Photoacoustic Effect of Near-Infrared Absorbing Organic Molecules via Click Chemistry.

Near-infrared dyes were developed to be contrast agents due to their ability to improve the productivity of photoacoustic (PA) imaging and photothermal therapy (PTT) treatments. During the article, we described in detail the PA and PT effects of a category of organic molecules. F4-TCNQ could potentially cause a red-shift in the peak PA intensity. The results show that the PTT intensity of the near-infrared dyes with phenyl groups were higher than near-infrared dyes with thiophene groups. We also investigated the photodynamic treatment effect of C1b to demonstrate that these dyes are highly desirable in biochemistry. The high photoacoustic intensity of the organic molecules and the good yield of reactive oxygen species could indicate that these dyes have good potential for a wide range of imaging applications. Finally, we embedded the dye (C1b) in a liposomal hydrophobic phospholipid bilayer (C1b⊂L) to facilitate the application of hydrophobic dyes in biomedical applications, which can be absorbed by cells with good compatible and high stability for the imaging of cellular PA.

Inhibition of WNK3 Kinase Signaling Reduces Brain Damage and Accelerates Neurological Recovery After Stroke.

BACKGROUND AND PURPOSE: WNK kinases, including WNK3, and the associated downstream Ste20/SPS1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress responsive 1 (OSR1) kinases, comprise an important signaling cascade that regulates the cation-chloride cotransporters. Ischemia-induced stimulation of the bumetanide-sensitive Na(+)-K(+)-Cl(-) cotransporter (NKCC1) plays an important role in the pathophysiology of experimental stroke, but the mechanism of its regulation in this context is unknown. Here, we investigated the WNK3-SPAK/OSR1 pathway as a regulator of NKCC1 stimulation and their collective role in ischemic brain damage. METHOD: Wild-type WNK3 and WNK3 knockout mice were subjected to ischemic stroke via transient middle cerebral artery occlusion. Infarct volume, brain edema, blood brain barrier damage, white matter demyelination, and neurological deficits were assessed. Total and phosphorylated forms of WNK3 and SPAK/OSR1 were assayed by immunoblotting and immunostaining. In vitro ischemia studies in cultured neurons and immature oligodendrocytes were conducted using the oxygen-glucose deprivation/reoxygenation method. RESULTS: WNK3 knockout mice exhibited significantly decreased infarct volume and axonal demyelination, less cerebral edema, and accelerated neurobehavioral recovery compared with WNK3 wild-type mice subjected to middle cerebral artery occlusion. The neuroprotective phenotypes conferred by WNK3 knockout were associated with a decrease in stimulatory hyperphosphorylations of the SPAK/OSR1 catalytic T-loop and of NKCC1 stimulatory sites Thr(203)/Thr(207)/Thr(212), as well as with decreased cell surface expression of NKCC1. Genetic inhibition of WNK3 or small interfering RNA knockdown of SPAK/OSR1 increased the tolerance of cultured primary neurons and oligodendrocytes to in vitro ischemia. CONCLUSIONS: These data identify a novel role for the WNK3-SPAK/OSR1-NKCC1 signaling pathway in ischemic neuroglial injury and suggest the WNK3-SPAK/OSR1 kinase pathway as a therapeutic target for neuroprotection after ischemic stroke.

Chronic high fat diet induces cardiac hypertrophy and fibrosis in mice.

BACKGROUND: Obesity can cause pathological changes in organs. We determined the effects of chronic high fat diet (HFD) and intermittent fasting, a paradigm providing organ protection, on mouse heart. METHODS: Seven-week old CD1 male mice were randomly assigned to control, HFD and intermittent fasting groups. Control mice had free access to regular diet (RD). RD was provided every other day to mice in the intermittent fasting group. Mice in HFD group had free access to HFD. Their left ventricles were harvested 11 months after they had been on these diet regimens. RESULTS: HFD increased cardiomyocyte cross-section area and fibrosis. HFD decreased active caspase 3, an apoptosis marker, and the ratio of microtubule-associated protein 1A/1B-light chain 3 (LC3) II/LC3I, an autophagy marker. HFD increased the phospho-glycogen synthase kinase-3ß (GSK-3ß) at Ser9, a sign of GSK-3ß inhibition. Nuclear GATA binding protein 4 and yes-associated protein, two GSK-3ß targeting transcription factors that can induce hypertrophy-related gene expression, were increased in HFD-fed mice. Mice on intermittent fasting did not have these changes except for the increased active caspase 3 and decreased ratio of LC3II/LC3I. CONCLUSIONS: These results suggest that chronic HFD induces myocardial hypertrophy and fibrosis, which may be mediated by GSK-3ß inhibition.

Docosahexaenoic acid reduces ER stress and abnormal protein accumulation and improves neuronal function following traumatic brain injury.

In this study, we investigated the development of endoplasmic reticulum (ER) stress after traumatic brain injury (TBI) and the efficacy of post-TBI administration of docosahexaenoic acid (DHA) in reducing ER stress. TBI was induced by cortical contusion injury in Sprague-Dawley rats. Either DHA (16 mg/kg in DMSO) or vehicle DMSO (1 ml/kg) was administered intraperitoneally at 5 min after TBI, followed by a daily dose for 3-21 d. TBI triggered sustained expression of the ER stress marker proteins including phosphorylated eukaryotic initiation factor-2α, activating transcription factor 4, inositol requiring kinase 1, and C/EBP homologous protein in the ipsilateral cortex at 3-21 d after TBI. The prolonged ER stress was accompanied with an accumulation of abnormal ubiquitin aggregates and increased expression of amyloid precursor protein (APP) and phosphorylated tau (p-Tau) in the frontal cortex after TBI. The ER stress marker proteins were colocalized with APP accumulation in the soma. Interestingly, administration of DHA attenuated all ER stress marker proteins and reduced the accumulation of both ubiquitinated proteins and APP/p-Tau proteins. In addition, the DHA-treated animals exhibited early recovery of their sensorimotor function after TBI. In summary, our study demonstrated that TBI induces a prolonged ER stress, which is positively correlated with abnormal APP accumulation. The sustained ER stress may play a role in chronic neuronal damage after TBI. Our findings illustrate that post-TBI administration of DHA has therapeutic potentials in reducing ER stress, abnormal protein accumulation, and neurological deficits.

Glutamate transporter type 3 mediates isoflurane preconditioning-induced acute phase of neuroprotection in mice.

A pre-exposure to isoflurane reduces ischemic brain injury in rodents (isoflurane preconditioning). This neuroprotection has acute and delayed phases. Our previous in vitro studies suggest that the acute phase may involve excitatory amino acid transporters (EAATs). We determine whether this protection involves EAAT3, the major neuronal EAAT. Adult male EAAT3 knockout mice and their wild-type littermates were exposed or were not exposed to 1.5% isoflurane for 30 min. Sixty minutes later, they were subjected to a 90- or 60-min middle cerebral arterial occlusion (MCAO). Their neurological outcomes were evaluated 24h after the MCAO. In another experiment, cerebral cortex was harvested for Western blotting at 30 min after animals were exposed to 1.5% isoflurane for 30 min. Here, we showed that isoflurane reduced brain infarct volumes and improved neurological functions of wild-type mice after a 90-min MCAO. However, isoflurane pre-exposure did not change the neurological outcome of EAAT3 knockout mice no matter whether the MCAO was for 90 min or 60 min. Isoflurane increased phospho-Akt, a survival-promoting protein, in the wild-type mice but not in the EAAT3 knockout mice. The isoflurane-induced neuroprotection in the wild-type mice was abolished by LY294004, an Akt activation inhibitor. LY294004 alone did not affect the neurological outcome of the wild-type or EAAT3 knockout mice after focal brain ischemia. These results suggest that the isoflurane preconditioning-induced acute phase of neuroprotection involves EAAT3. The downstream event includes Akt activation.

Electroacupuncture preconditioning-induced neuroprotection may be mediated by glutamate transporter type 2.

Electroacupuncture has been shown to induce a preconditioning effect in the brain. The mechanisms for this protection are not fully elucidated. We hypothesize that this protection is mediated by excitatory amino acid transporters (EAATs) that have been shown to be neuroprotective. To test this hypothesis, two-month old male Sprague-Dawley rats and EAAT type 3 (EAAT3) knockout mice received or did not receive 30-min electroacupuncture once a day for five consecutive days. They were subjected to a 120-min middle cerebral arterial occlusion (MCAO) at 24h after the last electroacupuncture. Neurological outcome was assessed 2days after the MCAO. Brain tissues were harvested at 24h after the last electroacupuncture for Western blotting. Rats subjected to electroacupuncture at the Baihui acupoint had smaller brain infarct volumes and better neurological deficit scores than control rats. Electroacupuncture increased EAAT type 2 (EAAT2) in the cerebral cortex, tended to increase EAAT3 in the hippocampus, and had no effect on EAAT type 1 expression. Dihydrokainate, an EAAT2 inhibitor, worsened the neurological outcome of rats with electroacupuncture pretreatment. Electroacupuncture pretreatment at the Baihui acupoint increased EAAT2 in the cerebral cortex and improved the neurological outcome of EAAT3 knockout mice. Together, our results suggest that EAAT2 may mediate the electroacupuncture preconditioning-induced neuroprotection.

Chronic intermittent fasting improves cognitive functions and brain structures in mice.

Obesity is a major health issue. Obesity started from teenagers has become a major health concern in recent years. Intermittent fasting increases the life span. However, it is not known whether obesity and intermittent fasting affect brain functions and structures before brain aging. Here, we subjected 7-week old CD-1 wild type male mice to intermittent (alternate-day) fasting or high fat diet (45% caloric supplied by fat) for 11 months. Mice on intermittent fasting had better learning and memory assessed by the Barnes maze and fear conditioning, thicker CA1 pyramidal cell layer, higher expression of drebrin, a dendritic protein, and lower oxidative stress than mice that had free access to regular diet (control mice). Mice fed with high fat diet was obese and with hyperlipidemia. They also had poorer exercise tolerance. However, these obese mice did not present significant learning and memory impairment or changes in brain structures or oxidative stress compared with control mice. These results suggest that intermittent fasting improves brain functions and structures and that high fat diet feeding started early in life does not cause significant changes in brain functions and structures in obese middle-aged animals.

Glutamate transporter type 3 knockout leads to decreased heart rate possibly via parasympathetic mechanism.

Parasympathetic tone is a dominant neural regulator for basal heart rate. Glutamate transporters (EAAT) via their glutamate uptake functions regulate glutamate neurotransmission in the central nervous system. We showed that EAAT type 3 (EAAT3) knockout mice had a slower heart rate than wild-type mice when they were anesthetized. We design this study to determine whether non-anesthetized EAAT3 knockout mice have a slower heart rate and, if so, what may be the mechanism for this effect. Young adult EAAT3 knockout mice had slower heart rates than those of their littermate wild-type mice no matter whether they were awake or anesthetized. This difference was abolished by atropine, a parasympatholytic drug. Carbamylcholine chloride, a parasympathomimetic drug, equally effectively reduced the heart rates of wild-type and EAAT3 knockout mice. Positive immunostaining for EAAT3 was found in the area of nuclei deriving fibers for vagus nerve. There was no positive staining for the EAATs in the sinoatrial node. These results suggest that EAAT3 knockout mice have a slower heart rate at rest. This effect may be caused by an increased parasympathetic tone possibly due to increased glutamate neurotransmission in the central nervous system. These findings indicate that regulation of heart rate, a vital sign, is one of the EAAT biological functions.

Isoflurane postconditioning reduces ischemia-induced nuclear factor-κB activation and interleukin 1ß production to provide neuroprotection in rats and mice.

Application of isoflurane, a volatile anesthetic, after brain ischemia can reduce ischemic brain injury in rodents (isoflurane postconditioning). This study is designed to determine whether isoflurane postconditioning improves long-term neurological outcome after focal brain ischemia and whether this protection is mediated by attenuating neuroinflammation. Adult male Sprague-Dawley rats were subjected to a 90-min middle cerebral arterial occlusion (MCAO). Isoflurane postconditioning was performed by exposing rats to 2% isoflurane for 60min immediately after the MCAO. Isoflurane postconditioning reduced brain infarct volumes, apoptotic cells in the ischemic penumbral brain tissues and neurological deficits of rats at 4weeks after the MCAO. Isoflurane postconditioning reduced brain ischemia/reperfusion-induced nuclear transcription factor (NF)-κB (NF-κB) activation as well as interleukin 1ß (IL-1ß) and interleukin-6 production in the ischemic penumbral brain tissues at 24h after the MCAO. IL-1ß deficient mice had smaller brain infarct volumes and better neurological functions than wild-type mice at 24h after a 90-min focal brain ischemia. Isoflurane posttreatment failed to induce neuroprotection in the IL-1ß deficient mice. Our results suggest that isoflurane postconditioning improved long-term neurological outcome after transient focal brain ischemia. This protection may be mediated by inhibiting NF-κB activation and the production of the proinflammatory cytokine IL-1ß.

Isoflurane induces learning impairment that is mediated by interleukin 1ß in rodents.

Postoperative cognitive decline is a clinical syndrome. Volatile anesthetics are commonly used during surgery. It is conceivable that volatile anesthetics may contribute to postoperative cognitive decline. Isoflurane can impair cognitive functions of animals under certain conditions. However, the mechanisms for this impairment are not clear. Here, male 18-month old Fisher 344 rats or 10-week old mice were exposed to 1.2 or 1.4% isoflurane for 2 h. Our studies showed that isoflurane impaired the cognitive functions of the rats in Barnes maze. Isoflurane-exposed rats had reduced freezing behavior during the training sessions in the fear conditioning test. This isoflurane effect was attenuated by lidocaine, a local anesthetic with anti-inflammatory property. Rats that had training sessions and were exposed to isoflurane 30 min later had freezing behavior similar to that of control animals. Isoflurane increased the expression of interleukin 1ß (IL-1ß), interleukin-6 and activated caspase 3 in the hippocampus of the 18-month old rats. IL-1ß positive staining was co-localized with that of NeuN, a neuronal marker. The increase of IL-1ß and activated caspase 3 but not interleukin-6 was attenuated by lidocaine. Isoflurane also impaired the cognitive functions of 10-week old C57BL/6J mice and increased IL-1ß in their hippocampi. However, isoflurane did not affect the cognitive functions of IL-1ß deficient mice. Our results suggest that isoflurane impairs the learning but may not affect the recall of the aged rats. IL-1ß may play an important role in this isoflurane effect.

Contribution of microRNA-203 to the isoflurane preconditioning-induced neuroprotection.

A prior exposure to isoflurane, a common volatile anesthetic, provides neuroprotection (isoflurane preconditioning). To determine the role of microRNAs in this protection, we performed microRNA array assay on cerebral cortex harvested from rats exposed to isoflurane or isoflurane-exposed rat B35 neuron-like cells. We showed that isoflurane significantly increased microRNA-203 expression in B35 neuron-like cells. The microRNA-203 expression in rat cerebral cortex also trended to increase after isoflurane exposure. Over-expression of microRNA-203 increased the tolerance of B35 cells to oxygen-glucose deprivation and the expression of phospho-Akt, a protein kinase that promotes cell survival. Isoflurane preconditioning also reduced the injury of these cells after oxygen-glucose deprivation. These results suggest that isoflurane preconditioning-induced neuroprotection may involve increased expression of microRNA-203. This finding provides the initial evidence that micoRNA-203 is a target for isoflurane in the brain.

Delayed treatment with lidocaine reduces mouse microglial cell injury and cytokine production after stimulation with lipopolysaccharide and interferon γ.

BACKGROUND: Neuroinflammation is an important pathological process for almost all acquired neurological diseases. Microglial cells play a critical role in neuroinflammation. We determined whether lidocaine, a local anesthetic with anti-inflammatory property, protected microglial cells and attenuated cytokine production from activated microglial cells. METHODS: Mouse microglial cultures were incubated with or without 1 µg/mL lipopolysaccharide and 10 U/mL interferon γ (IFNγ) for 24 hours in the presence or absence of lidocaine for 1 hour started at 2, 3, or 4 hours after the onset of lipopolysaccharide and IFNγ stimulation. Lactate dehydrogenase release and cytokine production were determined after the cells were stimulated by lipopolysaccharide and IFNγ for 24 hours. RESULTS: Lidocaine dose-dependently reduced lipopolysaccharide and IFNγ-induced microglial cell injury as measured by lactate dehydrogenase release. This effect was apparent with lidocaine at 2 µg/mL (30.3% ± 5.8% and 23.1% ± 9.7%, respectively, for stimulation alone and the stimulation in the presence of lidocaine, n = 18, P = 0.025). Lidocaine applied at 2, 3, or 4 hours after the onset of lipopolysaccharide and IFNγ stimulation reduced the cell injury. This lidocaine effect was not affected by the mitochondrial K(ATP) channel inhibitor 5-hydroxydecanoate. Similar to lidocaine, QX314, a permanently charged lidocaine analog that usually does not permeate through the plasma membrane, reduced lipopolysaccharide and IFNγ-induced microglial cell injury. QX314 also attenuated the stimulation-induced interleukin-1ß production. CONCLUSIONS: Delayed treatment with lidocaine protects microglial cells and reduces cytokine production from these cells. These effects may involve action site(s) on the cell surface.

Isoflurane preconditioning reduces oxygen-glucose deprivation-induced neuronal injury via B-cell lymphoma 2 protein.

A brief exposure to isoflurane prior to brain ischemia reduces ischemic brain injury in rodents. Here we showed that exposure of rat cerebral cortical neuronal cultures to 2% isoflurane for 30 min at 24 h before a 2-h oxygen-glucose deprivation (OGD) reduced the OGD-induced cell injury. This effect was abolished by HA14-1, a selective inhibitor of B-cell lymphoma 2 (Bcl-2) protein. Bcl-2 is well-known for its anti-apoptotic property. HA14-1 alone did not change OGD-induced cell injury. OGD reduced the expression of Bcl-2 in these neurons. This reduction was attenuated by isoflurane preconditioning. These results suggest that isoflurane preconditioning-induced neuroprotection is mediated by Bcl-2.

Inhibition of isoflurane-induced increase of cell-surface redistribution and activity of glutamate transporter type 3 by serine 465 sequence-specific peptides.

Excitatory amino acid transporters (EAAT) transport glutamate into cells to regulate glutamate neurotransmission and to maintain nontoxic extracellular glutamate levels for neurons. We showed previously that the commonly used volatile anesthetic isoflurane increases the transporting activity of EAAT3, the major neuronal EAAT. This effect requires a protein kinase C (PKC) α-mediated and S465-dependent EAAT3 redistribution to the plasma membrane. Thus, we hypothesize that specific peptides can be designed to block this effect. We conjugated a 10-amino acid synthetic peptide with a sequence identical to that of EAAT3 around the S465 to a peptide that can facilitate permeation of the plasma membrane. This fusion peptide inhibited the isoflurane-increased EAAT3 activity and redistribution to the plasma membrane in C6 cells and hippocampus. It did not affect the basal EAAT3 activity. This peptide also attenuated isoflurane-induced increase of PKCα in the immunoprecipitates produced by an anti-EAAT3 antibody. A scrambled peptide that has the same amino acid composition as the S465 sequence-specific peptide but has a random sequence did not change the effects of isoflurane on EAAT3. The S465 sequence-specific peptide, but not the scrambled peptide, is a good PKCα substrate in in vitro assay. These peptides did not affect cell viability. These results, along with our previous findings, strongly suggest that PKCα interacts with EAAT3 to regulate its functions. The S465 sequence-specific peptide may interrupt this interaction and is an effective inhibitor for the regulation of EAAT3 activity and trafficking by PKCα and isoflurane.

Glutamate transporter type 3 knockout reduces brain tolerance to focal brain ischemia in mice.

Excitatory amino-acid transporters (EAATs) transport glutamate into cells under physiologic conditions. Excitatory amino-acid transporter type 3 (EAAT3) is the major neuronal EAAT and also uptakes cysteine, the rate-limiting substrate for synthesis of glutathione. Thus, we hypothesize that EAAT3 contributes to providing brain ischemic tolerance. Male 8-week-old EAAT3 knockout mice on CD-1 mouse gene background and wild-type CD-1 mice were subjected to right middle cerebral artery occlusion for 90 minutes. Their brain infarct volumes, neurologic functions, and brain levels of glutathione, nitrotyrosine, and 4-hydroxy-2-nonenal (HNE) were evaluated. The EAAT3 knockout mice had bigger brain infarct volumes and worse neurologic deficit scores and motor coordination functions than did wild-type mice, no matter whether these neurologic outcome parameters were evaluated at 24 hours or at 4 weeks after brain ischemia. The EAAT3 knockout mice contained higher levels of HNE in the ischemic penumbral cortex and in the nonischemic cerebral cortex than did wild-type mice. Glutathione levels in the ischemic and nonischemic cortices of EAAT3 knockout mice tended to be lower than those of wild-type mice. Our results suggest that EAAT3 is important in limiting ischemic brain injury after focal brain ischemia. This effect may involve attenuating brain oxidative stress.

Delayed treatment with isoflurane attenuates lipopolysaccharide and interferon gamma-induced activation and injury of mouse microglial cells.

BACKGROUND: Isoflurane pretreatment can induce protection against lipopolysaccharide and interferon gamma (IFNgamma)-induced injury and activation of mouse microglial cells. This study's goal was to determine whether delayed isoflurane treatment is protective. METHODS: Mouse microglial cells were exposed to various concentrations of isoflurane for 1 h immediately after the initiation of lipopolysaccharide (10 or 1000 ng/ml) and IFNgamma (10 U/ml) stimulation or to 2% isoflurane for 1 h at various times after initiation of the stimulation. Nitrite production, lactate dehydrogenase release, and cell viability measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were assessed after stimulation with lipopolysaccharide and IFNgamma for 24 h. Inducible nitric oxide synthase (iNOS) protein expression was quantified by Western blotting. The iNOS expression in mouse brain was also studied. RESULTS: Isoflurane applied 0 and 2 h after the initiation of lipopolysaccharide and IFNgamma stimulation improved cell viability. Isoflurane at 2%, but not at 1 or 3%, reduced the lipopolysaccharide and IFNgamma-induced nitrite production and decreased cell viability. Aminoguanidine, an iNOS inhibitor, also attenuated this decreased cell viability. Chelerythrine and bisindolylmalemide IX, protein kinase C inhibitors, abolished isoflurane effects on cell viability and iNOS expression after lipopolysaccharide and IFNgamma application. Isoflurane also decreased lipopolysaccharide-induced iNOS expression in mouse brain. Late isoflurane application to microglial cells reduced lipopolysaccharide and IFNgamma-induced lactate dehydrogenase release that was not inhibited by aminoguanidine. CONCLUSIONS: These results suggest that delayed isoflurane treatment can reduce lipopolysaccharide and IFNgamma-induced activation and injury of microglial cells. These effects may be mediated by protein kinase C.

Isoflurane induces a postconditioning effect on bovine pulmonary arterial endothelial cells exposed to oxygen-glucose deprivation.

Application of volatile anesthetics during the onset of reperfusion reduced ischemia-induced cardiac and brain injury (anesthetic postconditioning). This study was designed to evaluate whether volatile anesthetics induced a postconditioning effect in endothelial cells. Bovine pulmonary arterial endothelial cell (BPAEC) cultures were exposed to oxygen-glucose deprivation, a condition to simulate ischemia in vitro, for 3 h. The volatile anesthetics isoflurane and desflurane were applied during the early phase of simulated reperfusion. Cell injury was quantified by lactate dehydrogenase (LDH) release and flow cytometrical measurement after annexin V and propidium iodide staining. Oxygen-glucose deprivation and the subsequent simulated reperfusion increased LDH release and annexin V-positive staining cells, a characteristic of cell apoptosis. Posttreatment with isoflurane, but not desflurane, reduced this cell injury. This protection was apparent even when 2% isoflurane was applied at 60 min after the onset of reperfusion. The isoflurane postconditioning effect was abolished by glybenclamide, a general ATP sensitive K(+) (K(ATP)) channel blocker, 5-hydroxydecanoate, a mitochondrial K(ATP) channel blocker, and chelerythrine, a protein kinase C inhibitor. Diazoxide, a mitochondrial K(ATP) channel activator, applied at the onset of reperfusion also decreased oxygen-glucose deprivation-induced endothelial cell injury. This diazoxide-induced protection was abolished by chelerythrine and 5-hydroxydecanoate. We conclude that isoflurane induced a postconditioning effect in BPAEC. The effective time window of isoflurane postconditioning was from 0 to 60 min after the onset of reperfusion. This isoflurane postconditioning effect may be mediated by mitochondrial K(ATP) channels and PKC. PKC may be downstream of mitochondrial K(ATP) channels for this isoflurane effect.

Protective effects of TREK-1 against oxidative injury induced by SNP and H2O2.

AIM: TREK-1 (TWIK-related K+ channel-1) is a 2-pore-domain K+ channel subtype. The present study investigated the role of TREK-1 in cell death induced by oxidative stress. METHODS: The cell viability of wild-type Chinese hamster ovary (CHO) and TREK-1-transfected CHO cells (TREK-1/CHO cells) was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in the presence of sodium nitroprusside (SNP) or hydrogen peroxide (H2O2). Apoptosis of wild-type CHO and TREK-1/CHO cells was detected using Hoechst33342 staining. RESULTS: Both SNP and H2O2 caused dose- and time-dependent growth inhibition of wild-type CHO and TREK-1/ CHO cells. Following a 12 h exposure to SNP, the 50% inhibition (IC(50)) values for wild-type CHO and TREK-1/CHO cells were calculated as 0.69 mmol/L and 1.14 mmol/L, respectively. The IC(50) values were 0.07 mmol/L and 0.09 mmol/L in H2O2-treated wild-type CHO and TREK-1/CHO cells, respectively, following 12 h exposure to H2O2. Moreover, SNP/H2O2 induced less apoptosis in TREK-1/ CHO cells than that in wild-type CHO cells (P<0.05). CONCLUSION: The results demonstrated that TREK-1 played a protective role against oxidative injury.

Postconditioning with isoflurane reduced ischemia-induced brain injury in rats.

BACKGROUND: Preexposure of brain to isoflurane, a commonly used anesthetic, induces ischemic tolerance. This phenomenon is called isoflurane preconditioning. However, it is not known whether isoflurane application after ischemia provides neuroprotection. METHODS: Corticostriatal slices (400 microm) freshly prepared from adult male Sprague-Dawley rats were subjected to a 15-min oxygen-glucose deprivation (OGD; to simulate ischemia in vitro). Isoflurane was applied after OGD. Brain slices were harvested 2 h after OGD for measuring 2,3,5-triphenyltetrazolium chloride (TTC) conversion to quantify cell injury. Adult male Sprague-Dawley rats were also subjected to middle cerebral arterial occlusion for 90 min and then treated with or without 2% isoflurane for 60 min started at the onset of reperfusion. The infarct volumes, neurologic deficit scores, and performance on rotarod were evaluated at 24 h after the onset of reperfusion. RESULTS: Isoflurane applied immediately after the 15-min OGD for 30 min dose-dependently reversed the OGD-induced decrease of TTC conversion. The TTC conversion was 34 +/- 16% and 58 +/- 28% of the control, respectively, for OGD alone and OGD plus 2% isoflurane (P < 0.05, n = 12). Application of 2% isoflurane for 30 min started at 10 min after the OGD also reduced the OGD-decreased TTC conversion. The presence of 0.3 microm glibenclamide, a general adenosine 5'-triphosphate-sensitive potassium channel blocker, or 500 microm 5-hydroxydecanoic acid, a mitochondrial adenosine 5'-triphosphate-sensitive potassium channel blocker, during the application of 2% isoflurane abolished the isoflurane preservation of TTC conversion. Application of isoflurane during reperfusion also improved neurologic outcome after brain ischemia. CONCLUSIONS: The results suggest that isoflurane administrated after OGD or brain ischemia provides neuroprotection. Mitochondrial adenosine 5'-triphosphate-sensitive potassium channels may be involved in this protection.

Isoflurane preconditioning increases B-cell lymphoma-2 expression and reduces cytochrome c release from the mitochondria in the ischemic penumbra of rat brain.

We and others have shown that prior exposure to the volatile anesthetic isoflurane induces ischemic tolerance in the brain. Our results also suggest that isoflurane preconditioning reduces cell apoptosis in the penumbral region of rat brain. We designed this study to determine whether isoflurane preconditioning decreased mitochondria-dependent cell apoptosis. Adult male Sprague-Dawley rats were exposed to or not exposed to 2% isoflurane for 30 min at 24 h before the permanent middle cerebral arterial occlusion. Western blotting was used to quantify protein expression in the cytosolic and mitochondrial fractions of non-ischemic brain cortex and brain cortex in the ischemic core and penumbra. Isoflurane preconditioning significantly decreased the infarct volume of cerebral cortex and improved neurological outcome. Isoflurane increased the expression of the antiapoptotic B-cell lymphoma-2 (Bcl-2) proteins in the cerebral cortex of rats without brain ischemia. Rats preconditioned with isoflurane before brain ischemia had increased Bcl-2 expression in the penumbra. Isoflurane preconditioning reduced the release of cytochrome c from the mitochondria and the activation of caspase 3 in the penumbra. However, isoflurane preconditioning did not alter the translocation of Bid and Bax from the cytosol to the mitochondria, identified mechanisms for Bcl-2 to block the release of cytochrome c from the mitochondria. Our results suggest that isoflurane preconditioning increases Bcl-2 expression to block the release of cytochrome c from the mitochondria to decrease the cell apoptosis in the penumbra.