Modeling microcirculatory disorders using an ultraviolet laser]

Reactions of microvessels and mast cells to laser irradiation were studied in rat mesentery by applying the method of intravital microscopy. An ultra-violet laser (gamma=337 nm) was used. The diameter of the laser beam was changed from 2 to 100 mum. Different irradiation doses provoked either an increase of vascular permeability or thrombus formation or hemorrhage. Apart from the vascular wall injury, factors accompanying the damage of red blood cells and other cells of the blood may play a role in the process of thrombus formation. Changes of the vascular diameter and permeability after laser irradiation of mast cells are probably connected with the release of histamine and serotonin contained in them.     

Leukocyte adhesion and microvessel permeability

To investigate the direct effect of leukocyte adherence to microvessel walls on microvessel permeability, we developed a method to measure changes in hydraulic conductivity (L(p)) before and after leukocyte adhesion in individually perfused venular microvessels in frog mesentery. In 19 microvessels that were initially free of leukocyte sticking or rolling along the vessel wall, control L(p) was measured first with Ringer-albumin perfusate. Blood flow was then restored in each vessel with a reduced flow rate in the range of 30-116 microm/s to facilitate leukocyte adhesion. Each vessel was recannulated in 45 min. The mean number of leukocytes adhering to the vessel wall was 237 +/- 22 leukocytes/mm(2). At the same time, L(p) increased to 4.7 +/- 0.5 times the control value. Superfusion of isoproterenol (10 microM) after leukocyte adhesion brought the increased L(p) back to 1.1 +/- 0.2 times the control in 5-10 min (n = 9). Superfusing isoproterenol before leukocyte adhesion prevented the increase in L(p) (n = 6). However, the number of leukocytes adhering to the vessel wall was not significantly affected. These results demonstrated that leukocyte adhesion caused an increase in microvessel permeability that could be prevented or restored by increasing cAMP levels in endothelial cells using isoproterenol. Thus cAMP-dependent mechanisms that regulate inflammatory agent-induced increases in permeability also modulate leukocyte adhesion-induced increases in permeability but act independently of mechanisms that regulate leukocyte adhesion to the microvessel wall. Application of ketotifen, a mast cell stabilizer, and desferrioxamine mesylate, an iron-chelating reagent, attenuated the increase in L(p) induced by leukocyte adhesion, suggesting the involvement of oxidants and the activation of mast cells in leukocyte adhesion-induced permeability increase. Furthermore, with the use of an in vivo silver stain technique, the locations of the adherent leukocytes on the microvessel wall were identified quantitatively in intact microvessels.     

Physiological roles of endogenous nitric oxide in lymphatic pump activity of rat mesentery in vivo

Physiological roles of endogenous nitric oxide (NO) in the lymphatic pump activity of rat mesenteries in vivo were evaluated using an intravital video microscope system. Changes in the pumping frequency (F), the end diastolic diameter (EDD), and the end systolic diameter (ESD) of the mesenteric lymph microvessels were measured with the microscope system and then the pump flow index (PFI) was calculated. A 15-min superfusion of 30 microM N(omega)-nitro-L-arginine methyl ester (L-NAME) in the mesenteries caused significant increases of F and PFI and a significant decrease of the EDD and ESD. Simultaneous superfusion of 1 mM L-arginine with 30 microM L-NAME produced a significant reversal of the L-NAME-mediated increase of F and decrease of ESD. A 15-min superfusion of 100 microM aminoguanidine caused no significant effects on F, EDD, and ESD of the mesenteric lymph vessels in vivo. These findings suggest that endogenous NO has physiologically modulated the lymphatic pump activity in rat mesentery in vivo and that the production and release of NO may be mediated by constitutive NO synthase but not by inducible NO synthase.     

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.     

Use of the television analysing system in the studies on microcirculation]

In the experiments on anesthetized rats the television analysing system (LEITZ-TAS) was used for evaluation of quantitative structure-functional characteristics of microcirculation under intravital conditions and the development of microvessel network, for measuring geometric parameters of microvessels and blood flow change in them, as well as to define the degree and spreading of the disturbances in the vessel wall permeability.     

Nitric oxide in the lymphatic microvessel regulation

Topical application of sodium nitroprusside on rat mesentery has a marked influence on lymph microvessels function. The drug causes a dilation of majority of lymphangions and decrease of the pacemaker activity of the vessel wall structures and valves. These changes do not lead to lymph stasis, and lymph flow velocity does not reduce. The non-selective inhibitor of NO synthase (N-nitro-L-arginine) intensifies vasomotions of lymph microvessels, modulates phasic contractile activity and increases lymph flow velocity. There is a time dependent dynamic of changes in action of N-nitro-L-arginine. During inhibition of endogenous NO synthesis the responses of lymph microvessels on sodium nitroprusside application are modified: the constriction of majority lymphangions and activation of valve work are observed.     

Homocysteine causes cerebrovascular leakage in mice

Elevated plasma homocysteine (Hcy) is associated with cerebrovascular disease and activates matrix metalloproteinases (MMPs), which lead to vascular remodeling that could disrupt the blood-brain barrier. To determine whether Hcy administration can increase brain microvascular leakage secondary to activation of MMPs, we examined pial venules by intravital video microscopy through a craniotomy in anesthetized mice. Bovine serum albumin labeled with fluorescein isothiocyanate (BSA-FITC) was injected into a carotid artery to measure extravenular leakage. Hcy (30 microM/total blood volume) was injected 10 min after FITC-BSA injection. Four groups of mice were examined: 1) wild type (WT) given vehicle; 2) WT given Hcy (WT + Hcy); 3) MMP-9 gene knockout given Hcy (MMP-9-/- + Hcy); and 4) MMP-9-/- with topical application of histamine (10(-4) M) (MMP-9-/- + histamine). In the WT + Hcy mice, leakage of FITC-BSA from pial venules was significantly (P < 0.05) greater than in the other groups. There was no significant leakage of pial microvessels in MMP-9-/- + Hcy mice. Increased cerebrovascular leakage in the MMP-9-/- + histamine group showed that microvascular permeability could still increase by a mechanism independent of MMP-9. Treatment of cultured mouse microvascular endothelial cells with 30 microM Hcy resulted in significantly greater F-actin formation than in control cells without Hcy. Treatment with a broad-range MMP inhibitor (GM-6001; 1 microM) ameliorated Hcy-induced F-actin formation. These data suggest that Hcy increases microvascular permeability, in part, through MMP-9 activation.     

Age-dependent mitogenesis in normal connective tissue cells

We have previously described ways to use the mesentery for studies of proliferation in intact tissue. Here we have studied a weak and a strong mitogenic response in the mesentery of rats aged 6--42 weeks, induced by a single intraperitoneal injection of saline or the mast-cell-degranulating drug 48/80. Proliferation was measured by the specific DNA activity, the fraction of fibroblast- and mesothelial-like cells in the S+G2 cell-cycle-phases, and the mitotic index. We also counted the relative number of mast cells in the tissue (normalized to 5,000 fibroblast-like and mesothelial-like cells), since this might influence 48/80-induced mast-cell-mediated proliferation. In old animals there was a decline in the proliferative response and the time required to initiate DNA synthesis was prolonged. This appears to be the first report of such an age-dependent proliferation characteristic in the mesentery, and probably in any connective tissue. The normalized number of mast cells in the mesentery also declined with increasing age.     

Dilatation of arterioles in the frog submaxillary muscle under the influence of visible light]

Frog denervated submaxillar muscle arterioles dilates under the influence of visible light. In darkness the vessel returns to tis initial condition. The degree of dilatation is in the main inversely proportional to the initial diameter of the vessel. Besides, the vessel reactivity also depends on the thickness of its wall. Thick-walled arterioles are the most reactive ones. Their diameter becomes 2 -3 times greater under the influence of visible light. This light-evoked vasodilatation should be taken into account during intravital microscopy, as such a considerable dilatation of arterioles caused by light changes their reactivity considerably.     

肠系膜微血管通透速度的图象分析

本文应用微循环活体观察和荧光示踪技术,通过计算机数字图象处理对荧光素钠（ＦＩＮａ）和ＦＩＴＣ－Ｄｅｘｔｒａｎｓ（ＦＤ－４,ＭＷ４０００,ＦＤ－１５０,ＭＷ１５００００）在肠系膜微血管的通透过程进行了定量研究,建立了正常大鼠肠系膜微血管对不同分子量荧光物质的通透方程,得到了通透系数及对通透系数评价的定量方法。结果证实：颈动脉注射荧光素钠后４ｓ即可在微血管中见到,扩散速度很快,通透系数为３．３６２×１０－８ｃｍ２／ｓ。注射ＦＤ－４后１５ｓ可在微血管中见到,扩散速度比荧光素钠慢１倍左右,通透系数为１．７１８×１０－８ｃｍ２／ｓ。注射ＦＤ－１５０后８ｓ可在微血管中见到,扩散速度很慢,约为荧光素钠扩散速度的１／７０,通透系数为０．０４８６×１０－８ｃｍ２／ｓ。荧光物质的渗出部位主要在毛细血管及细静脉,在细动脉则极少见到。证实我们计算得出的通透系数能够较好地反映肠系膜微血管通透的实际情况,所采用的评价方法及得到的通透方程可以用于微血管物质交换参数的定量评价。     

Microcirculatory bed of the heart during peripheral arteriovenous shunting

In the experiment performed on 108 test and 28 control dogs, by means of injection and histological methods, the effect of arteriovenous fistula on the cardiac microcirculatory bed has been studied. The arteriovenous blood shunting results in plethora and stasis in the cardiac microcirculatory bed, in dilatation of all its links, in aggregation and adhesion of the blood formed elements, in increased permeability of microvessels, in diapedesis of erythrocytes and in myocardial edema, in winding microvessels, in development of the venoarterial reaction, in hypertrophy of myocytes, recalibration and sclerosis of the microvascular walls.     

Maturation of adult rat peritoneal and mesenteric mast cells

Summary Repopulation and maturation of rat mesenteric and peritoneal mast cells were studied after mast cell depletion by intraperitoneal injection of distilled water. Immature mast cells were first identified in the mesentery and peritoneal fluid 5 and 6 days, respectively, after water injection. The most immature mast cells that could be identified contained a few orthochromatic granules. Upon maturation, the granules became metachromatic and increased in size and number. Heparin, revealed by toluidine blue staining and berberine sulfate fluorescence, appeared simultaneously with orthophthaldialdehyde (OPT)-induced histamine fluorescence. Paraformaldehyde-induced serotonin fluorescence appeared somewhat later. Repopulation of mesentery and peritoneal fluid by mast cells seemed to be independent of each other and to occur from undifferentiated precursor cells.     

Microcirculatory changes in experimental trigeminal neuralgia in rats

Biomicroscopic studies of mesentery in trigeminal neuralgia rats caused by creation of a generator of pathologically enhanced excitation in trigeminal nerve caudal nucleus (injection 0.25-1.0 DLM Tetanus toxin) have shown the microcirculatory disorders, venular permeability, mast cells degranulation, and an increase in lymphatic contractile activity. Microcirculatory disorders intensity and adaptation reaction appearance correlated with trigeminal neuralgia clinical picture.     

Correlation between activity of tissue basophils and leukocytes and permeability of mesenteric venules of rats in experimental peritonitis

Calculation of adhered leukocytes, degranulated mast cells and leaked mesentery venules have been prepared in 1, 2, 4 and 8 hours after induction of inflammation. Number of adhered to the venular walls leukocytes enhanced with constant speed for the first 4 hours. The speed of the appearance of new mast cells with the signs of the degranulation in the period was raised. Alteration of the number (share) of leak vessels had parallel dynamic. This data show that in this model of inflammation the main role in disturbances of the permeability is played by products of the degranulation of must cells. In 4 hours and more predominant significance in genesis of disturbances of transport function of vessels wall begin to acquire metabolites of activated leukocytes--free radicals of the oxygen. Apparently this substances played an important role in the mechanism of releasing of the thrombosis.     

Fahraeus-effect-reversal (FER) in compaction stasis (CS): microrheological and haemodynamic consequences of intravascular sedimentation of red cell aggregates

Red cell aggregate sedimentation under gravitation produces pronounced and rapid "phase separation effects" culminating in "compaction stasis" (CS), i.e. almost complete stuffing of microvessels by RBC. This can be readily observed and monitored in microvessels of vertically placed mesentery preparations by a horizontally aimed intravital microscope as shown by (GOBEL et al. VIRCHOW's Arch., 1988,). "Layered flow", floatational plasma skimming and progressive increase in local tube hematocrit (HT) up to 100% ("compaction stasis") occurs during induced low flow states in vivo (here preferentially in postcapillary venules), as well as in vitro (non-permeable artificial micro tube networks). Quantitative densitometry and velocimetry in vertically placed microvessels demonstrates that the process of RCA sedimentation results in progressive vertical skewing of the velocity profile, culminating in standstill of the RBC-sediment in the dependent vessel half, with superfluent plasma and small aggregates in the upper vessel half. The theory of compaction stasis is developed: in striking contrast to the situation under high shear conditions, red cells travel on slower trajectories than plasma: due to "red cell undervelocity" the average residence times of RBC in a venule is much higher than that of plasma. Consequently, CS can be explained as the result of a FAHRAEUS effect reversal since the principle of mass conservation requires that HT much greater than HD. Network aspects and hemodynamic consequences are also incorporated into the theory.     

Substance P induces degranulation of mast cells and leukocyte adhesion to venular endothelium

Substance P (SP), one of the established neurotransmitters, evokes an immunoinflammatory response involving leukocyte adhesion to venular endothelium and the degranulation of mast cells. The pathogenetic relationship between these responses, however, remains unresolved. In this study, we propose to examine the changes associated with the activation of mast cells, as well as leukocyte adhesion to venular endothelium by in vivo observation of the rat mesentery. The use of an in vitro assay for intracellular Ca²⁺ mobilization and the degranulation of mast cells demonstrated the significant upper shift of concentration response to SP (10⁻⁴–10⁻⁵ M). In vivo experiments on the mesenteric microcirculation also showed that SP induced a significant increase in the number of degranulated mast cells as well as in the number of leukocytes adherent to the venular wall. Tranilast, a mast cell stabilizer, as well as SP antagonist (CP-96,345) significantly attenuated the extent of mast cell degranulation and leukocyte adhesion elicited by SP. Although an immunoneutralization against CD18 by WT-3 significantly attenuated the leukocyte adhesion, it had no influence on the mast cell degranulation after SP superfusion. These separate in vivo observations show that SP induces leukocyte adhesion to the venular endothelium, possibly through the degranulation of mast cells.     

The importance of mast cells for the neutrophil influx in immune complex-induced peritonitis in mice

The role of mast cells in polymorphonuclear leukocyte (PMN) influx in Ag-antibody complex-induced peritonitis was evaluated in mast cell-deficient WBB6F1-W/Wv (W/Wv) mice and their normal littermates, WBB6F1-+/+ (+/+). Peritoneal cell influx was evaluated after i.p. injection of preformed immune complexes. The first significant elevation in the PMN count over PBS-treated controls in +/+ mice was observed 2 h after stimulation. During the period of maximum leukocyte concentrations (6 to 10 h), the increase in total cell count was 5-fold and in PMN 25-fold. In W/Wv mice the PMN influx started 2 h later than in the +/+ mice, and the maximum response (8 to 10 h) was only 50% of that in controls. Reconstitution of mast cells in W/Wv mice for 2 wk or more restored the PMN response to immune complexes. Mast cell release due to AG-antibody complexes was evaluated by measuring fluorescence intensity after berberine sulfate staining for heparin in mast cells from unstimulated as well as stimulated +/+ mice. There was a significant decrease in fluorescence intensity as early as 15 min after stimulation. By 30 min the fluorescence intensity had declined by 65%. This indicates extensive mast cell release that started before PMN mobilization. These experiments demonstrate that mast cells make a significant contribution to immune complex-induced inflammation.     

Propagation properties of vasomotion at terminal arterioles and precapillaries in the rabbit mesentery

A vasomotion activity in the mesentery of anesthetized rabbits were studied by simultaneous measurements of inside diameters at multiple sites in arterioles, precapillaries and their bifurcations. A frame-by-frame diameter determination technique was used with a microcomputer-assisted laser video disk recorder and video-image analysis system. Simultaneous intensity profiles across microvessels were continuously obtained. Applying an automatic wall surface tracer and a graphic editor to construct temporal sequences of intensity profiles, we obtained digitized data of inside diameters of microvessels, and implemented cross-correlation analysis between data sets to calculate phase differences of vasomotion at separated sites. The present analysis of the propagation of vasomotion showed that the vasomotion originated from the orifice of precapillaries at the bifurcations, spreading downstream in the precapillary. The vasomotion wave spreads both upstream and downstream along single arterioles from various origins of the vasomotion activity. The propagation velocity of vasomotion was 0.17 +/- 0.03 mm/sec (n = 19), and it became significantly slower through the branching points than along the arterioles. It is suggested that the vasomotion in terminal arterioles and precapillaries may spread through some mechanical transmission factors.     

Quantification of the Blood Platelet Reactivity in the ADP-Induced Model of Non-Lethal Pulmonary Thromboembolism in Mice with the Use of Laser Doppler Flowmetry

IntroductionThe paper describes an alternative method for quantification of in vivo ADP-induced thromboembolism. The aim of the studies was to develop a method of quantification which would not require either extravasation or labelling of platelets. Our proposed approach is based on the monitoring of changes of blood flow with the use of laser Doppler flowmetry.ResultsThe injection of ADP resulted in a dose-dependent reduction of the blood flow in the mesentery. These responses were fully attributable to blood platelet aggregation, as shown by the lack of the effect in platelet-depleted mice, and significantly reduced responses in mice pretreated with cangrelor and eptifibatide. No platelet aggregate formation in mesenteric vessels was revealed by intravital microscopy, while ex vivo imaging showed accumulation of fluorescent labelled platelets in the lung.ConclusionsInjection of ADP to the venous system results in the formation of platelet aggregates predominantly in the lung. This results in reversible blood flow cessation in peripheral blood vessels. The measurement of this blood flow cessation in the mesentery allows indirect measurement of ADP-induced pulmonary thromboembolism. We suggest that this approach can be useful for in vivo screening for antiplatelet drug candidates.