Nitric oxide down-regulates caveolin-1 expression in rat brains during focal cerebral ischemia and reperfusion injury

As a signalling molecule of the integral membrane protein family, caveolin participates in cellular signal transduction via interaction with other signalling molecules. The nature of interaction between nitric oxide (NO) and caveolin in the brain, however, remains largely unknown. In this study we investigated the role(s) of NO in regulating caveolin-1 expression in rat ischemic brains with middle cerebral artery occlusion (MCAO). Exposure to 1 h ischemia induced the increases in neuronal nitric oxide synthase (nNOS) and NO concentration with concurrent down-regulation of caveolin-1 expression in the ischemic core of rat brains. Subsequent 24 h or more reperfusion time led to an increase in inducible NOS (iNOS) expression and NO production, as well as a decline of caveolin-1 protein at the core and penumbra of the ischemic brain. Afterwards, NOS inhibitors and an NO donor were utilized to clarify the link between NO production and caveolin-1 expression in the rats with 1 h ischemia plus 24 h reperfusion. N(G)-nitro-l-arginine methyl ester (L-NAME, a non-selective NOS inhibitor), N(6)-(1-iminoethyl)-lysine (NIL, an iNOS inhibitor), and 7-nitroindazole (7-NI, a nNOS inhibitor) prevented the loss of caveolin-1 in the core and penumbra of the ischemic brain, whereas l-N(5)-(1-iminoethyl)-ornithine (L-NIO, an endothelial NOS inhibitor) showed less effect than the other NOS inhibitors. S-Nitroso-N-acetylpenicillamine (SNAP, a NO donor) down-regulated the expression of caveolin-1 protein in normal and ischemic brains. These results, when taken together, suggest that NO modulates the expression of caveolin-1 in the brain and that the loss of caveolin-1 is associated with NO production in the ischemic brain.     

P-glycoprotein trafficking at the blood-brain barrier altered by peripheral inflammatory hyperalgesia

J. Neurochem. (2012) 122, 962-975. ABSTRACT: P-glycoprotein (ABCB1/MDR1, EC 3.6.3.44), the major efflux transporter at the blood-brain barrier (BBB), is a formidable obstacle to CNS pharmacotherapy. Understanding the mechanism(s) for increased P-glycoprotein activity at the BBB during peripheral inflammatory pain is critical in the development of novel strategies to overcome the significant decreases in CNS analgesic drug delivery. In this study, we employed the λ-carrageenan pain model (using female Sprague-Dawley rats), combined with confocal microscopy and subcellular fractionation of cerebral microvessels, to determine if increased P-glycoprotein function, following the onset of peripheral inflammatory pain, is associated with a change in P-glycoprotein trafficking which leads to pain-induced effects on analgesic drug delivery. Injection of λ-carrageenan into the rat hind paw induced a localized, inflammatory pain (hyperalgesia) and simultaneously, at the BBB, a rapid change in colocalization of P-glycoprotein with caveolin-1, a key scaffolding/trafficking protein. Subcellular fractionation of isolated cerebral microvessels revealed that the bulk of P-glycoprotein constitutively traffics to membrane domains containing high molecular weight, disulfide-bonded P-glycoprotein-containing structures that cofractionate with membrane domains enriched with monomeric and high molecular weight, disulfide-bonded, caveolin-1-containing structures. Peripheral inflammatory pain promoted a dynamic redistribution between membrane domains of P-glycoprotein and caveolin-1. Disassembly of high molecular weight P-glycoprotein-containing structures within microvascular endothelial luminal membrane domains was accompanied by an increase in ATPase activity, suggesting a potential for functionally active P-glycoprotein. These results are the first observation that peripheral inflammatory pain leads to specific structural changes in P-glycoprotein responsible for controlling analgesic drug delivery to the CNS.     

不同亚型一氧化氮合酶在脑缺血/再灌注早期的表达变化

目的:观察脑缺血/再灌注(CI/R)早期缺血区脑组织的内皮型一氧化氮合酶(eNOS)与神经型一氧化氮合酶(nNOS)表达的变化。方法:健康wistar大鼠60只,体重200～280g,由中国医科大学动物中心提供,雌雄各半。随机分为6组(n=10):假手术组、缺血1h组、缺血2h组、再灌注0.5h组、再灌注1h组、再灌注2h组。采用线栓法制作大鼠CI/R模型,免疫组化方法检测缺血区脑组织的eNOS与nNOS蛋白表达情况。结果:与假手术组比较,CI/R模型大鼠脑组织血管内皮细胞内eNOS表达在缺血1h内升高,之后到再灌注2h内持续降低。而nNOS的表达在缺血到再灌注2h内持续上升。结论:CI/R模型中缺血区脑组织的eNOS与nNOS的变化趋势不同,表明一氧化氮在缺血性脑损伤病理过程的作用与一氧化氮合酶亚型的变化有关。     

Inducible nitric oxide synthase expression in the ischemic core and penumbra after transient focal cerebral ischemia in mice

The present observations examined the hypothesis that the iNOS expression in the ischemic penumbra after a transient focal ischemic insult is involved in the recruitment of penumbra into infarction. The middle cerebral artery in mice was occluded for 2 h by an intraluminal filament and then recirculated. The measurement of iNOS activity, iNOS protein formation and NO concentration in the ischemic core and penumbra, and the determination of infarct volume were performed at 6, 12, 24 and 48 h after reperfusion. iNOS protein and iNOS enzymatic activity appeared at 6 h, peaked at 24 h, and declined at 48 h in the penumbra after reperfusion. iNOS protein was not detectable in contralateral area and in sham-operated brains. The time course of iNOS protein, enzymatic activity and NO concentration in the penumbra but not in the core matched the process of infarct maturation. Treatment with iNOS inhibitor aminoguanidine (100 mg.kg(-1), i.p.) at 6 and 12 h after reperfusion inhibited iNOS activity by 88.0 +/- 10.4% and reduced NO concentration by 48.5 +/- 8.3% in the penumbra, and lessened infarct size by 48.8 +/- 7.2%. The iNOS activity and NO level in the core were not affected by the administration of aminoguanidine. These results suggest that iNOS expression in the ischemic penumbra is involved in the recruitment of penumbra into infarction and thereby contributing to the enlargement of infarct.     

Microsphere embolism-induced endothelial nitric oxide synthase expression mediates disruption of the blood-brain barrier in rat brain

Microsphere embolism (ME)-induced up-regulation of endothelial nitric oxide synthase (eNOS) in endothelial cells of brain microvessels was observed 2-48 h after ischemia. eNOS induction preceded disruption of the blood-brain barrier (BBB) observed 6-72 h after ischemia. In vascular endothelial cells, ME-induced eNOS expression was closely associated with protein tyrosine nitration, which is a marker of generation of peroxynitrite. Leakage of rabbit IgG from microvessels was also evident around protein tyrosine nitration-immunoreactive microvessels. To determine whether eNOS expression and protein tyrosine nitration in vascular endothelial cells mediates BBB disruption in the ME brain, we tested the effect of a novel calmodulin-dependent NOS inhibitor, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), which inhibits eNOS activity and, in turn, protein tyrosine nitration. Concomitant with inhibition of protein tyrosine nitration in vascular endothelial cells, DY-9760e significantly inhibited BBB disruption as assessed by Evans blue (EB) excretion. DY-9760e also inhibited cleavage of poly (ADP-ribose) polymerase as a marker of the apoptotic pathway in vascular endothelial cells. Taken together with previous evidence in which DY-9760e inhibited brain edema, ME-induced eNOS expression in vascular endothelial cells likely mediates BBB disruption and, in turn, brain edema.     

大鼠局灶性脑缺血后一氧化氮合酶基因表达的变化

目的:观察大鼠局灶性脑缺血后3种类型一氧化氮合酶(NOS)mRNA表达的变化.方法:大鼠随机分为正常对照组、缺血后2、6、12、24 h组,以逆转录-聚合酶链反应(RT-PCR)法分别检测缺血脑组织NOS基因表达的变化.结果:脑缺血后eNOS、nNOSmRNA表达增强,分别于2、6 h达高峰;iNOS mRNA表达亦增高,但在缺血后12 h达高峰.结论:大鼠脑缺血早期eNOS和nNOS占主要地位,缺血后期iNOS占主要地位.     

Exogenous nitric oxide negatively regulates c-Jun N-terminal kinase activation via inhibiting endogenous NO-induced S-nitrosylation during cerebral ischemia and reperfusion in rat hippocampus

Nitric oxide (NO), synthesized from l -arginine by NO synthases, is a small endogenous free radical with multiple functions. The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in mediating apoptosis in cerebral ischemia and reperfusion. In this study, we found that the NO donor sodium nitroprusside (SNP) can decrease the damage of hippocampal neurons induced by cerebral ischemia and reperfusion. Our current study demonstrates that SNP can suppress the phosphorylation of JNK3 by suppressing the increased S-nitrosylation of JNK3 induced by cerebral ischemia and reperfusion. In contrast, dithiothreitol reversed the effect of SNP on S-nitrosylation of JNK3. Furthermore, the inhibitor of nNOS (7-NI) and the inhibitor of iNOS (AMT) can decrease JNK3 phosphorylation through decreasing S-nitrosylation of JNK3. Our data suggest that endogenous NO synthesized by NO synthases can increase JNK3 phosphorylation by means of S-nitrosylation during global ischemia/reperfusion in rat hippocampus. However, the exogenous NO (SNP) can reverse the effect of endogenous NO by inhibiting S-nitrosylation of JNK3. Together, these results suggest that the exogenous NO may provide a new clue for stroke therapy.     

大鼠脑缺血再灌注时水通道蛋白4表达与血脑屏障通透性变化

目的:研究大鼠脑缺血/再灌注时脑组织中水通道蛋4(AQP4)表达与脑水肿、血脑屏障通透性间关系。方法:采用大鼠大脑中动脉线栓缺血模型,免疫组化法、蛋白印迹法测定AQP4表达,干湿重法测定脑水含量以评价脑水肿,伊文氏蓝(EB)法测定血脑屏障通透性。结果:脑缺血后再灌注4～6h,AQP4表达上调,至12h上调显著,48～72h达高峰。脑水含量、EB含量均与此趋势相一致,且AQP4表达与脑水含量、EB含量呈显著正相关(P0.05)。结论:AQP4表达参与了缺血性脑水肿的产生,且与BBB通透性改变呈正相关。     

Interaction of caveolin-1, nitric oxide, and nitric oxide synthases in hypoxic human SK-N-MC neuroblastoma cells

Neuroblastoma cells are capable of hypoxic adaptation, but the mechanisms involved are not fully understood. We hypothesized that caveolin-1 (cav-1), a plasma membrane signal molecule, might play a role in protecting neuroblastoma cells from oxidative injury by modulating nitric oxide (NO) production. We investigated the alterations of cav-1, cav-2, nitric oxide synthases (NOS), and NO levels in human SK-N-MC neuroblastoma cells exposed to hypoxia with 2% [O2]. The major discoveries include: (i) cav-1 but not cav-2 was up-regulated in the cells exposed to 15 h of hypoxia; (ii) NO donor 1-[N, N-di-(2-aminoethyl) amino] diazen-1-ium-1, 2-diolate up-regulated the expression of cav-1, whereas the non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester and inducible NOS (iNOS) inhibitor 1400W each abolished the increase in cav-1 expression in the hypoxic SK-N-MC cells. These results suggest that iNOS-induced NO production contributes to the up-regulation of cav-1 in the hypoxic SK-N-MC cells. Furthermore, we studied the roles played by cav-1 in regulating NO, NOS, and apoptotic cell death in the SK-N-MC cells subjected to 15 h of hypoxic treatment. Both cav-1 transfection and cav-1 scaffolding domain peptide abolished the induction of iNOS, reduced the production of NO, and reduced the rates of apoptotic cell death in the hypoxic SK-N-MC cells. These results suggest that increased expression of cav-1 in response to hypoxic stimulation could prevent oxidative injury induced by reactive oxygen species. The interactions of cav-1, NO, and NOS could be an important signal pathway in protecting the neuroblastoma cells from oxidative injury, contributing to the hypoxic tolerance of neuroblastoma cells.     

Ascorbic acid and N-acetyl cysteine prevent uncoupling of nitric oxide synthase and increase tolerance to ischemia/reperfusion injury in diabetic rat heart

Abstract

Oxidative stress may cause a loss of tetrahydrobiopterin (BH4), a co-factor of nitric oxide synthase (NOS), decrease the bioavailability of NO and aggravate ischemia/reperfusion (I/R) injury in diabetic heart. We hypothesized that ascorbic acid (AA) and N-acetyl cysteine (NAC) protect the diabetic heart from I/R injury by increasing BH4/dihydrobiopterin (BH2) ratio and inhibiting uncoupling of NOS. Diabetes mellitus was induced in rats by streptozotocin treatment, and the hearts were isolated and perfused. BH4 and BH4/BH2 ratio decreased in the diabetic heart associated with increased production of superoxide and nitrotyrosine (NT). Treatment with AA or NAC significantly increased BH4/BH2 ratio in the diabetic heart associated with decreased production of superoxide and NT and increased generation of nitrate plus nitrite (NOx). Pre-treatment with AA or NAC before 30 min ischemia followed by 120 min reperfusion improved left ventricular (LV) function and reduced infarct size in the diabetic but not non-diabetic hearts. The NOS inhibitor, L-NAME, inhibited the increase in the generation of superoxide, NT and NOx, but aggravated LV function and increased infarct size in the diabetic heart. L-NAME also abrogated the increase in NOx and improvement of LV function and the infarct size-limiting effect induced by AA or NAC in the diabetic heart. These results suggest that AA and NAC increase BH4/BH2 ratio and prevent NOS uncoupling in the diabetic heart. Resultant increase in the bioavailability of NO renders the diabetic heart toleratant to I/R injury.     

Role of nitric oxide in the reperfusion induced injury in hyperthyroid rat hearts

We recently reported that hyperthyroidism affects the heart response to ischemia/reperfusion. A significant tachycardia during reperfusion was associated with an increase in the oxidative stress of hearts from T₃-treated animals. In the present study we checked the possible role of nitric oxide (NO) in this major stress induced by the hyperthyroid state. We compared the functional recovery from ischemia/reperfusion of Langendorff preparations from euthyroid (E) and hyperthyroid (H, ten daily intraperitoneal injections of T₃, 10 μg/100 g body weight) rats, in the presence and in the absence of 0.2 mM N^ω-nitro-L-arginine (L-NNA). At the end of the ischemia/reperfusion protocol (10 min preischemic perfusion, 20 min global ischemia, 30 min reperfusion) lipid peroxidation, antioxidant capacity (C_A) and susceptibility to in vitro oxidative stress were determined on heart homogenates. The main effect of hyperthyroidism on the reperfusion functional response was confirmed to be a strong tachycardic response (154% recovery at 25 min reperfusion) accompanied by a low recovery in both left ventricular diastolic pressure (LVDP) and left ventricular dP/dt_max. This functional response was associated with a reduction in C_A and an increase in both lipid peroxidation and susceptibility to oxidative stress. Perfusion of hearts with L-NNA per se had small but significant negative chronotropic and positive inotropic effects on preischemic performance of euthyroid rat hearts only. More importantly, L-NNA perfusion completely blocked the reperfusion tachycardic response in the hyperthyroid rats. Concomitantly, myocardium oxidative state (lipid peroxidation, C_A and in vitro susceptibility to oxidative stress) of L-NNA perfused hearts was similar to that of E animals. These results suggest that the higher reperfusion-induced injury occurring in hyperthyroid animals is associated with overproduction of nitric oxide.     

Genetic engineering, expression, and activity of a fusion protein of a human neurotrophin and a molecular Trojan horse for delivery across the human blood-brain barrier

Neurotrophins, such as brain derived neurotrophic factor (BDNF), do not cross the blood-brain barrier (BBB). Certain monoclonal antibodies (MAb) to the human insulin receptor (HIR) do cross the BBB via receptor-mediated transport, and can act as a molecular Trojan horse to ferry across the BBB an attached drug. A genetically engineered fusion protein was produced whereby the amino terminus of human BDNF is fused to the carboxyl terminus of the heavy chain of a chimeric HIRMAb. The HIRMAb-BDNF fusion protein reacted equally with antibodies to human IgG and BDNF. The bi-functionality of the fusion protein was retained as the affinity of the fusion protein for the HIR was identical to that of the chimeric HIRMAb, and the affinity of the fusion protein for the trkB receptor was identical to that of BDNF. The fusion protein was equi-potent with BDNF in a neuroprotection assay in human neural cells. The pharmacokinetics (PK) of the fusion protein was examined in the adult Rhesus monkey. The mean residence time (MRT) of the fusion protein in blood was >100-fold longer than the MRT of BDNF. Therapeutic levels of BDNF were produced in primate brain following the intravenous administration of the fusion protein. A fusion protein tandem vector was engineered that allowed for isolation of a CHO cell line that produced the fusion protein at high levels in serum free medium. Neurotrophins, such as BDNF, can be re-formulated to enable these molecules to cross the human BBB, and such fusion proteins represent a new class of human neurotherapeutics.     

Characterization of cerebral glucose dynamics in vivo with a four-state conformational model of transport at the blood-brain barrier

Determination of brain glucose transport kinetics in vivo at steady-state typically does not allow distinguishing apparent maximum transport rate (T(max)) from cerebral consumption rate. Using a four-state conformational model of glucose transport, we show that simultaneous dynamic measurement of brain and plasma glucose concentrations provide enough information for independent and reliable determination of the two rates. In addition, although dynamic glucose homeostasis can be described with a reversible Michaelis-Menten model, which is implicit to the large iso-inhibition constant (K(ii)) relative to physiological brain glucose content, we found that the apparent affinity constant (K(t)) was better determined with the four-state conformational model of glucose transport than with any of the other models tested. Furthermore, we confirmed the utility of the present method to determine glucose transport and consumption by analysing the modulation of both glucose transport and consumption by anaesthesia conditions that modify cerebral activity. In particular, deep thiopental anaesthesia caused a significant reduction of both T(max) and cerebral metabolic rate for glucose consumption. In conclusion, dynamic measurement of brain glucose in vivo in function of plasma glucose allows robust determination of both glucose uptake and consumption kinetics.     

Different mechanisms influencing permeation of PDGF-AA and PDGF-BB across the blood-brain barrier

Platelet-derived growth factor (PDGF) exerts neurotrophic and neuromodulatory effects on the CNS. To determine the permeability of the blood-brain barrier (BBB) to PDGF, we examined the blood-to-brain influx of radioactively labeled PDGF isoforms (PDGF-AA and PDGF-BB) by multiple-time regression analysis after intravenous (i.v.) injection and by in-situ perfusion, and also determined the physicochemical characteristics which affect their permeation across the BBB, including lipophilicity (measured by octanol:buffer partition coefficient), hydrogen bonding (measured by differences in octanol : buffer and isooctane : buffer partition coefficients), serum protein binding (measured by capillary electrophoresis), and stability of PDGF in blood 10 min after i.v. injection (measured by HPLC). After i.v. bolus injection, neither 125I-PDGF-AA nor 125I-PDGF-BB crossed the BBB, their influx rates being similar to that of the vascular marker 99mTc-albumin. 125I-PDGF-AA degraded significantly faster in blood than 125I-PDGF-BB. PDGF-BB, however, was completely bound to a large protein in serum whereas PDGF-AA showed no binding. Thus, degradation might explain the poor blood-to-brain influx of PDGF-AA, whereas protein binding could explain the poor influx of circulating PDGF-BB. Despite their lack of permeation in the intact mouse, both 125I-PDGF-AA and 125I-PDGF-BB entered the brain by perfusion in blood-free buffer, and the significantly faster rate of 125I-PDGF-AA than 125I-PDGF-BB may be explained by the lower hydrogen bonding potential of 125I-PDGF-AA. Thus, the lack of significant distribution of PDGF from blood to brain is not because of the intrinsic barrier function of the BBB but probably because of degradation and protein binding. Information from these studies could be useful in the design of analogues for delivery of PDGF as a therapeutic agent.     

Engineering and expression of a chimeric transferrin receptor monoclonal antibody for blood-brain barrier delivery in the mouse

Protein therapeutics may be delivered across the blood-brain barrier (BBB) by genetic fusion to a BBB molecular Trojan horse. The latter is an endogenous peptide or a peptidomimetic monoclonal antibody (MAb) against a BBB receptor, such as the insulin receptor or the transferrin receptor (TfR). Fusion proteins have been engineered with the MAb against the human insulin receptor (HIR). However, the HIRMAb is not active against the rodent insulin receptor, and cannot be used for drug delivery across the mouse BBB. The rat 8D3 MAb against the mouse TfR is active as a drug delivery system in the mouse, and the present studies describe the cloning and sequencing of the variable region of the heavy chain (VH) and light chain (VL) of the rat 8D3 TfRMAb. The VH and VL were fused to the constant region of mouse IgG1 heavy chain and mouse kappa light chain, respectively, to produce a new chimeric TfRMAb. The chimeric TfRMAb was expressed in COS cells following dual transfection with the heavy and light chain expression plasmids, and was purified by protein G affinity chromatography. The affinity of the chimeric TfRMAb for the murine TfR was equal to the 8D3 MAb using a radio-receptor assay and mouse fibroblasts. The chimeric TfRMAb was radio-labeled and injected into mice for a pharmacokinetics study of the clearance of the chimeric TfRMAb. The chimeric TfRMAb was rapidly taken up by mouse brain in vivo at a level comparable to the rat 8D3 MAb. In summary, these studies describe the genetic engineering, expression, and validation of a chimeric TfRMAb with high activity for the mouse TfR, which can be used in future engineering of therapeutic fusion proteins for BBB drug delivery in the mouse.     

Role of ERK1/2 in the anti-apoptotic and cardioprotective effects of nitric oxide after myocardial ischemia and reperfusion

Objective: Experimental results from cultured cells suggest that there is cross-talk between nitric oxide (NO) and extracellular signal-regulated kinase (ERK) in their anti-apoptotic effect. However, the cross-talk between these two molecules in either direction has not been confirmed in the whole organ or whole animal level. The aim of the present study was to determine whether ERK may play a role in the anti-apoptotic and cardioprotective effects of NO in myocardial ischemia/reperfusion (MI/R). Methods: Isolated perfused mouse hearts were subjected to 20 min of global ischemia and 120 min of reperfusion and treated with vehicle or an NO donor (SNAP, 10 μM) during reperfusion. To determine the role of ERK1/2 in the anti-apoptotic and cardioprotective effects of NO, hearts were pre-treated (10 min before ischemia) with U0126, a selective MEK1/2 inhibitor (1 μM). Results: Treatment with SNAP exerted significant cardioprotective effects as evidenced by reduced cardiac apoptosis (TUNEL and caspase 3 activity, p < 0.01), and improved cardiac functional recovery (p < 0.01). In addition, treatment with SNAP resulted in a 2.5-fold increase in ERK activation when compared with heart receiving vehicle. Pre-treatment with U0126 slightly increased post-ischemic myocardial apoptosis but had no significant effect on cardiac functional recovery in this isolated perfused heart model. However, treatment with U0126 completely blocked SNAP-induced ERK activation and markedly, although not completely, inhibited the cardioprotection exerted by SNAP. Conclusion: These results demonstrate that nitric oxide exerts its anti-apoptotic and cardioprotective effects, at least in part, by activation of ERK in ischemic/reperfused heart. The first two authors contribute equally to this study.     

大鼠心脏缺血-再灌注损伤对心肌L-Arg/NO途径的影响

为探讨大鼠心脏缺血-再灌注损伤(IRI)期间一氧化氮(NO)生成增加的环节和过程。本实验用离体灌流大鼠心脏,预灌流15 min,停灌45 min,取30 ml KH 液循环灌流15 min,观察冠脉流出液中细胞胞浆酶(LDH)、蛋白质、肌红蛋白漏出量和NO     

一氧化氮在大鼠肢体缺血再灌注后肺损伤中的作用

在大鼠肢体缺血再灌注（LIR）损伤模型上,观察应用一氧化氮合酶（NOS）抑制剂氨基胍（AG）及一氧化氮（NO）合成前体物质L－精氨酸（L－Arg)对大鼠骨骼肌和肺组织的NOS活性、NO含量、丙二醛（MDA）、髓过氧化物酶（MPO）和湿／干重（W／D）值的影响以及肺磷脂酰胆碱（PC）的改变,并观察了肺组织在光镜下形态学的变化。结果显示,与对照组比较,LIR组骨骼肌和肺组织NOS活性均增强,MDA值、MPO活性增加,W／D值增大,肺PC含量降低;光镜下,肺间质多形核粒细胞（PMN）聚集和浸润,肺间隔面密度值增加。给予AG后,与LIR组相比NOS活性降低,NO产生下降,而MPO活性、W／D比值增加,肺PC含量进一步降低;镜下PMN聚集和浸润增加,肺间隔面密度值增大。而给予L－Arg后能 减轻LIR引起的上述变化。上述结果提示,LIR后2h时,骨骼肌和肺组织NOS活性增加,NO产生增多;内源性NO可能在LIR所诱发的早期急性肺损伤中起保护作用。     

The relationship between erythropoietin pretreatment with blood-brain barrier and lipid peroxidation after ischemia/reperfusion in rats

Blood-brain barrier (BBB) leakage plays a role in the pathogenesis of many pathological states of the brain including ischemia and some neurodegenerative disorders. In recent years, erythropoietin (EPO) has been shown to exert neuroprotection in many pathological conditions including ischemia in the brain. This study aimed to investigate the effects of EPO on BBB integrity, infarct size and lipid peroxidation following global brain ischemia/reperfusion in rats. Wistar male rats were divided into four groups (each group n=8); Group I; control group (sham-operated), Group II; ischemia/reperfusion group, Group III; EPO treated group (24 h before decapitation--000 U/kg r-Hu EPO i.p.), Group IV; EPO+ ischemia/reperfusion group (24 h before ischemia/reperfusion--3000 U/kg r-Hu EPO i.p.). Global brain ischemia was produced by the combination of bilateral common carotid arteries occlusion and hemorrhagic hypotension. Macroscopical and spectrophotometrical measurement of Evans Blue (EB) leakage was observed for BBB integrity. Infarct size was calculated based on 2,3,5-triphenyltetrazolium chloride (TTC) staining. Lipid peroxidation in the brain tissue was determined as the concentration of thiobarbituric acid-reactive substances (TBARS) for each group. Ischemic insult caused bilateral and regional BBB breakdown (hippocampus, cortex, corpus striatum, midbrain, brain stem and thalamus). EPO pretreatment reduced BBB disruption, infarct size and lipid peroxide levels in brain tissue with 20 min ischemia and 20 min reperfusion. These results suggest that EPO plays an important role in protecting against brain ischemia/reperfusion through inhibiting lipid peroxidation and decreasing BBB disruption.