Morphologic basis of increased vascular permeability induced by acetyl glyceryl ether phosphorylcholine

The potent vasoactive and leukotactic properties of acetyl glyceryl ether phosphorylcholine (AGEPC) were further characterized histologically. After intravenous infusion of colloidal carbon and local injection of AGEPC, microscopic examination of rat cremaster muscle and skin revealed histamine-like vascular labeling restricted to postcapillary venules. Ultrastructural studies demonstrated subendothelial carbon accumulation in labeled venules. In rat skin, vascular labeling with colloidal carbon was an equally sensitive indicator of AGEPC-induced vasoactivity as was Evans blue dye extravasation for the assessment of AGEPC-induced increased vascular permeability (i.e., 1 pmole of AGEPC consistently initiated both vascular labeling and increased vascular permeability). In addition to its potent vasoactive effects in rabbits and rats, concomitant leukocyte emigration was observed in venules within 15 minutes after intradermal injection of AGEPC. In rabbit skin, AGEPC was equally as potent for the induction of leukocyte infiltrates as for the stimulation of increased vascular permeability. However, the vasoactive properties of AGEPC appeared to be neutrophil independent as well as independent of mast cell and platelet stimulation; these data suggest that AGEPC may act upon the microvasculature by direct stimulation of the venular endothelial cells. Thus, the putative role of AGEPC as a potent inflammatory mediator includes both the vasoactive and the leukotactic aspects of the acute inflammatory process.     

Edema and hemoconcentration in mice experimentally infected with Vibrio vulnificus.

Vibrio vulnificus (lactose-positive Vibrio), a recently recognized pathogenic marine species, produced extreme hemoconcentration and death within 3 to 6 h after subcutaneous or intraperitoneal injection of 10(8) viable cells into mice; hemotocrit values approached 70% (normal, 45%). About 1 ml of edema fluid accumulated at the site of each subcutaneous injection, and locally increased vascular permeability was demonstrated by a skin bluing assay, using Evans blue dye. A corresponding fluid accumulation did not occur in the peritoneal cavity after an intraperitoneal injection. Filter-sterilized supernatants of cultures grown under a variety of conditions did not produce local edema or lethality, nor did whole Vibrio cells killed by a variety of methods or disrupted by sonic oscillation. Edema fluids collected from infected mice and sterilized by filtration had no effect when they were injected subcutaneously or intraperitoneally into mice. Inocula of 10(9) viable cells of V. vulnificus contained within a diffusion chamber implanted subcutaneously did not produce skin bluing, edema, or lethality; Vibrio cells remained viable and virulent within these chambers for at least 2 weeks. These experiments suggested that vascular permeability changes in V. vulnificus infections may not be attributable to a diffusible toxin and may require direct contact between host cells and viable Vibrio cells.     

Effect of Bordetella pertussis extract and vasoactive amines on vascular permeability

Vasxular permeability to Evans blue dye and 131-I-labeled human serum albumin was studied in normal mice and in mice treated with alkaline saline extracts (SE) from Bordetella pertussis cells. Skin sites inoculated intracutaneously with small doses of histamine, serotonin, or a combination of these 2 substances were more permeable in SE-treated mice than in normal animals. Intravenously administered catecholamines were able to reduce in varying degrees the vascular permeability induced by serotonin or by histamine in normal mice; in SE-treated mice the catecholamines were less effective. The relative effectiveness of intravenously administered catecholamines to reduce vascular permeability in normal or SE-treated mice was ranked as follows: isoproterenol greater than epinephrine greater than norepinephrine. When catecholamines were given concomitantly with histamine and serotonin in the skin test site, the permeability in both normal and SE-treated mice was again reduced or blocked, but isoproterenol was only weakly effective in this instance. Their relative effectiveness was epinephrine greater than norepinephrine greater than isoproterenol. The possible explanations for these results are discussed.     

Paf induces rat plasma extravasation and releases eicosanoids during anaphylaxis

The effects of anaphylaxis on vascular protein extravasation in selected tissues and on the release of prostaglandins in the peritoneal cavity were studied in sensitized rats. Extravasation of Evans blue dye was used as a measure of vascular permeability. Specific antigen challenge increased by 279, 297, 328, 250, and 192% the protein extravasation in the trachea, upper and lower bronchi, pancreas, and duodenum, respectively, but did not modify significantly the vascular permeability of the lung parenchyma, heart, liver, and kidney. Extravasation of Evans blue dye also was increased by 43-fold in the peritoneal cavity. Pretreatment of the animals with indomethacin (10 mg/kg) did not modify significantly the protein extravasation of the trachea, upper and lower bronchi, pancreas, and duodenum induced by anaphylaxis. Pretreatment with a mixture of mepyramine (3 mg/kg) and methysergide (2.5 mg/kg) reduced by 62, 66, and 40% the protein extravasation in the trachea, upper bronchi, and peritoneal cavity, respectively, in similar conditions. The PAF antagonist BN-52021 (5 mg/kg) very strongly reduced the protein extravasation elicited by anaphylaxis in the trachea, upper and lower bronchi, pancreas, and duodenum by 72, 87, 82, 67, and 85%, respectively, and by 53% in the peritoneal cavity. Anaphylaxis also increased the concentrations of thromboxane B₂ and leukotriene B₄ in the peritoneal exudates, but prostaglandin E₂ levels were not affected. Pretreatment with BN-52021 reduced by 29 and 75 % the level of thromboxane B₂ and leukotriene B₄ in the exudates. These results suggest that PAF, histamine, and serotonin mediate the protein extravasation associated with anaphylaxis, whereas prostaglandins are likely to play a minor role in this reaction.     

Correlation between enhanced vascular permeability, up-regulation of cellular adhesion molecules and monocyte adhesion to the endothelium in the retina during the development of fatal murine cerebral malaria.

The relationships between increased vascular permeability to protein, monocyte adherence to the endothelium, and expression of the cell adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the central nervous system microvasculature were studied during the progression of fatal murine cerebral malaria. CBA mice were inoculated with Plasmodium berghei ANKA, and changes in the retinal microvasculature were examined on days 3, 5, and 7 postinoculation (p.i.). Evans blue dye and horseradish peroxidase (HRP) were administered intravenously to assess vascular permeability to macromolecules macroscopically and by light and electron microscopy. ICAM-1 and VCAM-1 expression were examined by immunohistochemistry. HRP leakage into the retinal parenchyma was seen macroscopically at a low level on day 3 p.i., increasing progressively at day 5 (the earliest time at which cerebral symptoms were observed) and day 7 (the day on which animals showed severe behavioral abnormalities and died). The inner retinal vascular plexus showed a slight increase in vascular permeability to intravenous Evans blue at day 3 p.i. and congestion, monocyte adherence to the endothelium, and increased vascular permeability to both Evans blue and HRP at day 7 p.i. Electron microscopic observations were consistent with these findings and also revealed disrupted light junctions and the coating of monocytes and endothelium with HRP at day 7 p.i. Immunohistochemical staining and densitometry showed a progressive increase from day 3 to day 7 p.i. in the densities of ICAM-1 and VCAM-1 on the venular endothelium of the inner retinal vascular plexus, with the appearance of adherent ICAM-1+ monocytes at the terminal stage of the disease. None of the pathological changes associated with the inner retinal plexus were seen at any stage in the outer retinal plexus. These results suggest the following sequence of events in the inner retinal vessels, particularly the venules, during the progression of fatal murine cerebral malaria: 1) a mild increase in vascular permeability at approximately day 3 p.i., 2) a progressive increase in endothelial expression of the cell adhesion molecules ICAM-1 and VCAM-1, commencing at approximately day 3 p.i., 3) monocyte adhesion to the endothelium starting at approximately day 5 p.i., and 4) frank disruption of endothelial integrity at the terminal stage (day 7 p.i.), leading to edema and hemorrhage. Similar changes in cerebral vessels may underlie the neurological complications of the disease.     

The effects of adrenalectomy and corticosterone on vascular permeability responses in the skin of the rat.

The vascular permeability response induced in the rat by intracutaneous histamine or serotonin is noticeably influenced by previous adrenalectomy or treatment with corticosterone. Permeability responses were demonstrated by the local exudation of circulating Evans blue at sites of intracutaneous injection of the above permeability factors, the intensity of the responses being assessed by the diameter of the blue lesions as well as by the amount of exuded dye. In adrenalectomized rats maintained for 72-80 h on 0-9% solution of NaCl, the permeability response to histamine was enhanced about 10-fold, that to serotonin about 5-fold. When rats were given subcutaneous corticosterone, 1-0 mg/animal 1 h before testing, the responses to the same 2 permeability factors were decreased about 10-fold and 5-fold respectively. Corticosterone also decreased the enhanced responses in adrenalectomized rats to levels somewhat below those in mock adrenalectomized controls. The results support the proposal from other work that vascular exudation in experimental inflammation is regulated by an anti-inflammatory factor that accumulates in injured tissues and owes its effect to release of corticosteroids.     

Inflammation in Healing: I. Time-Course and Mediation of Exudation in Wound Healing in the Rat

The time-course of increased vascular permeability in excised wounds in the skin of the rat was measured by injecting Evan''s blue i.v. at various times after wounding and extracting the dye which exuded into the injured tissues during various short intervals of time. A single peak of increased permeability occurred within the first hour after which exudation rapidly diminished to a low level which then persisted for at least 5 days after wounding.In animals with circulating colloidal carbon only venules in the edge and floor of wounds exhibited increased permeability during the phase of maximum exudation, after which leakage occurred only from capillaries in the advancing edge of the healing granulation tissue.In controlled experiments, using rats matched for weight, the exudation in the first 30 min. after wounding was partially suppressed by inhibitors of histamine and serotonin, but not by anti-inflammatory agents which inhibit the kinins and/or kallikrein.It is concluded that, in the absence of exogenous irritation, the inflammatory component of the process of healing after cutaneous wounding is evanescent, slight in magnitude, and mediated by histamine and serotonin.     

Demonstration of vascular permeability changes in cattle infected with bovine ephemeral fever virus

Five cattle infected with bovine ephemeral fever virus were necropsied on the day after onset of clinical disease, when clinical signs of lameness were most severe. Gross lesions observed included a serofibrinous polyserositis involving the synovial, pericardial, thoracic and abdominal cavities. The associated histological changes consisted primarily of oedema and an influx of neutrophils into affected tissues and fluids. In a further eight infected cattle, increases in permeability of vessels associated with serosal surfaces were demonstrated by labelling with either colloidal carbon or Evans blue. Intravenous injections of carbon provided both macroscopic and histological labelling of affected vessels. Evans blue appeared to be more sensitive than carbon but did not provide a histological marker of vascular permeability and provided labelling of tissues rather than individual vessels. The main sites of increased permeability were synovial, pericardial, thoracic and abdominal serosae.     

Bone Morphogenetic Protein-Modulator BMPER Regulates Endothelial Barrier Function

The endothelium serves as a selective barrier and controls the exchange of nutrients, hormones, and leukocytes between blood and tissues. Molecular mechanisms contributing to the pathogenesis of endothelial barrier dysfunction remain incompletely understood. Accumulating evidence implicates bone morphogenetic protein (BMP)-modulator BMPER as a key regulator in endothelial biology. Herein, we analyze the impact of BMPER in the control of endothelial barrier function. To assess the role of BMPER in vascular barrier function in mice, we measured the leakage of Evans blue dye from blood into interstitial lung tissue. BMPER^+/? mice exhibited a significantly higher degree of vascular leak compared with wild-type siblings. In accordance with our in vivo observation, siRNA-based BMPER knockdown in human umbilical endothelial cells increased endothelial permeability measured by FITC-dextran passage in transwell assays. Mechanistically, BMPER knockdown reduced the expression of VE-cadherin, a pivotal component of endothelial adherens junctions. Conversely, recombinant human BMPER protein upregulated VE-cadherin protein levels and improved endothelial barrier function in transwell assays. The effects of BMPER knockdown on VE-cadherin expression and endothelial permeability were induced by enhanced BMP activity. Supporting this notion, activation of BMP4-Smad-Id1 signaling reduced VE-cadherin levels and impaired endothelial barrier function in vitro. In vivo, Evans blue dye accumulation was higher in the lungs of BMP4-treated C57BL/6 mice compared to controls indicating that BMP4 increased vascular permeability. High levels of BMPER antagonized BMP4-Smad5-Id1 signaling and prevented BMP4-induced downregulation of VE-cadherin and endothelial leakage, suggesting that BMPER exerts anti-BMP effects and restores endothelial barrier function. Taken together, this data demonstrates that BMPER-modulated BMP pathway activity regulates VE-cadherin expression and vascular barrier function.     

Assessing vascular permeability during experimental cerebral malaria by a radiolabeled monoclonal antibody technique

Vascular endothelial integrity, assessed by Evans blue dye extrusion and radiolabeled monoclonal antibody leakage, was markedly compromised in the brain, lung, kidney, and heart during Plasmodium berghei infection, a well-recognized model for human cerebral malaria. The results for vascular permeability from both methods were significantly (P < 0.001) related.     

Effect of age on transvascular fluid movement.

Swellings of the mouse tail and ear were produced by subjecting them to subatmospheric pressures of minus 40 to minus 80 mmHg for 15-60 min. Increase in volume was measured volumetrically in the tail and gravimetrically in the ear. Blood volume increases in the tail, as measured with 51Cr erythrocytes, contributed a minor part of the fluid increase. Comparison of mice from 3 to 36 wk in age showed a large decrease of fluid movement with age, with major changes during the growth period. Study of permeability of the ear under decreased pressure,to intravenously administred Evans blue, showed no influence of age on permeability to the protein-bound dye. Measurement of transmission of the applied negative pressure through the skin, and of compliance of the tissues of the ear and tail in mice of different age groups, indicated that these factors were not responsible for the observed changes with age.     

Quantitation of phototoxic hyperemia and permeability to protein: I. Inhibition by histamine (H1 and H2) and serotonin receptor antagonists in pig skin

Exposure of pig skin treated with a solution of anthracene to long-wave ultraviolet radiation (UVA) produced an inflammatory response which consisted of erythema and increased vascular permeability. The erythema was measured by the increase in ⁵¹Cr-RBC content of the skin; permeability of the microvasculature was determined by measurement of the accumulation of ¹²⁵I-albumin. Within the first 100 seconds (2.6 × 10³ J/M²) of irradiation, the ⁵¹Cr-RBC content of the skin increased to twice normal and remained at that level despite continued irradiation. The dermal vasculature became increasingly permeable through 1000 sec of irradiation at which time the ¹²⁵I-albumin content was ten times normal. Pretreatment of pigs with the histamine receptor antagonists pyrilamine (H₁) or cimetidine (H₂) had no effect in blocking the photobiologic increase in blood content but showed a significant inhibition of the increased permeability to ¹²⁵I-albumin. The serotonin receptor antagonist methysergide had the same permeability inhibiting effects but was about ten times more active. Thus, in the pig, the erythema occurring during the anthracene-UVA reaction is not mediated by receptors for histamine (H₁ or H₂), or by serotonin, whereas the increased vascular permeability to ¹²⁵I-albumin occurring during the same phototoxic reaction is mediated by histamine and serotonin receptors.     

IL-1 and IFN-gamma increase vascular permeability.

We examined the effect of cytokines on vascular permeability in vivo. Wistar rats received intradermal injections of various cytokine preparations and the permeability index (delta PI) was calculated from the difference between the absorption values of cytokine- and vehicle-treated skin sections after the extraction of accumulated Evans blue vital dye. Injections of a mixture of recombinant interleukin-1 alpha and beta (Il-1 alpha, IL-1 beta) and interferon-gamma (IFN-gamma) caused a maximal increase of permeability after 30 min (delta PI = 2). The administration of single recombinant cytokines revealed that the increase of permeability is mainly due to the action of IL-1 beta (delta PI = 1.4) and IFN-gamma (delta PI = 2.9) (P less than 0.001) at doses of 1-20 microU per injection site. IL-1 alpha slightly increased vascular permeability, whereas recombinant IL-2 and recombinant tumour necrosis factor alpha had no significant effects. Histological observations revealed significantly increased numbers of degranulated mast cells in skin sections pretreated with IL-1 beta (P less than 0.005) or IFN-gamma (P less than 0.001). The cytokine-mediated rise of vascular permeability could be suppressed by pretreatment of the animals with the vasoactive amine antagonizing drugs methysergide, pizotifen and cyproheptadine. Our experiments indicate an important role of IL-1 beta and IFN-gamma as vasoactive substances besides their function as hormone-like messengers between leucocytes.     

Mustard oil-induced cutaneous inflammation in the pig

Recent findings indicate that chemical stimulation of the porcine skin with capsaicin evokes a flare response similar to that observed in man. The aim of the present study was to elucidate whether chemical stimulation of cutaneous capsaicin-sensitive nerve endings with mustard oil produces neurogenic inflammatory reactions in the pig.The application of mustard oil onto the abdominal skin of domestic pigs resulted in a pronounced flare response. After a previous intravenous injection of a solution of Evans blue, the skin area in contact with the irritant turned dark blue, indicating a marked extravasation of albumin. Quantitative estimation of the dye content of the skin supported this conclusion. The technique of vascular labelling revealed a delicate network of small subepidermal blood vessels in histological preparations after the application of mustard oil following a previous intravenous injection of colloidal silver. Labelled blood vessels were not noted outside the treated area.The present results show that mustard oil produces a strong cutaneous inflammatory response in the pig, and suggest that the porcine skin provides a valuable model for study of the significance of capsaicinsensitive sensory nerves in vascular and other cutaneous reactions.     

Enhanced vascular permeability in rat skin induced by sensory nerve stimulation: evaluation of the time course and appropriate stimulation parameters

Activation of nociceptors causes them to secrete neuropeptides. The binding of these peptides to receptors on blood vessels causes vasodilation and increased vascular permeability that allows loss of proteins and fluid (plasma extravasation, PE); this contributes to inflammation. This study defines the relationship between electrical activation of nociceptors and PE and evaluates the time course of this response in the skin of rats. We measured the time course and extent of PE by digital imaging of changes in skin reflectance caused by leakage of Evans Blue (EB) dye infused in the circulatory system before stimulation. Stimulation of the exclusively sensory saphenous nerve caused the skin to become dark blue within 2 min due to accumulation of EB. While PE is usually measured after 5–15 min of electrical stimulation, we found that stimulation for only 1 min at 4 Hz produced maximum PE. This response was dependent on the number of electrical stimuli at least for 4 Hz and 8 Hz stimulation rates. Since accumulation of EB in the skin is only slowly reversible, to determine the duration of enhanced vascular permeability we administered EB at various times after electrical stimulation of the saphenous nerve. PE was only observed when EB was infused within 5 min of electrical stimulation but could still be observed 50 min after capsaicin (1%, 25 μl) injection into the hind paw. These findings indicate that enhanced vascular permeability evoked by electrical stimulation persists only briefly after release of neuropeptides from nociceptors in the skin. Therefore, treatment of inflammation by blockade of neuropeptide release and receptors may be more effective than treatments aimed at epithelial gaps. We propose, in models of stimulation-induced inflammation, the use of a short stimulus train.     

The direct demonstration of histamine release in allergic reactions in the skin using a skin chamber technique.

We have adapted a skin chamber technique to permit sampling of fluid at the skin window sites of pollen antigen-induced allergic reactions. Low background levels of histamines are formed in control chambers, whereas significantly increased (p less than 0.01) amounts are found within 30 min following ragweed application in sensitized subjects.     

Modeling the Evans Blue Dilution Method for the Measurement of Plasma Volume in Small Animals: A New Optimized Method

The measurement of plasma volume (V_p) in humans and animals is frequently performed by the Evans blue dye dilution method. However, after injection of Evans blue into the circulation, no steady state is observed because of delayed mixing and progressive leakage of dye out of vascular space. Various methods of calculation have been proposed, either with a single blood sampling 5–10 min after dye injection (Single point method), or with extrapolation at time zero of a logarithmic decay (Log linear method). We propose a method based on a two-compartment hypothesis taking into account the initial mixing and the leakage phase in the time course of dye concentration. Nineteen Sprague–Dawley rats were studied in various conditions and blood sampling was performed before and 2, 4 and 6 min after injection of 200 μg Evans blue. A mathematical model was designed to describe the two-compartment hypothesis and allowed the calculation of V_p and K_out (rate of disappearance of dye from vascular space). A Bland and Altman representation evidenced an overestimation of V_p with previous methods and the great dispersion of results with the single point method, especially when using the 6 min point. Calculation of K_out revealed more accurate with the model than the Log linear method, especially when the mixing rate is slow. We suggest using the two-compartment model to measure V_p with Evans blue technique in rats. This method also allows precise evaluation of the rate of dye leakage, which could be a good marker of vascular permeability to albumin.     

Studies on vascular permeability increasing factors involved in 48-hour homologous PCA in the mouse ear

Several attempts were made to elucidate the possible role of histamine, serotonin, leukotrienes C4 (LTC4) and D4 (LTD4), and prostaglandin E1 (PGE1) as vascular permeability increasing factors involved in 48-hour homologous passive cutaneous anaphylaxis (PCA) in the mouse ear. Increased vascular permeability in the mouse ear caused by the mediator injection or PCA was assessed quantitatively by measuring the amount of extravasated dye. In skin reactions, all of the mediators used in the present study significantly increased vascular permeability. The most potent mediator was serotonin, which increased the vascular permeability from a concentration of 10(-8) g/ml, and the activity was about 100 times higher than that of histamine on a weight basis. Vascular permeability increasing activity of LTC4 was about 10 times higher than that of histamine, and LTD4 and PGE1 were also more potent than histamine. Increases of vascular permeability caused by histamine, serotonin, LTC4 and LTD4 were significantly potentiated by injecting 10(-6) g/ml of PGE1 simultaneously. Histamine-, serotonin- and LTC4-induced skin reactions in the mouse ear were suppressed significantly by the administrations of chlorpheniramine, methysergide and FPL 55712, respectively. In contrast, though chlorpheniramine and methysergide suppressed also mouse ear PCA (about 50 and 40%, respectively), neither FPL 55712, indomethacin nor BW 755C suppressed it. These results strongly suggest that the most important mediator involved in mouse ear PCA is histamine and that serotonin also plays an important role in the increase of vascular permeability caused by PCA. Despite their potent vascular permeability increasing activity LTC4, LTD4 and PGE1 do not seem to play an important role in mouse ear PCA.     

Evans蓝血管显影法在脊髓损伤研究中的应用

为了观察脊髓损伤缺血区的血管变化,本研究就灵敏有效的血管显影方法进行了探索。由于Evans蓝可与血浆白蛋白结合,而且血管内皮细胞上有血浆白蛋白受体,所以Evans蓝可能用于显示血管,并具有不同于常用方法的独特优点。我们尝试了Evans蓝液的不同配方及在染液静脉灌注前或灌注后固定的两种固定方法。Evans蓝液的不同配方为单纯Evans蓝、Evans蓝加明胶以及Evans蓝加明胶及铬明矾。我们观察到每一种配方都有其优缺点,而且能彼此互补。有的方法可进行免疫组化双标染色。由于许多物质与脊髓血管的病理变化有关,大多可用免疫组化染色予以显示,故此对脊髓损伤研究非常重要。     

Ovalbumin aerosol challenge in actively sensitized guinea pigs: relationship between airway microvascular leakage and airflow obstruction

Airway inflammation is a common feature of asthma, and one of the cardinal features of inflammation is increased microvascular permeability. We investigated the characteristics of inhaled ovalbumin challenge-induced airflow obstruction and airway microvascular leakage in vivo in mechanically ventilated guinea pigs actively sensitized to ovalbumin. A method was used to quantify both airflow obstruction and airway microvascular leakage in order to investigate the relationship between these 2 pathophysiological features in the same animal. Airway microvascular leakage was assessed by Evans blue dye extravasation into airway tissues. Actively sensitized guinea pigs developed both acute airflow obstruction (increased lung resistance and reduced dynamic lung compliance) and Evans blue dye extravasation in response to exposure to aerosolised ovalbumin. Evans blue dye extravasation was preferentially distributed in the distal airways and correlated with airflow obstruction. The results show that inhaled allergen induced both acute airflow obstruction and airway microvascular leakage.