Variable restriction of albumin diffusion across inflamed cerebral microvessels of the anaesthetized rat.

1. The possibility of restricted diffusion of macromolecules in single cerebral venular capillaries that have become leaky due to inflammation was investigated by comparing the permeabilities to Lucifer Yellow (457 Da; PLY) and rhodamine-labelled albumin (69 kDa; PRh-A). 2. The dyes were trapped between two micro-occlusion probes and the permeabilities were measured from the rates of decrease in dye fluorescence at low intraluminal hydrostatic pressure. 3. Removal of one probe had little effect on PLY but did reduce PRh-A, consistent with the influence of convection on diffusion through 22 nm wide transendothelial slits 1 micron deep. 4. Direct comparisons were made over time between PLY and PRh-A in six vessels while hydrostatic pressure effects were controlled. In all vessels PRh-A:PLY varied from being similar to the ratio of the free diffusion coefficients to virtually zero even though PLY remained high. The question of the source of this variable restriction to albumin is discussed in terms of the secretion and sloughing of glycosaminoglycans and the possible role of transient formation of transendothelial gaps.     

Histamine-stimulated prostaglandin synthesis in rat brain microvessels

The histamine-sensitivity of prostacyclin and prostaglandin synthesis was investigated in isolated brain microvessels prepared from normal and hypoxic exercised rats. 10^–4 M histamine stimulated thein vitro synthesis of all components of arachidonate cascade. The chronic hypoxic exercise also resulted in an enhanced production of each fraction. Hypoxia and histamine showed an additive effect in the synthesis of PGE₂ only. The possible molecular mechanism induced by hypoxia and histamine is discussed.     

Molecular forms of butyrylcholinesterase in rat brain microvessels

Molecular forms of butyrylcholinesterase (BuChE) were studied in microvessels isolated from rat brain, using sedimentation analysis in sucrose gradients. Three forms, G1, G2 and G4, were found with sedimentation coefficients close to 3S, 6S and 9S, respectively. The relative proportion of the 3 forms was 19% for the monomer, 33% for the dimer and 46% for the tetramer. This sedimentation pattern of BuChE forms appears to be characteristic of cerebral microvessels and may represent distinct functional features of the blood-brain barrier.     

Potassium transport in isolated cerebral microvessels from the rat

Microvessels have been prepared from the gray matter of a rat brain by a technique involving density gradient centrifugation. A suspension of these vessels, largely capillaries, was incubated in vitro in order to investigate K transport. The flux of K (as 86Rb) into and out of endothelial cells was estimated. Potassium influx was sensitive to temperature and pH of the medium, and was markedly inhibited by 1 mM ouabain (45%). Ouabain did not inhibit K efflux, as anticipated, when Na-K pumps are mainly located on the abluminal plasma membrane of the endothelial cell. The ouabain-sensitive K influx was measured at varying external concentrations of K. The Km of ouabain-sensitive K influx was 2.95 mM, which is similar to the affinity of the transport carrier of K, found in in vivo studies of K efflux from brain to the blood system. Both 1 mM furosemide and 5 mM barium chloride inhibited part of the ouabain-insensitive K influx. Potassium efflux was not influenced by furosemide, but was somewhat reduced by barium chloride. Noradrenalin (10(-3) mM) and histamine (0.1 mM) did not significantly affect the influx of K.     

Major localization of aminopeptidase M in rat brain microvessels

The localization of two enkephalin-hydrolysing aminopeptidases i.e. aminopeptidase M (aminopeptidase N, EC 3.4.11.2) relatively insensitive to puromycin (Ki = 78 microM), and a puromycin-sensitive aminopeptidase (Ki = 1 microM) was studied in rat brain. The two aminopeptidases were differentially identified and/or localized using polyclonal anti-aminopeptidase M antibodies displaying anticatalytic activity and the inhibitors puromycin, bestatin and amastatin. Microvessels represent a major localization of cerebral aminopeptidase M as shown by the intense immunostaining of their walls in sections from various regions as well as in a fraction isolated from cerebral cortex homogenates by a sieving procedure. As compared to the starting homogenate, aminopeptidase M activity was enriched about twenty fold in this microvascular fraction. Aminopeptidase M was identified in this fraction by comparing the inhibitory potencies of antibodies and peptidase inhibitors towards the hydrolysis of [tyrosyl-3,5-3H, Met5]enkephalin to those found for the purified enzyme. A rather high aminopeptidase M activity was also localized in choroid plexuses. Following differential and gradient centrifugation analysis of cerebral cortex homogenates, aminopeptidase M activity was also enriched (by five to six fold) in fractions containing synaptic membranes. No significant soluble aminopeptidase M activity could be detected. These data suggest a dual localization of cerebral aminopeptidase M in microvessels and synaptic membranes consistent with its roles in preventing the access of circulating peptides to brain as well as in inactivating neuropeptides released from cerebral neurones. In comparison, puromycin-sensitive aminopeptidase activity, which is about 100 fold higher than aminopeptidase M activity in brain, was relatively low in microvessels and non-detectable in fractions enriched in synaptic membranes, being almost entirely restricted to soluble fractions.     

Oxygen tension in rat cerebral cortex microvessels in acute anemia

Polarigraphic microelectrodes were used to study the distribution of oxygen tension (pO(2)) in arterioles (lumen diameters 8-80 microm) and venules (lumen diameters 8-120 microm) in the rat cerebral cortex during acute reductions in blood hemoglobin ([Hb]). Isovolumic hemodilution with 5% albumin solution was performed in steps from an initial [Hb] of 14.1 +/- 0.3 g/dl (control) to 9.8 +/- 0.3 g/dl (step 1), 6.6 +/- 0.4 g/dl (step 2), and 4.6 +/- 0.3 g/dl (step 3). Mild anemia (step 1, hematocrit 30%) led to an increase in pO(2) in the arterial side of the microcirculatory bed, with virtually no change in pO(2) in the venous side. Step 2 (hematocrit 20%) was accompanied by a further insignificant increase in pO(2) in arterioles, while there was a significant reduction (on average to 32 mmHg) in venules. Step 3 (hematocrit 13-14%) led to a (statistically insignificant) increase in pO(2) in arterioles. pO(2) in venules decreased, on average, to 27 mmHg; the proportion of smallest venules with low pO(2) values (less than 20 mmHg) increased to 31% (from 3% in controls). In some capillaries, pO(2) was 5-10 mmHg, which was an indicator of the presence of hypoxic zones in brain tissues. These zones primarily arose close to the smallest capillary and venous microvessels, with slowed or impaired blood flow.     

Leakiness of rat brain microvessels to fluorescent probes following craniotomy

The effects of craniotomy and/or histamine treatment upon brain microvascular permeability was studied in Wistar rats. Extravasation of circulating Na-fluorescein (MW 376) and of FITC-albumin (MW 69,000) was observed through a cranial window using intravital fluorescence microscopy. Simple exposure of the pial microvessels induced formation of discrete spots of fluorescent material around venules, but not around arterioles or capillaries. The average number of leaky spots to Na-fluorescein and to FITC-albumin was 4.3 and 1.8 per 10 mm2, respectively, 35 min after exposure. Pretreatment of the rats with either indomethacin (a cyclo-oxygenase inhibitor) or promethazine (a histamine H1-receptor blocker) did not reduce the number of leaky sites, whereas pretreatment with a combination of the two drugs had a significant protective effect. Administration of histamine (10(-4) M) to the exposed brain surface for 5 min increased the number of leaky sites to Na-fluorescein and FITC-albumin 3.2 and 3.6 times, respectively. It is concluded that exposure of the brain surface induces release of histamine and cyclo-oxygenase metabolites, and that these inflammatory mediators elicit formation of leaky sites in brain venules.     

Improved method of ink-gelatin perfusion for visualising rat retinal microvessels

To visualize completely rat retinal microvessels, the gelatin-ink perfusion condition was systematically optimized using von Willebrand factor (vWf) immunostaining as control. Whether the vessel showed by the new perfusion condition can be used for double label with neurons or glial cells in the same retina was also tested. Our results showed that infusing rats first with 20 ml of 37 degrees C ink plus 3% gelatin at 140% rat mean arterial pressure (MAP), and subsequently with 20 ml of 37 degrees C ink plus 5% gelatin at 180% rat MAP allowed the ink to completely fill the rat retinal microvessels. Rat retinal microvessels labeled by the perfusion method were more in number than that by vWf immunostaining. Moreover, our data, for the first time, displayed that the improved gelatin-ink perfusion had no effect on and caused no contamination to the following fluorogold labeling or immunostaining of retinal neurons or glial cells in the same tissue. These data suggest that the improved gelatin-ink perfusion technique is a superior method for morphological characterization of rat retinal microvessels, compatible to the double labeling of glial cells and neurons, and it extends the practical scale of the classic method.     

Novel routes of albumin passage across the glomerular filtration barrier

Albuminuria is a hallmark of kidney diseases of various aetiologies and an unambiguous symptom of the compromised integrity of the glomerular filtration barrier. Furthermore, there is increasing evidence that albuminuria per se aggravates the development and progression of chronic kidney disease. This review covers new aspects of the movement of large plasma proteins across the glomerular filtration barrier in health and disease. Specifically, this review focuses on the role of endocytosis and transcytosis of albumin by podocytes, which constitutes a new pathway of plasma proteins across the filtration barrier. Thus, we summarize what is known about the mechanisms of albumin endocytosis by podocytes and address the fate of the endocytosed albumin, which is directed to lysosomal degradation or transcellular movement with subsequent vesicular release into the urinary space. We also address the functional consequences of overt albumin endocytosis by podocytes, such as the formation of pro‐inflammatory cytokines, which might eventually result in a deterioration of podocyte function. Finally, we consider the diagnostic potential of podocyte‐derived albumin‐containing vesicles in the urine as an early marker of a compromised glomerular barrier function. In terms of new technical approaches, the review covers how our knowledge of the movement of albumin across the glomerular filtration barrier has expanded by the use of new intravital imaging techniques.     

两种显示大鼠不同组织微血管方法的比较

目的 比较CD34免疫组织化学法和硝基四氮唑蓝/5-溴-4-氯-3-吲哚磷酸盐(NBT/BCIP)酶组织化学法显示不同组织微血管的特性. 方法 选取大鼠小肠、肾、脾、肝、心、肺和皮肤等7种组织,通过形态学分析和显微计数,分析上述两种方法的区别. 结果 CD34免疫组织化学法对所取各器官组织内微血管均能显示,NBT/BCIP酶组织化学法能显示心、肠、肾、肝组织内微血管,而不能显示皮肤、脾和肺组织内微血管;对同一组织同一切面,CD34免疫组织化学法显示微血管密度显著性低于NBT/BCIP酶组织化学法. 结论 CD34免疫组织化学法适于各种组织内微血管的显示,而NBT/BCIP酶组织化学法能够更大密度地显示部分器官组织内的微血管.     

内毒素休克时大鼠肠系膜微血管对去甲肾上腺素反应性的变化

本文应用微循环显微也视录象技术,研究内毒素休克大鼠在休克不同时期肠系膜微血管对去甲肾上腺素(NA)反应性的变化。发现局部滴用由低至高浓度NA使微血管口径呈剂量依赖性缩小,对照组在相当于休克相应时间三次观察收缩反应曲线无明显差异(P>0.05),而休克早期微血管对NA的缩血管反应明显,晚期收缩反应减弱(均P<0.01);同时,休克早期浓度—反应曲线右移,EC_(50)减小,反应阈值降低;休克晚期浓度—反应曲线左移,EC_(50)增大,反应阈值增高;但对照组三次观察无明显变化。提示内毒素休克早期微血管对NA的反应性增高,晚期反应性降低,一、二级细动脉的变化尤为显著。可能是休克早期的代偿与晚期的失代偿机理之一。     

Action of vasopressin on neurons and microvessels in the rat hippocampal slice

Summary Arginine vasopressin is reported to have an excitatory effect on hippocampal neurons in the slice preparation. However, vasopressin also has a classic vasopressor action on mammalian blood vessels. We used the rat hippocampal slice to examine the effects of this peptide on central neural and vascular targets. The hippocampus is densely vascularized and pyramidal cells are enmeshed in a network of microvessels. Vasopressin increased the excitability of impaled neurons without substantially altering membrane potential or resistance. The peptide also caused pronounced vasoconstriction in penetrating microvessels when applied at micromolar concentrations. The concerted action of vasopressin on neurons and blood vessels and the physical proximity of these cell types suggest mechanisms whereby these responses may be associated.     

ET-1在大鼠纹状体微血管表达的增龄变化

目的 :研究内皮素 1 (ET 1 )在纹状体微血管表达的年龄变化 ,为探讨纹状体易卒中机制、揭示脑血管病发病机理提供参考资料。方法 :应用SABC免疫组化染色法及图像分析等方法观测 1～ 2月龄、6～ 7月龄及 >2 4月龄三组共 3 6只Wistar大鼠纹状体微血管ET 1的表达。结果 :随月龄增加 ,ET 1在纹状体、颞叶皮质、海马微血管的表达增加 ,>2 4月龄组大鼠纹状体、海马、颞叶皮质微血管ET 1阳性反应强度与其他两组间存在显著差异 (P <0 .0 5 ) ,同月龄组大鼠纹状体、颞叶皮质、海马微血管的ET 1表达有显著差异 (P <0 .0 5 ) ,其中纹状体阳性反应强度最高。结论 :ET 1的分布及其表达的增龄变化 ,可能与该区易发生脑血管病有关。     

Clostridium perfringens epsilon-toxin increases permeability of single perfused microvessels of rat mesentery

Epsilon-toxin, the primary virulence factor of Clostridium perfringens type D, causes mortality in livestock, particularly sheep and goats, in which it induces an often-fatal enterotoxemia. It is believed to compromise the intestinal barrier and then enter the gut vasculature, from which it is carried systemically, causing widespread vascular endothelial damage and edema. Here we used single perfused venular microvessels in rat mesentery, which enabled direct observation of permeability properties of the in situ vascular wall during exposure to toxin. We determined the hydraulic conductivity (L(p)) of microvessels as a measure of the response to epsilon-toxin. We found that microvessels were highly sensitive to toxin. At 10 microg ml(-1) the L(p) increased irreversibly to more than 15 times the control value by 10 min. At 0.3 microg ml(-1) no increase in L(p) was observed for up to 90 min. The toxin-induced increase in L(p) was consistent with changes in ultrastructure of microvessels exposed to the toxin. Those microvessels exhibited gaps either between or through endothelial cells where perfusate had direct access to the basement membrane. Many endothelial cells appeared necrotic, highly attenuated, and with dense cytoplasm. We showed that epsilon-toxin, in a time- and dose-dependent manner, rapidly and irreversibly compromised the barrier function of venular microvessel endothelium. The results conformed to the hypothesis that epsilon-toxin interacts with vascular endothelial cells and increases the vessel wall permeability by direct damage of the endothelium.     

Pituitary adenylate cyclase-activating polypeptide inhibits the cyclooxygenase pathway of rat cerebral microvessels

The presence of nerve endings containing pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) around cerebral microvessels suggests that these peptides have regulatory roles in the cerebral microcirculation. Prostanoids synthesized by the cerebrovascular endothelium have a determining role in the regulation of the brain circulation. In the present study, the effects of PACAP and VIP on the cyclooxygenase pathway of cerebral microvessels were investigated. The isolated microvessels were incubated with 1-¹⁴C-arachidonic acid and different concentrations of the peptides. The prostanoids formed were separated by means of overpressure thin-layer chromatography, and were quantitatively determined by liquid scintillation. Higher concentrations (10^–7 and 10^–6 mol L^–1) of PACAP significantly inhibited the activity of the cyclooxygenase pathway, whereas VIP had no significant effect on it. As regards the cyclooxygenase metabolites, the syntheses of thromboxane A₂ and prostaglandin D₂ were inhibited significantly. PACAP and VIP are known to increase the intracellular cAMP level in the cerebral microvessels and in the present experiments the protein kinase A inhibitor H-89 attenuated the effect of PACAP on prostanoid synthesis. It is concluded that the cyclooxygenase pathway of rat cerebral microvessels is more sensitive to PACAP than to VIP. The inhibitory effect of PACAP on prostanoid synthesis is mediated via a cAMP-dependent pathway. By inhibiting the formation of vasoactive prostanoids, PACAP can decrease the vasoreactivity of the microvessels.