Cytidine-5'-diphosphocholine affects CTP-phosphocholine cytidylyltransferase and lyso-phosphatidylcholine after transient brain ischemia

Cytidine-5'-diphosphocholine (CDP-choline, also referred as citicoline), the key intermediate in phosphatidylcholine (PtdCho) synthesis, provided significant benefit in experimental central nervous system (CNS) injury including cerebral ischemia. CDP-choline is synthesized by CTP:phosphocholine cytidylyltransferase (CCT), the key rate-limiting enzyme in PtdCho synthesis. Phospholipase A(2) (PLA(2)) hydrolyzes PtdCho to produce free fatty acids and lyso-PtdCho, an inhibitor of CCT. We investigated the status of CCT and lyso-PtdCho after 10-min transient brain ischemia in gerbils with reperfusion up to 2 days. Ischemia with no reperfusion resulted in loss of CCT activity in cytosol (408 +/- 8 pmol/min/mg protein compared to sham 695 +/- 45; P < 0.01) and membrane (383 +/- 61 compared to sham 532 +/- 54; P < 0.05). CCT activity remained low over 24-hr reperfusion, and returned to sham levels at Day 2 in membrane but remained low in cytosol. CDP-choline significantly increased CCT activity in cytosol at 1 hr reperfusion (saline, 339 +/- 35 compared to CDP-choline, 430 +/- 70; P < 0.05) and in membrane at 6 hr (saline, 381 +/- 32 compared to CDP-choline, 489 +/- 50; P < 0.01) and 24 hr (saline, 417 +/- 24 compared to CDP-choline, 594 +/- 45; P < 0.01), but had no effect on CCT activity at Day 2. Lyso-PtdCho increased at 1-hr reperfusion (219 +/- 5 nmol/g tissue compared to sham, 92 +/- 8; P < 0.01), and remained elevated over 2 days. CDP-choline attenuated lyso-PtdCho levels at 1-hr reperfusion (162 +/- 21, P < 0.01 compared to saline). These data indicate that PtdCho synthesis is impaired after brain ischemia, and CDP-choline may increase PtdCho levels by attenuating the loss of CCT activity and lyso-PtdCho formation.     

阿司匹林对沙土鼠全脑缺血-再灌注后脑内一氧化氮合酶及一氧化氮的影响

目的观察阿司匹林对沙土鼠全脑缺血-再灌注后的脑保护作用及其与脑内一氧化氮合酶及一氧化氮水平变化的关系。方法采用夹闭双侧颈总动脉的方法,制备沙土鼠短暂性全脑缺血-再灌注模型。27只健康雄性蒙古沙土鼠随机分为假手术组、脑缺血-再灌注组和阿司匹林治疗组,观察缺血7min再灌注24h后沙土鼠脑组织的病理学改变,以及一氧化氮合酶与一氧化氮水平的变化。结果病理学检查结果显示,沙土鼠脑缺血7min再灌注24h后海马CA1区缺血性损害明显,脑组织内一氧化氮合酶及一氧化氮水平显著升高(P<0.01);与脑缺血-再灌注组相比,阿司匹林治疗组沙土鼠的病理损害较轻,一氧化氮合酶与一氧化氮水平明显下降(P<0.01)。结论阿司匹林可显著减轻脑缺血-再灌注后的脑损伤,其作用机制可能与抑制一氧化氮合酶与一氧化氮水平上升有关。     

Citicoline mechanisms and clinical efficacy in cerebral ischemia

Citicoline, an intermediate in the biosynthesis of phosphatidylcholine (PtdCho), has shown beneficial effects in various CNS injury models and neurodegenerative diseases. PtdCho hydrolysis by phospholipase A(2) (PLA(2)) after cerebral ischemia and reperfusion yields arachidonic acid (ArAc) and lyso-PtdCho. ArAc oxidative metabolism results in formation of reactive oxygen species and lipid peroxides. Lyso-PtdCho could inhibit activity of cytidine triphosphate-phosphocholine cytidylyltransferase (the rate-limiting enzyme in PtdCho biosynthesis), resulting in impaired PtdCho synthesis. Citicoline significantly increased glutathione levels and attenuated release of ArAc and the loss of PtdCho, cardiolipin, and sphingomyelin following transient cerebral ischemia. These effects could be explained by an effect of citicoline on PLA(2). Based on these observations, a mechanism has been hypothesized. This Mini-Review summarizes recent experimental data on the effects of citicoline in cerebral ischemia and evaluates several factors that might have hindered efficacy of citicoline in stroke clinical trials in the United States. Clinical stroke trials of citicoline in Europe and Japan have demonstrated beneficial effects. U.S. trials shown only marginal effects, which might be due to the 24 hr time window, the dose and route of administration, and the stringency of the primary outcome parameters. Recent evaluation of U.S. clinical data suggests that reduction of infarct growth may be a more sensitive measure of the citicoline effect than improvement on the NIH Stroke Scale (NIHSS) by > or =7 points. The citicoline neuroprotective mechanism has not been clearly identified, and its potential in stroke treatment might still be fully recognized in the United States. The clinical efficacy of citicoline should be examined further in light of the recent phase III stroke clinical trials and experimental data for cerebral ischemia.     

Immediate or delayed mild hypothermia prevents focal cerebral infarction

The protective effect of mild hypothermia was studied in rodent models of both permanent and transient focal cerebral ischemia. In Expt. 1, Wistar rats were exposed to 6 h permanent ischemia by bilateral occlusion of both common carotid arteries and right middle cerebral artery. In Expt. 2, animals were exposed to 3 h transient ischemia followed by 21 h reperfusion, and in Expt. 3, 3 h transient ischemia was followed by 69 h of reperfusion. Expt. 4 used 3 h transient ischemia followed by 3 h reperfusion. In Expt. 1, animals maintained at 37 degrees C rectal (normothermia) suffered a mean infarct volume (+/- S.D.) of 142 +/- 44 mm3 (n = 6), which was reduced for those exposed to permanent hypothermic (32 degrees C) ischemia to 56 +/- 64 mm3 (n = 10) (P less than 0.05). In Expt. 2, normothermic ischemia and reperfusion resulted in an infarction of 211 +/- 35 mm3 (n = 6). Intra-ischemic hypothermia (32 degrees C) followed by 21 h of normothermic reperfusion resulted in 17 +/- 12 mm3 of infarction (n = 9) (P less than 0.001). Hypothermia for either the first or second 1.5 h of the 3 h ischemic insult reduced the infarct volume to 116 +/- 76 mm3 (n = 6) (P less than 0.05) or 108 +/- 73 mm3 (n = 7) (P less than 0.01), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluating the efficacy of citicoline in embolic ischemic stroke in rats: neuroprotective effects when used alone or in combination with urokinase

The combination of thrombolysis with neuroprotection, because of different mechanisms, would be expected to show better results when used after onset of focal ischemia. In this study we report our experience with the neuronal protective effects of citicoline alone and in combination with urokinase in a model of focal ischemia. Both medications were injected 2 h after onset of a focal occlusion of the middle cerebral artery (MCA) in rats. Focal ischemia was produced with embolization of a clot into the origin of the MCA. This produces a large infarction involving the cortex and the basal ganglia. Animals were observed for neuronal deficts at 2 and 24 h after surgery and were sacrificed 72 h after onset of ischemia. Saline-treated animals showed a large infarction involving the cerebral cortex and basal ganglion in most animals (volume 33.1 +/- 9.7%). Animals treated with citicoline alone were divided in two groups. The first group of animals were treated with a single injection (300 mg/kg, ip) of the medication 2 h after the arterial occlusion. The second group was treated with the active medication intermittently (3 x 300 mg/kg, ip) over a 72-h period. There was a significant decrease in the neuronal damage in the cortex in the animals treated with citicoline (single dose, 20.9 +/- 9.7%, P = 0.01; intermittent injection, 18.9 +/- 11.4%, P < 0.008). The last experiment evaluated the usefulness of the combination of citicoline with intraarterial urokinase. The combination showed significantly more protection than with urokinase or citicoline alone (volume 13.6 +/- 9.1%, P < 0.001). We conclude from our experiments that citicoline may offer significant neuronal protection that may be further enhanced with the addition of a thrombolytic agent.     

Effect of piribedil, a D-2 dopaminergic agonist, on dopamine, amino acids, and free radicals in gerbil brain after cerebral ischemia

The excitatory amino acids (EAA) are involved in the pathogenesis of the cerebral ischemia. Moreover, several investigators have demonstrated that a considerable amount of dopamine (DA) is released in the striatum after ischemia reperfusion/insult (IRI). Recently, studies have demonstrated in vitro, that D-2 agonist, at the level of striatum and retina, may represent a powerful signal to inhibit release of excitatory amino acids implicated in cerebral ischemia. Therefore we have been incited to test, in vivo, the action of a D-2 agonist, piribedil, on gerbil brain after IRI. We have used the Stroke Index (SI); then to precise the mechanism of action, we have determined the levels of dopamine, EAA, and hydroxyl-free radicals (·OH), in striatum, hippocampus, and hemisphere. Piribedil, administered at dose of 10 mg/kg, per os, 60 min before induction of transient cerebral ischemia in gerbils, presents a neuroprotective effect, as measured by SI and significantly reverses the increase of DA, EAA, and ·OH induced by IRI. The mechanism of action of piribedil could be related to its D-2 agonist property.     

Effects of tempol, a membrane-permeable radical scavenger, in a gerbil model of brain injury

There is evidence that the excessive generation of reactive-oxygen radicals contributes to the brain injury associated with transient, cerebral ischemia. This study investigates the effects of tempol, a small, water-soluble molecule, that crosses biological membranes, on the brain injury caused by bilateral occlusion and reperfusion of both common carotid arteries in the gerbil (BCO). Treatment of gerbils with tempol (30 mg/kg i.p. at 30 min prior to reperfusion and at 1 and 6 h after the onset of reperfusion) reduced the formation of post-ischemic brain oedema. Tempol also attenuated the increase in the cerebral levels of malondialdehyde (MDA) and the hippocampal levels of myeloperoxidase (MPO) caused by cerebral ischemia and reperfusion. The immunohistochemical analysis of the hippocampal region of brains subjected to ischemia-reperfusion exhibited positive staining for nitrotyrosine (an indicator of the generation of peroxynitrite) and poly(ADP-ribose) synthetase (PARS) (an indicator of the activation of this nuclear enzyme secondary to single strand breaks in DNA). In gerbils subjected to BCO, which were treated with tempol, the degree of staining for nitrotyrosine and PARS was markedly reduced. Tempol increased survival and reduced the hyperactivity (secondary to the ischemia-induced neurodegeneration) caused by cerebral ischemia and reperfusion. The loss of neurons from the pyramidal layer of the CA1 region caused by ischemia and reperfusion was also attenuated by treatment of gerbils with tempol. This is the first evidence that the membrane-permeable, radical scavenger tempol reduces the cerebral injury caused by transient, cerebral ischemia in vivo.     

Activation of mitogen-activated protein kinases after transient forebrain ischemia in gerbil hippocampus.

We investigated the expression, activation, and distribution of c-Jun N-terminal kinases (JNKs), p38 mitogen-activated protein kinases (p38s) and extracellular signal-regulated kinases (ERKs) using Western blotting and immunohistochemistry in gerbil hippocampus after transient forebrain ischemia to clarify the role of these kinases in delayed neuronal death (DND) in the CA1 subfield. Immunoblot analysis demonstrated that activities of JNK, p38, and ERK in whole hippocampus were increased after 5 min of global ischemia. We used an immunohistochemical study to elucidate the temporal and spatial expression of these kinases after transient global ischemia. The immunohistochemical study showed that active JNK and p38 immunoreactivities were enhanced at 15 min of reperfusion and then gradually reduced and disappeared in the hippocampal CA1 region. On the other hand, in CA3 neurons, active JNK and p38 immunoreactivities were enhanced at 15 min of reperfusion and peaked at 6 hr of reperfusion and then gradually reduced but was continuously detected 72 hr after ischemia. Active ERK immunoreactivity was observed transiently in CA3 fibers and dentate gyrus. Pretreatment with SB203580, a p38 inhibitor, but not with PD98059, an ERK kinase 1/2 inhibitor, reduced ischemic cell death in the CA1 region after transient global ischemia by inhibiting the activity of p38. These findings indicate that the p38 pathway may play an important role in DND during brain ischemia in gerbil. Components of the pathway are important target molecules for clarifying the mechanism of neuronal death.     

基质细胞衍生因子1及其受体CXCR4对人骨髓间充质干细胞向

背景：近年研究发现移植的骨髓间充质干细胞能向颅内创伤、脑卒中、炎症和变性疾病等病灶部位迁移,进而发挥治疗作用,但对于其向病灶定向迁移的具体机制还不十分清楚。 目的：探讨基质细胞衍生因子1及其受体 CXCR4在移植的骨髓间充质干细胞趋向缺血脑组织迁移中的作用。 设计、时间及地点：细胞学体内实验,于2008-02/2009-02在解放军第三军医大学新桥医院中心实验室进行。 材料：骨髓标本取自解放军第三军医大学附属新桥医院血液科收治的15~40岁正常或原发病未累及骨髓患者,三四月龄健康雄性SD大鼠72只由解放军第三军医大学野战外科研究所实验动物中心提供。 方法：密度梯度离心贴壁筛选法分离纯化、体外培养人骨髓间充质干细胞。54只大鼠参照Nagasawa线栓法制备局灶性脑缺血再灌注模型,剩余18只作为假手术组,仅插入线栓10 mm。模型组及假手术组大鼠各取9只,分别于造模后第2,4,8天,采用Real-time PCR和免疫组织化学法定量分析缺血脑组织基质细胞衍生因子1表达变化。剩余36只脑缺血再灌注模型鼠随机分为细胞移植组、溶液对照组,18只/组,于再灌注后24 h分别从尾静脉缓慢注入1 mL人骨髓间充质干细胞悬液(含2×109 L-1个细胞)或1 mL PBS。 主要观察指标：人骨髓间充质干细胞CXCR4 mRNA和蛋白的表达,缺血再灌注后脑组织基质细胞衍生因子1 mRNA和蛋白表达变化,免疫组织化学检测人骨髓间充质干细胞向缺血脑组织的迁移和分布。 结果：RT-PCR结果发现人骨髓间充质干细胞表达CXCR4 mRNA,免疫细胞化学染色发现CXCR4主要表达于人骨髓间充质干细胞的胞膜和胞浆。脑缺血再灌注损伤后2,4,8 d,趋化因子基质细胞衍生因子1 mRNA水平呈上升趋势,与假手术组比较差异有显著性意义(P < 0.05)。经静脉移植的人骨髓间充质干细胞定向迁移到脑损伤区域,并大量分布于基质细胞衍生因子1高表达的缺血半暗区,损伤侧大脑半球人骨髓间充质干细胞数量显著高于对侧半球(P < 0.01)。 结论：基质细胞衍生因子1及其受体CXCR4参与并促进人骨髓间充质干细胞向脑缺血再灌注损伤区的迁移。     

Electron microscopic investigation of the cerebral cortex after cerebral ischemia and reperfusion in the gerbil

Prompt dendritic damage has been observed in the hippocampus of the gerbil brain after transient cerebral ischemia. In the present study, we studied the frontoparietal cortex of the gerbil brain electron microscopically after brief bilateral carotid occlusion to assess the vulnerability of dendritic processes. After ischemia for 5 min, there was swelling of the periphery of dendrites accompanied by swelling of mitochondria, cytoplasmic vacuolation and disintegration of microtubules in layer I, which spread to layer III after ischemia for 20 min. After reperfusion for 3-24 h following ischemia for 20 min, swelling in the periphery of dendrites and of mitochondria inside receded but vacuole formation and disintegration of microtubules propagated proximally. In neuronal perikarya, polyribosomal disaggregation was observed after ischemia for 20 min and persisted thereafter, while fragmentation of rough endoplasmic reticulum (ER) and microvacuolation occurred after reperfusion for 3 h. Electron-dense clumping of neuronal perikarya was observed after reperfusion for 6 h particularly in layers III and Vb, which increased in number for up to 72 h. The observed progressive damage in dendrites may be common to neurons vulnerable to cerebral ischemia and may significantly contribute to development of delayed neuronal death.     

Focal ischemia enhances choline output and decreases acetylcholine output from rat cerebral cortex

Choline concentration is rate limiting in the synthesis of acetylcholine. There is a negative arteriovenous difference for choline concentration across the brain, indicating the steady output of choline from this organ. Cerebral ischemia may increase extracellular choline concentration by interfering with its removal by the circulation and by enhancing its net production from phospholipids. We tested this hypothesis in six rats subjected to middle cerebral artery occlusion. We determined choline and acetylcholine output from the ischemic cerebral cortex by analyzing their concentrations in the fluid contained in cortical cups by gas chromatography-mass spectrometry. Mean +/- SEM choline output over 40 minutes before ischemia (baseline value) was 31.1 +/- 1.6 pmol/min/cm2. During ischemia, mean +/- SEM choline output rose to 100.8 +/- 13, 97.3 +/- 12.7, 100 +/- 22.4, and 93.1 +/- 16.9 pmol/min/cm2 in four consecutive 10-minute periods, respectively. Mean +/- SEM acetylcholine output was 15.6 +/- 1.1 before and 5.9 +/- 1.2, 8.3 +/- 2.6, 8.6 +/- 2.1, and 13.7 +/- 4.6 pmol/min/cm2 in the four 10-minute collection periods during ischemia. All four choline values and the first acetylcholine value during ischemia were significantly different from their respective baseline values. We conclude that ischemia induces an increase in extracellular choline concentration with possible implications for acetylcholine metabolism. The attending transient decline in acetylcholine output may be due to impaired release due to local hypoxia or to decreased acetylcholine synthesis.     

Up-regulation of the peripheral-type benzodiazepine receptor expression and [(3)H]PK11195 binding in gerbil hippocampus after transient forebrain ischemia

In mammalian CNS, the peripheral-type benzodiazepine receptor (PTBR) is localized on the outer mitochondrial membrane within the astrocytes and microglia. The main function of PTBR is to transport cholesterol across the mitochondrial membrane to the site of neurosteroid biosynthesis. The present study evaluated the changes in the PTBR density, gene expression and immunoreactivity in gerbil hippocampus as a function of reperfusion time after transient forebrain ischemia. Between 3 to 7 days of reperfusion, there was a significant increase in the maximal binding site density (B(max)) of the PTBR antagonist [(3)H]PK11195 (by 94-156%; P < 0.01) and PTBR mRNA levels (by 1.8- to 2.9-fold; P < 0.01). At 7 days of reperfusion, in the hippocampal CA1 (the brain region manifesting selective neuronal death), PTBR immunoreactivity increased significantly. Increased PTBR expression after transient forebrain ischemia may lead to increased neurosteroid biosynthesis, and thus may play a role in the ischemic pathophysiology.     

The effect of the calcium antagonist nimodipine on the gerbil model of experimental cerebral ischemia

The gerbil model was used to assess the therapeutic effects of the calcium antagonist nimodipine on cerebral ischemia. Transient cerebral ischemia was produced in each gerbil by bilateral common carotid occlusion of 10-, 15- or 20-min duration. Nimodipine (0.01 or 0.1 mg/kg) was administered intraperitoneally just before the carotid occlusion or 10-30 min after the removal of the arterial clips. Morbidity of each animal was evaluated using the stroke index, and the sum of stroke indices was calculated for evaluating the overall morbidity during a particular period of reperfusion. Mortality was observed for 24 hours after clip removal. Although, depending on the timing of the drug administration, the low-dose (0.01 mg/kg) nimodipine worsened the morbidity in the gerbils with 10-min ischemia, the high-dose (0.1 mg/kg) of the drug had a clear beneficial effect on the mortality associated with cerebral ischemia. These results are considered worthwhile for further trials to assess the usefulness of nimodipine as a therapeutic agent in the management of the acute ischemic stroke.     

Lipoxygenase metabolites of arachidonic acid and the development of ischaemic cerebral oedema

This study examined the changes in cerebral blood flow, water content, and lipoxygenase metabolites (leukotrienes) following bilateral carotid artery occlusion (BCO) and reperfusion in the gerbil. The effect of inhibiting lipoxygenase with nordihydroguaretic acid (NDGA) was also examined. BCO caused cerebral blood flow (measured using H2 clearance) to decline from 23.5 +/- 1.9 to 4.5 +/- 1.9 ml/min/100 gm. Reperfusion increased flow to 27.9 +/- 4 ml/min/100 gm at 10 min, which declined to 13.7 +/- 1.3 ml/min/100 gm at 50 min. Concomitant oedema measurement revealed brain specific gravity decreasing to 1.0402 +/- 0.0014 at 10 min and to 1.0325 +/- 0.0006 at 50 min reperfusion (nonoccluded controls). Leukotriene B4 (LTB4) increased from 26.8 +/- 4.6 to 33.5 +/- 2.1 pg/mg protein 10 min after reperfusion (p less than 0.05), but declined to 21.8 +/- pg/mg protein by 100 min (vs nonischaemic control = 21.3 +/- 2.9 pg/mg protein). Activation of arachidonate metabolism was confirmed by significantly increased 6 keto PGF1 alpha. Pretreatment of the animals with NDGA did not alter CBF, but increased specific gravity above saline-treated controls at 50 min of reperfusion (NDGA = 1.0370 +/- 0.002 vs control = 1.0325 +/- 0.0006, p less than 0.05). Similarly, NDGA blunted the increase in LTB4 formation 10 min after reperfusion (control = 26.8 +/- 4.6 pg/mg protein vs NDGA = 29.7 +/- 2.9 pg/mg protein, p = N.S.). These findings indicate that LTB4 production is stimulated by BCO and reperfusion in the gerbil, and that this stimulation occurs early on in the reperfusion. Further, we observe that the lipoxygenase inhibitor NDGA limits the formation of ischaemic cerebral oedema.(ABSTRACT TRUNCATED AT 250 WORDS)     

巴曲酶对脑缺血再灌注细胞外液单胺类及其代谢产物的影响——微透析研究

目的巴曲酶对脑缺血再灌流损伤的保护机理。方法采用脑内微透析技术结合高灵敏度的高压液相色谱－电化学检测手段（ＨＰＬＣ－ＥＤ），测定前脑缺血３０ｍｉｎ再灌注１２０ｍｉｎ时的纹状体细胞外液（ＥＣＦ）的ＤＡ、５－ＨＴ和ＮＥ及其代谢产物（５－ＨＩＡＡ）和ＨＶＡ的变化和巴曲酶的影响。结果显示脑缺血时，ＥＣＦＤＡ、ＮＥ及５－ＨＴ明显升高，巴曲酶能显著地降低脑缺血时ＥＣＦＤＡ及再灌注时ＥＣＦＨＶＡ和５－ＨＩＡＡ的水平。结论巴曲酶影响单胺神经递质是对脑缺血再灌注损伤起保护作用的机理之一     

Window of opportunity of cerebral hypothermia for postischemic white matter injury in the near-term fetal sheep.

Postresuscitation cerebral hypothermia is consistently neuroprotective in experimental preparations; however, its effects on white matter injury are poorly understood. Using a model of reversible cerebral ischemia in unanesthetized near-term fetal sheep, we examined the effects of cerebral hypothermia (fetal extradural temperature reduced from 39.4 +/- 0.1 degrees C to between 30 and 33 degrees C), induced at different times after reperfusion and continued for 72 hours after ischemia, on injury in the parasagittal white matter 5 days after ischemia. Cooling started within 90 minutes of reperfusion was associated with a significant increase in bioactive oligodendrocytes in the intragyral white matter compared with sham cooling (41 +/- 20 vs 18 +/- 11 per field, P < 0.05), increased myelin basic protein density and reduced expression of activated caspase-3 (14 +/- 12 vs 91 +/- 51, P < 0.05). Reactive microglia were profoundly suppressed compared with sham cooling (4 +/- 6 vs 38 +/- 18 per field, P < 0.05) with no effect on numbers of astrocytes. When cooling was delayed until 5.5 hours after reperfusion there was no significant effect on loss of oligodendrocytes (24 +/- 12 per field). In conclusion, hypothermia can effectively protect white matter after ischemia, but only if initiated early after the insult. Protection was closely associated with reduced expression of both activated caspase-3 and of reactive microglia.     

Folic acid deficiency increases delayed neuronal death, DNA damage, platelet endothelial cell adhesion molecule-1 immunoreactivity, and gliosis in the hippocampus after transient cerebral ischemia

Folic acid deficiency increases stroke risk. In the present study, we examined whether folic acid deficiency enhances neuronal damage and gliosis via oxidative stress in the gerbil hippocampus after transient forebrain ischemia. Animals were exposed to a folic acid-deficient diet (FAD) for 3 months and then subjected to occlusion of both common carotid arteries for 5 min. Exposure to an FAD increased plasma homocysteine levels by five- to eightfold compared with those of animals fed with a control diet (CD). In CD-treated animals, most neurons were dead in the hippocampal CA1 region 4 days after ischemia/reperfusion, whereas, in FAD-treated animals, this occurred 3 days after ischemia/reperfusion. Immunostaining for 8-hydroxy-2'-deoxyguanosine (8-OHdG) was performed to examine DNA damage in CA1 neurons in both groups after ischemia, and it was found that 8-OHdG immunoreactivity in both FAD and CD groups peaked at 12 hr after reperfusion, although the immunoreactivity in the FAD group was much greater than that in the CD group. Platelet endothelial cell adhesion molecule-1 (PECAM-1; a final mediator of neutrophil transendothelial migration) immunoreactivity in both groups increased with time after ischemia/reperfusion: Its immunoreactivity in the FAD group was much higher than that in the CD group 3 days after ischemia/reperfusion. In addition, reactive gliosis in the ischemic CA1 region increased with time after ischemia in both groups, but astrocytosis and microgliosis in the FAD group were more severe than in the CD group at all times after ischemia. Our results suggest that folic acid deficiency enhances neuronal damage induced by ischemia.     

Leukocyte-endothelium interactions in pial venules during the early and late reperfusion period after global cerebral ischemia in gerbils.

The contribution of leukocytes to secondary brain damage after cerebral ischemia is still under discussion. The purpose of the present study was to examine the pial microcirculation after global cerebral ischemia while focusing on leukocyte-endothelium interactions during the early and late reperfusion period of up to 4 days. A closed cranial window technique that leaves the dura mater intact was used. Global cerebral ischemia of 15 minutes' duration was induced in male Mongolian gerbils (n = 91). Pial microcirculation was observed by intravital fluorescence microscopy. Leukocyte-endothelium interactions (LEIs) in pial venules, vessel diameters, capillary density, and regional microvascular blood flow measured by laser Doppler flowmetry were quantified during 3 hours of reperfusion and in intervals up to 4 days after ischemia. Within 3 hours of reperfusion, the number of leukocytes (cells/100 microm x minute) rolling along or adhering to the venular endothelium increased from 0.1 +/- 0.2 to 28.4 +/- 17.4 (P < 0.01 vs. control) and from 0.2 +/- 0.2 to 4.0 +/- 3.8 (P < 0.05), respectively. There was no capillary plugging by leukocytes; capillary density remained unchanged. In the late reperfusion period, at 7 hours after ischemia, LEIs had returned to baseline values. Furthermore, from 12 hours to 4 days after ischemia, no LEIs were observed. Changes in regional microvascular blood flow did not correlate with LEIs. Global cerebral ischemia of 15 minutes' duration induces transient LEIs that reach a maximum within 3 hours of reperfusion and return to baseline at 7 hours after ischemia. LEIs are not related to changes in microvascular perfusion, which suggests mainly that the expression of adhesion receptors is necessary to induce LEIs rather than rheologic factors. It seems unlikely that this short-lasting activation of leukocytes can play a role in the development of secondary brain damage.     

Neuroprotection by 2-h postischemia administration of two free radical scavengers, alpha-phenyl-n-tert-butyl-nitrone (PBN) and N-tert-butyl-(2-sulfophenyl)-nitrone (S-PBN), in rats subjected to focal embolic cerebral ischemia

Oxygen free radical generation may have important secondary damaging effects after the onset of cerebral ischemia. Free radical scavengers have been used successfully in attenuating neuronal damage in the reperfusion period in transient forebrain ischemia. There are limited data on effectiveness in models of focal ischemia. Two free radical scavengers, alpha-phenyl-n-tert-butyl-nitrone (PBN) and N-tert-butyl-(2-sulfophenyl)-nitrone (S-PBN), have been shown to reduce oxidative-stress-induced neuronal injury. Whereas PBN has been demonstrated to reduce infarct volume in focal ischemia, neuroprotection has not been evaluated with S-PBN. The present study was designed to evaluate the neuroprotective effect of PBN and S-PBN compared to vehicle in a focal embolic middle cerebral artery (MCA) cerebral ischemia model in rats. Wistar rats were randomly divided into three groups (n = 10 each group). Animals in the control group received vehicle and those in the treatment groups were treated with PBN or S-PBN (both 100 mg/kg/day x 3 days, intraperitoneally) starting 2 h after the introduction of an autologous thrombus into the right-side MCA. The neurological outcome was observed and compared before and after treatment and between groups. The percentage of cerebral infarct volume was estimated from 2,3, 5-triphenyltetrazolium chloride stained coronal slices 72 h after the ischemic insult. Two-hour postischemia administration of PBN or S-PBN significantly improved neurobehavioral scores at 24 h following MCA embolization (both P < 0.01). The percentage of infarct volume for animals receiving vehicle was 32.8 +/- 9.4%. Two-hour delayed administration of PBN and S-PBN achieved a 35.4% reduction in infarct volume in treatment groups when compared with animals receiving vehicle (PBN vs control, 21.2 +/- 10.9% vs 32.8 +/- 9.4%; P < 0.05; S-PBN vs control, 21.2 +/- 13.1%, (P < 0.05). These data indicate that free radical generation may be involved in brain damage in this model and 2-h delayed postischemia treatment with PBN and S-PBN may have neuroprotective effects in focal cerebral ischemia. As S-PBN does not normally cross the blood-brain barrier, the neuroprotection evident in this study may be explained by entry into the brain via damaged vessels.