Anandamide enhances expression of heat shock protein 72 to protect against ischemia–reperfusion injury in rat heart

Anandamide (AEA), one of endocannabinoids, has been reported to exhibit a cardioprotective ability to limit the damage produced by ischemia–reperfusion injury. AEA reportedly enhanced heat shock protein 72 (HSP72) and HSP25 expression in lungs to protect against lung inflammation. This study tested the hypothesis that intravenously injected AEA would induce HSP72 in the heart and thus render cardioprotection against ischemia–reperfusion injury in rats. Cardiac expression of HSPs was quantitatively evaluated in rats by Western blot analysis. That intravenously injected AEA 1 mg/kg in vivo induced expression of HSP72, which peaked at 24 h after administration. The enhancement of HSP72 by AEA was blocked by cannabinoid 2 (CB₂) receptor antagonist AM630, but not cannabinoid 1 (CB₁) receptor antagonist AM251. Therefore, the rats were induced with a 30-min coronary occlusion followed by a 120-min reperfusion in vivo at 24 h after administration of drugs or vehicle, and then the infarct size was measured. AEA reduced myocardial infarct size compared to control group. Pretreatment with AM630 but not AM251 abolished the infarct size-limiting effect of AEA. Further study demonstrated pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, Akt inhibitor MK-2206 and AM630 attenuated phosphorylation of Akt and AEA-induced HSP72 expression. The results suggest that AEA is cardioprotective against ischemia–reperfusion insult through its induction of HSP72, which might be mediated by the PI3K/Akt signaling pathway. These effects were mediated by CB₂ but not CB₁ receptors.     

L-丝氨酸对永久性大脑中动脉栓塞大鼠的神经保护作用

目的: 研究L-丝氨酸对大鼠永久性脑梗死的神经保护作用、治疗剂量及有效治疗时间窗,并探讨相关作用机制。方法: 制作大鼠永久性大脑中动脉栓塞(pMCAO)模型,腹腔注射L-丝氨酸,通过神经行为学评分、脑梗死体积测定和尼氏染色法,观察L-丝氨酸的治疗剂量效应(56 mg/kg、168 mg/kg和504 mg/kg治疗组)和治疗时间窗(1 h、3 h、6 h、12 h和24 h治疗组);并测定丝氨酸消旋酶抑制剂对L-丝氨酸疗效的影响。利用激光多普勒血流监测仪观察缺血区血供及L-丝氨酸对缺血区局部脑血流量的影响。结果: 与pMCAO组相比,L-丝氨酸于pMCAO后3 h使用,168 mg/kg和504 mg/kg两个剂量都能较好地降低神经行为学评分,减少脑梗死体积,抑制海马CA1区神经细胞的丢失。在治疗时间窗的研究中,L-丝氨酸在pMCAO后6 h内治疗具有明显的神经保护作用,12 h及以后使用,神经保护作用不明显。丝氨酸消旋酶抑制剂不改变L-丝氨酸的疗效。脑缺血30 min时注射L-丝氨酸可明显增加缺血区局部脑血流量,并且这一作用不受甘氨酸受体阻断剂士的宁的影响。结论: L-丝氨酸对永久性脑梗死具有神经保护作用,其机制可能部分与增加缺血区皮质的血供有关。     

Intracerebroventricular injection of a neuropeptide Y-Y1 receptor agonist increases while BIBP3226, a Y1 antagonist,reduces the infarct volume following transient middle cerebral artery occlusion in rats

Recent studies using middle cerebral artery occlusion in the rat have suggested a role of neuropeptide Y in ischemic pathophysiology. In this study, we investigated the effects of an i.c.v. injection of a neuropeptide Y-Y2 receptor agonist, neuropeptide Y 3-36, a Y1 receptor agonist, [Leu(31),Pro(34)]-neuropeptide Y, or a Y1 receptor antagonist, BIBP3226, on infarct volume and hemodynamic parameters following middle cerebral artery occlusion. Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion for 2 h. A single i.c.v. injection of neuropeptide Y 3-36 (15 microg/kg), [Leu(31),Pro(34)]-neuropeptide Y (30 microg/kg), or BIBP3226 (5, 15, or 45 microg/kg) was given at 30 min of ischemia. Blood pressure, heart rate, and regional cerebral perfusion were monitored during ischemia and reperfusion. The rats were decapitated after 70 h of reperfusion, and their brains were cut into 2-mm-thick coronal slices before reaction with a 2% solution of 2,3,5-triphenyltetrazolium chloride to reveal the infarct. When compared with an infarct volume of 17.4+/-4.4% of the ipsilateral hemisphere following injection of neuropeptide Y 3-36, administration of the Y1 receptor analogs significantly modified the infarct volume (ordinary one-way analysis of variance (ANOVA), P<0.0001). [Leu(31),Pro(34)]-neuropeptide Y increased the infarct volume to 32.0+/-4.1% (Student-Newman-Keuls post-test, P<0.01), whereas BIBP3226 at 15 microg/kg decreased the infarct volume to 6.5+/-1.0% (post-test P<0.05). Although there was no major difference in the hemodynamic parameters among the groups, injection of [Leu(31),Pro(34)]-neuropeptide Y tended to further reduce cerebral perfusion during ischemia, while injection of BIBP3226 at 15 microg/kg appeared to have the opposite effect. In addition to glutamate, calcium ion and nitric oxide, activation of the neuropeptide Y-Y1 receptors may mediate cerebral damage during focal ischemia. Conversely, inhibiting the Y1 receptors may protect the brain against ischemic injury. Further studies are warranted to confirm the neuroprotective potential of neuropeptide Y-Y1 receptor inhibition.     

Cannabinoid CB2 receptor activation reduces mouse myocardial ischemia-reperfusion injury: involvement of cytokine/chemokines and PMN

In this study, we have assessed the activation of the cannabinoid CB2 receptor (CB2-R) in a model of mouse myocardial ischemia/reperfusion (I/R). The results show that treatment of animals with WIN55212-2, a CB1/CB2-R agonist, given 30 min before induction of I/R, significantly reduced the extent of infarct size (IS) in the area at risk, as measured 2.5 h later, with almost a 51% inhibition observed at the dose tested of 3.5 mg/kg intraperitoneally (i.p.). The protective effect of WIN55212-2 was almost abolished by the selective CB2-R antagonist AM630 (1 mg/kg i.p.) and not affected by the selective CB1-R antagonist AM251 (3 mg/kg i.p.). The CB2-R antagonist administered alone produced a slight but significant (P<0.05) increase in IS compared with vehicle alone. The protection afforded by WIN55212-2 was paralleled by lower values of myeloperoxidase activity and interleukin-1beta and of the CXC chemokine ligand 8 into the injured tissue. In conclusion, we demonstrate for the first time that exogenous and endogenous CB2-R activation reduces the leukocyte-dependent myocardial damage associated with an I/R procedure.     

Increased expression of cysteinyl leukotriene receptor-1 in the brain mediates neuronal damage and astrogliosis after focal cerebral ischemia in rats

Cysteinyl leukotrienes are potent pro-inflammatory mediators. Cysteinyl leukotriene receptor 1 is one of the two cysteinyl leukotriene receptors cloned. We recently reported that cysteinyl leukotriene receptor 1 antagonists protected against cerebral ischemic injury, and an inducible expression of cysteinyl leukotriene receptor 1 was found in neuron- and glial-appearing cells after traumatic injury in human brain. To determine the role of cysteinyl leukotriene receptor 1 in ischemic brain injury, we investigated the temporal and spatial profile of cysteinyl leukotriene receptor 1 expression in rat brain from 3 h to 14 days after 30 min of middle cerebral artery occlusion, and observed the effect of pranlukast, a cysteinyl leukotriene receptor 1 antagonist, on the ischemic injury. We found that cysteinyl leukotriene receptor 1 mRNA expression was up-regulated in the ischemic core both 3-12 h and 7-14 days, and in the boundary zone 7-14 days after reperfusion. In the ischemic core, cysteinyl leukotriene receptor 1 was primarily localized in neurons 24 h, and in macrophage/microglia 14 days after reperfusion; while in the boundary zone it was localized in proliferated astrocytes 14 days after reperfusion. Pranlukast attenuated neurological deficits, reduced infarct volume and ameliorated neuron loss in the ischemic core 24 h after reperfusion; it reduced infarct volume, ameliorated neuron loss and inhibited astrocyte proliferation in the boundary zone 14 days after reperfusion. Thus, we conclude that cysteinyl leukotriene receptor 1 mediates acute neuronal damage and subacute/chronic astrogliosis after focal cerebral ischemia.     

Topiramate reduces non-convulsive seizures after focal brain ischemia in the rat

Acute "silent" seizures after brain injury are associated with a worsening of patient outcome and are often refractory to anti-epileptic drug (AED) therapy. In the present study we evaluated topiramate (TPM, 1-30 mg/kg, i.v.) in a rodent model of spontaneous non-convulsive seizure (NCS) activity induced by focal cerebral ischemia. For seizure detection, electroencephalographic (EEG) activity was continuously recorded for 24h in male Sprague-Dawley rats subjected to permanent middle cerebral artery occlusion (MCAo). Infarct volume, neurological deficit, and NCS were evaluated by an experimenter blinded to the treatment group. All vehicle treated rats (7/7) exhibited NCS following MCAo. TPM treatment, delivered at 20 min post-occlusion and prior to onset of NCS activity, dose-dependently reduced the incidence of NCS (ED(50)=21.1mg/kg). The highest dose of TPM tested (30 mg/kg) exhibited maximal reductions of 76% in the number of NCS/rat (vehicle=22.1+/-5.3, TPM=4.4+/-3.2, P<0.05), 80% in the total time of NCS (vehicle=1259+/-337 s, TPM=253+/-220 s, P<0.05), 20% in core brain infarction (vehicle=45+/-1%, TPM=36+/-4%, percent of ipsilateral volume corrected for swelling, P<0.05), and 38% in neurological deficit score (vehicle=7.4+/-1.2, TPM=4.6+/-1.5, P<0.05). Despite efficacy as a pre-seizure treatment, TPM was not effective when delivered immediately following onset of the first NCS event (36+/-5 min post-MCAo). In conclusion, TPM exhibited significant efficacy for the prophylactic treatment of brain-injury induced NCS and represents a novel class of AED for treatment of this type of silent brain seizure.     

Modulation of the balance between cannabinoid CB(1) and CB(2) receptor activation during cerebral ischemic/reperfusion injury

Cannabinoid receptor activation has been shown to modulate both neurotransmission (CB(1)) and neuroinflammatory (CB(2)) responses. There are conflicting reports in the literature describing the influence of cannabinoid receptor activation on ischemic/reperfusion injury. The goal of this study was to evaluate how changing the balance between CB(1) and CB(2) activation following cerebral ischemia influences outcome. CB(1) and CB(2) expression were tested at different times after transient middle cerebral artery occlusion (MCAO) in mice by real-time RT-PCR. Animals subjected to 1 h MCAO were randomly assigned to receive different treatments: a CB(1) antagonist, a CB(2) antagonist, a CB(2) agonist, a CB(1) antagonist plus CB(2) agonist, a CB(2) antagonist plus CB(2) agonist or an equal volume of vehicle as control. Cerebral blood flow was continuously monitored during ischemia; cerebral infarction and neurological deficit were tested 24 h after MCAO. Cerebral CB(1) and CB(2) mRNA expression undertook dynamic changes during cerebral ischemia. The selective CB(1) antagonist significantly decreased cerebral infarction by 47%; the selective CB(2) antagonist increased infarction by 26% after 1 h MCAO followed by 23 h reperfusion in mice. The most striking changes were obtained by combining a CB(1) antagonist with a CB(2) agonist. This combination elevated the cerebral blood flow during ischemia and reduced infarction by 75%. In conclusion, during cerebral ischemia/reperfusion injury, inhibition of CB(1) receptor activation is protective while inhibition of CB(2) receptor activation is detrimental. The greatest degree of neuroprotection was obtained by combining an inhibitor of CB(1) activation with an exogenous CB(2) agonist.     

Interleukin-1 receptor antagonist decreases the number of necrotic neurons in rats with middle cerebral artery occlusion.

Marked increases in the brain expression of interleukin (IL)-1 have been reported in rats after permanent occlusion of a large cerebral artery. Interactions between endothelial cells and leukocytes have been implicated in the pathogenesis of several types of ischemic injury to the myocardium and other organs. In this study we asked whether inhibiting the effects of IL-1 would affect the outcome of an experimental brain infarct. Adult male Wistar rats (n = 13) with permanent occlusion of the middle cerebral artery were given IL-1 receptor antagonist. A second group (n = 13) with the same type of brain injury was given a placebo. A third group, subjected to a sham operation, was given either IL-1 receptor antagonist (n = 2) or a placebo (n = 2). Experiments were terminated after either 24 hours or 7 days. Compared with the control group, animals treated with IL-1 receptor antagonist improved their neurological score (P < 0.05), experienced less pronounced changes in body weight (P < 0.05), and had fewer necrotic neurons (P < 0.001) and fewer leukocytes in the ischemic hemisphere (P < 0.001) as well as a smaller area of pallor (P < 0.05) in the ischemis hemisphere. The results suggest that inhibiting the proinflammatory effects of IL-1 with a receptor antagonist is an effective way of influencing the leukocyte responses elicited by an arterial occlusion. Such leukocyte inhibition seemingly attenuates the number of necrotic neurons resulting from the occlusion of a large brain artery.     

Neuroprotective effects of olprinone after cerebral ischemia/reperfusion injury in rats

Olprinone hydrochloride, a specific phosphodiesterase III inhibitor, has anti-inflammatory effects in addition to its inotropic and vasodilatory effects. The purpose of this study was to examine the beneficial effects of olprinone on cerebral ischemia reperfusion injury. In the present study, we examined the detailed mechanisms underlying the inhibitory effects of olprinone on inflammatory and apoptotic responses induced by middle cerebral artery occlusion (MCAo) in rats. Focal cerebral ischemia was induced by transient MCAo in the right hemisphere, via the external carotid artery into the internal carotid to block the origin of the median carotid artery. The rats were subjected to artery occlusion (2 h) followed by reperfusion (22 h). Olprinone was administered 5 min before reperfusion. MCAo-induced cerebral ischemia was associated with an increase in inducible nitric oxide synthase expression, nitrotyrosine formation, as well as IL-1β expression and ICAM-1 expression in ischemic regions. Olprinone treatment showed marked reduction in infarct size compared with control rats. These expressions were markedly inhibited by olprinone treatment. We also demonstrated that olprinone reduces levels of apoptosis (TUNEL, Bax and Bcl-2 expression) resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Based on these findings we propose that olprinone would be useful in lowering the risk of damage or improving function in ischemia-reperfusion brain injury-related disorders.     

Protective effects of PJ34, a novel, potent inhibitor of poly(ADP-ribose) polymerase (PARP) in in vitro and in vivo models of stroke

Focal cerebral ischemia activates the nuclear protein poly(ADP-ribose) polymerase (PARP) by single DNA strand breaks which leads to energy depletion and cell necrosis. Deletion or inhibition of PARP protects against ischemic brain injury. Here we examined the neuroprotective effect of PJ34, a novel potent inhibitor of PARP in vitro and in vivo. Serum-free primary neuronal cultures derived from rat cortex (E15-17) and kept in culture for 10 days were exposed to oxygen glucose deprivation (OGD) in vitro. Neuronal injury was quantified by LDH release after 24 h. Pretreatment with 30-1000 nM PJ34 significantly protected from OGD-induced cell injury in a dose-dependent manner. For in vivo experiments SV/129 mice were treated with PJ34 (50 microg) by intraperitoneal injection 2 h before 1 h middle cerebral artery occlusion (MCAo) and again 6 h later. Twenty-three h after reperfusion ischemic injury was significantly decreased compared to vehicle-treated controls (infarct volume reduction of 40%, p<0.05). Similarly, in a rat model of MCAo (2 h occlusion followed by up to 22 h reperfusion), PJ34 administration (10 mg/kg i.v.) significantly reduced infarct size, and the effect of the drug was maintained even if it was given as late as 10 min prior to reperfusion time. PJ34 significantly protected in a 4 h, but not in a 24 h permanent occlusion model. In conclusion, PJ34, a novel, potent inhibitor of PARP exerts massive neuroprotective agents, with a significant therapeutic window of opportunity. The present work strengthens the concept that pharmacological PARP inhibition may be a suitable approach for the treatment of acute stroke in man.     

Suramin reduces infarct volume in a model of focal brain ischemia in rats

Extracellular adenosine 5'-triphosphate (ATP) provides excitatory transmission in the central nervous system. Stimulation by ATP of ionotropic ligand-gated ion channel purinoceptors (P_2X) leads to increased intracellular calcium levels, and activation of P_2X receptors may be involved in the process of excitotoxic neuronal injury caused by stroke. Suramin, as an agent that is known to block P_2X receptors at a specific concentration, was assessed for its neuroprotective potential in a model of experimental stroke in the rat. We propose that the effectiveness of suramin is limited to those concentrations where it is an effective P_2X receptor antagonist. Focal brain ischemia was produced by unilateral occlusion and transection of the middle cerebral artery (MCAT) and bilateral occlusion of the common carotid arteries (CCA). Thirty-four male Sprague-Dawley rats were randomly separated into five groups. Changes in regional cerebral blood flow in the ischemic region were verified by laser Doppler flowmetry. All rats received, over a period of 30 min before MCAT, a dose of suramin at 0 (saline), 25, 50, 100, or 250 mg/kg intravenously in a volume of 1 ml using an infusion pump. Six hours after ischemia onset, evaluation of the neurologic status and cerebral blood flow was followed by morphometric analysis of infarct volume. During the surgical procedure mean arterial pressure, blood gases (PaCO₂, PaO₂), and pH were monitored. A dose-dependent decrease in infarct volume (slope –0.049, SE 0.009, P<0.001) was observed in groups treated over the dose range of 0 to 100 mg/kg (r ²=0.55). Suramin at a dose of 100 mg/kg significantly decreased infarct volume (n=9, P<0.001) and edema volume (P=0.003). The neuroprotective effect of suramin at a dose of 100 mg/kg was supported by an improved neurologic score in this group (median 0) compared with a median of 3 in control animals (P=0.02). These findings indicate that suramin at 100 mg/kg is an effective pre-treatment neuroprotective agent. As the estimated brain concentration of 10 μM (from McNally et al., 2000, Life Sci 67:1847–1857) is the IC₅₀ for suramin-mediated P_2X antagonism, these results suggest that interference with the ATP excitatory system could provide neuroprotection from brain ischemia. Electronic Publication     

Intravenous administration of thioredoxin decreases brain damage following transient focal cerebral ischemia in mice

Thioredoxin (TRX) is induced by a variety of oxidative stimuli and shows cytoprotective roles against oxidative stress. To clarify the possibility of clinical application, we examined the effects of intravenously administered TRX in a model of transient focal cerebral ischemia in this study. Mature male C57BL/6j mice received either continuous intravenous infusion of recombinant human TRX (rhTRX) over a range of 1-10 mg/kg, bovine serum albumin, or vehicle alone for 2 h after 90-min transient middle cerebral artery occlusion (MCAO). Twenty-four hours after the transient MCAO, the animals were evaluated neurologically and the infarct volumes were assessed. Infarct volume, neurological deficit, and protein carbonyl contents, a marker of protein oxidation, in the brain were significantly ameliorated in rhTRX-treated mice at the dose of 3 and 10 mg/kg versus these parameters in control animals. Moreover, activation of p38 mitogen-activated protein kinase, whose pathway is involved in ischemic neuronal death, was suppressed in the rhTRX-treated mice. Further, rhTRX was detected in the ischemic hemisphere by western blot analysis, suggesting that rhTRX was able to permeate the blood-brain barrier in the ischemic hemisphere. These data indicate that exogenous TRX exerts distinct cytoprotective effects on cerebral ischemia/reperfusion injury in mice by means of its redox-regulating activity.     

脑的不对称性影响大鼠局灶性脑缺血的结局

为评价左右侧大脑中动脉闭塞(MCAO)对右利大鼠神经行为功能和脑梗死体积的影响,本研究应用四足动物觅食实验筛选右利爪雄性SD大鼠24只,随机分为经左、右侧插线组各12只,8%水合氯醛腹腔注射(300mg/kg)麻醉,线栓法经左、右侧颈外-内动脉插入头端涂有多聚赖氨酸的4-0尼龙线,建立大鼠MCAO缺血2h模型,再灌注72h后评价动物的神经行为功能,测量脑梗死体积。结果表明,所有动物在脑缺血2h神经功能缺损评分最高,再灌注1、24、48和72h经左侧MCAO大鼠显著高于经右侧MCAO大鼠(P<0.05),后者功能明显优于前者,脑梗死体积经左侧插线的大鼠显著大于经右侧插线的大鼠(P<0.05)。研究结果提示,大鼠主侧半球大脑中动脉缺血后,神经功能缺损和脑梗死体积较对侧严重,脑的不对称性影响大鼠局灶性脑缺血的最终结局。     

Extension of the neuroprotective time window for thiazolidinediones in ischemic stroke is dependent on time of reperfusion

Stroke is a leading cause of death and disability but has limited therapeutic options. Thiazolidinediones (TZDs), agonists for the nuclear receptor, peroxisome proliferator-activated receptor (PPAR)γ, reduce infarct volume and improve neurologic function following transient middle cerebral artery occlusion (MCAO) in rats. Translation of these findings into clinical therapy will require careful assessment of dosing paradigms and effective time windows for treatment. Understanding the mechanisms by which TZDs protect the brain provides insight into how time windows for neuroprotection might be extended. We find that two TZDs, pioglitazone and rosiglitazone, significantly reduce infarct volume at doses similar to those used clinically (1 mg/kg for pioglitazone and 0.1 mg/kg for rosiglitazone). We also find that pioglitazone reduces infarction volume in a transient, but not a permanent MCAO model suggesting that reperfusion plays an important role in TZD mediated neuroprotection. Since PPARγ agonists reduce inflammation and oxidative stress, both of which are exacerbated by reperfusion, we hypothesized that TZDs would be most effective if administered prior to reperfusion. We administered TZDs 3 h after MCAO and found that infarction volume and neurologic function are significantly improved in animals reperfused at 3 h and 15 min (after TZD treatment), but not in animals reperfused at 2 h (before TZD treatment) when assessed either 24 h or 3 weeks after MCAO. While TZDs reduce intercellular adhesion molecule (ICAM) expression to a similar extent regardless of the time of reperfusion, leukocyte entry into brain parenchyma is more dramatically reduced when reperfusion is delayed until after drug treatment. The finding that delaying reperfusion until after TZD treatment is beneficial despite a longer period of ischemia, is dramatic given the widely held view that duration of ischemia is the most important determinate of injury.     

Neuroprotective effect of Coenzyme Q10 on ischemic hemisphere in aged mice with mutations in the amyloid precursor protein

This study was designed to test whether Coenzyme Q10 (CoQ10) supplementation has neuroprotective effect in aged, double-transgenic amyloid precursor protein (APP)/presenilin 1 (PS1), single transgenic APP and PS1 mice exposed to ischemic injury of the brain. Forty-eight mice (12 each of APP/PS1, APP, PS1 and wild-type) were studied. Half of each genotype groups (n=6 per group) was treated with CoQ10 (1200 mg/kg/day) after ischemic injury and the other half with placebo. Magnetic resonance (MR) images were used to measure the volume of induced infarction (IFV), as well as the volume of the hemispheres and hippocampi. Significantly greater volumes of infarction and lesser volumes of hemisphere/hippocampus on the ischemic side were observed in APP/PS1 and APP mice than in PS1 and wild-type mice. This is consistent with amplification of the effect of ischemia in APP carriers. After 28 days of CoQ10 treatment, APP/PS1 or APP mutations have smaller infarct volumes, while the volumes of hemisphere and hippocampus on the infarcted side were larger than those treated with placebo. No differences between CoQ10- and placebo-treated groups in volumes of infarct, hemisphere and hippocampus were observed in PS1 and wild-type mice. We conclude that CoQ10 has a protective effect on the brain from infarction and atrophy induced by ischemic injury in aged and susceptible transgenic mice.     

Treatment with the snail peptide CGX-1007 reduces DNA damage and alters gene expression of c-fos and bcl-2 following focal ischemic brain injury in rats

Delayed cell death following ischemic brain injury has been linked to alterations in gene expression. In this study we have evaluated the upregulation of several genes associated with delayed cell death (c-fos, bax, and bcl-2) during the initial 24 h of transient middle cerebral artery occlusion (MCAo) in the rat and the effects of postinjury treatment with the NR2B subunit specific NMDA receptor antagonist CGX-1007 (Conantokin-G, Con-G). C-fos mRNA levels peaked at 1 h postinjury in both cortical and subcortical ischemic brain regions (30-fold increase), remained elevated at 4 h and returned to within normal, preinjury levels 24 h postinjury. The increase in mRNA levels correlated to increased protein expression in the entire ipsilateral hemisphere at 1 h. Regions of necrosis at 4 h were void of C-Fos immunoreactivity with continued upregulation in surrounding regions. At 24 h, loss of C-Fos staining was observed in the injured hemisphere except for sustained increases along the border of the infarct and in the cingulate cortex of vehicle treated rats. CGX-1007 treatment reduced c-fos expression throughout the infarct region by up to 50%. No significant differences were measured in either bcl-2 or bax mRNA expression between treatment groups. However, at 24 h postinjury CGX-1007 treatment was associated with an increase in Bcl-2 immunoreactivity that correlated to a reduction in DNA fragmentation. In conclusion, CGX-1007 effectively attenuated gene expression associated with delayed cell death as related to a neuroprotective relief of cerebral ischemia.     

The FAAH inhibitor URB-597 interferes with cisplatin- and nicotine-induced vomiting in the Suncus murinus (house musk shrew)

Considerable evidence implicates the endocannabinoid system as a neuromodulator of nausea and vomiting. The action of anandamide (AEA) can be prolonged by inhibiting its degradation, through the use of URB597 (URB), a Fatty Acid Amide Hydrolase (FAAH) enzyme inhibitor. Here we present evidence that the FAAH inhibitor, URB, interferes with cisplatin- and nicotine-induced vomiting in the Suncus murinus. In Experiment 1, shrews were injected with URB (0.9 mg/kg) or vehicle 120 min prior to the behavioral testing. They received a second injection of AEA (5 mg/kg) or vehicle 15 min prior to being injected with cisplatin (20 mg/kg) or saline and the number of vomiting episodes were counted for 60 min. In Experiment 2, shrews were injected with vehicle or URB (0.9 mg/kg) 120 min prior to receiving an injection of nicotine (5 mg/kg) or saline and the number of vomiting episodes were counted for 15 min. Experiment 3 evaluated the potential of the CB₁ antagonist, SR141716, to reverse the effect of URB on nicotine-induced vomiting. URB attenuated vomiting produced by cisplatin and nicotine and the combination of URB + AEA suppressed vomiting produced by cisplatin. The effect of URB on nicotine-induced vomiting was reversed by SR141716. These data suggest that the EC system plays a tonic role in the regulation of toxin-induced vomiting.     

Concentration-dependent dual effect of anandamide on sensory neuropeptide release from isolated rat tracheae

Most actions of anandamide (AEA) are mediated by the cannabinoid 1 (CB(1)) receptor activation, but on sensory neurones it is also an agonist on the vanilloid subtype 1 receptor (VR(1)). The aim of the present study was to analyse the effect of AEA (10(-6)-10(-4) M) on inhibitory CB(1) and excitatory VR(1) receptors by measuring sensory neuropeptide release such as somatostatin, substance P and calcitonin gene-related peptide, from isolated rat tracheae. AEA (10(-6) M) vas without significant effect, 10(-5) M inhibited neuropeptide release, which was abolished by the G protein-coupled receptor blocker pertussis toxin (100 ng/ml) and the CB(1) receptor antagonist SR141716A (5x10(-7) M). High concentrations of AEA (5x10(-5) M, 10(-4) M) increased the release of the peptides and this inhibition was prevented by the competitive VR(1) antagonist capsazepine (10(-5) M). These results indicate a dual, concentration-dependent action of AEA on CB(1) receptors and VR(1) on peripheral sensory nerve terminals.     

Overexpressed protein disulfide isomerase in brains of patients with sporadic Creutzfeldt-Jakob disease

Cannabinoids produce antinociception via specific cannabinoid receptor activation, but there are also non-receptor mediated effects like for example the activation of the arachidonic acid cascade. Here we investigate the influence of cannabinoids (CB) on sleep duration after isoflurane anesthesia. We found that the CB receptor agonists R(-)-7-hydroxy-delta-6-tetra-hydrocannabinol-dimethylheptyl (HU-210) (0.1 mg/kg), 2-O-arachidonoylglycerylether (30 mg/kg) and arachidonyl-2-chloroethylamide (3 mg/kg) significantly prolong the duration of isoflurane induced sleep in mice (P<0.05). This effect was absent when co-injecting the selective CB(1) antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (1 mg/kg). Furthermore, HU-210 was ineffective in CB(1) receptor knockout mice (CB(1)-/-). Our behavioral tests (tail flick, rotarod) indicate that the sleep latency can be prolonged even at low drug dosages which do not influence thermal nociception. In the chosen dosages thimerosal (20 mg/kg), 2-AG (10 mg/kg), R(1)-methanandamide (R(1)-MAEA) (10 mg/kg) and flurbiprofen (27 mg/kg) were ineffective to increase sleep duration.     

辣椒素受体拮抗剂AMG517在小鼠脑缺血/再灌注损伤中通过调节炎症因子释放发挥神经保护作用#br#

目的 观察辣椒素受体(TRPV1)拮抗剂AMG517在小鼠脑缺血/再灌注损伤中的作用及相关机制。方法 40只雄性C57BL/6小鼠随机分为假手术组(sham,n=10)、赋形剂（vehicle）+缺血/再注灌注组(vehicle,n=10)、辣椒素+缺血/再注灌注组(capsaicin,n=10)和AMG517+缺血/再注灌组(AMG517,n=10)。采用大脑中动脉闭塞(MCAO)诱导缺血/再灌注损伤,再灌注72 h进行神经行为学评分;同时检测各组小鼠梗死体积、脑水肿、TRPV1 mRNA表达和血清肿瘤坏死因子-α(TNF-α)浓度及白细胞介素-10(IL-10)浓度。结果 与vehicle组相比,AMG517显著减小鼠脑梗死体积(P<0.01)。AMG517给药后也可显著降低小鼠神经行为学评分(P<0.01)。与sham组比较,vehicle组TRPV1 mRNA表达显著增加(P<0.01)。AMG517给药后可显著增加抗炎性细胞因子IL-10浓度,并降低炎性细胞因子TNF-α浓度(P<0.05)。结论 AMG517可改善小鼠缺血/再灌注损伤,可能是通过缓解炎症发挥神经保护作用。