Terminal hepatic venule injury in liver biopsies of allogeneic haematopoietic stem cell recipients-a study of 63 cases.

AIMS: Subtle lesions of terminal hepatic venules (THVs) may be overlooked in liver biopsies from haematopoietic stem cell transplant (HSCT) receipients when graft-versus-host disease is the clinical concern. The aim of this study was to evaluate the frequency of THV injury resembling sinusoidal obstruction syndrome (SOS). METHODS AND RESULTS: Sixty-three consecutive biopsies from allogeneic HSCT recipients were scored for injured THVs. Forty-nine (78%) biopsies had injured THVs, and 10 (16%) were diagnosed with SOS (mean ± standard deviation of injured THVs/biopsy: 90 ± 9%). Biopsies diagnosed with other diseases also had injured THVs (36 ± 33%). Biopsies from patients with cyclophosphamide plus fractionated total body irradiation conditioning and biopsies taken within 100 days post-HSCT had significantly more occluded THVs (respectively: 40 ± 38%, P = 0.0188; and 35 ± 35%, P = 0.0076) than those with other conditioning regimens or in biopsies taken >100 days post-HSCT. All biopsies taken at any time in the 6-year post-HSCT period had similar amounts of THV phlebosclerosis (23 ± 25%). CONCLUSIONS: Our results demonstrate a high incidence of THV injuries resembling SOS in post-HSCT liver biopsies. THV injuries were detectable for several years post-HSCT, and were concurrent with other diagnoses. Our results also suggest that SOS may be underdiagnosed.

Quinic acid derivative KZ-41 exhibits radiomitigating activity in preclinical models of radiation injury.

Acute radiation syndrome is induced when a significant portion of the body receives high-dose, as well as high-dose rate, radiation. We have previously identified a quinic acid-based derivative, KZ-41, that protects from radiation injury. Further preclinical efficacy studies were conducted to determine the radiomitigating activity of KZ-41. C57BL/6 mice received total body irradiation (TBI-LD80/30, ¹³7Cs; ∼2 min) followed by either normal saline or KZ-41 (100 mg/kg sc ∼26 h post-TBI). KZ-41 increased 30-day survival by approximately 45% compared with vehicle controls (P < 0.05). To further investigate the potential radiomodulating mechanisms of KZ-41, we developed a combined radiation and vascular injury model. C57BL/6 mice surgically fixed with dorsal windows for dermal vasculature imaging received either sham or TBI (¹³7Cs; 6 Gray). Postcapillary venule injury was induced (24, 48, 72, and 96 h post-TBI) followed by imaging at 5 min and 24 h to assess clot formation and blood flow. Impairment in flow (P < 0.05) and clot formation (P < 0.05) were observed as early as 48 and 72 h, respectively. Thus, vascular injury 72 h post-TBI was used to evaluate intervention (KZ-41; 100 mg/kg i.p. at 12, 36, and 60 h post-TBI) on radiation-induced changes in both flow and clot formation. KZ-41, although not improving flow, increased clot formation (P < 0.05). Platelet counts were lower in both irradiated groups compared with sham controls (P < 0.05). In summary, KZ-41 exerts radiomitigating activity in lethally irradiated mice. Imaging results suggest KZ-41 exerts radiomitigating activity through mechanisms involving promotion of initial clot formation and vascular flow restoration. The imaging model described herein is useful for further examination of radiation-induced vascular injury repair mechanisms.

AT1 receptor blockade prevents microvascular dysfunction induced by ischemia/reperfusion injury.

Ischemia/reperfusion (I/R) in post-arterior post-capillary venules induces an acute inflammatory response, characterized by increased adherence and emigration of leukocytes and vascular permeability, all of which play important roles in cardiovascular disease. The aim of this study was to determine the roles of angiotensin II and AT1 receptor blockade in microvascular I/R injury in rats. Rats were anesthetized and intubated, then the peritoneum was opened and the mesentery was revealed. Small post-capillary venules were examined by in vivo fluorescence microscopy. The flow of erythrocytes and leukocytes was observed under the microscope and video recorded for later dynamic analyses. The superior mesenteric artery (SMA) was ligated with polyethylene tubing and released to induce I/R (20 min of ischemia/60 min of reperfusion). Subsequently, leukocyte adhesion, emigration and albumin leakage were compared with those of non-I/R controls. I/R injury was significantly suppressed by superfusing tissues with the AT1 receptor antagonist losartan (LO; 10 microM). The beneficial effects of LO were inhibited by topical application of either the bradykinin B2 receptor antagonist HOE140 (10 nM) or nitric oxide (NO) synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME 10 microM). The effects of LO were lost in the presence of AT2 receptor blocker PD 123319 (PD). In conclusion, LO suppressed and protected against I/R injuries. The possible interaction between AT1 and AT2 receptors was also suggested.

Sarpogrelate hydrochloride, an antagonist of 5-hydroxytryptamine receptor, improves skin flap survival and suppresses thrombus formation in injured microvessels of rabbits.

The authors carried out two studies to examine the effects of the 5-hydroxytryptamine receptor antagonist, sarpogrelate hydrochloride (SH), on flap necrosis. The first study measured survival rates and included histologic examination of random-pattern skin flaps in rabbits. The second study assessed the time to complete obstruction of blood flow with the artificial thrombus formation method, and the time for leukocytes to adhere to the endothelium of microvessels in a rabbit-ear chamber. The treatment groups were injected with SH. The survival rates of skin flaps in the SH-treated group were increased significantly. Histologic examination of vessels in the control group revealed that vessels in the deep dermis were obstructed completely by thrombi, whereas such vessels were not obstructed in the SH-treated group. Thrombus formation time and leukocyte adhesion time in the SH-treated group were prolonged and decreased, respectively. Thus, SH maintained flap circulation and promoted flap survival by preventing thrombus formation.

Microvascular consequences of thrombosis in small venules: an in vivo microscopic study using a novel model in the ear of the hairless mouse.

Little is known of the development of chronic microvascular alterations following small vessel thrombosis, which is probably due to the lack of appropriate experimental models. Herein we report the first results on thrombosis-associated long-term changes of microvascular permeability and vessel tortuosity and diameter and blood perfusion using the ear of the hairless mouse, and intravital fluorescence microscopy. Thrombosis was induced photochemically in small venules (diameter: 75 to 100 microm) using light/dye exposure (fluorescein isothiocyanate-dextran 150,000), and the microcirculation compromised by the blockade of blood drainage was analyzed before and 30 minutes after induction of thrombosis as well as repeatedly over a 28-day observation period. Thrombus formation resulted in a marked increase (p<0.05) of microvascular permeability (0.85+/-0.11) when compared with baseline values (0.46+/-0.04). Permeability remained elevated (p<0.05) at days 1 (0.67+/-0.07), 3 (0.58+/-0.02), and 7 (0.60+/-0.06), but returned to normal after 28 days (0.43+/-0.03). Tortuosity, diameter, and red blood cell velocity of venular segments, located upstream of thrombus formation, were found unchanged during the entire 28-day observation period. This was probably due to the fact that blood flow from the thrombosis-affected tissue was frequently drained into nonaffected tissue via preexisting "through-fare" channels, serving as venulo-venular collaterals. In accordance, in 10 to 20% of these venular segments the direction of blood perfusion was found changed, while those changes were only rarely observed in venular vessel segments of the nonthrombotic contralateral ears. We conclude that thrombosis in small cutaneous venules is primarily characterized by an increased vascular permeability, reflecting an inflammatory response, similar to what is known from thrombophlebitis in patients. The model presented herein may be a versatile tool to study pathogenesis of chronic microcirculatory derangements in microthrombosis and their prevention by novel therapeutic strategies.

Mechanisms of parenchymal cell death in-vivo after microvascular hemorrhage.

OBJECTIVE: In vitro studies suggest that microhemorrhages with escape of red cells into the tissue may be cytotoxic to parenchymal cells due to oxygen free radical formation. We examined in the rat mesentery the impact of microhemorrhages on parenchymal cell death, as detected by propidium iodide staining, using an intravital approach. METHODS AND RESULTS: Postcapillary venules were punctured with a closed-end micropipette, permitting escape of blood cells and plasma into the mesentery interstitium. Over a period of 2 h, no significant increase in parenchymal cell death was encountered in tissues with hemorrhagic sites compared with nonhemorrhagic control sites. Interstitial microinjections of plasma derived from whole blood incubated for several hours with and without a combination of sodium azide (2 mM) and hydrogen peroxide (1 mM) led to significantly increased levels of cell death compared to control experiments. Interventions against the hydroxyl radical with dimethylthiourea (DMTU, 2 mM) or 2,2'-dipyridyl (DPD, 2 mM), a lipid soluble iron chelator, provided no protective effect against the parenchymal cell death. DMTU slightly delayed tile cytotoxic reaction. CONCLUSIONS: These observations suggest that a newly formed microhemorrhage is not necessarily cytotoxic to parenchymal tissue cells. Interstitial microinjections of plasma, derived from whole blood after prolonged exposure to oxygen free radicals or just aging under in vitro conditions, may be cytotoxic to mesenteric parenchymal cells without effective blockade by interventions against the hydroxyl radical.

Laser fluence for permanent damage of cutaneous blood vessels.

Treatment of vascular disorders may be improved by a more thorough understanding of laser-blood vessel interaction. In this study, the probability of permanent damage to a given type and size of blood vessel was determined as a function of fluence at the top (superficial edge) of the vessel lumen. A 532 nm wavelength, 10 ms pulse duration, 3 mm spot size laser was used to perform approximately 250 irradiations of subdermal blood vessels in the hamster dorsal skin flap preparation. The radiant exposure required for a 50% probability of permanent damage was calculated using a probit analysis of experimental results. Threshold radiant exposure increased with larger blood vessel diameters and was greater for arterioles than venules. Monte Carlo modeling of a typical blood vessel geometry revealed that fluence at the top of the blood vessel lumen was amplified by a factor of approximately 2.4 over tissue surface radiant exposure, due to light scattering in the tissue and internal reflection at the skin-air interfaces.

Endogenous nitric oxide protects against thromboembolism in venules but not in arterioles.

Because nitric oxide (NO) inhibits aggregation and adhesion of blood platelets, NO may play a role in platelet-vessel wall interactions. Therefore, the purpose of this study was to investigate the involvement of endogenous NO in thromboembolic processes, as induced by wall puncture, in rabbit mesenteric arterioles and venules (diameters 20 to 43 microm). In venules, inhibition of NO synthase by superfusion of the mesentery with N omega-nitro-L-arginine (L-NA; 0.1 mmol/L) significantly increased the duration of embolization (from 50 seconds to 511 seconds) and the number of emboli produced (from 2 to 11 emboli per vessel), while the median period of time needed to produce an embolus was not influenced. On the contrary, in arterioles, L-NA had no significant effect on embolization (duration of embolization: 426 seconds in the control and 382 seconds in the L-NA group, with 20 and 12 emboli per vessel, respectively). Addition to the L-NA superfusate of L-arginine (L-ARG; 1 mmol/L), the active precursor for endogenous NO synthesis, resulted in a complete reversal of the L-NA effects in venules, while addition of the inactive D-arginine (D-ARG; 1 mmol/L) had no effect. Addition of L-ARG and D-ARG had no significant effect in arterioles. Addition to the L-NA superfusate of the exogenous NO donor sodium nitroprusside (0.1 micromol/L) also resulted in reversal of the L-NA effects in venules, while in arterioles, it slightly but significantly decreased embolization duration. The differences in effect of L-NA on embolization between arterioles and venules were not caused by differences in fluid dynamic conditions. It is concluded that the role of endogenous NO in inhibiting thromboembolic processes is more important in venules than in arterioles.

Thrombogenicity of ionic and nonionic contrast media tested in a laser induced rat thrombosis model.

Contrast media are used as substances for visualization of vascular system. But, their administration is often associated with thromboembolic complications. The purpose of this study is to evaluate the thrombogenic action of ionic and non-ionic contrast media on thrombus formation. The experimental destruction of endothelial cells by Laser injury leads to thrombus and emboli formation. Two ionic and two non-ionic contrast media were injected intravenously via penis vein and tested at various dosages (1.0 and 2.5 ml/kg) 5, 30, 45 and 65 minutes after injection. The administration of these contrast media decreases the number of Laser injuries required to induce thrombus formation, increases the number of emboli which detached from thrombus and prolongs duration of embolization (p < or = 0.05). These experimental results suggest that ionic and non-ionic contrast media induce thrombogenic effects. This thrombogenicity was the greatest for non-ionic contrast media. It was observed the decrease of the white cells, red cells and platelets.

Different roles of prostaglandins in thromboembolic processes in arterioles and venules in vivo.

The involvement of prostaglandins in thromboembolic processes, as induced by wall puncture, was studied in rabbit mesenteric arterioles and venules using intravital videomicroscopy. Inhibition of prostaglandin formation with aspirin (100 mg/kg, i.v.) significantly increased in arterioles duration of embolization (from 91 to 200 s) and number of emboli produced (from 4 to 8.5 per vessel), while rate of embolus production was not influenced. In venules, aspirin only influenced embolization rate (a significant decrease from one embolus/14 s to one/23 s). Specific blockade of TXA2-receptors by sulotroban (30 mg/kg, i.v.) only influenced the arteriolar reaction: it significantly decreased embolization duration (from 560 to 218 s) and number of emboli produced (from 23 to 10 emboli per vessel), without affecting embolization rate. These findings indicate that both platelet activating and inhibiting prostaglandins play a more important role in thromboembolism in arterioles than in venules; this suggests a difference in prostaglandin synthetic capacity between arteriolar and venular endothelium.

[Changes in the drainage system of the eye during hydromonitoring exposure]. / Izmeneniia drenazhnoi sistemy glaza pri gidromonitornom vozdeistvii.

A hydromonitoring exposure with a high-velocity pulsed liquid jet aimed into Schlemm's canal lumen and a similar exposure with a syringe at a 60 H pressure on piston were carried out in isolated eyes. Examinations of stained sections showed that hydromonitoring exposure at a pressure of 1 to 5 techn. atm. induced destruction of Schlemm's canal internal wall and cyclodialysis. If a higher pressure was used, corneal stratification was observed. Hydromonitoring exposure at a pressure of less than 1 techn. atm. or an exposure with a syringe induced deformation of Schlemm's canal internal wall, dilatation of intertrabecular fissures, and ruptures of individual trabecular layers.

Antithrombotic effect of argatroban on the pial vessels of the rat: a study with He-Ne laser-induced thrombus formation.

The antithrombotic effect of the synthetic thrombin inhibitor (2R,4R)-4-methyl-1-[N2-(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfon yl)-L-arginyl]-2-piperidinecarboxylic acid monohydrate (argatroban) was investigated in cerebral vessels of the rat. An occlusive thrombus was formed in pial vessels using a He-Ne laser in a closed cranial window technique. Argatroban retarded the formation of thrombi in a dose-dependent manner. The antithrombotic effect of a single intravenous dose of argatroban at 0.5 mg/kg was diminished after 30 min in arterioles and after 50 min in venules, respectively. The antithrombotic activity was maintained, however, by continuous intravenous infusion (2 mg/kg/h).

Changes in the microvasculature after mechanical pressure on the hamster cheek pouch.

The reaction of the microvasculature in the periodontal ligament to mechanical pressure is considered a very important phenomenon with respect to the biological background to orthodontic tooth movement. For clarification of the microvascular reaction to mechanical pressure, an experimental model that incorporated a hamster cheek pouch was established. This in vivo model solves some of the problems of other experimental models of the microvasculature of the periodontal ligament. Blood plasma permeation in this model was examined by means of a blue dye (pontamine sky blue), and histological observation was performed by light and transmission electron microscopy. Increased vascular permeability was observed within 30 min after removal of the mechanical stimulation. The threshold weight resulting in dye leakage was between 1 and 5 g applied for 60 min. From the histological observations, some large gaps between the endothelial cells in venules were found, while most basement membranes remained undisturbed. These observations suggest that the plasma leakage was due to mild traumatic injury to the endothelial cells. Both leukocytes and platelets were observed in the gaps between the endothelial cells in the venule. These types of microvascular reactions to mechanical pressure could initiate tissue changes in the periodontal ligament during orthodontic tooth movement.

Individual and combined effects of a thromboxane receptor antagonist and different antithrombotic agents in a rat microcirculatory thrombosis model.

The antithrombotic effect of a thromboxane A2 receptor antagonist (HN-11501:5-[2-(4-chlorophenylsulfonylamino)-ethyl]-2-thienylox y-acetic acid) alone and in combination with other antithrombotic agents has been studied in an experimental thrombosis model in which laser lesions are used to induce a defined thrombosis in rat mesenteric venules. The thromboxane receptor antagonist showed a significant and dose-dependent antithrombotic effect if given orally. The strongest additive thrombosis-inhibiting effect was observed after oral administration of HN-11501 at a dose of 2.5 mg/kg together with an intravenous infusion of 1 microgram/kg/h of a prostacyclin analogue (cicaprost). An additive antithrombotic effect was also observed after oral application of 2.5 mg/kg of HN-11501 and intravenous injection of 0.2 mg/kg of a low molecular weight heparin (Fraxiparine). The combination of 2.5 mg/kg of HN-11501 orally with an intravenous injection of 0.1 mg/kg molsidomine also had a significant additive effect. No significant additive effect was observed when 2.5 mg/kg of HN-11501 and 10 mg/kg of acetylsalicylic acid were orally administered simultaneously.

Thromboembolic reaction following wall puncture in arterioles and venules of the rabbit mesentery.

The walls of rabbit mesenteric arterioles and venules (diameter 20 to 40 microns) were punctured with glass micropipets (tip diameter 6 to 8 microns). Thromboembolic reactions resulting from this standardized, small mechanical vessel wall injury could be quantified in vivo with the use of intravital video-microscopy. Following induction of the injury thrombus growth started immediately (less than 0.1 s). Bleeding times were short, on the average less than 2 s, and did not differ between arterioles and venules. The duration of the embolization process was significantly longer in arterioles than in venules (median 101 and 17 s, respectively), and more emboli were produced in arterioles than in venules (median 6 and 1, respectively). Arteriolar thrombi were more effective in plugging the punctured holes than venular thrombi. The differences in thromboembolic reaction between arterioles and venules, as found in the present study, can probably not be explained by fluid dynamic factors.

[Age and features of thrombus formation in the rat]. / Vozrastnye osobennosti tromboobrazovaniia u krys.

With the aid of a laser-induced thrombosis model, the process of thrombus formation was studied in the microvessels of the small intestine mesentery of 3-4- and 12-14-month old rats. Venules and arterioles were laser-irradiated. The thrombus formation developed faster in both types of vessels in elder rats. The data obtained suggest an increase of the thrombotic potential of the mesentery microvessels in the course of ageing in rats.

Influence of ADP, blood flow velocity, and vessel diameter on the laser-induced thrombus formation.

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.

An ultrastructural analysis of endothelial change paralleling platelet aggregation in a light/dye model of microvascular insult.

Those microvascular endothelial events that parallel the evolution of platelet aggregation were evaluated in a well-controlled animal model. Cat pial microvessels were observed through a cranial window while local platelet aggregation was produced by intravenous injection of sodium fluorescein and simultaneous exposure of the pial vessels to light from a filtered mercury lamp that excited the fluorescein. The vessels were fixed in situ when the in vivo observations of a preselected vessel indicated early, intermediate, or advanced aggregation in that vessel. The preselected vessel was then harvested for ultrastructural study together with adjacent vessels from the illuminated field. These vessels and appropriate controls were compared in semiserial thin sections. The onset of platelet aggregation in both venules and arterioles was accompanied by focal endothelial lucency, vacuole formation, luminal membrane rupture, and swelling of the nuclear envelope. These changes were not found in control material. With intermediate aggregation these changes were more common, while with advanced aggregation these abnormalities occurred together with focal endothelial denudation. Thus, in this model denudation occurred only with advanced aggregation and was not a prerequisite for aggregation.

[Bradykinin, thrombin and prostaglandins as modulators of microthrombogenesis in local injury of the blood vessel wall]. / Bradikinin, trombin i prostaglandiny kak moduliatory protsessa mikrotromboobrazovaniia pri lokal'nom povrezhdenii sosudistoi stenki.

The microthrombogenesis parameters were studied after local laser injury to the rat mesenterial venule in the presence of infusion of bradykinin (20 micrograms/kg/min) and thrombin (10 Units/kg/min). Bradykinin diminished and thrombin, on the contrary, increased the hemostasis parameters, this effect being observed only during the first minutes after the drug infusion. Activation of the blood kallikrein-kinin system preceded the action of thrombin. It is suggested that apart from prostaglandins, the component of the Hageman factor system may play an important role in the control of microhemostasis in the injured vascular wall.