三七总皂苷对脑缺血再灌注大鼠神经血管单元超微结构的影响

为了观察脑缺血再灌注(cerebral ischemia reperfusion, CIR)大鼠缺血灶周边脑组织不同时间点神经血管单元(neurovascular unit, NVU)超微结构变化,研究三七总皂苷(Panax notoginseng saponins, PNS)对脑缺血再灌注大鼠脑组织NVU超微结构的影响,本研究采用改良Zea Longa法制作局灶性大脑中动脉闭塞(MCAO)模型,缺血2 h后再灌注;采用Longa法评分标准检测各组大鼠术后4 h神经功能评分,随之各组进行干预,分别在缺血再灌注后24 h、72 h、7 d、3周进行神经功能评分和透射电镜下观察各组大鼠缺血灶周边脑组织的NVU超微结构变化。研究结果表明,干预前即术后4 h治疗组和对照组神经功能评分比较无明显差异;PNS干预后治疗组大鼠神经功能评分逐渐改善,缺血再灌注后24 h与对照组比较,差异无统计学意义(p>0.05),再灌注72 h、7 d、3周的大鼠神经缺损评分与同时间点对照组相比差异具有统计学意义(p<0.05)。电镜观察发现再灌注24 h、72 h、7 d、3周治疗组大鼠脑组织NVU超微结构的病理形态损伤均较同时间点对照组明显减轻。本研究结论认为,PNS通过整合促进脑缺血后NVU的神经元、胶质细胞和微血管的修复,改善神经功能缺损症状,对脑缺血具有保护作用。     

铜纳米颗粒对缺血性卒中后神经血管单元的保护作用

目的:研究铜纳米颗粒(copper nanoparticle,Cu-nps)对缺血性卒中后神经血管单元(neurovascular units,NVU)的保护作用。方法:采用加热搅拌法合成Cu-nps。体内建立大鼠短暂大脑中动脉阻塞/再灌注模型(transient middle cerebral artery occlusion / reperfusion,tMCAO),实验分为正常组(sham)、模型组(tMCAO)、给药组(Cu-nps),检测各组脑梗死面积、神经凋亡情况、血脑屏障(blood-brain barrier,BBB)完整性以及相关蛋白表达。结果:制备出的Cu-nps以Cu²⁺、80 nm左右的粒径均匀存在,具有良好的生物相容性。Cu-nps靶向脑缺血部位受损神经元,提高神经元细胞活力,降低活性氧,减少神经元凋亡和脑梗死面积,降低伊文思蓝染料渗漏量和炎症因子表达。结论:Cu-nps可以减少氧化应激,保护BBB完整性,降低神经胶质细胞活化,保护NVU功能,从而降低脑缺血再灌注损伤。     

Neurovascular Unit: a Focus on Pericytes

The blood–brain barrier (BBB) is a highly specialized system that controls the exchanges between the blood and the central nervous system (CNS). This barrier shields the CNS from toxic substances in the blood and provides nutrients to CNS, thus playing an essential role in the maintenance of homeostasis. The anatomical basis of the BBB is formed by the endothelial cells of brain microvasculature, with elaborated tight and adherens junctions, which together with pericytes, the basement membrane, and astrocytes, as well as neurons, microglia and oligodendrocytes form the neurovascular unit. The interaction between all these components guarantees a proper environment for neural function and a restricted permeability and transport. Pericytes were initially reported by Rouget in 1873 and since then they have been recognized as an important component of the BBB, despite the difficulty of their identification. Diverse functions have been assigned to pericytes, including a role in BBB properties, hemostasis, and angiogenesis, as well as a contractile, immune, and phagocytic function. These cells are also seen like multipotent cells and so with a great potential for therapy. Here, we review the neurovascular unit composition and the interplay between the diverse components, addressing pericytes with a particular detail.     

IGF-1对缺血性脑损伤大鼠脑内神经发生的影响

目的:建立大鼠单侧局灶脑缺血模型,观察胰岛素样生长因子-1(IGF-1)对局灶脑缺血后的鼠脑神经发生及增殖后细胞生存的影响.方法:用健康雄性SD大鼠建立大脑中动脉阻塞(MCAO)模型,随机分成假手术组,缺血对照组和IGF-1治疗组.各组再按不同的治疗时间分为7d、14d、28d、42d组.免疫组化法观察BrdU、PSA-NCAM的变化,免疫双标法观察BrdU/PSA-NCAM、BrdU/MAP2和BrdU/GFAP的共同表达变化.结果:BrdU标记细胞和PSA-NCAM标记细胞计数均在缺血后第7d最多,分别是缺血对照组的4.0倍和1.8倍,是假手术组的9.9倍和5.4倍.BrdU和PSA-NCAM双标细胞在缺血发生后双侧SVZ和DG区可以检测到,于第7d计数最多,之后逐渐降低;而BrdU和MAP2以及BrdU和GFAP双标细胞却从第14d开始逐渐增多,直到第42d.随着BrdU/PSA-NCAM双标阳性表达的逐渐降低,BrdU/MAP2双标阳性表达逐渐增高,呈现此消彼涨的变化.结论:IGF-1侧脑室注射后,在早期(7d内)诱导了缺血性脑损伤后神经细胞的增殖;在中期(7d-14d)诱导了新生细胞的迁移;在后期(14d后)伴随着迁移的进行新生细胞逐渐发生了分化.     

大蒜素对全脑缺血再灌注大鼠脑保护机制的研究

目的：通过大蒜素预处理,观察全脑缺血再灌注大鼠海马区ICAM-1 的表达,从而探讨大蒜素的脑保护机制。方法：雄性 Wistar 大鼠30 只,随机分为5 组：假手术组、缺血再灌注组、缺血再灌注+ 大蒜素10、20、30 mg/kg 组。采用四血管闭塞法制备大 鼠全脑缺血再灌注模型,于再灌注24 h 取出海马,硫堇染色观察海马组织的形态学改变,免疫组织化学染色测定海马CA1 区 ICAM-1 免疫反应阳性细胞面积和积分光密度值。结果：通过给予大鼠全脑缺血8 min 再灌注24 h处理,海马CA1 区组织形态学 改变显著,神经元密度明显降低;ICAM-1的表达显著增加。静脉给予大蒜素可使缺血再灌注海马组织形态学改变明显改善,存活 神经元数目增加,ICAM-1 表达显著较少。结论：大蒜素可以通过减少ICAM-1 的表达抑制全脑缺血再灌注后的炎症损失从而发 挥脑保护作用。     

Neuroprotective Effects of Ischemic Preconditioning in Brain Mitochondria Following Cerebral Ischemia

Numerous studies support the hypothesis that reperfusion following cerebral ischemia contributes substantially to ischemic injury and that mitochondrial dysfunction plays a central role. Defining the mechanisms by which mitochondrial dysfunction occurs may be important for the development of new therapies against delayed neuronal cell death. Ischemic preconditioning (IP) increases an organ's resistance to ischemic injury. There are two windows for IPC, one that requires several hours to develop and another one with a rapid setting (rapid window). However, the rapid window only provides neuroprotection for few days. We have recently determined that this lack of chronic protection by the rapid window was due to lack of protection against mitochondrial dysfunction.     

远端缺血后处理对脑缺血保护作用的研究进展

近20多年来人们对脑缺血损伤的保护研究很多,但真正能将脑缺血保护从基础研究应用到临床治疗的措施甚少。多数基础研究表明缺血预处理对大鼠脑缺血具有保护作用,然而由于脑缺血的不可预见性,研究者们将目标转向了缺血后处理。远端缺血后处理是指在非缺血器官交替实施短时间的缺血和再灌注后对缺血器官所产生的作用。由于脑组织本身对缺血的敏感,很难控制缺血后处理的程度,因此远端缺血后处理被应用到脑缺血的保护研究具有很强的临床应用价值,其机制可能与氧自由基、神经传导、蛋白质、内质网应激、Akt信号通路、线粒体途径、mitoKATP和阿片受体有关。本文主要就近几年远程缺血后处理对脑缺血保护的概念、实施方法、保护作用及分子机制做一综述。     

Potassium Buffering in the Neurovascular Unit: Models and Sensitivity Analysis

Astrocytes are critical regulators of neural and neurovascular network communication. Potassium transport is a central mechanism behind their many functions. Astrocytes encircle synapses with their distal processes, which express two potassium pumps (Na-K and NKCC) and an inward rectifying potassium channel (Kir), whereas the vessel-adjacent endfeet express Kir and BK potassium channels. We provide a detailed model of potassium flow throughout the neurovascular unit (synaptic region, astrocytes, and arteriole) for the cortex of the young brain. Our model reproduces several phenomena observed experimentally: functional hyperemia, in which neural activity triggers astrocytic potassium release at the perivascular endfoot, inducing arteriole dilation; K⁺ undershoot in the synaptic space after periods of neural activity; neurally induced astrocyte hyperpolarization during Kir blockade. Our results suggest that the dynamics of the vascular response during functional hyperemia are governed by astrocytic Kir for the fast onset and astrocytic BK for maintaining dilation. The model supports the hypothesis that K⁺ undershoot is caused by excessive astrocytic uptake through Na-K and NKCC pumps, whereas the effect is balanced by Kir. We address parametric uncertainty using high-dimensional stochastic sensitivity analysis and identify possible model limitations.     

Potassium Buffering in the Neurovascular Unit: Models and Sensitivity Analysis

Astrocytes are critical regulators of neural and neurovascular network communication. Potassium transport is a central mechanism behind their many functions. Astrocytes encircle synapses with their distal processes, which express two potassium pumps (Na-K and NKCC) and an inward rectifying potassium channel (Kir), whereas the vessel-adjacent endfeet express Kir and BK potassium channels. We provide a detailed model of potassium flow throughout the neurovascular unit (synaptic region, astrocytes, and arteriole) for the cortex of the young brain. Our model reproduces several phenomena observed experimentally: functional hyperemia, in which neural activity triggers astrocytic potassium release at the perivascular endfoot, inducing arteriole dilation; K⁺ undershoot in the synaptic space after periods of neural activity; neurally induced astrocyte hyperpolarization during Kir blockade. Our results suggest that the dynamics of the vascular response during functional hyperemia are governed by astrocytic Kir for the fast onset and astrocytic BK for maintaining dilation. The model supports the hypothesis that K⁺ undershoot is caused by excessive astrocytic uptake through Na-K and NKCC pumps, whereas the effect is balanced by Kir. We address parametric uncertainty using high-dimensional stochastic sensitivity analysis and identify possible model limitations.     

In Vivo Targeting of the Neurovascular Unit: Challenges and Advancements

Cellular and Molecular Neurobiology - The blood–brain barrier (BBB) is essential for the homeostasis of the central nervous system (CNS). Functions of the BBB are performed by the...     

Effects of Metabotropic Glutamate Receptor Stimulation on Cerebral O2 Consumption and Blood Flow During Focal Cerebral Ischemia in Rats

This investigation was performed to evaluate the effects of ACPD [(1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid], a metabotropic glutamate receptor agonist, on cerebral O₂ consumption during focal cerebral ischemia. Male Wistar rats were placed in control (n = 7) and ACPD (n = 7) groups under isoflurane anesthesia. Twenty minutes after middle cerebral artery (MCA) occlusion, gauze sponges with 10^–5 M ACPD or normal saline were placed on the ischemic cortex (IC) for a period of 40 min and were changed every 10 min. One hour after MCA occlusion, regional cerebral blood flow (rCBF) was determined using the C¹⁴-iodoantipyrine autoradiographic technique. Regional arterial and venous oxygen saturation were determined using microspectrophotometry. There were no statistical differences in vital signs, blood gases, and hemoglobin between the groups. In the control group, the cerebral blood flow and oxygen consumption of the IC were significantly lower than the contralateral cortex (rCBF: 45 ± 11 vs. 110 ± 11 ml/min/100 g, O₂ consumption: 2.9 ± 0.4 vs. 5.4 ± 1.1 ml O₂/min/100 g). ACPD did not change regional cerebral blood flow of the IC, but did significantly increase the oxygen extraction (7.8 ± 0.2 vs. 6.9 ± 0.3 ml O₂/100 ml) and oxygen consumption of the IC (4.3 ± 1.5 vs. 2.9 ± 0.4) compared to the control IC. Our data demonstrated that topical application of 10^–25 M ACPD to the ischemic area worsened cerebral O₂ balance. These data suggest that metabotropic glutamate receptors are not maximally activated during ischemia in the temporal cortex.     

大鼠肢端缺血预处理对脑缺血后炎症反应的影响

目的通过观察肢端缺血预处理（1imbisehemicpreconditioning,LIP）对大鼠脑缺血性损伤后重要炎症因子表达的影响,探讨LIP诱导的脑缺血耐受与炎症反应之间的关系。方法选取72只SD大鼠,实验组（LIP组）30只、缺血组30只和对照组12只。实验组和缺血组设立5个时间点：6h、12h、24h、48h和72h,每点6只。通过线栓法建立大鼠大脑中动脉阻塞（middlecerebralarteryocclusion,MCAO）的局灶性脑缺血模型及LIP法建立脑缺血耐受模型,采用HE观察每组大鼠的脑组织形态学改变、QRT—PCR和ELISA方法检测脑组织中炎症因子IL-17及IL-6的表达变化。结果实验组脑组织学病理改变明显轻于缺血组。与缺血组相比：实验组的IL-17和IL-6的基因和蛋白表达在整体水平均呈下降趋势;mRNA水平提示实验组在缺血12h、24h和48h后脑组织中IL-17、IL-6的表达量显著减少（P〈0．01）;蛋白水平提示实验组在缺血24h和48h后脑组织中的IL-6以及在缺血12h、24h和48h后脑组织中IL-17的表达量均降低（P〈0．05）。结论LIP诱导脑缺血耐受,可以减轻脑缺血后的炎症反应,对缺血性脑损伤有一定的保护作用。     

Linolenic Acid Provides Multi-cellular Protective Effects After Photothrombotic Cerebral Ischemia in Rats

Alpha-linolenic acid (LIN) has been shown to provide neuroprotective effects against cerebral ischemia. LIN is a potent activator of TREK-1 channel and LIN-induced neuroprotection disappears in Trek1?/? mice, suggesting that this channel is directly related to the LIN-induced resistance of brain against ischemia. However, the cellular mechanism underlying LIN induced neuroprotective effects after ischemia remains unclear. In this study, using a rat photochemical brain ischemia model, we investigated the effects of LIN on the protein abundance of astrocytic glutamate transporter and AQP4, microglia activation, cell apoptosis and behavioral recovery following ischemia. Administration of LIN rescued the protein abundance of astrocytic glutamate transporter GLT-1, decreased the protein abundance of AQP4 and brain edema, inhibited microglia activation, attenuated cell apoptosis and improved behavioral function recovery. Meanwhile, TREK-1 was widely distributed in the cortex and hippocampus, primarily localized in astrocytes and neurons. LIN could potentiate the TREK-1 mediated astrocytic passive conductance and hyperpolarize the membrane potential. Our results suggest that LIN provides multiple cellular neuroprotective effects in cerebral ischemia. TREK-1 may serve as a promising multi-mechanism therapeutic target for the treatment of stroke.     

Polyamine Changes in Reversible Cerebral Ischemia

Putrescine, spermidine, and spermine levels were measured in the cortex, caudoputamen, and hippocampus of rats during 30 min of severe forebrain ischemia (induced by occlusion of both carotid and vertebral arteries) and subsequent recirculation. During ischemia, polyamine levels did not change significantly. During postischemic recirculation, however, putrescine levels dramatically increased whereas those of spermine and spermidine did not change, with the exception of the severely damaged caudoputamen, where the concentration declined after 24 h. The increase of putrescine is explained by postischemic activation of ornithine decarboxylase and inhibition of S-adenosylmethionine decarboxylase. It is suggested that the accumulation of putrescine during postischemic recirculation may be responsible for the delayed neuronal death occurring after ischemia.     

The Roles of Autophagy in Cerebral Ischemia

Recent studies indicate the existence of autophagy in cerebral ischemia, but the functions of autophagy in this setting remain unclear. Here we discuss the role of autophagy in cerebral ischemia based on our own publication and the literature on this subject. We propose that oxidative and endoplasmic reticulum (ER) stresses in cerebral ischemia-hypoxia are potent stimuli of autophagy in neurons. We also reviewed evidence suggesting autophagosomes may have a shorter half-life in neurons and that a fraction of LC3 protein is degraded within autolysosomes, leading to a smaller detectable amount of LC3-II in the brain while there are clear indications of on-going autophagy. Finally, we suggest autophagy is an important modifier of cell death and survival, interacting with necrosis and apoptosis in determining the outcomes and final morphology of deceased neurons.     

Effects of Transient Cerebral Ischemia on the Expression of DNA Methyltransferase 1 in the Gerbil Hippocampal CA1 Region

DNA methylation is a key epigenetic modification of DNA that is catalyzed by DNA methyltransferases (Dnmt). Increasing evidences suggest that DNA methylation in neurons regulates synaptic plasticity as well as neuronal network activity. In the present study, we investigated the changes in DNA methyltransferases 1 (Dnmt1) immunoreactivity and its protein levels in the gerbil hippocampal CA1 region after 5 min of transient global cerebral ischemia. CA1 pyramidal neurons were well stained with NeuN (a neuron-specific soluble nuclear antigen) antibody in the sham-group, Four days after ischemia–reperfusion (I–R), NeuN-positive (⁺) cells were significantly decreased in the stratum pyramidale (SP) of the CA1 region, and many Fluro-Jade B (a marker for neuronal degeneration)⁺ cells were observed in the SP. Dnmt1 immunoreactivity was well detected in all the layers of the sham-group. Dnmt1 immunoreactivity was hardly detected only in the stratum pyramidale of the CA1 region from 4 days post-ischemia; however, at these times, Dnmt1 immunoreactivity was newly expressed in GABAergic interneurons or astrocytes in the ischemic CA1 region. In addition, the level of Dnmt1 was lowest at 4 days post-ischemia. In brief, both the Dnmt1 immunoreactivity and protein levels were distinctively decreased in the ischemic CA1 region 4 days after transient cerebral ischemia. These results indicate that the decrease of Dnmt1 expression at 4 days post-ischemia may be related to ischemia-induced delayed neuronal death.     

丹酚酸A对大鼠脑缺血/再灌注损伤及抗氧化酶活性的影响

目的:探讨丹酚酸A对大鼠脑缺血/再灌注(cerebral ischemia/reperfusion,CI/R)损伤及抗氧化酶活性的影响。方法:采用大鼠脑中动脉闭塞(middle cerebral arteryocclusion,MCAO)2 h再灌注24 h模型。实验终末,检测脑梗死面积,脑水肿以及评价神经功能损伤,并进一步分析脑组织中三种抗氧化酶的活性水平。结果:与模型组相比,丹酚酸A组大鼠脑梗死面积显著减少(P0.05),水肿程度显著减轻(P0.05),神经功能学评分显著下降(P0.05)。模型组再灌注24 h后,SOD,GSH-PX及CAT活性显著下降(P0.05);丹酚酸A组SOD,GSH-PX及CAT活性则显著升高(P0.05)。结论:丹酚酸A对大鼠CI/R损伤具有保护作用,可能与CI/R损伤时的脑组织SOD,GSH-PX及CAT活性显著升高相关。