusion through downregulation of aquaporin 4 expression

Ginsenoside Rg1 is regarded as one of main bioactive compounds responsible for pharmaceutical actions of ginseng with little toxicity and has been shown to have possibly neuroprotective effects. However, the mechanism of its neuroprotection for acute ischemic stroke is still elusive. The purpose of present study is thus to assess the neuroprotective effects of the ginsenoside Rg1 against blood brain barrier disruption and neurological injury in a rat model of cerebral ischemia/reperfusion, and then to explore the mechanisms for these neuroprotective effects by targeting aquaporin 4. Focal cerebral ischemia was induced by middle cerebral artery occlusion. Neurological examinations were performed by using Longa's 5-point scale. Evans blue dye was used to investigate the effects of ginsenoside Rg1 on blood brain barrier permeability. Immunohistochemical analysis and real-time fluorescence quantitative polymerase chain reaction were used to assess aquaporin 4 expression. As a result, general linear model with repeated measures analysis of variance for neurological scores at 5 repeated measures showed that ginsenoside Rg1-treated group could significantly reduce the changing trend of neurological deficit scores when compared with the middle cerebral artery occlusion model group (p < 0.05). Compared with the middle cerebral artery occlusion model group, ginsenoside Rg1 group has significantly decreased Evans blue content and reduced aquaporin 4 expression at each time point (p < 0.05). In conclusion, ginsenoside Rg1 as a ginsenoside neuroprotective agent could improve neurological injury, attenuate blood brain barrier disruption and downregulate aquaporin 4 expression induced by cerebral ischemia/reperfusion insults in rats.     

The contribution of thioredoxin-2 reductase and glutathione peroxidase to H2O2 detoxification of rat brain mitochondria

Brain mitochondria are not only major producers of reactive oxygen species but they also considerably contribute to the removal of toxic hydrogen peroxide by the glutathione (GSH) and thioredoxin-2 (Trx2) antioxidant systems. In this work we estimated the relative contribution of both systems and catalase to the removal of intrinsically produced hydrogen peroxide (H₂O₂) by rat brain mitochondria. By using the specific inhibitors auranofin and 1-chloro-2,4-dinitrobenzene (DNCB), the contribution of Trx2- and GSH-systems to reactive oxygen species (ROS) detoxification in rat brain mitochondria was determined to be 60 ± 20% and 20 ± 15%, respectively. Catalase contributed to a non-significant extent only, as revealed by aminotriazole inhibition. In digitonin-treated rat hippocampal homogenates inhibition of Trx2- and GSH-systems affected mitochondrial hydrogen peroxide production rates to a much higher extent than the endogenous extramitochondrial hydrogen peroxide production, pointing to a strong compartmentation of ROS metabolism. Imaging experiments of hippocampal slice cultures showed on single cell level substantial heterogeneity of hydrogen peroxide detoxification reactions. The strongest effects of inhibition of hydrogen peroxide removal by auranofin or DNCB were detected in putative interneurons and microglial cells, while pyramidal cells and astrocytes showed lower effects. Thus, our data underline the important contribution of the Trx2-system to hydrogen peroxide detoxification in rat hippocampus. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Overexpression of HSPA12B protects against cerebral ischemia/reperfusion injury via a PI3K/Akt-dependent mechanism

Background and purpose: HSPA12B is a newly discovered member of the Hsp70 family proteins. This study investigated the effects of HSPA12B on focal cerebral ischemia/reperfusion (I/R) injury in mice. Methods: Transgenic mice overexpressing human HSPA12B (Tg) and wild-type littermates (WT) were subjected to 60 min of middle cerebral artery occlusion to induce ischemia and followed by reperfusion (I/R). Neurological deficits, infarct volumes and neuronal death were examined at 6 and 24 hrs after reperfusion. Blood–brain-barrier (BBB) integrity and activated cellular signaling were examined at 3 hrs after reperfusion. Results: After cerebral I/R, Tg mice exhibited improvement in neurological deficits and decrease in infarct volumes, when compared with WT I/R mice. BBB integrity was significantly preserved in Tg mice following cerebral I/R. Tg mice also showed significant decreases in cell injury and apoptosis in the ischemic hemispheres. We observed that overexpression of HSPA12B activated PI3K/Akt signaling and suppressed JNK and p38 activation following cerebral I/R. Importantly, pharmacological inhibition of PI3K/Akt signaling abrogated the protection against cerebral I/R injury in Tg mice. Conclusions: The results demonstrate that HSPA12B protects the brains from focal cerebral I/R injury. The protective effect of HSPA12B is mediated though a PI3K/Akt-dependent mechanism. Our results suggest that HSPA12B may have a therapeutic potential against ischemic stroke.     

Neuroprotective effect of glutamate-substituted analog of gramicidin A is mediated by the uncoupling of mitochondria

BackgroundReactive oxygen species are grossly produced in the brain after cerebral ischemia and reperfusion causing neuronal cell death. Mitochondrial production of reactive oxygen species is nonlinearly related to the value of the mitochondrial membrane potential with significant increment at values exceeding 150 mV. Therefore, limited uncoupling of oxidative phosphorylation could be beneficial for cells exposed to deleterious oxidative stress-associated conditions by preventing excessive generation of reactive oxygen species.MethodsProtonophoric and uncoupling activities of different peptides were measured using pyranine-loaded liposomes and isolated mitochondria. To evaluate the effect of glutamate-substituted analog of gramicidin A ([Glu1]gA) administration on the brain ischemic damage, we employed the in vitro model of neuronal hypoxia using primary neuronal cell cultures and the in vivo model of cerebral ischemia induced in rats by the middle cerebral artery occlusion.Results[Glu1]gA was the most effective in proton-transferring activity among several N-terminally substituted analogs of gramicidin A tested in liposomes and rat brain and liver mitochondria. The peptides were found to be protective against ischemia-induced neuronal cell death and they lowered mitochondrial membrane potential in cultured neurons and diminished reactive oxygen species production in isolated brain mitochondria. The intranasal administration of [Glu1]gA remarkably diminished the infarct size indicated in MR-images of a brain at day 1 after the middle cerebral artery occlusion. In [Glu1]gA-treated rats, the ischemia-induced brain swelling and behavioral dysfunction were significantly suppressed.ConclusionsThe glutamate-substituted analogs of gramicidin A displaying protonophoric and uncoupling activities protect neural cells and the brain from the injury caused by ischemia/reperfusion.General significance[Glu1]gA may be potentially used as a therapeutic agent to prevent neuron damage after stroke.     

Optimization of cyclic sulfamide derivatives as 11β-hydroxysteroid dehydrogenase 1 inhibitors for the potential treatment of ischemic brain injury

The 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), a cortisol regenerating enzyme that amplifies tissue glucocorticoid levels, plays an important role in diabetes, obesity, and glaucoma and is recognized as a potential therapeutic target for various disease conditions. Moreover, a recent study demonstrated that selective 11β-HSD1 inhibitor can attenuate ischemic brain injury. This prompted us to optimize cyclic sulfamide derivative for aiming to treat ischemic brain injury. Among the synthesized compounds, 6e has an excellent in vitro activivity with an IC₅₀ value of 1 nM toward human and mouse 11β-HSD1 and showed good 11β-HSD1 inhibition in ex vivo study using brain tissue isolated from mice. Furthermore, in the transient middle cerebral artery occlusion model in mice, 6e treatment significantly attenuated infarct volume and neurological deficit following cerebral ischemia/reperfusion injury. Additionally, binding modes of 6e for human and mouse 11β-HSD1 were suggested.     

Redox-Optimized ROS Balance and the relationship between mitochondrial respiration and ROS

The Redox-Optimized ROS Balance [R-ORB] hypothesis postulates that the redox environment [RE] is the main intermediary between mitochondrial respiration and reactive oxygen species [ROS]. According to R-ORB, ROS emission levels will attain a minimum vs. RE when respiratory rate (VO₂) reaches a maximum following ADP stimulation, a tenet that we test herein in isolated heart mitochondria under forward electron transport [FET]. ROS emission increased two-fold as a function of changes in the RE (~ 400 to ~ 900 mV·mM) in state 4 respiration elicited by increasing glutamate/malate (G/M). In G/M energized mitochondria, ROS emission decreases two-fold for RE ~ 500 to ~ 300 mV·mM in state 3 respiration at increasing ADP. Stressed mitochondria released higher ROS, that was only weakly dependent on RE under state 3. As a function of VO₂, the ROS dependence on RE was strong between ~ 550 and ~ 350 mV·mM, when VO₂ is maximal, primarily due to changes in glutathione redox potential. A similar dependence was observed with stressed mitochondria, but over a significantly more oxidized RE and ~ 3-fold higher ROS emission overall, as compared with non-stressed controls. We conclude that under non-stressful conditions mitochondrial ROS efflux decreases when the RE becomes less reduced within a range in which VO₂ is maximal. These results agree with the R-ORB postulate that mitochondria minimize ROS emission as they maximize VO₂ and ATP synthesis. This relationship is altered quantitatively, but not qualitatively, by oxidative stress although stressed mitochondria exhibit diminished energetic performance and increased ROS release.     

Regulation and actions of activin A and follistatin in myocardial ischaemia–reperfusion injury

Activin A, a member of the transforming growth factor-β superfamily, is stimulated early in inflammation via the Toll-like receptor (TLR) 4 signalling pathway, which is also activated in myocardial ischaemia–reperfusion. Neutralising activin A by treatment with the activin-binding protein, follistatin, reduces inflammation and mortality in several disease models. This study assesses the regulation of activin A and follistatin in a murine myocardial ischaemia–reperfusion model and determines whether exogenous follistatin treatment is protective against injury. Myocardial activin A and follistatin protein levels were elevated following 30 min of ischaemia and 2 h of reperfusion in wild-type mice. Activin A, but not follistatin, gene expression was also up-regulated. Serum activin A did not change significantly, but serum follistatin decreased. These responses to ischaemia–reperfusion were absent in TLR4^−/− mice. Pre-treatment with follistatin significantly reduced ischaemia–reperfusion induced myocardial infarction. In mouse neonatal cardiomyocyte cultures, activin A exacerbated, while follistatin reduced, cellular injury after 3 h of hypoxia and 2 h of re-oxygenation. Neither activin A nor follistatin affected hypoxia-reoxygenation induced reactive oxygen species production by these cells. However, activin A reduced cardiomyocyte mitochondrial membrane potential, and follistatin treatment ameliorated the effect of hypoxia-reoxygenation on cardiomyocyte mitochondrial membrane potential. Taken together, these data indicate that myocardial ischaemia–reperfusion, through activation of TLR4 signalling, stimulates local production of activin A, which damages cardiomyocytes independently of increased reactive oxygen species. Blocking activin action by exogenous follistatin reduces this damage.     

Neuroprotection by tempol in a model of iron-induced oxidative stress in acute ischemic stroke

Studies suggest iron exacerbates the damage caused by ischemic stroke. Our aim was to elucidate the effect of iron overload on infarct size after middle cerebral artery occlusion (MCAO) and to evaluate the efficacy of tempol, a superoxide dismutase mimetic, as a neuroprotective agent. Rats were administered iron +/- tempol before MCAO; control rats received saline. The middle cerebral artery was occluded for 24 h, and the size of the resultant infarct was assessed and expressed as the percentage of the hemisphere infracted (%HI). Iron treatment increased infarct size compared with control (51.83 +/- 3.55 vs. 27.56 +/- 3.28%HI iron treated vs. control, P = 0.01); pretreatment with tempol reversed this (51.83 +/- 3.55 vs. 26.09 +/- 9.57%HI iron treated vs. iron + tempol treated, P = 0.02). We hypothesized that reactive oxygen species (ROS) were responsible for the iron-induced damage. We measured ROS generated by exogenous iron in brain and peripheral vasculature from rats that had not undergone MCAO. There was no increase in ROS production in the brain of iron-treated rats or in brain slices incubated with iron citrate. However, ROS generation in carotid arteries incubated with iron citrate was significantly increased. ROS generation from the brain was assessed after MCAO by dihydroethidine staining; there was a dramatic increase in the ROS generation by the brain in the iron-treated rats compared with control 30 min after MCAO. We propose that iron-induced ROS generation in the cerebral vasculature adds to oxidative stress during an ischemic episode after the disruption of the blood-brain barrier.     

Effect of photoperiod on growth of the plants,and sesamin content and CYP81Q1 gene expression in the leaves of sesame (Sesamum indicum L.)

Sesamin is a major lignan constituent of sesame (Sesamum indicum) seed and considered responsible for a number of beneficial human health effects. We previously reported that sesamin is present in sesame leaves, and proposed use of sesame leaves as a sesamin-containing material. This study focused on the possibility that both leaf yield and sesamin content would be increased with increasing photoperiod. Additionally, it was hypothesized that sesamin content would be affected by photoperiod in relation to CYP81Q1 gene expression. We thus investigated the effect of photoperiod on growth and leaf sesamin content in relation to CYP81Q1 gene expression to confirm our hypothesis. Under short-day (SD) condition, increase of leaf area was suppressed due to the phase transition from vegetative to reproductive growth, which resulted in reduction of leaf yield. Under long-day (LD) conditions, vegetative growth was continued, and both leaf area and yield increased as photoperiod increased up to 24 h (continuous light). Sesamin accumulated particularly in the leaves of plants grown under a 24-h photoperiod for 4 weeks. High expression level of the CYP81Q1 gene in those plants indicates that photoperiod-dependent differences in leaf sesamin content correlate with differences in CYP81Q1 gene expression levels. We conclude that cultivation under continuous light enables high-yield production of sesame leaves containing distinctively high levels of sesamin.