Identification of first candidate genes for creativity: a pilot study

Nitric oxide (NO) can be neuroprotective or neurotoxic during cerebral ischemia, depending on the NO synthase (NOS) isoform involved. In addition to neurotoxic effect in ischemic brain, inducible NOS (iNOS) also adversely affect ischemic outcome by blocking neurogenesis. In the present study, therefore, we studied the chronological and spatial change of the distribution of iNOS and cell proliferation in subventricular zone (SVZ) after transient focal cerebral ischemia. After 90 min of transient middle cerebral artery occlusion (tMCAO), iNOS-positive cells decreased in the ischemic core at 1 to 21 days, and increased in the ipsilateral periischemic area at 1 and 3 days. 5-Bromodeoxyuridine (BrdU)-positive cells appeared in the ischemic core at 3 to 21 days, appeared in the periischemic area at 3 and 7 days, and increased in the ipsilateral SVZ at 7 days. ED-1-positive cells appeared in the ischemic core at 3 to 21 days, and some of them were double positive with BrdU or iNOS, but the majority were BrdU-negative. The present study suggests that astrocytes are born within the periischemic area at early stage after tMCAO and migrate from SVZ into periischemic area at later stage, and that time-dependent and spatial changes of iNOS expression may be involved in the proliferation and differentiation of adult neurogenesis after focal cerebral ischemia.     

Up-regulation of low-density lipoprotein receptor expression in the ischemic core and the peri-ischemic area after transient MCA occlusion in rats

Low-density lipoprotein (LDL) receptor is involved in cholesterol metabolism of CNS as a receptor of apolipoprotein E (ApoE), which plays an important role in regenerative process after brain ischemia. Temporal and spatial changes of LDL receptor were investigated after 90 min of transient middle cerebral artery occlusion (MCAO) in relation to those of microtubule-associated protein 2 (MAP2) and ApoE. In the ischemic core, LDL receptor became positive at 1 d after transient MCAO, which was not double positive for MAP2 or ApoE, and disappeared in 7 and 56 d. In the peri-ischemic area, LDL receptor became observed at 7 d, which peaked at 21 d, most of which were double positive for MAP2. The number of LDL receptor and ApoE double-positive cells increased at 7 d and decreased at 21 d with the shift of LDL receptor immunoreactivity from cytoplasm at 7 d to dendrites at 21 d in the peri-ischemic area. These results suggest that LDL receptor, interacting with ApoE, is profoundly involved in lipid transport of CNS for tissue repair in the peri-ischemic area after brain ischemia.     

Neuronal apolipoprotein E is not synthesized in neuron after focal ischemia in rat brain

Apolipoprotein E (ApoE) is a major apolipoprotein in the central nervous system (CNS) that plays an important role in Alzheimer's disease. It may also be involved in other CNS disorders including ischemic injury. We investigated the changes of ApoE protein and mRNA expression in the brain with middle cerebral artery occlusion (MCAO) to clarify its origin after focal ischemia in rats. Increased ApoE immunoreactivity was recognized in astrocytes 3-14 days after MCAO in the affected side of cortex, and in neurons 4-14 days after MCAO in the same area. ApoE immunoreactivity was also detected in macrophages in the ischemic core 3-14 days after MCAO. In contrast, ApoE mRNA was expressed in astrocytes and macrophages, but not in neurons. These results suggested that neuronal ApoE was not synthesized in neurons, but derived from astrocytes.     

Neuronal apolipoprotein E is not synthesized in neuron after focal ischemia in rat brain

Abstract

Apolipoprotein E (ApoE) is a major apolipoprotein in the central nervous system (CNS) that plays an important role in Alzheimer's disease. It may also be involved in other CNS disorders including ischemic injury. We investigated the changes of ApoE protein and mRNA expression in the brain with middle cerebral artery occlusion (MCAO) to clarify its origin after focal ischemia in rats. Increased ApoE immunoreactivity was recognized in astrocytes 3-14 days after MCAO in the affected side of cortex, and in neurons 4-14 days after MCAO in the same area. ApoE immunoreactivity was also detected in macrophages in the ischemic core 3-14 days after MCAO. In contrast, ApoE mRNA was expressed in astrocytes and macrophages, but not in neurons. These results suggested that neuronal ApoE was not synthesized in neurons, but derived from astrocytes.     

Different expression of low density lipoprotein receptor and ApoE between young adult and old rat brains after ischemia

OBJECTIVES: Reduction of brain plasticity underlies the poor outcome of aged stroke patients. The molecular mechanism of plasticity reduction by aging is uncertain, but disturbed lipid metabolism may be implicated. METHODS: We investigated the expression of low density lipoprotein receptors (LDL-R) and apolipoprotein E (ApoE), both of which play active roles in lipid metabolism in young adult and old rat brains after ischemia. RESULTS: LDL-R, trivially expressed in the sham-operated brain neurons, was increased from day 1 and became prominent at days 7 and 21 at the peri-ischemic cortex. The magnitude was smaller in the old than in the young adult rats. ApoE was increased in the astrocytes and neurons of the peri-ischemic cortex at day 1, which became further pronounced in the neurons but not in the astrocytes at days 7 and 21. ApoE expression was again less prominent in the old animals at days 7 and 21. DISCUSSION: As ApoE-containing lipoprotein is recruited via LDL-R, the present results suggest that old brains had less capability to induce LDL-R, which resulted in impaired recruitment of lipoprotein after the ischemic injury. Impaired lipid recruitment causes disturbance of synaptogenesis and thus brain plasticity reduction. This molecular mechanism may result in poor functional recovery of aged stroke patients.     

卒中后抑郁与脑卒中患者载脂蛋白E水平对照研究

目的:探讨卒中后抑郁(PSD)患者载脂蛋白E(ApoE)水平特点,为PSD的诊断提供新的客观依据。方法:采用实时荧光定量PCR技术和酶联免疫吸附法(ELISA)检测PSD患者及卒中后非抑郁患者ApoE水平。结果:PSD组ApoE基因mRNA表达量低于卒中组,差异具有统计学意义(P<0.01);PSD组血清ApoE水平高于卒中组,差异具有统计学意义(P<0.05)。结论:PSD患者外周血ApoE基因mRNA表达和血清ApoE水平与卒中非抑郁患者不同。     

阿司匹林和氯吡格雷联合应用 对小鼠脑缺血恢复期RAGE及 sRAGE表达的影响

目的 探索阿司匹林和氯吡格雷联合应用对小鼠大脑中动脉远端缺血再灌注模型恢复期晚期糖基化终末产物受体（receptor for advanced glycation end products,RAGE）及可溶性晚期糖基化终末产物受体（soluble receptor for advanced glycation end products,sRAGE）表达的影响及其机制。 方法 60只C57BL/6J雄性小鼠随机分为假手术组、溶剂组、阿司匹林和氯吡格雷联合组（双抗组）,每组20只。通过压迫大脑中动脉远端制作脑缺血再灌注模型（缺血60?min再灌注）,再灌注即刻灌胃,溶剂组给予饮用水100?μL,双抗组给予阿司匹林（剂量12?mg/kg,每只实际用量0.3?mg）与氯吡格雷（剂量12?mg/kg,每只实际用量0.3?mg）混悬液100?μL,每日1次,连续21?d。缺血1、3、5、7、9、11、14、21?d对小鼠进行神经功能评价。缺血21?d取材,酶联免疫吸附法检测小鼠血清sRAGE表达水平;免疫印记检测脑组织RAGE表达水平;实时荧光定量PCR检测CD16、CD32、CD11b、诱导型一氧化氮合成酶（inducible nitric-oxide synthase,iNOS）、CD206、精氨酸酶1（arginase1,Arg1）、转化生长因子β（transforming growth factor β,TGF-β）、几丁质酶样蛋白（chitinase-like 3,Chil3/Ym1/2）等炎症因子及RAGE的mRNA表达情况;免疫荧光染色标记RAGE、神经元特异核蛋白（neuronal nuclei,NeuN）、胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP）,观察RAGE与神经元和星形胶质细胞共定位情况。 结果 双抗组小鼠3?d时胡须碰触评分和神经功能总分、9?d时神经功能总分优于溶剂组,差异有统计学意义（P=0.0067、0.0140、0.0406）。缺血再灌注21?d时,双抗组小鼠血清sRAGE表达水平高于溶剂组（1.099±0.541?ng/mL?vs.?0.319±0.341?ng/mL,P=0.0120）;脑组织iNOS（1.250±0.318 vs.?1.843±0.301,P=0.0164）及RAGE（2.105±0.300?vs.?2.732±0.249,P=0.0071）的mRNA相对表达较溶剂组下降。在小鼠脑梗死周边区RAGE与神经元具有共定位,与星形胶质细胞无共定位;双抗组RAGE+NeuN+细胞数量少于溶剂组（328.798±35.183个/平方毫米?vs.?814.437±165.758个/平方毫米,P=0.0012）。 结论 阿司匹林和氯吡格雷联合应用可能通过下调RAGE、上调sRAGE的表达水平,减轻炎症反应,从而发挥脑保护作用。     

Up-regulation of neuropilin-1 in neovasculature after focal cerebral ischemia in the adult rat.

During development, neuropilin-1 is a receptor for semaphorin 3a-mediated axonal guidance and for vascular endothelial growth factor (VEGF) promotion of angiogenesis. The authors measured neuropilin-1 expression in the adult ischemic brain using Northern blot, in situ hybridization, and immunohistochemistry. Neuropilin-1 mRNA was significantly up-regulated as early as 2 hours and persisted at least 28 days after focal cerebral ischemia. Acute up-regulation of neuropilin-1 mRNA primarily localized to the ischemic neurons. A marked increase in both mRNA and protein of neuropilin-1 was detected in endothelial cells of cerebral blood vessels at the border and in the core of the ischemic lesion 7 days after ischemia, and neuropilin-1 gene expression persisted on these vessels for at least 28 days after ischemia. In these areas, neovascularization was detected using three-dimensional reconstructed images obtained from laser scanning confocal microscopy. Activated astrocytes also exhibited neuropilin-1 immunoreactivity during 7 to 28 days of ischemia. Double immunofluorescent staining showed colocalization of neuropilin-1 and VEGF to cerebral blood vessels and activated astrocytes. These data suggest that in addition to its role in axonal growth, up-regulation of neuropilin-1, in concert with VEGF and its receptors, may contribute to neovascular formation in the adult ischemic brain.     

Traumatic brain injury induces biphasic upregulation of ApoE and ApoJ protein in rats

Apolipoproteins play an important role in cell repair and have been found to increase shortly after traumatic brain injury (TBI). In addition, apolipoproteins reduce amyloid-beta (Abeta) accumulation in models of Alzheimer's disease. Considering that TBI induces progressive neurodegeneration including Abeta accumulation, we explored potential long-term changes in the gene and protein expression of apolipoproteins E and J (ApoE and J) over 6 months after injury. Anesthetized male Sprague-Dawley rats were subjected to parasagittal fluid-percussion brain injury and their brains were evaluated at 2, 4, 7, 14 days, and 1 and 6 months after TBI. In situ hybridization, Western blot, and immunohistochemical analysis demonstrated that although there was a prolonged upregulation in both the gene expression and protein concentration of ApoE and J after injury, these responses were uncoupled. Upregulation of ApoE and J mRNA expression lasted from 4 days to 1 month after injury. In contrast, a biphasic increase in protein concentration and number of immunoreactive cells for ApoE and ApoJ was observed, initially peaking at 2 days (i.e., before increased mRNA expression), returning to baseline by 2 weeks and then gradually increasing through 6 months postinjury. In addition, ApoE and J were found to colocalize with Abeta accumulation in neurons and astrocytes at 1-6 months after injury. Collectively, these data suggest that ApoE and J play a role in the acute sequelae of brain trauma and reemerge long after the initial insult, potentially to modulate progressive neurodegenerative changes.     

短暂脑缺血后成年和老年大鼠大脑髓鞘相关蛋白基因表达变化

目的探讨短暂脑缺血后成年和老年大鼠大脑髓鞘相关蛋白基因表达变化及其意义。方法以线栓法制作成年和老年SD大鼠短暂脑缺血模型（阻塞60min再灌注1d、7d和14d）；采用5末端标记地高辛的寡核苷酸探针荧光原位核酸分子杂交检测短暂脑缺血大鼠大脑梗死中心区、梗死周边区和梗死对侧区PLP mRNA和MyT1 mRNA照射后表达变化，并计数阳性细胞数量。结果（1）短暂脑缺血后成年和老年大鼠大脑皮质梗死中心区PLP mRNA和MyT1 mRNA阳性细胞数明显减少，两组之间比较无显著差异。（2）短暂脑缺血后早期成年和老年大鼠梗死周边区PLP mRNA和MyT1 mRNA阳性细胞数无明显变化，之后（7d和14d）逐渐增加，成年大鼠PLP mRNA和MyT1 mRNA阳性细胞数增加更明显。结论成年大鼠短暂脑缺血后急性期梗死周边区髓鞘相关蛋白基因表达强于老年大鼠，提示年龄是影响少突胶质前体细胞参与脑缺血后损伤修复的因素之一。     

Eag1, Eag2, and SK3 potassium channel expression in the rat hippocampus after global transient brain ischemia

Transient global brain ischemia causes delayed neuronal death in the hippocampus that has been associated with impairments in hippocampus-dependent brain function, such as mood, learning, and memory. We investigated the expression of voltage-dependent Kcnh1 and Kcnh5, ether à go-go-related Eag1 and Eag2 (K(V) 10.1 and K(V) 10.2), and small-conductance calcium-activated SK3 (K(Ca) 2.3, Kcnn3) K(+) channels in the hippocampus in rats after transient global brain ischemia. We tested whether the expression of these channels is associated with behavioral changes by evaluating the animals in the elevated plus maze and step-down inhibitory avoidance task. Seven or tweny-eight days after transient global brain ischemia, one group of rats had the hippocampus bilaterally dissected, and mRNA levels were determined. Seven days after transient global brain ischemia, the rats exhibited a decrease in anxiety-like behavior and memory impairments. An increase in anxiety levels was detected 28 days after ischemia. Eag2 mRNA downregulation was observed in the hippocampus 7 days after transient global brain ischemia, whereas Eag1 and SK3 mRNA expression remained unaltered. This is the first experimental evidence that transient global brain ischemia temporarily alters Eag2. The number of intact-appearing pyramidal neurons was substantially decreased in CA1 and statistically measurable in CA2, CA3, and CA4 hippocampal subfields compared with sham control animals 7 or 28 days after ischemia. mRNA expression in the rat hippocampus. The present results provide further information for the characterization of the physiological role of Eag2 channels in the central nervous system.     

Cystatin C and apolipoprotein E immunoreactivities in CA1 neurons in ischemic gerbil hippocampus

The distribution patterns of cystatin C and apolipoprotein E (apo E) were studied immunocytochemically in the gerbil hippocampus before and after 5 min ischemia. In the controls, cystatin C was distributed mainly in astrocytes. In addition, a large number of dots positive for cystatin C were observed around the outlines of neuronal perikarya in the CA1 subfields. One day after ischemia, cystatin C-positive stainings outlining neuronal cell bodies disappeared. On the fourth day, intense stainings for cystatin C appeared in atrophied pyramidal neurons and these stainings in neurons disappeared by the 14th day. A remarkable increase in the number of cystatin C-positive astrocytes occurred on the fourth day and thereafter these spread over the whole of the CA1 subfield. Apo E was also distributed in astrocytes in the control specimens. From the fourth day, extra- and/or intracellular distribution of apo E-immunoreactivities was noted in the stratum pyramidale. Apo E-positive astrocytes disappeared transiently on the fourth day and then reappeared and increased remarkably by the 14th day. These findings indicate that cystatin C and apo E are involved in the degeneration process of brain neuronal cells.     

Focal ischemia induces transient expression of IL-6 in the substantia nigra pars reticulata

We examined the expression of IL-6 in the substantia nigra pars reticulata (SNr) at various time points after transient (3 h) middle cerebral artery occlusion (MCAO) in rats. The animals were killed at 1, 3, 7 or 14 days following operation. Coronal brain sections were processed for immunohistochemistry with antibodies against GFAP, OX-42 and IL-6 and for Nissl staining. Microglial activation was detected 3 and 7 days after ischemia. Reactive astrocytes have been found 7 and 14 days after ischemia. IL-6 expression was detected 3 and 7 days after ischemia. IL-6-positive cells beared the typical morphology of neurons. Distribution of IL-6-positive cells within the SNr was not homogenous. The lateral area of the SNr bears the highest number of IL-6-positive neurons while the central core bears the lowest. Quantification of intact neurons in the SNr 14 days after reperfusion shows that the highest amount of cell loss was found in the central core of the SNr and less neuronal cell loss was observed in the lateral area of the SNr. Thus, the SNr area with the highest IL-6 expression 3 and 7 days after ischemia bears the highest number of intact neurons 14 days after ischemia. This finding could be a clue for the neuroprotective role of IL-6 in the remote region SNr after focal cerebral ischemia.     

Expression of basic fibroblast growth factor receptor messenger RNA in the periinfarcted brain tissue

The present study examined whether expression of basic fibroblast growth factor receptor (bFGFR) messenger ribonucleic acid (mRNA) was upregulated by focal ischemia. We have studied the in situ hybridization autoradiography for bFGFR mRNA in the rat model of middle cerebral artery (MCA) occlusion. Male Wistar rats were used for occlusion of the left MCA, and were sacrificed 1, 3, 7 and 14 days after MCA occlusion. In situ hybridization was performed on the brain sections of these animals and sham controls by using 35S-labeled antisense and sense (control) RNA probes for rat bFGFR. Expression of bFGFR mRNA was observed in the periinfarcted area of the rats within 1-14 days after MCA occlusion. Expression was evident in the whole hemisphere of the infarcted side, especially at 1 and 3 days after ischemia, but no expression was detected in the contralateral side. On microautoradiograms, the signals of bFGFR mRNA were detected in both neurons and non-neural cells located in the periinfarcted area. Upregulation of bFGFR mRNA detected in the periinfarcted brain tissue suggests that receptor-mediated action of bFGF may be related to preservation of neurons injured by ischemia.     

Early increase of Nox4 NADPH oxidase and superoxide generation following endothelin-1-induced stroke in conscious rats

Oxidative stress contributes to the progression of brain injury following ischemic stroke and reperfusion. NADPH oxidase is a well-established source of superoxide in vascular disease, but its contribution to tissue injury following ischemic stroke has yet to be fully elucidated. Here we show the spatiotemporal profile of NADPH oxidase subunits Nox2 and Nox4 and concurrent superoxide generation following stroke induced by middle cerebral artery constriction in conscious rats. Nox2 mRNA was progressively up-regulated in both the ipsilateral cortex and the striatum from 6 hr to 7 days poststroke and reperfusion. Nox4 mRNA was also up-regulated transiently in the cortex at 6 hr poststroke but returned to control levels after this time. In situ detection of superoxide generation with dihydroethidium fluorescence revealed an increase in superoxide within the ischemic core at 6 hr poststroke that was mostly colocalized with the neuronal marker NeuN. By 24 hr, this increase in superoxide production had spread to the boundary zone of the infarct, whereas it disappeared in the ischemic core as neuronal numbers declined. Subsequently, superoxide within the ischemic core again increased at 7 days and was mostly colocalized with the activated microglia/macrophage marker OX-42. Immunoreactivity to Nox2 followed the same spatiotemporal pattern as that of OX-42 immunostaining poststroke. Clearly, NADPH oxidase is an important mediator of oxidative stress and contributes to the progression of brain damage beyond the infarct core, via the activation of two catalytic subunits, Nox2 and Nox4. Selectively blocking these subunits might be useful for intervening in the progression of stroke brain injury.     

Cerebral ischemia and Alzheimer's disease: the expression of amyloid-beta and apolipoprotein E in human hippocampus

Growing evidence suggests a synergistic and perhaps etiological relationship between vascular disease and Alzheimer's disease (AD), which is characterized by the progressive accumulation of amyloid-beta peptide (Abeta). Moreover, apolipoprotein E (ApoE) has also been shown to be associated with AD and cerebral ischemia. It seems that cerebral ischemia may play an important, both direct and indirect, role in the pathogenesis of AD. We investigated the expression and distribution of Abeta1-40, beta1-42 and ApoE in human hippocampus after cerebral ischemia in this study to determine the role of cerebral ischemia in Alzheimer's disease. Our study has demonstrated that the accumulation of both Abeta1-40 and beta1-42 were increased dramatically and consistently after cerebral ischemia. Neuronal ApoE immunoreactivity was also significantly increased in all ischemic groups compared with controls. The most likely stimulus for the increased Abeta1-40, Abeta1-42 and ApoE immunoreactivity in the CA1 and CA3 neurons is the ischemic conditions, and their upregulation, in turn, may partly explain the contribution of cerebral ischemia to the pathogenesis of AD. Therefore our observations provide a basis for establishing therapeutic strategies aimed at preventing ischemic insults and subsequent neurodegeneration in AD.     

IL-6 and PACAP Receptor Expression and Localization after Global Brain Ischemia in Mice

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts a neuroprotective action against ischemic damage. This action is mediated by the interleukin-6 (IL-6) pathway. However, as the expression patterns of PACAP receptors and IL-6 following ischemia are not understood, we evaluated them in the mouse hippocampus in response to ischemia induced by bilateral common carotid artery occlusion. Real-time PCR determination of PAC1R mRNA expression in the hippocampus was significantly elevated on day?7 after ischemia. VPAC1R mRNA expression was significantly decreased 3?days after the ischemic episode, while VPAC2R mRNA expression showed a nonsignificant tendency to increase on day?7. IL-6 mRNA expression was significantly increased on day?3 and peaked on day?7 after ischemia. The mRNA expression of activity-dependent neuroprotective protein, which is a neuroprotective factor stimulated by PACAP, remained virtually unchanged in response to ischemia. IL-6 immunoreactivity was detected in the CA1 pyramidal cell layer and colocalized with the neuronal marker NeuN on day?1 after ischemia. On day?3, irregularly shaped IL-6-immunopositive cells colocalized with the astrocytic marker glial fibrillary acidic protein but not with the microglial marker Iba1. PAC1R immunoreactivity co-labeled with IL-6 immunoreactivity. These results suggest that PACAP could stimulate IL-6 secretion by neurons during the acute phase after an ischemic episode and thereafter by astrocytes during the subacute phase.     

ERK对缺血缺氧性脑损伤后神经元凋亡的影响

目的研究在缺血缺氧性脑损伤后细胞外信号调节激酶(ERKs)对神经元凋亡的影响。方法建立光化学法诱导大鼠局灶性脑缺血模型,随机分为脑缺血组(缺血组和干预组)和假手术组,干预组于缺血前30min尾静脉注入U0126溶液,缺血组尾静脉注入相同体积不含U0126的DMSO稀释溶液;2,3,5-氯化(或溴化)三苯四氮唑(TTC)染色显示梗死灶;应用免疫组织荧光化学法检测梗死灶周围神经元核心抗原(NeuN)的表达及通过TUNEL方法检测神经元凋亡,并做NeuN与TUNEL的双标;用免疫印迹(Westernblot)方法观察损伤侧皮层NeuN、细胞周期蛋白D1(CyclinD1)和细胞周期蛋白E(CyclinE)蛋白的表达。结果缺血组的梗死体积明显大于干预组,在假手术组未见梗死灶;缺血组大鼠NeuN阳性细胞数和NeuN蛋白表达明显少于假手术组,TUNEL阳性细胞数和CyclinD1和CyclinE蛋白表达明显高于假手术组(P<0.05)。干预组NeuN阳性细胞数和NeuN蛋白表达亦少于假手术组,但多于缺血组,TUNEL阳性细胞数和CyclinD1及CyclinE蛋白表达亦多于假手术组,但少于缺血组(P<0.05)。结论ERK可通过对细胞周期的调控而对神经元凋亡产生影响,抑制脑缺血引起的pERK1/2磷酸化可部分抑制神经元凋亡,减少缺血梗死灶,对缺血性脑损伤起一定的保护作用。