代谢型谷氨酸受体阻断剂α-methyl-(4-tetrazolyl-phenyl)glycine对大鼠海马脑缺血耐受诱导的影响

实验采用大鼠四血管闭塞全脑缺血模型,用硫堇染色法和胶质纤维酸性蛋白(GFAP)免疫组化法,观察右侧脑室内注射Ⅱ型代谢型谷氨酸受体(metabotropic glutamate receptor 2/3,mGluR2/3)阻断剂α-methyl-(4-tetrazolyl-phenyl)glycine(MTPG)对海马CAl区神经元缺血耐受(BIT)诱导的影响,以探讨mGluR2／3在BIT诱导中的作用。54只大鼠推动脉凝闭后分为5组：(1)假手术组(n＝8)：游离双侧颈总动脉,但不夹闭;(2)单纯缺血组(n＝8)：夹闭双侧颈总动脉8min;(3)缺血预处理组(n＝8)：夹闭双侧颈总动脉3min作为脑缺血预处理(CIP),再灌注24h后再行夹闭8min;(4)MTPG 缺血预处理组(n＝22)：CIP前20min右侧脑室注射MTPG,其余步骤同缺血预处理组;MTPG的剂量分别为0．4、0．2、0．04和0．008mg,以观察其剂量效应关系;(5)MTPG 单纯缺血组(n＝8)：右侧脑室注射MTPG0．2mg 24h后,夹闭双侧颈总动脉8min。所有动物均在手术后或末次缺血后7d处死,取材观察。结果如下：(1)与假手术组相比,单纯8min缺血组海马CAl区组织学分级升高、锥体神经元密度降低,GFAP阳性表达增加(P＜0．05);(2)缺血预处理组的组织学分级、神经元密度及GFAP表达与假手术组相似,未见单纯缺血组的上述变化,表明CIP可防止后续8min缺血造成的神经元损伤;(3)MTPG 缺血预处理组海马CAl区组织学分级明显增加、锥体神经元密度降低,并且GFAP表达也明显增加(P＜0．05),这种变化与MTPG的剂量呈明显正相关,表明CIP对神经元的保护作用可被MTPG阻断;(4)MTPG 单纯缺血组海马CAl区组织学分级和神经元密度以及GFAP的表达与单纯缺血组相似。上述结果提示,3minCIP可诱导BIT的形成,MTPG可阻断CIP诱导BIT的作用,表明mGluR2／3参与BIT的诱导。     

全脑缺血模型中不同缺血时间参数对预处理保护效应的影响

目的：观察脑缺血预处理（CIP）的持续时间、CIP与后续损伤性缺血之间的间隔时间对CIP抗全脑缺血所致海马锥体神经元迟发性死亡（DND）作用的影响。方法：采用四血管闭塞法（4VO）,制作大鼠全脑缺血模型。脑组织切片硫堇染色法观察海马CA1区锥体神经元DND程度,确定组织学分级（HG）。结果：Sham组和3minCIP组海马未见DND。损伤性脑缺血组海马CA1区有明显的DND,其中6min、10min缺血组的HG为2～3级,15min缺血组的HG主要为3级。CIP＋损伤性脑缺血组中,3min-3d-6min（3minCIP后间隔三天给予6min损伤性脑缺血,下同）和3min-3d-10min组DND不明显,提示CIP可有效地保护海马CAl区神经元,防止6min或10min损伤性脑缺血诱导的DND。在3min-1d-10min组和3min-3d-15min组中,CIP的保护效应较3min-3d-10min组明显减弱。定量分析CIP对海马神经元的保护效应发现,3min-3d-6min组的神经元保护数（PN）和保护指数（PI）与3min-3d-10min组相比无明显差别（P〉0．05）;但3min-3d-10min组的神经元增长指数（GI）较3min一3d一6min组明显升高（P〈0．05）。结论：虽然3min-3d-6min组与3min-3d-10min组中CIP对神经元的保护作用相近,但3min-3d-10min组中,CIP的保护作用更容易被观察到,且CIP的保护潜能可得到最大程度的显现。应用3min-3d-10min组的时间参数建立全脑缺血耐受模型可以诱导出CIP最大的保护潜能。     

代谢性谷氨酸受体配体(s)-4C3HPG对大鼠脑缺血耐受性诱导的影响

目的：观察侧脑室注射代谢型谷氨酸受体1／5亚型（mGluR1/5)配体（s)-4C3HPG对海马脑缺血耐受（BIT）诱导的影响,以探讨mGLUR1/5在BIT诱导中的作用。方法：采用大鼠四血管闭塞全脑缺血模型（4－vessel occlusion,4VO),应用硫堇染色和GFAP免疫组化法。36只大鼠椎动脉凝闭后分为sham组、单纯缺血组、BIT组和（s)-4C3HPG组,其中（s)-4C3HPG组又按所给药物剂量不同,分为0．2、0．04和0．008mg三个亚组。所有动物均在手术后或末次缺血后7d处死取材观察。结果：（1）单纯8min缺血可使海马CA1区组织学分级升高、锥体神经元密度降低和胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP)阳性表达增加（P＜0．05vs sham）.(2)BIT组未见单纯缺血组的上述变化,表明CIP可防止后续8min缺血造成的神经元损伤。（3）CIP的这种保护作用可被（s)-4C3HPG阻断,表现为海马CA1区组织学分级升高和锥体神经元密度降低（P＜0．05 vs sham)。这种变化与（s)-4C3HPG的剂量呈现明显的相关性,即剂量越大,上述改变越明显。结论：（s)-4C3HPG可阻断CIP诱导BIT的作用,提示mGluR1/5参与BIT的诱导。     

神经元型一氧化氮合酶在Ⅱ组代谢型谷氨酸受体介导的脑缺血耐受中的作用

目的：探讨神经元型一氧化氮合酶（nNOS）催化产生的一氧化氮（NO）在Ⅱ组代谢型谷氨酸受体（mGluR2／3）介导的脑缺血预处理（CIP）保护机制中的作用。方法：36只永久凝闭椎动脉的SD大鼠随机分为6组（n=6）：sham、CIP、损伤性缺血、CIP4-损伤性缺血、MqPG＋CIP和MTPG＋CIP＋损伤性缺血组。采用硫堇染色和免疫组化观察海马CA1区迟发性神经元死亡（DND）和nNOS表达的变化。结果：与Sham组相比,CIP组海马nNOS表达出现一定程度的上调,而损伤性脑缺血组则出现nNOS表达的明显上调,预先给与CIP可一定程度上防止损伤性脑缺血所致的nNOS表达的过度升高。在MTPG4-CIP组,预先侧脑室注射mGluR2／3阻断剂MTPG,可阻断CIP引起的nNOS表达增加,但对神经元的存活无影响。而在MTPG＋CIP＋损伤性缺血组中,出现大量锥体神经元DND,同时nNOS的表达较MTPG＋CIP组明显增加,该增加为损伤性脑缺血所致,而非MTPG的作用。结论：nNOS催化产生的NO作为mGluR2／3的下游分子参与脑缺血预处理过程中mGluR2／3介导的脑缺血耐受的形成。     

金属硫蛋白-ⅢmRNA表达与神经元缺血性损伤的关系

目的：探讨大鼠前脑缺血／再灌注后海马结构MT-ⅢmRNA表达变化规律及其与神经元缺血性损伤之间的关系。方法：建立前脑缺血／再灌注模型,用原住杂交法检测海马结构MT-ⅢmRNA表达,并观察缺血后各时相点海马神经元的病理变化。结果：①前脑缺血／再灌注后72h海马CAl区开始出现神经元变性,96h更为明显,7d时CAl区神经元多已坏死;②前脑缺血／再灌注后海马CAl区锥体细胞和齿状回颗粒细胞内MT-ⅢmRNA表达逐渐增加,96h达高峰,7d又降低至缺血前水平。结论：前脑缺血／再灌注后,海马神经元MT—ⅢmRNA表达增加,可能对神经元缺血性损伤产生影响。     

大鼠侧脑室注射腺苷A1受体反义寡聚脱氧核苷酸抑制脑缺血耐受的诱导

目的:观察侧脑室注射腺苷A1受体(ARA1)反义寡聚脱氧核苷酸(As-ODN)对脑缺血预处理(CIP)脑保护作用的影响,进一步探讨腺苷A1受体在CIP脑保护作用中的作用。方法:将54只凝闭双侧椎动脉的Wistar大鼠分为Sham组、CIP组、损伤性脑缺血组、CIP 损伤性脑缺血组、双蒸水 CIP 损伤性脑缺血组、ARA1As-ODN组、ARA1As-ODN CIP组、和ARA1As-ODN CIP 损伤性脑缺血组。ARA1As-ODN的剂量分为10nmol/5μl和20nmol/5μl,溶于双蒸水中,侧脑室注射。所有动物均在Sham手术后或末次全脑缺血/再灌注后7d断头取脑,硫堇染色观察海马CA1区锥体神经元迟发性死亡(DND)情况。结果:Sham组和CIP组均未见DND。与Sham、CIP组相比,损伤性脑缺血组出现了明显的DND,表现为组织学分级(HG)升高和锥体神经元密度(ND)下降(P<0.05)。CIP可显著抑制损伤性脑缺血引起的DND。与CIP 损伤性缺血组相比,ARA1As-ODN CIP 损伤性脑缺血组出现了显著的DND,表现为HG升高、ND降低(P<0.05),这种变化与ARA1As-ODN的剂量呈明显正相关。结论:腺苷A1受体As-ODN可阻断CIP诱导的脑缺血耐受,进一步证实了腺苷A1受体表达上调参与CIP诱导的脑缺血耐受。     

肢体缺血预处理减轻大鼠海马缺血/再灌注损伤

目的:探讨肢体缺血预处理(LIP)对大鼠全脑缺血/再灌注损伤的影响.方法: 36只大鼠椎动脉凝闭后随机分为假手术(Control)组、脑缺血组、肢体缺血组、LIP 0 d组(LIP后即刻行脑缺血)、LIP 1 d组(LIP后1 d行脑缺血)和LIP 2 d组(LIP后2 d行脑缺血).重复夹闭大鼠双侧股动脉3次(每次10 min,间隔10 min)作为LIP,夹闭颈总动脉进行全脑缺血8 min后再灌注.硫堇染色观察海马CA1区组织学分级及锥体神经元密度以判断海马损伤程度.结果:脑缺血组海马CA1区锥体神经元损伤严重,与Control组比较,组织学分级明显升高,神经元密度明显降低(P<0.01).LIP 0 d组海马CA1区神经元损伤较脑缺血组明显减轻,组织学分级明显降低,神经元密度明显升高(P<0.01).而LIP 1 d组和LIP 2 d组大鼠海马CA1区锥体细胞缺失较多,仍有明显的组织损伤.结论:LIP可减轻随后立即发生的脑缺血/再灌注损伤,但对间隔1 d后的脑缺血/再灌注损伤无显著对抗作用.     

胶质细胞谷氨酸转运体-1反义寡核苷酸对脑缺血预处理神经保护效应的抑制作用

本研究应用胶质细胞谷氨酸转运体-1(glial glutamate transporter-1,GLT-1)的反义寡核苷酸(antisense oligo-deoxynucleotides,AS-ODNs)抑制Wistar大鼠GLT-1蛋白的表达,观察其对脑缺血预处理(cerebral ischemic preconditioning.CIP)增强脑缺血耐受作用的影响,探讨GLT-1在CIP诱导的脑缺血耐受中的作用.将凝闭双侧椎动脉的Wistar大鼠随机分为7组:(1)Sham组:只暴露双侧颈总动脉,不阻断血流;(2)CIP组:夹闭双侧颈总动脉3 min;(3)脑缺血打击组:夹闭双侧颈总动脉8 min;(4)CIP 脑缺血打击组:夹闭双侧颈总动脉3 min作为CIP,再灌注2 d后,夹闭双侧颈总动脉8min;(5)双蒸水组:于分离暴露双侧颈总动脉(但不夹闭)前12 h、后12 h及后36 h右侧脑室注射双蒸水,每次5 μL,其它同sham组;(6)AS-ODNs组:于分离暴露双侧颈总动脉(但不夹闭)前12 h、后12 h及后36 h右侧脑室注射GLT-1 AS-ODNs溶液,每次5 μL,其它同sham组,再根据AS-ODNs的剂量进一步分为9 nmol和18 nmol 2个亚组;(7)AS-ODNs CIP 脑缺血打击组:于CIP前12 h、后12 h及后36 h右侧脑室注射GLT-1 AS-ODNs溶液,每次5 μL,其它同CIP 脑缺血打击组,根据AS-ODNs的剂量进一步分为9 nmol和18 nmol 2个亚组.Western blot分析法观察GLT-1蛋白的表达,硫堇染色观察海马CA1区锥体神经元迟发性死亡(delayed neuronal death,DND)情况.Western blot分析显示,侧脑室注射GLT-1 AS-ODNs可剂量依赖性地抑制大鼠海马CA1区GLT-1蛋白表达.硫堇染色显示,sham组和CIP组海马CA1区未见明显的DND;脑缺血打击组海马CA1区有明显的DND:预先给予CIP可显著对抗脑缺血打击引起的DND,表明CIP可以诱导海马CA1区神经元产生缺血性耐受,对抗脑缺血打击引起的DND;而在GLT-1 AS-ODNs CIP 脑缺血打击组,侧脑室注射GLT-1 AS-ODNs后,大鼠海马CA1区出现了明显的DND,表明GLT-1 AS-ODNs通过抑制大鼠GLT-1蛋白表达从而减弱CIP对抗脑缺血打击的神经保护作用.以上结果进一步证实了GLT-1参与CIP诱导的脑缺血耐受.     

镁预处理对兔全脑缺血神经保护作用的实验研究

目的探讨硫酸镁预处理对兔全脑缺血神经保护作用及其机制.方法 45只兔随机分为3组:对照组(n=15)、缺血组(n=15)和镁预处理组(n=15).阻断血管,诱导全脑缺血,缺血时间为 6 min.测定缺血再灌注30 min 兔海马谷氨酸、天冬氨酸、γ-氨基丁酸和甘氨酸含量.检测缺血再灌注3 d 海马CA1区神经元密度和凋亡神经元密度.结果 (1)镁预处理组海马天冬氨酸和甘氨酸显著低于缺血组(P<0.01),而γ-氨基丁酸含量显著高于缺血组(P<0.05);(2)镁预处理组正常神经元密度显著高于缺血组 (P<0.01),缺血神经元密度显著低于缺血组(P<0.01);(3)镁预处理组凋亡神经元密度显著低于缺血组(P<0.01).结论 (1)硫酸镁预处理对兔全脑缺血有神经保护作用;(2)该保护作用的机制可能有:1)抑制兔全脑缺血再灌注30 min 海马天冬氨酸和甘氨酸的过度释放以及抑制γ-氨基丁酸的耗竭;2)抑制兔全脑缺血再灌注3 d 海马CA1区神经元凋亡.     

肾动脉内注射L-精氨酸抑制肾神经传入纤维的自发活动

研究旨在应用记录肾传人神经多单位和单位放电的方法,观察肾动脉内注射L—精氨酸对麻醉家兔肾神经传人纤维自发放电活动的影响。结果表明：(1）肾动脉内注射L—精氨酸(0．05、0．24和0．48mmol/kg)可呈剂量依赖性地抑制肾传人纤维的活动,而动脉血压不变;(2)静脉内预先注射一氧化氮合酶抑制剂L—NAME(0．11mmol/kg),可完全阻断L—精氨酸对肾传人纤维的抑制;(3)肾动脉注射一氧化氮(N0)供体SIN—1(3．75μmol/kg)也可抑制肾传入神经的活动。以上结果提示：肾动脉内应用N0前体L—精氨酸和N0供体SIN—1均可抑制肾传入纤维的自发活动。     

Differing Neurochemical and Morphological Sequelae of Global Ischemia: Comparison of Single- and Multiple-Insult Paradigms

The purpose of this investigation was to investigate pathomechanisms responsible for the deleterious effects of repeated episodes of brief forebrain ischemia. Halothane-anesthetized male Wistar rats were subjected to either (a) a single 15-min period or (b) three 5-min periods (separated by 1 h) of global forebrain ischemia by bilateral carotid artery occlusions plus hypotension (50 mm Hg), followed by various periods of recirculation. Brain temperature was normothermic throughout. In one series of rats, extracellular levels of glutamate, glycine, and gamma-aminobutyric acid (GABA) were measured in the dorsolateral striatum (n = 6-8 per group) and lateral thalamus (n = 4-6 per group) by microdialysis and HPLC before and during ischemia and during 3-5 h of recirculation. In a parallel series of rats (n = 6 per group), ischemic cell change was quantified at 2 (dark neurons), 24, or 72 h following either single or multiple ischemic insults. A single 15-min ischemic period led to massive glutamate release (13-fold increase; p = 0.001), which returned to normal by 20-30 min of recirculation and remained normal thereafter. By contrast, in rats with three 5-min periods of ischemia, the glutamate level rise with each repeated insult (four- to 4.5-fold; p < or = 0.02) was smaller than that observed during the single 15-min insult, but a late sustained rise (five- to six-fold; p < 0.05) occurred at 2-3 h of recirculation. Brief ischemia-induced elevations of glycine and GABA levels were detected in both the single- and multiple-insult groups, with normalization during recirculation. In contrast, the excitotoxic index, a composite measure of neurotransmitter release ([glutamate] x [glycine]/[GABA]), differed markedly following single versus multiple insults (p = 0.002 by repeated-measures analysis of variance) and increased by seven- to 12-fold (p < 0.05) at 1-3 h following the third insult. The total amount of glutamate released was 3.3-fold higher in the multiple-insult than in the single-insult group (p < 0.02). At 2 h of recirculation, histopathological analysis of dorsolateral striatum showed a significantly greater frequency of dark neurons in the multiple- than in the single-insult group (p < 0.05 by analysis of variance). In the thalamus, a higher frequency of ischemic neurons was seen in the multiple-than in the single-insult group at all intervals studied. Thus, in rats with multiple ischemic insults, accelerated ischemic damage was found in the striatum, and severe ischemic injury was documented in the thalamus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cerebral ischemic pre-conditioning enhances the binding characteristics and glutamate uptake of glial glutamate transporter-1 in hippocampal CA1 subfield of rats

Glial glutamate transporter-1 (GLT-1) is the predominant subtype of glutamate transporters which are responsible for the homeostasis of extracellular glutamate. Our previous studies have shown that up-regulation in GLT-1 protein expression matches brain ischemic tolerance induced by cerebral ischemic preconditioning (CIP). To specify the role of functional changes of GLT-1 in the induction of brain ischemic tolerance by CIP, the present study was undertaken to examine changes in the binding properties of GLT-1 (including maximum binding and affinity for glutamate) and in GLT-1 mediated glutamate uptake, using L-3H-glutamate assay in the rat hippocampus. The results indicated that CIP was able to increase the maximum binding and affinity, and uptake of GLT-1 for glutamate in hippocampal CA1 subfield either with or without the presence of the subsequent severe brain ischemic insult. Simultaneously, accompanied with the above changes, CIP significantly reduced the delayed neuronal death (DND) in this region induced by lethal global cerebral ischemia. It could be concluded that up-regulation in the maximum binding and affinity and glutamate uptake of GLT-1 contributed to the neuronal protection of CIP against global cerebral ischemic insult.     

Cerebral ischemic preconditioning reduces glutamate excitotoxicity by up-regulating the uptake activity of GLT-1 in rats

Our previous study has shown that cerebral ischemic preconditioning (CIP) can up-regulate the expression of glial glutamate transporter-1 (GLT-1) during the induction of brain ischemic tolerance in rats. The present study was undertaken to further explore the uptake activity of GLT-1 in the process by observing the changes in the concentration of extracellular glutamate with cerebral microdialysis and high-performance liquid chromatography. The results showed that a significant pulse of glutamate concentration reached the peak value of sevenfold of the basal level after lethal ischemic insult, which was associated with delayed neuronal death in the CA1 hippocampus. When the rats were pretreated 2 days before the lethal ischemic insult with CIP which protected the pyramidal neurons against delayed neuronal death, the peak value of glutamate concentration decreased to 3.9 fold of the basal level. Furthermore, pre-administration of dihydrokainate, an inhibitor of GLT-1, prevented the protective effect of CIP on ischemia-induced CA1 cell death. At the same time, compared with the CIP + Ischemia group, the peak value of glutamate concentration significantly increased and reached sixfold of the basal level. These results indicate that CIP induced brain ischemic tolerance via up-regulating GLT-1 uptake activity for glutamate and then decreasing the excitotoxicity of glutamate.     

Neuroprotection by the N-Methyl-d-Aspartate Receptor Antagonist CGP 40116: In Vivo and In Vitro Studies

Abstract: The goal of this study was to evaluate the effects of a novel competitive N -methyl- d -aspartate (NMDA) receptor antagonist, d -( E )-2-amino-4-methyl-5-phosphono-3-pentoic acid (CGP 40116), on neuronal damage in vivo and in vitro. We studied 20 rabbits that underwent a 2-h occlusion of the left internal carotid, anterior cerebral, and middle cerebral arteries followed by 4 h of reperfusion. Ten minutes after occlusion the animals were treated with either normal saline (n = 7) or CGP 40116 at two different doses (20 mg/kg, n = 6; 40 mg/kg, n = 7) administered over a 5-min period. Somatosensory evoked potentials were used to confirm adequate ischemia and neuronal injury was assessed by histopathology and magnetic resonance imaging. CGP 40116 decreased cortical ischemic neuronal damage by 74 and 77% (control, 37.8%± 13.1%; CGP 20 mg/kg, 9.9 ± 3.6%; CGP 40 mg/kg, 8.7 ± 3.7%; p < 0.01) and reduced cortical ischemic edema by 52 and 35% (control, 42.3 ± 10.4%; CGP 20 mg/kg, 20.1 ± 6.7%; CGP 40 mg/kg, 27.5 ± 13.3%; p < 0.05) but did not protect against striatal injury. We performed a second study using primary cell cultures from mouse neocortex to determine the effects of CGP 40116 on neuronal death induced by a 10-min exposure to 500 µ M NMDA or by 45 min of oxygen-glucose deprivation (OGD). Our results demonstrate that CGP 40116 was effective at attenuating neuronal death in a concentration-dependent manner (ED₅₀ of 3.2 µ M against NMDA toxicity and 23.1 µ M against OGD) as measured by lactate dehydrogenase levels 24 h after the insult. The neuroprotective effects of CGP 40116 in vivo and in vitro suggest it may be of potential clinical therapeutic value.     

Effects of L-NAME and L-arginine on ischemia-reperfusion injury in rat skeletal muscle

The involvement of nitric oxide in ischemia-reperfusion injury remains controversial and has been reported to be both beneficial and deleterious, depending on the tissue and model used. This study evaluated the effects of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine-methyl ester (L-NAME) and the substrate for nitric oxide synthase, L-arginine on skeletal muscle necrosis in a rat model of ischemia-reperfusion injury. The rectus femoris muscle in male Wistar rats (250 to 500 g) was isolated on its vascular pedicle and subjected to 4 hours of complete arteriovenous occlusion. The animals were divided into five groups: (1) sham-raised control, no ischemia, no treatment (n = 6); (2) 4 hours of ischemia (n = 6); (3) vehicle control, 4 hours of ischemia + saline (n = 6); (4) 4 hours of ischemia + L-arginine infusion (n = 6); and (5) 4 hours of ischemia + L-NAME infusion (n = 6). The infusions (10 mg/kg) were administered into the contralateral femoral vein beginning 5 minutes before reperfusion and during the following 30 to 45 minutes. Upon reperfusion, the muscle was sutured in its anatomic position and all wounds were closed. The percentage of muscle necrosis was assessed after 24 hours of reperfusion by serial transections, nitroblue tetrazolium staining, digital photography, and computerized planimetry. Sham (group 1) animals sustained baseline necrosis of 11.9 +/- 3.0 (percentage necrosis +/- SEM). Four hours of ischemia (group 2) significantly increased necrosis to 79.2 +/- 1.4 (p < 0.01). Vehicle control (group 3) had no significant difference in necrosis (81.17 +/- 5.0) versus untreated animals subjected to 4 hours of ischemia (group 2). Animals treated with L-arginine (group 4) had significantly reduced necrosis to 34.6 +/- 7.5 versus untreated (group 2) animals (p < 0.01). Animals infused with L-NAME (group 5) had no significant difference in necrosis (68.2 +/- 6.7) versus untreated (group 2) animals. L-Arginine (nitric oxide donor) significantly decreased the severity of muscle necrosis in this rat model of ischemia-reperfusion injury. L-arginine is known to increase the amount of nitric oxide through the action of nitric oxide synthase, whereas L-NAME, known to inhibit nitric oxide synthase and decrease nitric oxide production, had comparable results to the untreated 4-hour ischemia group. These results suggest that L-arginine, presumably through nitric oxide mediation, appears beneficial to rat skeletal muscle subjected to ischemia-reperfusion injury.     

Real-time monitoring of nitric oxide and blood flow during ischemia-reperfusion in the rat testis

In the present study, we attempted to clarify the role of nitric oxide (NO) and its release during the ischemia-reperfusion rat testis. Eight-week-old male Sprague-Dawley rats were divided into seven groups: age-matched control rats, ischemia (30 minutes)-reperfusion (30 minutes) rats without N^G-nitro-L-arginine methyl ester (L-NAME) and L-arginine (L-Arg) treatment, ischemia (30 minutes)-reperfusion (30 minutes) rats treated with L-NAME (10, 30, and 100 mg/kg), ischemia-reperfusion rats treated with L-Arg (10 and 30 mg/kg). Sixty minutes prior to induction of ischemia, L-NAME or L-Arg was administrated intraperitoneally. Real-time monitoring of blood flow and NO release were measured simultaneously with a laser Doppler flowmeter and an NO-selective electrode, respectively. NO₂-NO₃ and malonaldehyde (MDA) concentrations were measured in the experimental testes. Furthermore, we investigated possible morphological changes in the testis. Clamping of the testicular artery decreased blood flow to 5–20% of the basal level measured before clamping. Immediately following clipping of the artery, NO release rapidly increased. After removing the clip, NO release gradually returned to the basal level. This phenomenon was enhanced by treatment with L-Arg and inhibited by treatment with L-NAME. NO₂-NO₃ concentrations were increased by treatment with L-Arg and decreased by treatment with L-NAME, while MDA concentrations were increased by treatment with L-NAME and were decreased by treatment with L-Arg. In histological studies, the ischemia-reperfusion caused infiltration of leukocytes and a rupture of microvessels in the testis. Our data suggest that NO has cytoprotective effects on ischemia-reperfusion injury in the rat testis.     

Differential effects of sublethal ischemia and chemical preconditioning with 3-nitropropionic acid on protein expression in gerbil hippocampus

Pretreatment with a low dose of 3-nitropropionic acid (3-NPA) has been shown to induce ischemic tolerance in the gerbil hippocampus. It is well known that sublethal (2-min) ischemia also induces ischemic tolerance. To investigate the acquisition of ischemic tolerance with 3-NPA, we examined the protein expression after 3-NPA treatment in comparison with sublethal ischemia. Immunohistochemical studies revealed intense expression of Bcl-2 and Bcl-xL in the hippocampal CA1 area after 3-NPA treatment. Furthermore, the time course of the expression of Bcl-xL showed a similar pattern to the acquisition of ischemic tolerance by 3-NPA treatment. The induction of Bcl-xL occurred in the hippocampal CA1 area at 24 h after 3-NPA treatment, and significant induction was observed at 48 h. Western blot analysis of hippocampus harvested 48 h after the pretreatment, showed that the expression of Bcl-2 and Bcl-xL was significantly increased by either 3-NPA treatment or 2-min ischemia. However, PMCA1 and HSP70 protein expression increased only in the sublethal ischemia treated group. The difference between 3-NPA treated group and control group was not statistically significant. These results suggest that Bcl-2 and Bcl-xL are essential for acquisition of ischemic tolerance, while HSP70 and PMCA1 play important roles in the enhancement of ischemic tolerance.