缺氧对血脑屏障细胞分泌组织型纤溶酶原激活物的影响

目的：探讨缺氧对体外培养鼠脑微血管内皮细胞和星形胶质细胞分泌组织型纤溶酶原激活物（ｔｉｓｓｕｅ－ｔｙｐｅ ｐｌａｓｍｉｎｏｇｅｎ ａｃｔｉｖａｔｏｒ,ＴＰＡ）的影响。方法：分别对新生小鼠脑微血管内皮细胞、星状胶质细胞进行缺氧条件下培养,常规培养作为空白对照,每组各取８例,吸取培养液用酶联免疫吸附试验测试ＴＰＡ活性。结果：缺氧后内皮细胞ＴＰＡ活性明显增高（Ｐ＜０．０１）,星形胶质细胞ＴＰＡ活性无明显变化（Ｐ＞０．０５）。结论：体外培养鼠脑微血管内皮细胞和星形胶质细胞均能合成分泌ＴＰＡ;缺氧状态下脑微血管内皮细胞产生的ＴＰＡ活性增高。内皮细胞分泌的ＴＰＡ是脑缺氧缺血致不可逆神经元损伤的一个重要媒介,而星形胶质细胞分泌的ＴＰＡ则主要参与发育阶段需要细胞迁移的重建活动。     

The fine structure of blood vessels of the telencephalic germinal matrix in the human fetus

Blood vessels of the human telencephalic germinal matrix during the tenth through the twenty-second week of gestation have been examined by light and electron microscopy. In all fetuses studied the ependymal and sub-ependymal zones of the germinal matrix have a prominent vascular network. During the tenth and twelfth weeks of gestation, the endothelial cells are plump and display numerous organelles, junctional complexes, conspicuous luminal microvilli and stub-like abluminal projections. Coated and micropinocytotic vesicles were found both in the cytoplasm and on luminal and abluminal surfaces. In endothelial cells intracytoplasmic, membrane-limited, rod-shaped bodies were frequently observed. These bodies have been linked to endothelial thromboplastic and clotting activities and related to abnormal clotting status. Their role in the pathogenesis of subependymal germinal matrix hemorrhage in premature infants remains unknown. Pericytes apposing the endothelial cells were recognized in all gestational periods. The endothelial basal lamina and astrocytic end-feet are ill defined, and the extracellular space is pronounced. By the fifteenth and seventeenth weeks of gestation the endothelial cells are still large and now possess more numerous luminal microvilli and abluminal projections. At this stage the pericytes, basal lamina and astrocytic end-feet are all well developed, resulting in a decrease in the surrounding extracellular space. By the twenty-second week the endothelial cells possess few luminal and abluminal projections and the associated basal lamina, glia, pericytes and extracellular compartment appear mature. The relationship of the germinal matrix vasculature to the pathogenesis of subependymal hemorrhage is discussed.     

Expression of caveolin-1 in human brain microvessels

Caveolae are microinvaginations of the cell plasma membrane involved in cell transport and metabolism as well as in signal transduction; these functions depend on the presence of integral proteins named caveolins in the caveolar frame. In the brain, various caveolin subtypes have been detected in vivo by immunocytochemistry: caveolin-1 and -2 were found in rat brain microvessels, caveolin-3 was revealed in astrocytes. The aim of this study was to identify the site(s) of cellular expression of caveolin-1 in the microvessels of the human cerebral cortex by immunofluorescence confocal microscopy and immunogold electron microscopy. Since in the barrier-provided brain microvessels tight relations occur between the endothelium-pericyte layer and the surrounding vascular astrocytes, double immunostaining with caveolin-1 and the astroglia marker, glial fibrillary acidic protein, was also carried out. Immunocytochemistry by confocal microscopy revealed that caveolin-1 is expressed by endothelial cells and pericytes in all the cortex microvessels; caveolin-1 is also expressed by cells located in the neuropil around the microvessels and identified as astrocytes. Study of the cortex microvessels carried out by immunoelectron microscopy confirmed that in the vascular wall caveolin-1 is expressed by endothelial cells, pericytes, and vascular astrocytes, and revealed the association of caveolin-1 with the cell caveolar compartment. The demonstration of caveolin-1 in the cells of the brain microvessels suggests that caveolin-1 may be involved in blood-brain barrier functioning, and also supports co-ordinated activities between these cells.     

Morphological changes of neural and vascular peptides in human skin suction blister injury

In 30 patients with reflux oesophagitis, the mucosal microvessels have been studied by transmission electron microscopy. The aim of the study was to test the hypothesis that a microvascular injury is involved in reflux oesophagitis, and to clarify if the epithelial metaplasia is correlated with a contemporaneous modification of the microvasculature in Barrett's oesophagus. In squamous epithelium-lined mucosa, signs of microangiopathy were found in all patients and capillaries regularly showed an interrupted, duplicated, or thickened basal lamina. In areas of columnar metaplasia, capillaries showed an ectatic lumen and a thin basal lamina without duplications or interruptions; endothelial cells had a thin rim of cytoplasm with many fenestrations. These findings demonstrate that microangiopathy is associated with epithelial damage in reflux oesophagitis.     

The structure of endometrial microvessels

Recent studies have provided substantial evidence to highlight abnormalities in the structure of endometrial microvessels in users of progestogen-only contraception. Structural changes alone are unlikely to lead to breakthrough bleeding, but appear to be associated with a reduction in vessel integrity, and may reflect alterations in the control and growth of endometrial microvessels in those exposed to exogenous progestogens. In users of low-dose progestogens, immunohistochemical studies have demonstrated changes in superficial endometrial vascular morphology, density and in endometrial migratory cell populations. In Norplant users the endometrial endothelial basal lamina is deficient in the initial months of exposure, when bleeding problems are most common. The basal lamina refers to the very thin structure consisting mostly of collagen IV and laminin seen underlying endothelial and epithelial cells at the electron microscope level. In addition, hysteroscopic studies have demonstrated increased fragility of superficial vessels. Changes in endometrial microvascular anatomy associated with normal menstruation have been reviewed, and are compared with those seen following contraceptive steroid exposure. Likely mechanisms of breakthrough bleeding such as increased superficial vascular fragility and the possible alterations in endometrial vascular structure leading to this are discussed.     

Injury of brain microvessels with a helium-neon laser and Evans blue can elicit local platelet aggregation without endothelial denudation

A helium-neon laser was used to produce endothelial lesions and local platelet aggregation in brain microvessels (pial arterioles and venules) sensitized by intravascular Evans blue. Electron microscopy showed that the aggregates formed in the absence of endothelial denudation or exposure of basal lamina. Initial lesions consisted only of endothelial lucencies, vacuoles, and distended rough or smooth endoplasmic reticulum. Aggregates consisted not only of degranulating platelets, rounded platelets, and platelets with pseudopods, but also of masses of discoid platelets. Red blood cells were uninjured and did not form the nidus of these collections. The possibility that aggregation occurred as a result of direct injury to the platelet, and independently of the endothelial damage, is discussed and considered unlikely. Rather, the data are consonant with the hypothesis that in the microcirculation, endothelial injury can produce aggregation prior to exposure of basal lamina.     

Neurovascular coupling in the mammalian brain

Normal brain function requires proper supply of oxygen and glucose in a timely and local manner. This is achieved through an orchestrated intercellular communication between neurones, astrocytes and microvessels that results in a rapid and restricted increase in cerebral blood flow, a process known as neurovascular coupling. Astrocytic end-feet make close contacts with neuronal synapses and blood vessels and, given their ability to release vasoactive signals following neuronal activation, have been recognized as key intermediaries in the neurovascular response. Both dilating and constricting signals appear to be released from astrocytes upon increases in intracellular Ca(2+) concentration, and both dilatation and constriction of brain vessels have been observed in previous studies. In this article, we discuss the various astrocyte-derived vasodilating and vasoconstricting signals, their interactions and effects on astrocytes and vascular smooth muscle cells, and suggest the importance of the intrinsic properties of the latter cell type on the overall neurovascular response. We present a working model in which the rise in astrocytic Ca(2+) following neuronal activation leads not only to the rapid activation of calcium-activated K(+) channels in astrocytic end-feet, but also to their modulation by metabolites of the arachidonic acid pathway, which in general have been proposed to act on vascular smooth muscle cells rather than on astrocytes. We propose that this latter mechanism may in turn modulate K(+) signalling from astrocytes to smooth muscle cells, influencing the overall effects of the vasodilating and vasoconstricting signals released during neuronal activation.     

1型糖尿病小鼠胰岛微血管内皮细胞超微结构受损

目的:探讨1型糖尿病小鼠胰岛微血管内皮细胞的超微结构改变。方法:BALB/c小鼠随机分为糖尿病组和对照组,每组6只。应用免疫组化染色检测胰岛素及胰岛微血管血小板内皮细胞黏附分子-1(CD31)表达水平;应用透射电镜观察糖尿病小鼠胰岛β细胞及胰岛微血管超微结构变化。结果:糖尿病组小鼠胰岛β细胞数量、β细胞/α细胞数量比、β细胞分泌颗粒数量及胰岛素阳性染色面积百分比显著降低(P0.01)。糖尿病组小鼠胰岛微血管数量,CD31表达水平显著降低(P0.01);微血管内皮细胞及胰岛周细胞线粒体肿胀变性;微血管基膜厚度显著升高(P0.01)。结论:1型糖尿病小鼠胰岛微血管内皮细胞超微结构受损。     

Endothelial heterogeneity in the chick wing bud: A morphometric study

Summary The microvascular endothelium of the chick wing bud at stages 22, 27, and 32 was evaluated by ultrastructural morphometry. The rationale for this study is based on the hypothesis that endothelial cells exhibit variation in structure and function during cytodifferentiation. The microvessels had a luminal diameter range such that they were classified as capillaries. The thin continuous endothelium was devoid of a basal lamina. The endothelium had a very small number of plasmalemmal vesicles; vacuoles were however present for all stages and in some cases were abundant. The temporal findings were that endothelial cell thickness increases, plasmalemmal vesicle densities decrease, and the densities of cytoplasmic vacuoles increase. The spatial results were that endothelial cells in proximal regions of the limb have a greater thickness, contain fewer vesicles and have more vacuoles than those in distal regions. In general, these results indicate that endothelial ultrastructural heterogeneity occurs within a 31/2 day timespan of wing bud development. The discussion considers the results with regard to recent reports on endothelial cell heterogeneity.     

Brain microvessels: factors altering their patency after the occlusion of a middle cerebral artery (Wistar rat).

The progression from ischemic injury to pannecrosis that occurs in the rat brain several hours after occluding a large artery may be partly attributable to a worsening of the circulation through the microvessels. The objective of this study was to quantitate selected structural changes involving astrocytes and endothelial cells within an area of focal brain ischemia created by the occlusion of a middle cerebral artery. The magnitude of these structural changes was correlated with alterations in the patency to a circulating macromolecule through the microvessels (< or = 15 mu in diameter) located within the territory of the occluded artery. One hundred eighty-five adult male Wistar rats had the right middle cerebral artery occluded after threading a nylon monofilament through the external carotid artery. Experiments were terminated by either cardiovascular perfusion or decapitation and immersion fixation at intervals ranging between 30 minutes and 7 days after the arterial occlusion. Randomly selected animals from each experimental subgroup were injected intravenously with horseradish peroxidase (molecular weight 44 kd) approximately 20 minutes before death. The progressive decline in the area fraction comprised by the vessels filled with horseradish peroxidase was preceded at 30 to 60 minutes by an increase in the surface area occupied (on a cross-section of a microvessel) by endothelial cells (both nucleus and cytoplasm). This was followed by an increase of 23.7% in the mean diameter of astrocytes nuclei and a decrease of approximately 35% in lumenal surface of the microvessels. These observations suggest that the occlusion of a large cerebral artery causes prompt swelling of endothelial cells and astrocytes; both of these early biological responses may interfere with erythrocyte circulation and oxygen delivery, which (after the arterial occlusion) are entirely dependent on the circulation provided by the collateral arterial connections. Through its interference with microvascular patency and oxygen delivery, cell swelling may influence the rate at which neurons become necrotic. In this model of brain infarct the number of necrotic neurons peaks approximately 72 hours after middle cerebral artery occlusion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Light and electron microscopic immunohistochemical demonstration of GABA-immunoreactive astrocytes in the brain stem of the rat

Summary An antiserum against -aminobutyric acid (GABA) was used in an immunohistochemical investigation of the nature of GABA-immunoreactive profiles in various regions of the brain stem of the rat. In accordance with findings in previous biochemical studies, but at discrepancy with recent immunohistochemical results, GABA-like immunoreactivity was demonstrated not only in neurons but also in glial cells. Electron microscopy revealed that the GABA-positive glial cells were astrocytes. Cells identified as oligodendrocytes were unlabelled as were pericytes and endothelial cells. In the labelled astrocytes, immunohistochemical reaction product was seen throughout the nucleus and cytoplasm, and in thin sheet-like processes surrounding neuronal elements and in end-feet lining the basal membrane of capillaries. These observations support the concept that astrocytes play a role in the metabolism of GABA. It is suggested that the failure to demonstrate the presence of GABA in glial cells in previous immunohistochemical studies may have been due to some factor in the tissue preparation. However, it is also possible that labelled glial cells may have been mistaken for small labelled neurons.     

Ultrastructural observations of astrocyte end-feet in the rat central nervous system

Astrocytic end-feet in the rat CNS were studied by thin-section electron microscopy. Astrocyte processes that enclose neuronal elements extended to blood vessels and the pia mater, where the processes expanded to form end-feet or glial limiting membranes. At the end-feet, cell junctions such as gap junctions and desmosome-like junctions were formed between the astrocyte processes. The end-foot plasma membrane facing the basal lamina was undercoated with electron-dense, layered materials, with an internal substructure of filamentous networks, with which bundles of glial filaments (GFs) appeared to be closely associated via fine filamentous structures, often showing a hemi-desmosome-like appearance. In specimens treated with Triton X-100, the internal substructure of the undercoat was better visualized and the association with GFs was well preserved. At the end-feet, some unique tubular structures were found in spatial relationship to the plasmalemmal undercoat. Plectin visualized by immunofluorescence was localized to astrocytes and their processes, especially at the end-feet facing the pia mater. Immunoelectron microscopy located plectin on fine filamentous structures lying between GFs and the plasmalemmal undercoat. These observations suggest that plasmalemmal undercoats at the astrocyte end-feet may serve as attachment sites of GFs to the plasma membrane and that plectin may be involved in such attachment.     

Ultrastructural and permeability features of microvessels in the hippocampus, cerebellum and pons of senescence-accelerated mice (SAM)

We previously reported that the accumulation of blood-borne radiolabelled serum albumin in brain parenchyma increased with aging, especially in senescence-accelerated mice (SAMP8), which showed age-related deficits in learning and memory. In this study, in order to examine morphological events related to the age-related increase of the brain accumulation of serum albumin, the transvascular passage of blood-borne horseradish peroxidase (HRP) and ultrastructural features of microvessels were examined in the hippocampus, cerebellum and pons of SAMP8 and SAMR1 (control) mice. Ultrastructural examination of the hippocampus showed that the staining for HRP was occasionally spreading throughout the parajunctional cytoplasm of the endothelial cell of aged SAMP8 mice, but not in young SAMP8 mice nor in SAMR1 mice. The number of vessels showing the staining reaction for HRP in the parajunctional cytoplasm of the endothelial cells in aged SAMP8 mice increased significantly compared with that in the others. Electron microscopic morphometry showed that there were no significant differences among the number of HRP-positive vesicles per unit area of the endothelial cell cytoplasm in young and old mice of both strains. The staining reaction for HRP was not seen in the basal lamina of microvessels and the perivascular neuropil in all mice examined. Perivascular lipofuscin-like granules and collagen deposits, swelling of astroglial perivascular endfeet and perivascular cells containing foamy, lipid-like droplets were frequently found in several brain regions of aged SAMP8 mice. The perivascular cells with a few lipid-like droplets and more electron-homogeneous lysosomes were occasionally seen in SAMR1 and young SAMP8, while the other findings were scarcely observed in SAMR1 and young SAMP8 mice.These findings suggest that the blood-brain barrier to HRP was preserved in microvessels in three brain regions of SAM mice but the blood microvessels showed some age-related ultrastructural alterations in SAMP8 brains. Uncontrolled passage of HRP through the parajunctional cytoplasm of the endothelial cells may partly contribute to the age-related increase of accumulation of serum albumin in SAMP8 brains.     

大鼠脑微血管内皮细胞的原代培养及其生物学行为初步探讨

本文旨在探讨建立稳定的大鼠原代脑微血管内皮细胞的培养方法,并对其血脑屏障特性进行初步研究。通过匀浆、葡聚糖高速梯度离心提取大鼠脑皮层微血管段,用胶原酶消化后,在37℃,5%CO2孵箱中进行原代及传代细胞培养,以跨内皮阻抗值分析其血脑屏障特性随传代次数的改变。结果证明:原代培养后7～10d沿微血管段生成单层"铺路石"样细胞,经VIII因子证实95%以上的培养细胞是血管内皮细胞,通过传代可以进一步纯化,得到稳定的脑微血管内皮细胞系,原代培养的脑微血管内皮细胞表现出很强的屏障特性,与内皮细胞株之间有显著差异,随着传代次数的增加脑微血管内皮细胞的跨内皮阻抗减弱。以上结果表明,采用匀浆、梯度离心、胶原酶消化是获取大鼠脑微血管内皮细胞稳定的方法,原代培养的脑微血管内皮细胞是研究血脑屏障的最佳技术手段。     

Fine structure of the blood—brain interface in the cuttlefishSepia officinalis (Mollusca, Cephalopoda)

Summary The blood—brain interface was studied in a cephalopod mollusc, the cuttlefishSepia officinalis, by thin-section electron microscopy. Layers lining blood vessels in the optic and vertical lobes of the brain, counting from lumen outwards, include a layer of endothelial cells and associated basal lamina, a layer of pericytes and a second basal lamina, and perivascular glial cells. The distinction between endothelial cells and pericytes breaks down in small vessels. In the smallest microvessels, equivalent to capillaries, and in venous channels, the endothelial and pericyte layers are discontinuous, but a layer of glial cells is always interposed between blood and neural tissue, except where neurosecretory endings reach the second basal lamina. In microvessels in which cell membranes of the entire perivascular glial sheath could be followed, the glial layer was apparently seamless, not interrupted by an intercellular cleft, inca 90% (27/30) of the profiles. Where a cleft did occur, it showed an elongated overlap zone between adjacent cells. The walls of venous channels are formed by lamellae of overlapping glial processes. In arterial vessels, the pericyte layer is thicker and more complete, with characteristic sinuous intercellular clefts. Arterioles are defined as vessels containing myofilaments within pericytes, and arteries those in which the region of the second basal lamina is additionally expanded into a wide collagenous zone containing fibroblast-like cells and cell processes enclosing myofilaments. The glio-vascular channels observed inOctopus brain are not a prominent feature ofSepia optic and vertical lobe. The organization of cell layers at theSepia blood—brain interface suggests that it is designed to restrict permeability between blood and brain.     

Histamine in brain capillaries

A high amount of histamine was found in capillaries isolated by subcellular fractionation from the brain. In view of the important effects of histamine on vascular permeability in peripheral vessels, it was thought that histamine had a similar function in the cerebral vasculature. Intracarotic histamine infusion resulted in an enhanced pinocytosis of endothelial cells and the oedematous swelling of the astrocytic end-feet system. [³H]-Histamine, injected in the cerebral ventricles, accumulated in the capillary wall. Histamine and cimetidine activated hydroxyfatty acid and prostaglandin D₂ synthesis in isolated brain capillaries.The possible function of the capillary histamine in the regulation of permeability of microvessels is discussed.     

大鼠脑微血管内皮细胞的培养

目的:探索一种简便易行、可培养出高纯度大鼠脑微血管内皮细胞的方法。方法:1月龄SD大鼠,解剖得到大脑皮质,密度离心法获得较纯的脑微血管段后进行原代培养,传代采用差速消化和贴壁方法进行纯化。通过形态学观察及第Ⅷ因子相关抗原免疫荧光检测对培养细胞进行鉴定。结果:密度离心法分离出的大量微血管段呈"串珠样"结构,培养24h可见短梭形、多角形细胞,8～10天基本融合。第2代细胞经免疫荧光染色,第Ⅷ因子相关抗原呈阳性,阳性率达90%。结论:原代细胞采用低分子量葡聚糖加Percoll密度离心法、传代细胞采用差速消化和差速贴壁法纯化可成功培养高纯度的脑微血管内皮细胞。     

Differential, strain-specific cellular and subcellular distribution of multidrug transporters in murine choroid plexus and blood-brain barrier

Multidrug transporters of the ATP-binding cassette family play an important role in regulating drug distribution and efflux in the brain, owing to their selective distribution in microvessels and choroid plexus. Their expression pattern and cellular distribution remain controversial, in part due to technical difficulties in localizing these membrane proteins in closely associated cells, such as endothelial cells and astrocytic end-feet at the blood-brain barrier. Here, we used high-resolution immunofluorescence staining with cell-type specific markers to investigate the distribution of major ATP-binding cassette transporters in mouse brain. We report that four ATP-binding cassette transporters, Mdr1, Mrp1, Mrp2 and Mrp5 can be detected in brain endothelial cells, forming three distinct layers, with Mdr1 and Mrp5 being located on the luminal side, Mrp1 on the abluminal (basal) side, and Mrp2 in between. Mrp3 and Mdr3 were undetectable. In choroid plexus, only Mrp1, Mrp2 and Mrp3 were detected, again with a differential distribution. Mrp1 was targeted basolaterally in epithelial cells, Mrp2 was restricted to endothelial cells, and Mrp3 was co-localized with zonula occludens-1 at tight junctions. Analysis of Mdr1a(0/0) and Mrp1(0/0) mice, generated in the FVB strain, revealed no major alteration in expression of the remaining transporters. An unexpected strain difference was unraveled, with wildtype FVB mice selectively lacking Mrp2 protein in brain, but not liver. In conclusion, these results indicate that ATP-binding cassette transporters provide multiple penetration barriers in the blood-brain barrier and choroid plexus, with a selective cellular and subcellular distribution, emphasizing their potential role for drug resistance in neurological disorders, such as epilepsy.     

舌乳头微血管和周围组织图像分析及其组织通道探讨

目的:分析舌乳头血管及其周围组织的图像和组织学,进而探讨舌乳头组织通道的形态学基础。方法:用Image-ProPlus医学图像系统测量正常舌及水肿舌的上皮层厚度、微血管袢顶到上皮层的距离、固有层和血管的面积以及上皮层和固有层色泽的RGB值,并进行组织切片观察。结果:两组舌微血管袢顶到上皮层均有一定距离,水肿组大于正常组,水肿组微血管在固有层内所占的面积明显小于正常组;上皮层和固有层的RGB值有较大差别,固有层G、B曲线的起伏与血管密度的多少相关。舌组织形态学显示上皮层细胞外间隙与固有层内均质样结构相通。结论:舌固有层内微血管袢顶到上皮层有一定空间,存在着弥散分布的有腔无壁的组织通道。     

Microvascular permeability.

This review addresses classical questions concerning microvascular permeabiltiy in the light of recent experimental work on intact microvascular beds, single perfused microvessels, and endothelial cell cultures. Analyses, based on ultrastructural data from serial sections of the clefts between the endothelial cells of microvessels with continuous walls, conform to the hypothesis that different permeabilities to water and small hydrophilic solutes in microvessels of different tissues can be accounted for by tortuous three-dimensional pathways that pass through breaks in the junctional strands. A fiber matrix ultrafilter at the luminal entrance to the clefts is essential if microvascular walls are to retain their low permeability to macromolecules. Quantitative estimates of exchange through the channels in the endothelial cell membranes suggest that these contribute little to the permeability of most but not all microvessels. The arguments against the convective transport of macromolecules through porous pathways and for the passage of macromolecules by transcytosis via mechanisms linked to the integrity of endothelial vesicles are evaluated. Finally, intracellular signaling mechanisms implicated in transient increases in venular microvessel permeability such as occur in acute inflammation are reviewed in relation to studies of the molecular mechanisms involved in signal transduction in cultured endothelial cells.