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Real Time In Vivo Imaging of Platelets During Thrombus Formation

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  • Intelligent Imaging Innovations
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... We have recently described a method to monitor in vivo calcium mobilization in fura-2-loaded platelets participating in the development of a thrombus (36,37). This method was used to compare in vivo platelet activation in BSDL-null mice and wild-type mice. ...
... Fura-2-loaded platelets (1 × 10 8 platelets/ml) were preincubated and stirred in a cuvette in a Perkin Elmer LS45 Fluorescence Spectrometer for 1 minute at 37°C with 2 mM of CaCl2; then 0.1 U of thrombin, 0.1 μg of V3-like loop peptide, or 0.5 μg of hBSDL was added and the fluorescence change recorded. The fluorescence emission following excitation at 340 nm and 380 nm was recorded at 510 nm (36,46). ...
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Bile salt-dependent lipase (BSDL) is an enzyme involved in the duodenal hydrolysis and absorption of cholesteryl esters. Although some BSDL is transported to blood, the role of circulating BSDL is unknown. Here, we demonstrate that BSDL is stored in platelets and released upon platelet activation. Because BSDL contains a region that is structurally homologous to the V3 loop of HIV-1, which binds to CXC chemokine receptor 4 (CXCR4), we hypothesized that BSDL might bind to CXCR4 present on platelets. In human platelets in vitro, both BSDL and a peptide corresponding to its V3-like loop induced calcium mobilization and enhanced thrombin-mediated platelet aggregation, spreading, and activated alpha(IIb)beta(3) levels. These effects were abolished by CXCR4 inhibition. BSDL also increased the production of prostacyclin by human endothelial cells. In a mouse thrombosis model, BSDL accumulated at sites of vessel wall injury. When CXCR4 was antagonized, the accumulation of BSDL was inhibited and thrombus size was reduced. In BSDL(-/-) mice, calcium mobilization in platelets and thrombus formation were attenuated and tail bleeding times were increased in comparison with those of wild-type mice. We conclude that BSDL plays a role in optimal platelet activation and thrombus formation by interacting with CXCR4 on platelets.
... Fluorescence data were captured digitally at up to 50 frames per second and analyzed as previously described (27). The kinetics of thrombus formation were analyzed by determining median fluorescence values over time in approximately 30 thrombi (30). ...
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Adhesion of platelets to an injured vessel wall and platelet activation are critical events in the formation of a thrombus. Of the agonists involved in platelet activation, thrombin, collagen, and vWF are known to induce in vitro calcium mobilization in platelets. Using a calcium-sensitive fluorochrome and digital multichannel intravital microscopy to image unstimulated and stimulated platelets, calcium mobilization was monitored as a reporter of platelet activation (as distinct from platelet accumulation) during thrombus formation in live mice. In the absence of vWF, platelet activation was normal, but platelet adherence and aggregation were attenuated during thrombus formation following laser-induced injury in the cremaster muscle microcirculation. In WT mice treated with lepirudin, platelet activation was blocked, and platelet adherence and aggregation were inhibited. The kinetics of platelet activation and platelet accumulation were similar in FcRgamma(-/-) mice lacking glycoprotein VI (GPVI), GPVI-depleted mice, and WT mice. Our results indicate that the tissue factor-mediated pathway of thrombin generation, but not the collagen-induced GPVI-mediated pathway, is the major pathway leading to platelet activation after laser-induced injury under the conditions employed. In the tissue factor-mediated pathway, vWF plays a role in platelet accumulation during thrombus formation but is not required for platelet activation in vivo.
... Finally, for each frame, the integrated fluorescence intensity was calculated as per following equation: integrated fluorescence intensity = sum Intensity of signal -(mean of the maximal background intensity × area of the signal). This calculation was performed for all frames in each thrombus and plotted versus time to provide the kinetics of thrombus formation (8,27,48,49). For multiple fluorescence channels, calculations of background were made independently for each channel. ...
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Thrombosis, or blood clot formation, and its sequelae remain a leading cause of morbidity and mortality, and recurrent thrombosis is common despite current optimal therapy. Protein disulfide isomerase (PDI) is an oxidoreductase that has recently been shown to participate in thrombus formation. While currently available antithrombotic agents inhibit either platelet aggregation or fibrin generation, inhibition of secreted PDI blocks the earliest stages of thrombus formation, suppressing both pathways. Here, we explored extracellular PDI as an alternative target of antithrombotic therapy. A high-throughput screen identified quercetin-3-rutinoside as an inhibitor of PDI reductase activity in vitro. Inhibition of PDI was selective, as quercetin-3-rutinoside failed to inhibit the reductase activity of several other thiol isomerases found in the vasculature. Cellular assays showed that quercetin-3-rutinoside inhibited aggregation of human and mouse platelets and endothelial cell-mediated fibrin generation in human endothelial cells. Using intravital microscopy in mice, we demonstrated that quercetin-3-rutinoside blocks thrombus formation in vivo by inhibiting PDI. Infusion of recombinant PDI reversed the antithrombotic effect of quercetin-3-rutinoside. Thus, PDI is a viable target for small molecule inhibition of thrombus formation, and its inhibition may prove to be a useful adjunct in refractory thrombotic diseases that are not controlled with conventional antithrombotic agents.
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Quercetin-3-rutinoside inhibits thrombus formation in a mouse model by inhibiting extracellular protein disulfide isomerase (PDI), an enzyme required for platelet thrombus formation and fibrin generation. Prior studies have identified PDI as a potential target for novel antithrombotic agents. Using a fluorescence enhancement-based assay and isothermal calorimetry, we show that quercetin-3-rutinoside directly binds to the b' domain of PDI with a 1:1 stoichiometry. The binding of quercetin-3-rutinoside to PDI induces a more compact conformation and restricts the conformational flexibility of PDI, as revealed by small angle xray scattering. The binding sites of quercetin-3-rutinoside to PDI were determined by studying its interaction with isolated fragments of PDI. Quercetin-3-rutinoside binds to the b'x domain of PDI. The infusion of the b'x fragment of PDI rescued thrombus formation that was inhibited by quercetin-3-rutinoside in a mouse thrombosis model. This b'x fragment does not possess reductase activity and, in the absence of quercetin-3-rutinoside, does not affect thrombus formation in vivo. The isolated b' domain of PDI has potential as an antidote to reverse the antithrombotic effect of quercetin-3-rutinoside by binding and neutralizing quercetin-3-rutinoside. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
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Key Points This is the first report to describe the influence of BAMBI on both hemostasis and thrombus stability. BAMBI present in the blood vessel wall (most likely the endothelium) rather than platelet BAMBI is required for thrombus stability.
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A new in vivo method to study the size and dynamics of a growing mural thrombus was set up in the rat femoral vein. The method uses a standardized crush injury to induce a thrombus, and a newly developed transilluminator combined with digital analysis of video recordings. Thrombi in this model formed rapidly, reaching a maximum size 391 ± 35 sec following injury, after which they degraded with a half-life of 197 ± 31 sec. Histological examination indicated that the thrombi consisted mainly of platelets. The quantitative nature of the transillumination technique was demonstrated by simultaneous measurement of the incorporation of 111In labeled platelets into the thrombus. Thrombus formation, studied at 30 min interval in both femoral veins, showed satisfactory reproducibility overall and within a given animal With this method we were able to induce a thrombus using a clinically relevant injury and to monitor continuously and reproducibly the kinetics of thrombus formation in a vessel of clinically and surgically relevant size
Article
The purposes of these studies were to produce a small animal model of arterial thrombosis for study of novel antithrombotic agents, to validate a simple temperature index of occlusive thrombosis, and to describe the composition of the thrombus. Small thermocouple transducers were fabricated from readily available materials. A thermocouple was inserted under a carotid artery of the anesthetized rat and vessel temperature was recorded continuously. Arterial injury was induced by FeCl3 solution applied topically to the artery above the thermocouple. To validate the relationship between thrombotic occlusion and vessel temperature, blood flow velocity, proximal to the injury, and temperature were recorded simultaneously. Temperature decreased rapidly when velocity averaged 24 +/- 12 percent of control and velocity did not differ from zero within 20 sec. In normal vessels, average flow velocity did not decrease significantly from control until a fixed stenosis decreased diameter by 78 percent. Average time to occlusion (TTO), signaled by the abrupt temperature inflection, ranged from 56 +/- 4 min to 14 +/- 1 min after 10 and 65 percent FeCl3 application respectively. Vessel segments were fixed at various times after FeCl3 exposure and examined by scanning electron microscopy. Endothelial damage was observed and was associated with thrombus composed of activated platelets, fibrin strands and entrapped erythrocytes. The results demonstrate that FeCl3 dose-dependently induced formation of an occlusive mixed thrombus that was indexed by monitoring the time between FeCl3 application and a rapid temperature decrease in the carotid artery of the rat.
Article
The antithrombotic effects of the xanthine derivative propentofylline (HWA 285) were investigated in the laser-induced thrombus model in healthy and diseased rat mesenteric arterioles. Laser-induced platelet thrombi were documented by the help of an intravital microscope and a video system. The statistical comparison of drug-treated and untreated animals, in respect to the number of laser shots necessary to induce a defined thrombus, served as a quantitative measure for a potential antithrombotic drug effect. Propentofylline and acetylsalicylic acid, in single dosages of 10 and 30 mg/kg orally, significantly increased the number of laser shots required to produce a defined thrombus. Moreover, the drug decreased the laser-induced thrombus formation in a chronic experiment in adjuvant arthritic rats. After oral administration of 30 mg/kg for 21 days, propentofylline and pentoxifylline reduced significantly the thrombus formation, whereas acetylsalicylic acid exerted no effect.
Article
This study demonstrates endothelium-dependent relaxation in the surface arterioles of the brain. A helium-neon laser was used to injure endothelium in situ following i.v. injection of Evans blue dye, which sensitizes the bed to the laser. Areas 18 or 36 micron in diameter were injured and no longer relaxed to either 1 ml of acetylcholine chloride or bradykinin triacetate, 80 micrograms/ml delivered for 60 seconds. Dilations to sodium nitroprusside (30 micrograms/ml) were unaffected. Normal responses to nitroprusside, plus electron microscopy, established that vascular smooth muscle was uninjured. Endothelium-dependent relaxation was impaired when only minor ultrastructural damage was present. Dilation was inhibited downstream and upstream as far as 80 micron from the center of the laser beam. This suggests a spread of endothelium injury around the site of laser impact. However, inhibition was somewhat more marked downstream than upstream, implying that a portion of the downstream response was dependent on a substance released from an upstream site. To date, very few studies have reported endothelium-dependent relaxation in vivo, especially in the microcirculation. The present study accomplishes this. Moreover, in contrast to in vitro observations of endothelium-dependent relaxation in large vessels, the in vivo elimination of endothelium-dependent relaxation in the microcirculation required neither removal of endothelium nor injury to large numbers of endothelium cells. Since endothelium-dependent relaxation in the microcirculation has now been demonstrated using three different techniques to injure endothelium, it is reasonable to conclude that the phenomenon is real.
Article
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.
Article
The laser-induced thrombus model in rat mesenteric arterioles and venules represents a reliable and reproducible in vivo method. It is suitable for basic investigations concerning factors involved in thrombus formation as well as for testing antithrombotic effects of drugs. The laser-induced thrombus formation depends significantly on the presence of ADP, as ADP-utilizing enzymes inhibit thrombosis in the animal model. The instrumental test set-up consists of a 4 W Argon laser (Spectra Physics, Mountain View, CA, USA), a ray adaptation and adjusting device (BTG, Munich, FRG), a microscope (ICM 405, Zeiss, Oberkochen, FRG), and a video system (Sony, Japan). RBC velocity data were recorded with the help of a modified dual-slit technique (acc. to Wayland and Johnson). Results were expressed as number of laser injuries necessary to produce a defined thrombus (minimum size: 1/4 of the vessel diameter) under constant conditions (effective capacity: 30 mW, exposure time: 1/5 sec). The number of laser lesions necessary to induce a defined thrombus decreased with an increase in arteriole diameter (10 to 20 micron) but increased again in larger arterioles and small arteries (greater than 25 micron). On the arteriolar side there are significant correlation coefficients between vessel diameter and RBC velocity (r = 0.69), vessel diameter and No. of laser injuries (r = 0.70), and RBC velocity and No. of laser injuries (r = 0.71). Due to relative low flow conditions in the venules, the number of laser injuries required to induce a defined thrombus does not significantly depend on the vessel diameter.
Article
Platelet aggregates form in arterial vessels following exposure of the vessels to a single pulse ruby-red laser beam. Usually, a small cluster of red blood cells occupies the center of the aggregate. It appears that endothelial damage is not required for the platelet aggregation, but that platelet activity is initiated by rupture of red blood cells, possibly through the release of ADP from the injured red cells. Also, no shape change is noted in platelets that adhere to the vessel wall or to each other.
Article
Microvascular injury was produced in rabbit mesentery and ear chamber preparations using a biolaser. Ultrastructural studies of the injury sites revealed damage to the endothelium which showed cytoplasmic swelling and flocculation. Complete destruction of the endothelium was not observed, indicating that subendothelial structures were not exposed to blood flow. Microvascular thrombi which occurred at the sites of injury were composed of platelets which in general were loosely packed and showed minor alterations in shape and internal structure. Erythrocytes appeared to be heavily damaged by the laser injury. Fibrin was not observed. It is concluded that ADP may be released from red blood cells, and possibly from other sources, as a result of the laser injury, and that the platelet thrombus formation is induced by ADP.
Article
A survey is made of the various approaches employed for the study of the microcirculation in striated muscle (cremaster) by in vivo microscopy in the rat, and the technique for opening the muscle pouch is described. When opened and extended the rat's cremaster, of 2.7 ± 0.4-cm diam and 175 ± 15 μm in thick ness, can be transilluminated in toto, and the branches of cremasteric artery and vein visualized to their final ramifications. It also allowed direct measurements of dynamic changes of lumen and wall of the small blood vessels to be made at high power and maximum resolution of the electrooptical image on the television screen. A platform microtensor device sustains the spread muscle on the stage of the microscope, and allows quantitative evaluation of microcirculatory variables to be made at known amounts of stretch of the host tissue. Radial spreading of the tissue with a stretch force equivalent to < 180 mg, alters neither size nor reactivity of muscular precapillary vessels, and no change of blood in capillary occurred from that seen in exposed, unopened cremaster. The method was applied to the mouse. Cremaster muscle in this rodent measured 1.7 ± 5 mm in diameter, and 98.5 ± 11.0 μm in thickness. The method for striated cremaster muscle, experimental set up and optics provide a clear field for observing the smallest blood vessels in this tissue, for over more than 2 hr, without significant changes in dynamic activity and flow characteristics.
Article
Physiologic and inflammatory leukocyte trafficking are controlled by adhesion molecules on leukocytes and endothelial cells. These adhesion molecules can be grouped into four major families: integrins, immunoglobulinlike molecules, selectins, and glycoproteins serving as selectin ligands. The recent advent of gene-targeting technology in embryonal stem cells has prompted the production of mutant mice that lack individual adhesion molecules or combinations thereof. Such gene-targeted mice permit studies into the roles of adhesion molecules in acute and chronic inflammation and of the regulation and interplay between different sets of adhesion receptors in vivo. Microcirculatory studies have been indispensable to our understanding of the importance of certain adhesion molecules for different steps of leukocyte recruitment in vivo. Targeting new genes and investigating mice with combined adhesion-molecule deficiencies will help to further clarify the molecular mechanisms of leukocyte trafficking in health and disease. This article reviews the insight gained through using adhesion molecule-deficient mice, emphasizing the role of microcirculatory studies in this research.
Article
LMWH (Fraxiparine), and NSAIDs (Aspirin, Feldene, Indocid and Profenid) injected together in doses, 1 mg/kg (Aspirin was used at 100 mg/kg), subcutaneously into rats 30 minutes before the thrombosis induction by LASER beams, increased the number of LASER beams required to induce platelet thrombus formation, decreased the number of emboli and reduced the duration of embolization, compared with control (p < or = 0.05). Of all the studied NSAIDs being injected either with LMWH or separately 30 minutes before the thrombosis stimulation by LASER only Aspirin appeared to potentiate the antithrombotic effect of Fraxiparine. Neither LMWH nor NSAIDs (except for Aspirin) at the dosages used modified aggregatory parameters compared with control. But it was observed the inhibition of platelet aggregation by the associations of Fraxiparine with Aspirin, Feldene or Profenid, tested in the whole blood 90 minutes after the drug injections.
Article
In the present study, we established an injury model of the mouse carotid artery. Complete removal of the endothelium was achieved with a flexible wire. A platelet monolayer covered the denuded surface, and damage to underlying medial smooth muscle cells (SMCs) was detected. Injection of [3H]thymidine was used to determine the replication index for medial SMCs, which was found to be 1.6% at 2 days after denudation and 9.8% at 5 days. SMCs were observed in the intima by day 8 (replication index, 66%), and by 2 weeks the intimal lesion had a similar cell content as the media. In most animals, repair of the endothelial lining was complete 3 weeks after injury. The present model will allow us to use transgenic animals to address questions relevant to vascular biology and atherosclerosis.
Article
We have developed a simplified technique for culturing human umbilical vein endothelial cells under shear flow conditions, using prefabricated glass microcapillary tubes ("microslides") with a well-defined rectangular cross section and good optical quality. These microslides have been incorporated into a controlled flow system for quantitative video-microscopic analysis of the adhesion of blood cells to endothelial cells. Microslides were pretreated with 3-aminopropyltriethoxy-silane, or gelatin, and then loaded with a suspension of endothelial cells. After the cells had settled and attached to the substrate, the microslides were inserted into a flow-based culture system. Medium was drawn through them at intervals or continuously until confluency was reached (approximately 24 hr). Cells were cultured at wall shear stresses over a range 0.06 to 2.2 Pa. For adhesion assays, the endothelialized microslides were attached to microscope slides, and suspensions of blood cells were drawn through at desired wall shear stresses (0.02-0.5 Pa). Adhesion of malarial-infected red blood cells and of neutrophilic granulocytes was quantitated by direct microscopic observation. The adhesive behavior of both cell types closely resembled that previously described by ourselves and others using flow chambers incorporating endothelial-coated glass coverslips. The use of microslides represents a significant simplification of methodology for endothelial cell growth and adhesion studies under flow conditions.
Article
1. Intravital microscopy of the rat mesentery was used to examine interactions between histamine and the chemoattractant leukotriene B4 (LTB4) with regard to leukocyte adhesion in postcapillary venules. 2. Topical administration of histamine caused a four fold potentiation of LTB4-induced leukocyte adhesion. 3. Histamine significantly increased the rolling leukocyte flux by 25%, and this effect of histamine on rolling was strictly blood flow-dependent, i.e. we found significant positive correlations between both blood flow and total leukocyte flux and between total and rolling leukocyte flux, while no changes in leukocyte rolling fraction or rolling velocity were observed. Furthermore, histamine caused a clear-cut increase in venular plasma protein leakage. 4. The platelet-activating factor (PAF) receptor antagonist WEB 2086, which effectively inhibited adhesion of leukocytes evoked by exogenous PAF, did not reduce the potentiating effect of histamine on LTB4-induced leukocyte adhesion. 5. The vasodilator acetylcholine (ACh) caused a moderate enhancement of LTB4 induced leukocyte adhesion in proportion to its blood flow-dependent 40% increase in rolling leukocyte flux. In contrast to histamine, ACh did not provoke vascular leakage of plasma proteins. 6. Taken together, our findings suggest that histamine plays an important pro-inflammatory role in tissues where leukocyte rolling is already present, by potentiating chemoattractant-induced firm leukocyte adhesion through a combination of microcirculatory changes such as increased rolling leukocyte flux and vascular permeability.
Article
Numerous clinically relevant animal models exist for thrombosis studies. Few of these are suitable for both arteries and veins. In this investigation, an established venous thrombosis model was adapted through minimal technical adjustments to allow also the study of arterial thrombosis. A standardized subintimal crush injury was performed to carotid arteries or femoral veins of hamsters. Thrombus volumes were then quantified by direct morphometric measurements from serial microscopic sections or by on-line image analysis of light intensity changes from transilluminated vessels. The platelet-rich mural thrombus, which was established within minutes of the trauma, disintegrated during the observation period. The life cycle of the thrombus was different in arteries and veins, but significant linear correlation (p < 0.01) was found in both types of vessel between thrombus volumes measured by the two techniques. The model can consequently be used for comparative in vivo thrombosis studies in small (approximately 1-mm) arteries and veins.
Article
The alteration of rheological blood properties as well as deterioration of vascular perfusion conditions and cell-cell interactions are major determinants of thrombus formation. Herein, we present an experimental model which allows for quantitative in vivo microscopic analysis of these determinants during both thrombus formation and vascular recanalisation. The model does not require surgical preparation procedures, and enables for repeated analysis of identical microvessels over time periods of days or months, respectively. After i.v. administration of FITC-dextran thrombus formation was induced photochemically by light exposure to individual arterioles and venules of the ear of ten anaesthetised hairless mice. In venules, epi-illumination induced rapid thrombus formation with first platelet deposition after 0.59 +/- 0.04 min and complete vessel occlusion within 7.48 +/- 1.31 min. After a 24-h time period, 75% of the thrombosed venules were found recanalised. Marked leukocyte-endothelial cell interaction in those venules indicated persistent endothelial cell activation and/or injury, even after an observation period of 7 days. In arterioles, epi-illumination provoked vasomotion, while thrombus formation was significantly (p <0.05) delayed with first platelet deposition after 2.32 +/- 0.22 min and complete vessel occlusion within 20.07 +/- 3.84 min. Strikingly, only one of the investigated arterioles was found recanalised after 24 h, which, however, did not show leukocyte-endothelial cell interaction. Heparin (300 U/kg, i.v.) effectively counteracted the process of thrombus formation in this model, including both first platelet deposition and vessel occlusion. We conclude that the model of the ear of the hairless mouse allows for distinct in vivo analysis of arteriolar and venular thrombus formation/recanalisation, and, thus, represents an interesting tool for the study of novel antithrombotic and thrombolytic strategies, respectively.
Article
This study was undertaken to evaluate in vivo the effect of recombinant hirudin (r-hirudin [HBW 023]), a potent thrombin inhibitor, on the process of microvascular thrombus formation and recanalization. Thrombosis was induced photochemically in distinct arterioles (n = 25) and venules (n = 30) of the ear of 16 hairless hr/hr mice (8 to 10 weeks old, 25 to 30 g of body weight). r-Hirudin (1 mg/kg of body weight) was administered intravenously directly before thrombus induction; saline-treated animals served as controls. Thrombus formation (i.e., first platelet deposition at the endothelial lining [FPD]; inner luminal diameter reduction to 50% [D/2]; complete vessel occlusion [CVO]), vessel recanalization, microcirculatory parameters, and leukocyte-endothelial cell interaction were analyzed by means of intravital fluorescence microscopy. Hirudin significantly delayed the process of thrombus formation compared with saline-treated controls in both arterioles (FPD: 381 +/- 80 vs 137 +/- 25 seconds, P < 0.05; D/2: 627 +/- 49 vs 501 +/- 71 seconds; CVO: 925 +/- 78 vs 854 +/- 60 seconds) and venules (FPD: 173 +/- 11 vs 59 +/- 4 seconds; D/2: 342 +/- 54 vs 228 +/- 27 seconds; CVO: 541 +/- 85 vs 344 +/- 43 seconds; P < 0.05). In addition, r-hirudin-treated animals showed an increased rate of vessel recanalization at 24 hours after thrombus induction (arterioles: 54% [7 of 13] vs 0% [0 of 12], P < 0.05; venules: 77% [10 of 13] vs 53% [9 of 17]), whereas microcirculatory parameters and leukocyte-endothelial cell interaction were not affected. Our data indicate that r-hirudin not only counteracts the process of thrombus formation but also promotes vessel recanalization, thus supporting its use in clinical microvascular surgery.
Article
The development of efficient transgenic technologies in mice has allowed the study of the consequences of genetic alterations on cardiovascular (patho)physiology, although the development of such models have been hampered by size limitation of species resulting in time-consuming, labor-intensive and costly analyses. This overview summarizes the murine models currently available for studying or manipulating angiogenesis, arterial stenosis, atherosclerosis, transplant arteriopathy, thrombosis, thrombolysis and bleeding and addresses techniques to evaluate vascular development during embryogenesis.
Article
The thrombotic risk associated with elevated plasma levels of clotting factor VIII (FVIII) was investigated in a mouse model of thrombophilia. After the intravenous injection of recombinant human FVIII and/or of purified FVIII-free human von Willebrand factor (vWF), a controlled mild injury was inflicted on the carotid artery of FVB mice by irradiation with filtered green light in combination with intravenous injection of the dye rose bengal. Formation of a platelet-rich thrombus was continuously monitored for 40 min via transillumination and the thrombus size was measured via image analysis. Administration of recombinant human FVIII at 40 microg/kg led to initial FVIII plasma activities equivalent to 250% of normal human plasma FVIII activity and significantly enhanced thrombus size. Immunohistochemical staining illustrated the accumulation of FVIII within the thrombi. Human vWF, even at 10 mg/kg, had no effect on thrombus formation. The thrombotic tendency induced by FVIII was significantly inhibited by the administration of human vWF in a dose-dependent manner. Separate plasma measurements revealed that human FVIII has comparable affinities for human and murine vWF but that human vWF does not effectively bind murine platelets. The inhibition by human vWF of the thrombotic tendency induced by human FVIII could therefore be explained by a lack of accumulation of FVIII within the developing thrombus because of the reduced affinity of human vWF for murine platelets and the reduced occupancy of murine von Willebrand factor by human FVIII after injection of human vWF. These results show that vWF actively participates in FVIII accumulation in the arterial thrombus and provide experimental evidence for epidemiological findings that elevated plasma FVIII levels are associated with an increased thrombotic risk, also in arteries.
Article
The role of fibrinolytic system components in thrombus formation and removal in vivo was investigated in groups of six mice deficient in urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), or plasminogen activator inhibitor-1 (PAI-1) (u-PA-/-, t-PA-/- or PAI-1-/-, respectively) or of their wild type controls (u-PA+/+, t-PA+/+ or PAI-1+/+). Thrombus was induced in the murine carotid artery by endothelial injury using the photochemical reaction between rose bengal and green light (540 nm). Blood flow was continuously monitored for 90 min on day 0 and for 20 min on days 1, 2 and 3. The times to occlusion after the initiation of endothelial injury in u-PA+/+, t-PA+/+ or PAI-1+/+ mice were 9.4+/-1.3, 9.8+/-1.1 or 9.7+/-1.6 min, respectively. u-PA-/- and t-PA-/- mice were indistinguishable from controls, whereas that of PAI-1-/- mice were significantly prolonged (1 8.4+/-3.7 min). Occlusion persisted for the initial 90 min observation period in 10 of 18 wild type mice and was followed by cyclic reflow and reocclusion in the remaining 8 mice. At day 1, persistent occlusion was observed in 1 wild type mouse, 8 mice had cyclic reflow and reocclusion and 9 mice had persistent reflow. At day 2, all injured arteries had persistent reflow. Persistent occlusion for 90 min on day 0 was observed in 3 u-PA-/-, in all t-PA-/- mice at day 1 and in 2 of the t-PA-/-mice at day 2 (p <0.01 versus wild type mice). Persistent patency was observed in all PAI-1-/- mice at day 1 and in 5 of the 6 u-PA-/- mice at day 2 (both p <0.05 versus wild type mice). In conclusion, t-PA increases the rate of clot lysis after endothelial injury, PAI-1 reduces the time to occlusion and delays clot lysis, whereas u-PA has little effect on thrombus formation and spontaneous lysis.
Article
Techniques of arterial injury commonly used in animals to mimic endovascular procedures are not suitable for small mouse arteries. This has limited examination of the response to arterial injury in genetically modified mice. We therefore sought to develop a model of transluminal injury to the mouse femoral artery that would be reproducible and result in substantial levels of intimal hyperplasia. Mice of the C57BL/6 strain underwent bilateral femoral artery denudation by passage of an angioplasty guidewire. Intimal hyperplasia was observed in 10% of injured arteries at 1 week, in 88% at 2 weeks, and in 90% at 4 weeks. The mean intimal-to-medial area ratio reached 1.1+/-0.1 at 4 weeks. No intimal proliferation was found in control sham-operated arteries. One hour after injury, the denuded surface was covered with platelets and leukocytes, predominantly neutrophils. This was associated with the accumulation of P-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. Expression of these adhesion molecules was not seen in the underlying medial smooth muscle cells. At 24 hours, few neutrophils remained on the denuded surface. At 1 week, macrophages and platelets were present in the vessel wall, partially covered by regenerated endothelium. Transluminal wire injury to the mouse femoral artery induces abundant intimal hyperplasia formation by 2 and 4 weeks and elicits the rapid accumulation of leukocytes and adhesion molecules on the denuded luminal surface. This model will be a valuable tool to study arterial injury in genetically modified mouse models.
Article
Vascular remodeling following arterial injury is characterized by an initial inflammatory reaction. Prior experiments using peritoneal inflammatory models have shown that the plasminogen system plays a role in the intensity of the inflammatory response. This study was undertaken to test the hypothesis that an absence of plasminogen would lead to a decrease in vascular remodeling. A left carotid artery injury was created with a flexible guidewire in both wild-type [Plg(+/+)] and plasminogen deficient [Plg(-/-)] mice. The right carotid artery was uninjured and used as a control. Three weeks postinjury, the mice were sacrificed and perfusion fixed, and the bilateral carotid arteries were sectioned for histological examination and collection of morphometric data. After arterial injury, electron microscopy of the acutely injured artery revealed that the endothelium was denuded, that there were breaks in the internal elastic membrane, and that there was disruption of the medial layer of smooth muscle cells. The intimal and medial areas were significantly increased between the uninjured and injured carotid arteries of both Plg(+/+) (+80% intimal, +41% medial, P < 0. 05) and Plg(-/-) [+48% intimal, +24% medial, P < 0.05) mice. However, although there was a significant increase in the adventitial area of Plg(+/+) mice (+18%, P < 0.05), there was no difference in Plg(-/-) mice (-6%). Interestingly, even after 3 weeks, four of six injured arteries in Plg(-/-) mice had persistent thrombus within the medial layer, whereas this was not found in any of the nine Plg(+/+) mouse arteries. Plasminogen deficiency inhibited the increase in adventitial area seen after injury in Plg(+/+) mice, but not the increase in intimal or medial areas. Not surprisingly, plasminogen-deficient mice also demonstrated a severe alteration in intramural thrombus clearance. Thus, specific aspects of the vascular remodeling response are dependent on plasminogen.
Article
ATX-S10(Na), a hydrophilic chlorin photosensitizer having an absorption maximum at 670 nm, is a candidate second-generation photosensitizer for use in photodynamic therapy (PDT) for cancer treatment. The effectiveness of PDT using ATX-S10(Na) and a diode laser for experimental tumors was evaluated in vitro and in vivo. In-vitro PDT using ATX-S10(Na) and the diode laser showed drug concentration-, laser dose- and drug exposure time-dependent cytotoxicity to various human and mouse tumor cell lines. In Meth-A sarcoma-implanted mice, optimal PDT conditions were found where tumors were completely eliminated without any toxicity. Against human tumor xenografts in nude mice, the combined use of 5 mg / kg ATX-S10(Na) and 200 J / cm(2) laser irradiation 3 h after ATX-S10(Na) administration showed excellent anti-tumor activity, and its efficacy was almost the same as that of PDT using 20 mg / kg porfimer sodium and a 100 J / cm(2) excimer dye laser 48 h after porfimer sodium injection. Microscopic observation of tumor tissues revealed that PDT using ATX-S10(Na) and the diode laser induced congestion, thrombus and degeneration of endothelial cells in tumor vessels, indicating that a vascular shutdown effect plays an important role in the anti-tumor activity of PDT using ATX-S10(Na) and the diode laser.
Article
Adhesion molecules of the selectin family (mainly P- and L-selectin) have been suggested to mediate interactions between platelets, leukocytes and endothelial cells in thrombus formation. The polysaccharide fucoidan has anticoagulative properties, but is also able to bind and block the function of the selectins. Here, we investigated in vivo (i) if fucoidan can prevent microvascular thrombus formation, and (ii) whether this is potentially mediated by the inhibition of P-and/or L-selectin. For this purpose, we used intravital microscopy in the mouse cremaster microcirculation in which thrombosis was induced photochemically by light exposure to individual arterioles and venules after intravenous (i.v.) injection of FITC-dextran. We found that intravenous administration of fucoidan significantly prolonged the time required for complete occlusion in arterioles and venules by almost seven- and nine-fold, respectively. In contrast, treatment with monoclonal antibodies against P- and L-selectin had no effect on the development of microvascular thrombosis. Fucoidan and also the anti-P-selectin antibody completely inhibited baseline venular leukocyte rolling in the cremaster muscle, indicating that these treatment regimes abolished P-selectin function. Importantly, fucoidan and the anti-P-selectin antibody had no effect on systemic platelet and leukocyte counts. On the other hand, we found that fucoidan treatment significantly altered coagulation parameters, including prothrombin time (Quick percentage), activated partial thromboplastin time (APTT) and thrombin clotting time (TCT), which may explain the potent in vivo anticoagulative effect of fucoidan observed here. Taken together, our novel findings suggest that fucoidan effectively prevents microvascular thrombus formation induced by endothelial damage in arterioles and venules in vivo. This protective effect of fucoidan is not attributable to inhibition of P- and L-selectin function but may instead be related to the anticoagulative capacity of fucoidan.
Article
A minimally invasive laser-induced injury model is described to study thrombus development in mice in vivo. The protocol involves focusing the beam of an argon-ion laser through a compound microscope on the vasculature of a mouse ear that is sufficiently thin such that blood flow can be visualized by intravital microscopy. Two distinct injury models have been established. The first involves direct laser illumination with a short, high-intensity pulse. In this case, thrombus formation is inhibited by the GPIIb/IIIa antagonist, G4120. However, the anticoagulants, hirulog, PPACK, and NapC2 have minimal effect. This indicates that thrombus development induced by this model mainly involves platelet interactions. The second model involves low-intensity laser illumination of mice injected with Rose Bengal dye to induce photochemical injury in the region of laser illumination. Thrombi generated by this latter procedure have a slower development and are inhibited by both anticoagulant and anti-platelet compounds.
Article
We have used confocal and widefield microscopy to image thrombus formation in real time in the microcirculation of a living mouse. This system provides high-speed, near-simultaneous acquisition of images of multiple fluorescent probes and of a brightfield channel. Vascular injury is induced with a laser focused through the microscope optics. We observed platelet deposition, tissue factor accumulation and fibrin generation after laser-induced endothelial injury in a single developing thrombus. The initiation of blood coagulation in vivo entailed the initial accumulation of tissue factor on the upstream and thrombus-vessel wall interface of the developing thrombus. Subsequently tissue factor was associated with the interior of the thrombus. Tissue factor was biologically active, and was associated with fibrin generation within the thrombus.
Article
We have developed novel instrumentation using confocal and widefield microscopy to image and analyze thrombus formation in real time in the microcirculation of a living mouse. This system provides high-speed, near-simultaneous acquisition of images of multiple fluorescent probes and a brightfield channel, and supports laser-induced injury through the microscope optics. Although this imaging facility requires interface of multiple hardware components, the primary challenge in vascular imaging is careful experimental design and interpretation. This system has been used to localize tissue factor during thrombus formation, to observe defects in thrombus assembly in genetically altered mice, to study the kinetics of platelet activation and P-selectin expression following vascular injury, to analyze leukocyte rolling on arterial thrombi, to generate three-dimensional models of thrombi, and to analyze the effect of antithrombotic agents in vivo.
Article
Platelet accumulation at sites of vascular injury is the primary event in arterial thrombosis. Initial platelet accrual into thrombi is mediated by interactions of platelet adhesion receptors with ligands on the injured endothelium or in the sub-endothelial matrix. The role of intracellular signals in initial platelet accumulation at sites of endothelial injury, however, is the subject of debate. We have used a newly discovered inhibitor of phosphodiesterase 3A (PDE3A) and the well-characterized PDE3A inhibitor, cilostazol, to modulate 3',5'-cyclic adenosine monophosphate (cAMP) levels in an in vivo model that enables the kinetic analysis of platelet accumulation. These studies demonstrate that elevation of basal cAMP levels results in an overall decline in platelet accumulation at the site of vascular injury. In particular, the initial rate of accumulation of platelets is inhibited by elevation of cAMP. Analysis of the kinetics of individual platelets at injury sites using intravital microscopy demonstrates that cAMP directs the rate at which platelets attach to and detach from thrombi. These studies demonstrate that cAMP in circulating platelets controls attachment to and detachment from sites of arteriolar injury. Thus, the status of the intracellular signaling machinery prior to engagement of platelet receptors influences the rate of platelet accumulation during thrombus formation.
Intravital microscopy is an extremely useful tool used as a qualitative and quantitative way of observing leukocyte-endothelial cell interactions in-vivo. This present article reviews the methods of the technique of intravital microscopy, in particular focussing on the mesentery and cremaster preparations. It focuses on how to actually carry out the experiments required to directly observe and localize the changes in the function of the microcirculation. Where necessary the reader is asked to refer to a selection of highly acclaimed publications, which should enable the reader to truly appreciate, and if necessary perform, the technique of intravital microscopy.
Article
Our understanding of hemorrhagic and thrombotic diseases has expanded with use of models aimed at studying the vasculature using a variety of different animals. Unlike in vitro experiments, these animal models enable the study of the broad continuum of biological consequences induced by alterations made to a single variable. This chapter briefly reviews animal models used in the study of thrombosis research, focusing primarily on the use of intravital fluorescence microscopy.
Article
Animal models are important tools in thrombosis research and preclinical drug development. Ferric chloride has been widely used to induce arterial thrombosis in a variety of species. However, almost all previous reports applied a very high concentration of ferric chloride (10-60%) to induce thrombus formation leading to difficulties in evaluating the efficacy of antithrombotic agents. Thus, the purpose of this study was to refine the ferric-chloride-induced thrombosis model to be better suited for thrombosis research. Dose-dependent study was used to identify a threshold concentration of ferric chloride sufficient for consistent occlusion (as reflected by the Doppler blood flow) of the carotid artery in C57BL/6 mice. Ferric chloride concentration at or about a threshold of 2.5% was found to be sensitive to anticoagulant (e.g., heparin) and antiplatelet (e.g., clopidogrel) agents. In contrast, the vessel rapidly occluded at or above 5% ferric chloride concentration despite pretreatment with the antithrombotic agents, even at doses that exerted maximal prolongation of tail bleeding time. Our study provides a simple, sensitive and highly controlled method for limiting vessel injury in mice to better detect the efficacy of antithrombotic drugs and/or evaluate therapeutic targets.
Article
Thrombus formation following vascular injury is an essential component of both hemostasis and pathologic vessel occlusion. This process occurs in a closed, pressurized environment in which blood flows rapidly over the injury site. Thrombus formation must occur quickly to reduce blood loss, but is carefully modulated to limit vessel occlusion. Circulating cells, plasma proteins, vessel wall components, and physical forces such as shear all influence thrombus formation. Historically, thrombus formation has been studied by isolating the separate components of blood involved in clot formation. With improved optical techniques, investigators have increasingly studied thrombus formation under conditions of flow in vitro and in live animals in vivo. Using multichannel fluorescence intravital videomicroscopy, the authors have studied the changes in the kinetics and deposition of platelets, fibrin, and tissue factor at the injury site during thrombosis in transgenic mice, bone marrow transplanted mice, and mice treated with pharmacological agents that modulate thrombosis. The differences in the kinetics of accumulation of the various components of thrombus in these mice have provided new insights about thrombus formation in arterioles. This review discusses the role of platelet intracellular signaling, P-selectin expression on platelets, and tissue factor-bearing microparticles in thrombus formation.
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Platelets are intimately involved in hemostasis and thrombosis. Under physiological conditions, circulating platelets do not interact with microvascular walls. However, in response to microvascular injury, platelet adhesion and subsequent thrombus formation may be observed in venules and arterioles in vivo. Numerous intravital video microscopy techniques have been described to induce and monitor the formation of microvascular thrombi. The mechanisms of microvascular injury vary widely among different models. Some models induce platelet activation with minimal effects on endothelium, others induce endothelial inflammation or injury, while other models lead to thrombus formation associated with endothelial denudation. The molecular mechanisms mediating platelet-vessel wall adhesive interactions differ among various models. In some instances, differences in responses between venules and arterioles are described that cannot be explained solely by hemodynamic factors. Several models for induction of microvascular thrombosis in vivo are outlined in this review, with a focus on the mechanisms of injury and thrombus formation, as well as on differences in responses between venules and arterioles. Recognizing these characteristics should help investigators select an appropriate model for studying microvascular thrombosis in vivo.