Effects of ATP-sensitive potassium channel activators diazoxide and BMS-191095 on membrane potential and reactive oxygen species production in isolated piglet mitochondria

Mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel openers protect the piglet brain against ischemic stress. Effects of mitoK(ATP) channel agonists on isolated mitochondria, however, have not been directly examined. We investigated the effects of K(ATP) channel openers and blockers on membrane potential and on the production of reactive oxygen species (ROS) in isolated piglet mitochondria. Diazoxide and BMS-191095, putative selective openers of mitoK(ATP), decreased the mitochondrial membrane potential (delta psi(m)). On a molar basis, diazoxide was less effective than BMS-191095. In contrast, diazoxide but not BMS-191095 increased ROS production by mitochondria. Since diazoxide also inhibits succinate dehydrogenase (SDH), we examined the effects of 3-nitropropionic acid (3-NPA), an inhibitor of SDH. 3-NPA failed to change the delta psi(m) but increased ROS production. Inhibitors of K(ATP) channels did not affect resting delta psi(m) or ROS production, but glibenclamide and 5-hydroxydecanoate (5-HD) blocked effects of diazoxide and BMS-191095 on delta psi(m) and diazoxide effects on ROS production. We conclude that BMS-191095 has selective effects on mitoK(ATP) channels while diazoxide also increases ROS production probably via inhibition of SDH.     

The mitochondrial K(ATP) channel opener BMS-191095 induces neuronal preconditioning

BMS-191095, reportedly a selective mitoK(ATP) channel opener which is free from the known side effects of the prototype mitoK(ATP) channel opener diazoxide, induced acute and delayed preconditioning against glutamate excitotoxicity and delayed preconditioning against oxygen-glucose deprivation in primary cultures of rat cortical neurons. BMS-191095 dose dependently depolarized the mitochondria, increased the phosphorylation of PKC isoforms, but had no detectable effects on the activation of MAP kinases and did not influence the expressions of HSP70 and Mn-SOD. In BMS-191095-preconditioned neurons the glutamate-induced free-radical production was abolished. Our data give the first evidence that selective opening of mitoK(ATP) channels with BMS-191095 leads to remarkable neuroprotection via mechanisms that involve mitochondrial depolarization, PKC activation and attenuated free radical production during neuronal stress.     

开放线粒体ATP敏感性钾通道对大鼠局灶性脑缺血再灌注损伤的保护作用

目的　研究开放线粒体 ATP敏感性钾通道对大鼠局灶性脑缺血再灌注损伤的作用。方法　采用线栓法建立大鼠局灶性脑缺血再灌注损伤模型 ,将 38只大鼠随机分成 4组 :假手术组 (S)、缺血组 (I)、缺血 二氮嗪组 (D)、缺血 二氮嗪 5 - HD组 (DH)。观察各组脑梗死体积和线粒体标志酶活性变化。结果　与 I组比较 ,D组脑梗死体积明显减少 (自 2 3.5 7± 7.83%减至 8.16± 3.81% ) ,线粒体标志酶活性明显增高 ,差异具有显著性 (P<0 .0 5 ) ,DH组与 I组比较无明显差异 (P>0 .0 5 )。结论　二氮嗪对大鼠局灶性脑缺血再灌注损伤具有保护作用 ,其机制与开放线粒体 ATP敏感性钾通道 ,维护线粒体功能有关     

ROS-independent preconditioning in neurons via activation of mitoK(ATP) channels by BMS-191095.

Previously, we have shown that the selective mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel opener BMS-191095 (BMS) induces neuronal preconditioning (PC); however, the exact mechanism of BMS-induced neuroprotection remains unclear. In this study, we have identified key components of the cascade resulting in delayed neuronal PC with BMS using isolated rat brain mitochondria and primary cultures of rat cortical neurons. BMS depolarized isolated mitochondria without an increase in reactive oxygen species (ROS) generation and induced rapid phosphorylation of Akt and glycogen synthase kinase-3beta. Long-term (3 days) treatment of neurons with BMS resulted in sustained mitochondrial depolarization, decreased basal ROS generation, and elevated ATP levels. This treatment also elicited almost complete protection against glutamate excitotoxicity, which could be abolished using the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin, but not with the superoxide dismutase (SOD) mimetic M40401. Long-term BMS treatment induced a PI3K-dependent increase in the expression and activity of catalase without affecting manganese SOD and copper/zinc-dependent SOD. Finally, the catalase inhibitor 3-aminotriazole dose-dependently antagonized the neuroprotective effect of BMS-induced PC. In summary, BMS depolarizes mitochondria without ROS generation, activates the PI3K-Akt pathway, improves ATP content, and increases catalase expression. These mechanisms appear to play important roles in the neuroprotective effect of BMS.     

Diazoxide preconditioning protects against neuronal cell death by attenuation of oxidative stress upon glutamate stimulation

We examined the effects of diazoxide, the putative mitochondrial adenosine triphosphate-sensitive potassium (mitoK(ATP)) channel opener, against glutamate excitotoxicity in primary cultures of rat cortical neurons. Cells were treated with diazoxide for 24 hr and then exposed to 200 microM glutamate. Cell viability was measured 24 hr after glutamate exposure. We found that treatment 24 hr before glutamate exposure with 250 and 500 microM diazoxide but not with another mitoK(ATP) channel opener, nicorandil, increased neuronal viability from 54 +/- 2% to 84 +/- 2% and 92 +/- 3%, respectively (n = 25-40). These effects were not inhibited by the putative mitoK(ATP) channel blocker 5-hydroxydecanoic acid. Diazoxide application increased production of reactive oxygen species (ROS) and coapplication of M40401, a superoxide dismutase mimetic, prevented delayed preconditioning. The 24 hr preconditioned neurons showed significantly reduced ROS production upon glutamate stimulation compared to that in untreated cells. These results suggest that diazoxide induces delayed preconditioning in cultured cortical neurons via increased ROS production and attenuation of oxidative stress upon glutamate stimulation.     

Reactive oxygen species mediate the neuroprotection conferred by a mitochondrial ATP-sensitive potassium channel opener during ischemia in the rat hippocampal slice

Reactive oxygen species (ROS) are known to mediate the protection conferred by the opening of mitochondrial ATP-sensitive potassium channels (mitoK(ATP)) during ischemia in heart, but this has not been demonstrated in brain. The present study examined whether ROS mediate the neuroprotection conferred by a mitoK(ATP) opener during ischemia in rat hippocampal slices. Ischemia was simulated by oxygen and glucose deprivation. The direct current potential and population spike were recorded in the stratum pyramidale of the CA1 region, and lactate dehydrogenase (LDH) efflux into the medium was assayed. ROS generation was measured spectrophotofluorometrically. Pretreatment of slices with diazoxide (DIA, 300 microM), a mitoK(ATP) opener, (i) prolonged the latency to ischemic depolarization and decreased its amplitude, (ii) delayed the onset of population spike disappearance and enhanced its recovery after reperfusion, (iii) decreased LDH efflux and (iv) increased ROS levels. The effects induced by DIA were attenuated by 5-hydroxydecanoic acid (200 microM), a mitoK(ATP) blocker. Pretreatment with N-2-mercaptopropionyl glycine (MPG, 500 microM), a ROS scavenger, also abrogated the effects induced by DIA, while treatment with MPG alone had no effect during normoxia and ischemia. These results indicate that ROS participate in the neuroprotection conferred by a mitoK(ATP) opener during ischemia.     

Effect of nicorandil on infarct volume and marker enzyme activity in mitochondria of rats with cerebral ischemia/ reperfusion injury

BACKGROUND: Recent studies have suggested that mitochondrial ATP-sensitive K channel openers could reduce myocardium infarct size, and protect the function of the mitochondria.OBJECTIVE: To investigate the changes of cerebral infarction volume and the activity of marker enzymes in brain mitochondria of rats given the ATP-sensitive K channel opener, nicorandil, before focal cerebral ischemia/reperfusion (I/R).DESIGN, TIME AND SETTING: Randomized, controlled animal experiment, completed at the Brain Scientific Research Center of the Affiliated Hospital of Qingdao University from July to November 2007.MATERIALS: Sixty healthy male Wistar rats weighing 280-300g. Nicorandil, 5-hydroxydecanoate (5-HD) and cytochrome C were purchased from Sigma in the USA. Standard malondialdehyde (MDA) and protein were purchased from Nanjing Jiancheng Biotechnology Institute.METHODS: Sixty rats were randomly divided into a sham operation group, a middle cerebral artery occlusion (MCAO) group, a nicorandil group and a nicorandil 5-HD group. MCAO for 2 hours was performed in the MCAO group, nicorandil group and nicorandil 5-HD group. A total of 5mL saline were given to the MCAO group before MCAO. The nicorandil group was injected with the ATP-sensitive K channel opener nicorandil 10mg/kg intraperitoneally 30 minutes before MCAO. The nicorandil 5-HD group was injected with 5-HD 10mg/kg intravenously 15 minutes before the same treatment as the nicorandil group.MAIN OUTCOME MEASURES: Infarct volume by total brain slice calculation, activities of succinate dehydrogenase (SDH) and cytochrome oxidase (CO), and content of MDA were observed at 22 hours of reperfusion after 2 hours MCAO.RESULTS: Sixty rats were included in the final analysis, without any loss. (1) Infarct volume: compared with the MCAO group and nicorandil 5-HD group, the percentage of infarct volume was significantly decreased in the nicorandil group (P<0.01). (2) The content of MDA, expression of SDH and CO in brain: the expressions of SDH and CO in the sham operation group were significantly lower than those in the MCAO, nicorandil and nicorandil 5-HD groups (P<0.01). The expressions of SDH and CO in the nicorandil group were significantly higher than those in the MCAO and nicorandil 5-HD groups (P<0.05). The content of MDA in the brain of the nicorandil group was significantly lower than those in the MCAO and nicorandil 5-HD groups (P<0.01).CONCLUSION: Nicorandil can significantly reduce the infarct volume in a rat MCAO model, increase the activity of the mitochondria and protect against cerebral I/R injury.     

Investigation of the subunit composition and the pharmacology of the mitochondrial ATP-dependent K+ channel in the brain

Selective activation of mitoK(ATP) channels can protect the brain or cultured neurons against a variety of anoxic or metabolic challenges. However, little is known about the subunit composition or functional regulation of the channel itself. In the present study, we sought to characterize the mitoK(ATP) channel in the mouse brain using overlapping approaches. First, we determined that mitochondria contain the pore-forming Kir6.1 and Kir6.2 subunits with Western blotting, immunogold electron microscopy and the identification of mitochondrial transport sequences. In contrast, we found no evidence for the presence of either known sulfonylurea receptors (SUR1 or SUR2) in the mitochondria. However, the ATP-dependent K (K(ATP)) channel inhibitor glibenclamide specifically binds to mitochondria in both neurons and astrocytes, and small molecular weight SUR2-like proteins were concentrated in mitochondria. In addition to mice, similar results were found in rats and pigs. Second, live respiring mitochondria were stained with the membrane potential sensitive dye MitoFluorRed and visualized by confocal microscopy. We investigated the effects of pharmacological closing and opening of the channel with glibenclamide and the specific mitoK(ATP) openers diazoxide and BMS-191095. Closing of the channel inhibited the energization of the mitochondria, which was reversed by the application of the mitoK(ATP) openers. We also found that blocking mitochondrial peroxynitrite formation with FP15 has a similar effect to blocking the mitoK(ATP) channels. We conclude that brain mitochondria contain functional K(ATP) channels. The pore-forming subunit of the channel can be either Kir6.1 or Kir6.2, and the SUR subunit may be a SUR2 splice variant or a similar protein.     

Cromakalin pretreatment affects mitochondrial structure and function in a rat model of ischemia/reperfusion injury

BACKGROUND: Mitochondrial structural changes and energy dysmetabolism frequently occur subsequent to cerebral ischemia. Adenosine triphosphate (ATP)-sensitive potassium channel openers exhibit protective effects on cerebral ischemia/reperfusion injury. OBJECTIVE: To validate the effects of cromakalin on mitochondrial structure and function in ischemic penumbra brain tissue in a rat model of middle cerebral artery occlusion (MCAO). DESIGN, TIME AND SETTING: The present single-factor analysis of variance, randomized, controlled, animal experiment was performed at the Institute of Brain Science, Affiliated Hospital of Qingdao University Medical College between October 2007 and March 2008. MATERIALS: Forty male, Wistar rats were randomly divided into four groups, with 10 rats per group: sham-operated, MCAO, MCAO ATP-sensitive potassium channel opener (cromakalin), and MCAO eromakalin ATP-sensitive potassium channel blocking agent (glibenclamide). METHODS: Focal cerebral ischemia/reperfusion injury was induced by MCAO in all groups except the sham-operated group. The MCAO cromakalin group was administered 10 mg/kg cromakalin (i.p.) prior to MCAO induction. The MCAO cromakalin glibenclamide group received an injection of 10 mg/kg cromakalin (i.v.), and subsequently an injection of 10 mg/kg cromakalin (i.p.) prior to MCAO induction. MAIN OUTCOME MEASURES: At 24 hours after cerebral ischemia/reperfusion injury, cellular apoptosis was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling technique. Cytochrome C expression was measured by immunohistochemistry. In addition, mitochondrial swelling, membrane fluidity, membrane phospholipid and malonaldehyde (MDA) contents, as well as Na -K -ATPase, Ca2 -ATPase, and superoxide dismutase (SOD) activities were determined. RESULTS: Compared with the sham-operated group, the three ischemia groups exhibited significantly elevated mitochondrial MDA content, reduced membrane phospholipid and ATP contents, down-regulated membrane fluidity, and reduced Na -K -ATPase, Ca2 -ATPase, and SOD activities (P < 0.05-0.01 ). In the MCAO eromakalin group, the number of apoptotic cells decreased, and cytochrome C expression, as well as MDA content, were reduced. However, ATP content and Na -K -ATPase, Ca2 -ATPase, and SOD activities significantly increased compared with the MCAO group (P < 0.05-0.01 ). Glibenclamide noticeably antagonized cromakalin protection of mitochondria.CONCLUSION: Pretreatment with the ATP-sensitive potassium channel opener cromakalin increased mitochondrial Na -K -ATPase, Ca2 -ATPase, and SOD activities, decreased neuronal apoptosis, and inhibited cytochrome C expression following MCAO.     

Diazoxide preconditioning attenuates global cerebral ischemia-induced blood-brain barrier permeability

Brain edema formation due to blood-brain barrier (BBB) disruption is a major consequence of cerebral ischemia. Previously, we demonstrated that targeting mitochondrial ATP-sensitive potassium channels (mitoK(ATP)) protects neuronal tissues in vivo and in vitro, however, the effects of mitoK(ATP) openers on cerebral endothelial cells and on BBB functions have never been examined. We investigated the effects of mitoK(ATP) channel opener diazoxide on BBB functions during ischemia/reperfusion injury (I/R). Rats were treated with 6, 20 or 40 mg/kg diazoxide ip for 3 days then exposed to global cerebral ischemia for 30 min. BBB permeability was assessed by administering Evan's-blue (EB) and Na-fluorescein (NaF) at the beginning of the 30 min reperfusion. I/R increased BBB permeability for the large molecular weight EB (ng/mg) in the cortex (control: 146 +/- 12, n = 7; I/R: 1049 +/- 152, n = 11) which was significantly attenuated in diazoxide-treated rats (575 +/- 99, n = 9; 582 +/- 104, n = 8; 20 and 40 mg/kg doses). Diazoxide pretreatment also significantly inhibited the extravasation of the low molecular weight NaF. Edema formation in the cortex was also decreased after diazoxide pretreatment. In cultured cerebral endothelial cells, diazoxide depolarized the mitochondrial membrane, suggesting a direct diazoxide effect on the endothelial mitochondria. Our results demonstrate that preconditioning of cerebral endothelium with diazoxide protects the BBB against ischemic stress.     

Redox properties of the adenoside triphosphate-sensitive K+ channel in brain mitochondria

Brain mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) opening by diazoxide protects against ischemic damage and excitotoxic cell death. Here we studied the redox properties of brain mitoK(ATP) . MitoK(ATP) activation during excitotoxicity in cultured cerebellar granule neurons prevented the accumulation of reactive oxygen species (ROS) and cell death. Furthermore, mitoK(ATP) activation in isolated brain mitochondria significantly prevented H2O2 release by these organelles but did not change Ca2+ accumulation capacity. Interestingly, the activity of mitoK(ATP) was highly dependent on redox state. The thiol reductant mercaptopropionylglycine prevented mitoK(ATP) activity, whereas exogenous ROS activated the channel. In addition, the use of mitochondrial substrates that led to higher levels of endogenous mitochondrial ROS release closely correlated with enhanced K+ transport activity through mitoK(ATP). Altogether, our results indicate that brain mitoK(ATP) is a redox-sensitive channel that controls mitochondrial ROS release.     

Nitrite does not provide additional protection to thrombolysis in a rat model of stroke with delayed reperfusion.

An adjuvant therapy to prolong the therapeutic window for stroke patients is urgently needed. This randomized, blinded, placebo-controlled study investigated adjuvant intravenous sodium nitrite with recombinant tissue plasminogen activator (rtPA) in middle cerebral artery occlusion (MCAO) with 6 and 2 h of ischemia followed by reperfusion in Sprague-Dawley rats (n=59). Quantitative diffusion, T(1)-, T(2)-weighted, and semiquantitative perfusion imaging were performed before and after reperfusion and at 48 h after ischemia to determine the spatiotemporal evolution of stroke. After 48 h animals were killed and examined to evaluate infarct size and evidence of hemorrhagic transformation. Factor VIII immunostaining was performed to assess vessel morphology. Nitrite treatment (6 h group: 37.5 micromol for more than 90 mins; 2 h group: 26.25 and 1.75 micromol for more than 60 mins) did not reduce infarct volume 48 h after MCAO compared with saline-treated placebo groups after 6 or 2 h of MCAO. Stroke progression from baseline to 48 h, based on the apparent diffusion coefficient and relative cerebral blood flow deficits before and after reperfusion and T(2)-weighted hyperintensity at 48 h, did not differ between treated and control animals. These results suggest that nitrite is not a protective adjuvant therapy to delayed rtPA administration after ischemic stroke in rats.     

Adenosine A(1) receptor antagonist and mitochondrial ATP-sensitive potassium channel blocker attenuate the tolerance to focal cerebral ischemia in rats.

Involvement of adenosine and adenosine triphosphate-sensitive potassium (KATP) channels in the development of ischemic tolerance has been suggested in global ischemia, but has not been studied extensively in focal cerebral ischemia. This study evaluated modulating effects of adenosine A1 receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and mitochondrial KATP channel blocker 5HD (5-hydroxydecanoate) on the development of tolerance to focal cerebral ischemia in rats. Preconditioning with 30-minute middle cerebral artery occlusion (MCAO) reduced cortical and subcortical infarct volume following 120-minute MCAO (test ischemia) given 72 hours later. The neuroprotective effect of preconditioning was attenuated by 0.1 mg/kg DPCPX given before conditioning ischemia (30-minute MCAO), but no influence was provoked when it was administered before test ischemia. DPCPX had no effect on infarct volume after conditioning or test ischemia when given alone. The preconditioning-induced neuroprotection disappeared when 30 mg/kg 5HD was administered before test ischemia. These results suggest a possible involvement of adenosine A1 receptors during conditioning ischemia and of mitochondrial KATP channels during subsequent severe ischemia in the development of tolerance to focal cerebral ischemia.     

Effects of intravenous dimethyl sulfoxide on ischemia evolution in a rat permanent occlusion model.

Dimethyl sulfoxide (DMSO) has a variety of biological actions that suggest efficacy as a neuroprotectant. We (1) tested the neuroprotective potential of DMSO at different time windows on infarct size using 2,3,5-triphenyltetrazolium staining and (2) investigated the effects of DMSO on ischemia evolution using quantitative diffusion and perfusion imaging in a permanent middle cerebral artery occlusion (MCAO) model in rats. In experiment 1, DMSO treatment (1.5 g/kg intravenously over 3 h) reduced infarct volume 24 h after MCAO by 65% (P<0.00001) when initiated 20 h before MCAO, by 44% (P=0.0006) when initiated 1 h after MCAO, and by 17% (P=0.11) when started 2 h after MCAO. Significant infarct reduction was also observed after a 3-day survival in animals treated 1 h after MCAO (P=0.005). In experiment 2, treatment was initiated 1 h after MCAO and maps for cerebral blood flow (CBF) and apparent diffusion coefficient (ADC) were acquired before treatment and then every 30 mins up to 4 h. Cerebral blood flow characteristics and CBF-derived lesion volumes did not differ between treated and untreated animals, whereas the ADC-derived lesion volume essentially stopped progressing during DMSO treatment, resulting in a persistent diffusion/perfusion mismatch. This effect was mainly observed in the cortex. Our data suggest that DMSO represents an interesting candidate for acute stroke treatment.     

The angiotensin II type 1-receptor blocker candesartan increases cerebral blood flow, reduces infarct size, and improves neurologic outcome after transient cerebral ischemia in rats.

The goal of the present study was to test the impact of administration time of the angiotensin II type 1-receptor blocker candesartan on cerebral blood flow (CBF), infarct size, and neuroscore in transient cerebral ischemia. Therefore, 1-hour middle cerebral artery occlusion (MCAO) was followed by reperfusion. Rats received 0.5-mg/kg candesartan intravenously 2 hours before MCAO (pretreatment), 24 hours after MCAO, every 24 hours after MCAO, or 2 hours before and every 24 hours after MCAO. Infarct size (mm3) and a neuroscore at day 7 were compared with controls. CBF was quantified by radiolabeled microspheres and laser-Doppler flowmetry. Compared with controls (95 +/- 8), infarct size in candesartan-treated groups was smaller (59 +/- 5, 68 +/- 10, 28 +/- 3, and 15 +/- 3, respectively; P<0.05). Although there was no difference in neuroscore between pretreatment and controls (1.55 +/- 0.18, 1.80 +/- 0.13), other treatment regimens resulted in improved neuroscores (1.33 +/- 0.16, 1.11 +/- 0.11, 0.73 +/- 0.15; P<0.05). CBF in pretreated animals at 0.5 hours after MCAO was significantly higher than in controls (0.58 +/- 0.09 mL x g(-1) x min(-1) and 44% +/- 7% of baseline compared with 0.49 +/- 0.06 mL x g(-1) x min(-1) and 37% +/- 6%, microspheres and laser-Doppler flowmetry; P<0.05). Thus, candesartan reduces infarct size even if administered only during reperfusion. Apart from pretreatment, other treatment regimens result in significantly improved neuroscores. In the acute phase of cerebral ischemia, candesartan increases CBF.     

The maxi-K channel opener BMS-204352 attenuates regional cerebral edema and neurologic motor impairment after experimental brain injury.

Large-conductance, calcium-activated potassium (maxi-K) channels regulate neurotransmitter release and neuronal excitability, and openers of these channels have been shown to be neuroprotective in models of cerebral ischemia. The authors evaluated the effects of postinjury systemic administration of the maxi-K channel opener, BMS-204352, on behavioral and histologic outcome after lateral fluid percussion (FP) traumatic brain injury (TBI) in the rat. Anesthetized Sprague-Dawley rats (n = 142) were subjected to moderate FP brain injury (n = 88) or surgery without injury (n = 54) and were randomized to receive a bolus of 0.1 mg/kg BMS-204352 (n = 26, injured; n = 18, sham), 0.03 mg/kg BMS-204352 (n = 25, injured; n = 18, sham), or 2% dimethyl sulfoxide (DMSO) in polyethylene glycol (vehicle, n = 27, injured; n = 18, sham) at 10 minutes postinjury. One group of rats was tested for memory retention (Morris water maze) at 42 hours postinjury, then killed for evaluation of regional cerebral edema. A second group of injured/sham rats was assessed for neurologic motor function from 48 hours to 2 weeks postinjury and cortical lesion area. Administration of 0.1 mg/kg BMS-204352 improved neurologic motor function at 1 and 2 weeks postinjury (P < 0.05) and reduced the extent of cerebral edema in the ipsilateral hippocampus, thalamus, and adjacent cortex (P < 0.05). Administration of 0.03 mg/kg BMS-204352 significantly reduced cerebral edema in the ipsilateral thalamus (P < 0.05). No effects on cognitive function or cortical tissue loss were observed with either dose. These results suggest that the novel maxi-K channel opener BMS-204352 may be selectively beneficial in the treatment of experimental TBI.     

Delayed neuroprotection induced by sevoflurane via opening mitochondrial ATP-sensitive potassium channels and p38 MAPK phosphorylation

This study aimed to investigate the role of p38 MAPK phosphorylation and opening of the mitoK_ATP channels in the sevoflurane-induced delayed neuroprotection in the rat brain. Adult male Sprague-Dawley rats (250–300 g) were randomly assigned into four groups: ischemia/reperfusion (Control), sevoflurane (Sevo), 5-hydroxydecanoate (5-HD) + sevoflurane (5-HD + Sevo) and 5-HD groups and were subjected to right middle cerebral artery occlusion (MCAO) for 2 h. Sevoflurane preconditioning was induced 24 h before MCAO in sevoflurane and 5-HD + sevoflurane groups by exposing the animals to 2.4% sevoflurane in oxygen for 60 min. In control and 5-HD groups: animals were exposed to oxygen for 60 min at 24 h before MCAO. A selective mitoK_ATP channel antagonist, 5-hydroxydecanoate (5-HD, 40 mg/kg, i.p.), was administered 30 min before sevoflurane/oxygen exposure in the 5-HD + sevoflurane and 5-HD groups, respectively. Neurological deficits scores and the protein expression of phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) were evaluated at 24 and 72 h after reperfusion. Cerebral infarct size was evaluated at 72 h after reperfusion by 2,3,5-triphenyltetrazolium chloride staining. Sevoflurane preconditioning produced marked improvement neurological functions and a reduction in brain infarct volumes than animals with brain ischemia only. Sevoflurane treatment also caused increased phosphorylation of p38 MAPK at 24 and 72 h after reperfusion. These beneficial effects were attenuated by 5-HD. Blockade of cerebral protection with 5-HD concomitant with decrease in p38 phosphorylation suggests that mitoK_ATP channels opening and p38 phosphorylation participate signal transduction cascade of sevoflurane preconditioning and p38 MAPK activation may be a downstream of opening mitoK_ATP channels.     

利用激光散斑成像技术观察尤瑞克林对脑梗死大鼠脑血流的影响

目的 利用激光散斑成像技术研究尤瑞克林对大鼠脑梗死后局部脑血流的影响.方法 成年雄性SD大鼠24只,线栓法制备大鼠永久性大脑中动脉梗死模型.激光散斑成像系统观测缺血半球皮质及大脑中动脉供血区血流,2,3,5-三苯基氯化四氮唑(TTC)染色法测定脑梗死体积,并进行神经功能评分.结果 皮质及大脑中动脉供血区血流在大剂量组第1天及第2天给药后均有明显改善,部分大脑皮质血管增粗,血流速度加快,小剂量组及生理盐水组无明显变化,脑缺血48 h后,大、小剂量尤瑞克林组及生理盐水组的梗死体积分别为10.14%±3.02%,25.99%±3.90%,27.10%±3.32%,大剂量组与生理盐水组比较差异有统计学意义(F=61.14,P<0.01),小剂量组与生理盐水组比较差异无统计学意义.缺血后4 h,大剂量组神经功能损伤明显改善,小剂量组及生理盐水组无明显改变,36 h各组间的神经功能评分差异无统计学意义.结论 尤瑞克林可以减少大鼠局灶性脑缺血后梗死体积,延缓神经功能损伤,其作用可能与促进侧支循环的开放,增加大脑皮质和缺血区血流有关.     

Pinacidil抑制线粒体和死亡受体通路减少大鼠脑缺血再灌注后神经元凋亡

目的探讨ATP敏感性钾通道开放剂pinacidil对大鼠脑缺血再灌注后神经元凋亡的保护作用及信号转导机制。方法100只Wistar雄性大鼠随机分为四组：A组（假手术组）、B组（缺血组）、C组（KATP开放剂处理组）及D组（KATP 开放剂和阻断剂处理组）。用线栓法制备大鼠大脑中动脉缺血（middle cerebral artery occlusion,MCAO）模型,用DNA断端末端标记法（tenninal-deoxynucleotidytransferase-mediated dUTP—biotin nick end labeling,TUNEL）检测神经元凋亡,用原位杂交方法检测caspase-3、caspase-8及caspase-9mRNA的表达。结果（1）C组12h、24h、48h、72h时间点的凋亡细胞数较B、D组显著减少（P〈0．05或P〈0．01）;B组和D组之间无显著性差异fP〉0．05）。（2）C组caspase-3mRNA和caspase-8mRNA在各时间点及caspase-9mRNA在12h、24h、48h、72h时间点的表达显著少于B组和D组（P〈0．01或P〈0．05）,B组和D组之间无显著性差异（P〉0．05）。结论K通道开放剂能显著减少大鼠脑缺血再灌注后的细胞凋亡及caspase-3、caspase-8及caspase-9 mRNA的表达。K通道开放剂可能通过抑制线粒体通路和死亡受体通路降低神经元凋亡,保护缺血再灌注损伤后的脑组织。     

Soluble epoxide hydrolase: a novel therapeutic target in stroke.

The P450 eicosanoids epoxyeicosatrienoic acids (EETs) are produced in brain and perform important biological functions, including protection from ischemic injury. The beneficial effect of EETs, however, is limited by their metabolism via soluble epoxide hydrolase (sEH). We tested the hypothesis that sEH inhibition is protective against ischemic brain damage in vivo by a mechanism linked to enhanced cerebral blood flow (CBF). We determined expression and distribution of sEH immunoreactivity (IR) in brain, and examined the effect of sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE) on CBF and infarct size after experimental stroke in mice. Mice were administered a single intraperitoneal injection of AUDA-BE (10 mg/kg) or vehicle at 30 mins before 2-h middle cerebral artery occlusion (MCAO) or at reperfusion, in the presence and absence of P450 epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH). Immunoreactivity for sEH was detected in vascular and non-vascular brain compartments, with predominant expression in neuronal cell bodies and processes. 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid butyl ester was detected in plasma and brain for up to 24 h after intraperitoneal injection, which was associated with inhibition of sEH activity in brain tissue. Finally, AUDA-BE significantly reduced infarct size at 24 h after MCAO, which was prevented by MS-PPOH. However, regional CBF rates measured by iodoantipyrine (IAP) autoradiography at end ischemia revealed no differences between AUDA-BE- and vehicle-treated mice. The findings suggest that sEH inhibition is protective against ischemic injury by non-vascular mechanisms, and that sEH may serve as a therapeutic target in stroke.