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Purpose: 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. Methods: 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. Results: 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. Conclusion: 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. (J Vasc Surg 1998;28:498-505.)

An intravital fluorescence microscopic method for measurement of changes in macromolecular permeability has been established in the mesenterial microcirculation of the rat. After exteriorization of the fat-free distal part of the ileal mesentery, a 1-hr period of stabilization was followed by the injection of FITC-labeled macromolecules. Five minutes later, histamine, leukotriene B₄, or leukotriene C₄ was topically applied to the tissue by means of a micromanipulator. Areas of 1 mm ² were videotaped with a SIT camera. The fluorescence intensity of these areas was measured by an analogous video image processing system and displayed as gray value histograms. The shift of the frequency of gray levels from lower to upper regions could be attributed to an increase in light intensity in the mesentery, indicating an increase in vessel wall permeability. The sites of action of histamine and leukotriene C₄ were very similar. Both mediators affected mainly the larger collecting venules. In contrast, leukotriene B₄ exerted its effect at postcapillary venules. Moreover, leukotriene B₄-induced extravasation was inhibited by superoxide dismutase, suggesting an involvement of oxygen radicals. The studies with histamine alone and with H₁- and H₂-antagonists demonstrated that histamine-induced extravasation in the rat mesentery was mediated by H₁-histamine receptors. The present study introduces an experimental model for the measurement of changes in macromolecular permeability, which is useful for studying mediator effects and their pharmacological inhibition in the microcirculation of the rat mesentery. Copyright 1995, 1999 Academic Press

Case reports are presented of two related patients suffering from a hereditary megaloblastoid hemolytic anemia which at the moment cannot be categorized into one of the well-known entities. The main characteristics of the disease consisted of constant jaundice, macrocytic normochromic anemia, marked hemolysis without a substantial decrease in osmotic resistance, increased iron turnover and hepatic hemosiderosis at a relatively young age. One patient had to undergo splenectomy due to hemolytic crises, the other one cholecystectomy due to gallstones.

Summary: It was demonstrated that the appearance of sickle-shaped erythrocytes can be induced by oxygenating the blood of European red deer (Cervus elaphus). The process was reversible. Blood cell counts with the exeption of a slight reduction in red cell number and protein fractions did not show any abnormal values. The blood, which was examined at least 18 h after sampling, exhibited considerable deviations of blood gas values. In addition, highly significant haemorheological changes due to the sickle-shaped and oxygenated erythrocytes were detected.

Objective and Design:To study the effects of tiaprofenic acid and doxycycline on urinary pyridinium crosslinks and paw swelling in adjuvant arthritic rats, and to gain additional information on the drugs' inhibitory potential vs. in vitro targets, such as enzyme activity of matrix metalloproteinases and cytokine generation.¶Material:124 male Wistar Lewis rats; for the in vitro studies human matrix metalloproteinases and human mononuclear cells were used.¶Treatment:Arthritis was induced by injection of complete Freund adjuvant. Drugs (2, 15, 50 mg tiaprofenic acid/kg; 5, 15, 30 mg doxycycline/kg) were administered daily p.o. until day 21. In the in vitro studies 10-1000 mmoles/l of these drugs were used.¶Methods:Urinary levels of pyridinoline and deoxypyridinoline, determined by HPLC/fluorescence, and paw volumes were the measurements in the rat study. In the in vitro studies enzyme activities were assessed using fluorogenic peptide substrates; cytokines were determined by ELISA.¶Results:On day 21 of disease crosslink excretion was about twofold higher compared to the healthy controls. After administering daily 15 or 30 mg/kg tiaprofenic acid p.o. this increase was almost completely prevented whereas the paw volumes were suppressed by about 50%. Up to 50 mg/kg doxycycline did not display significant suppressive effects on crosslinks and paw volumes. In vitro 50-100 μmol/l of both drugs inhibited the activities of selected metalloproteinases, but only doxycycline suppressed the generation of IL-1β/TNFα in human mononuclear cells, whereas tiaprofenic acid was virtually inactive in that model.¶Conclusions:In arthritic rats tiaprofenic acid has not only the capability to suppress paw inflammation, but also to prevent with high potency the excretion of pyridinium crosslinks. Doxycycline without inherent antiinflammatory activity does not exhibit such preserving effects on collagen degradation in this model. Thus the mode of action of cartilage protecting drugs within the complex pathogenesis of arthritis will need further elucidation.

Beside plasma viscosity and the functional state of the vessel wall, placental perfusion is determined by the concentration and behaviour of the blood cells. In order to evaluate the contribution of erythrocytes to placental flow in late pregnancy, we applied a new filtration technique allowing to analyze the passages of erythrocytes through a single pore. As a parameter of decreased deformability in late pregnancy, we found a significant increase in transit time (22.69 +/− 1.60 ms vs 24.03 +/− 2.40 ms; pore size 4.1 μm; hydrostatic pressure 100 Pa) correlating with the mean cell volume (r=0.763). Due to their size and rigidity, their property to adhere to altered endothelium and to release radicals and mediators, granulocytes were the second point of interest to investigate in late pregnancy. Granulocytes were more adherent in vitro when stimulated with PMA, FMLP or in the absence of these mediators. This was shown for BSA- and fibronectin- coated wells (BSA: PMA 72.6% vs 67.2%, FMLP 43.4% vs 36.1%, unstimulated 26.5% vs 15.4%; fibronectin: PMA 71.6% vs 66.7%, FMLP 44.7% vs 31.1%, unstimulated 25.7% vs 18.2%). Transit times of granulocytes through 8 μm pores were significantly shorter in the pregnant group at 400 Pa (5.02 +/− 0.50 ms vs 5.49 +/− 0.74 ms), nearly identical at 200 Pa (9.34 +/− 1.79 ms vs 9.57 +/− 1.65 ms) and impossible to measure at 100 Pa (frequent obstruction). Luminol-enhanced whole blood chemiluminescence of unstimulated granulocytes was decreased in the pregnant group (0.19 counts/cell/2s vs 0.33 counts/cell/2s) but exceeded values of the control group when stimulated with PMA or FMLP (PMA: 20.13 counts/cell/2s vs 15.90 counts/cell/2s; FMLP: 2.35 counts/cell/2s vs 1.94 counts/cell/2s). Our results may indicate that in times of insufficient placental flow, as it is seen in hypertension-related gestational disorders, a decreased erythrocyte deformability as well as an enhanced adhesivity and chemiluminescence of activated granulocytes may perpetuate and aggravate placental malperfusion.

For the determination of the ability of erythrocytes to deform but not to flow in the microcirculation, the method proposed primarily by Braasch [1] was essentially improved. A measuring device was constructed with which it is possible to produce a histogram of the passage times of a population of erythrocytes through a narrow pore in a membrane. With this apparatus we are able to quantify even discreet changes of the deformability. With this method, we measured the pore passage time of normal red blood cells and cells which are treated with Ca++. A part of the erythrocytes damaged through Ca++ were treated with pentoxifylline to quantify the improvement of deformability. For comparing with the data cited above, the deformability of patients with acute cerebral insult was determined.

SummaryThe 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.

Pentoxifylline (PTX) has been shown to combat effectively endotoxin induced symptoms of shock or inflammation by reducing both leukocyte activation and endogenous cytokine formation. With regard to blood perfusion, inflammation is defined as a local reaction to injury of the living microvasculature and its content. Leukocyte margination, rolling, adhesion, and emigration is mediated by adhesion molecules along the endothelium of postcapillary venules and is considered to be an important step in the inflammatory response. Changes in the vascular integrity can be estimated in terms of increased extravasation of macromolecules. Using intravital microscopy with the help of an analogous video image processing system we measured the effect of PTX on lipopolysaccharide (LPS, 15 mg/kg i.v.) induced leukocyte adhesion and extravasation of FITC-rat serum albumin (FITC-RSA) in rat mesenteric venules. The changes in vascular permeability correlates significantly (r=0.75) with a locally increased number of adherent leukocytes. PTX significantly inhibits both leukocyte adhesion and extravasation of FITC-RSA dose dependently. Our results indicate that PTX effectively preserves vascular integrity in the microcirculation by acting primarily on LPS-induced leukocyte adhesion.

Employing the “Singlepore Erythrocyte Rigidometer” (SER) in which small pore diameters were used, significant differences were measured between red blood cells from adjuvant arthritic rats compared to control from non-diseased animals. The results changed to opposite values using higher pore diameters, which may be due to differences in cell “rigidity” and MCV.

Employing the “Singlepore Erythrocyte Rigidometer” (SER) in which small pore diameters were used, significant differences were measured between red blood cells from adjuvant arthritic rats compared to control from non-diseased animals. The results changed to opposite values using higher pore diameters, which may be due to differences in cell “rigidity” and MCV.

Summary The effect of pentoxifylline on the deformability of red cells in sickle cell thalassaemia was investigated. The fluidity of the blood in sickle cell thalassaemia is disturbed and is accompanied by violent pains and irreversible tissue damage caused by capillary occlusions. After treating a 15-years-old female patient with pentoxifylline (2 g orally each day), the fluidity of the blood improved distinctly, and this correlated with a condition free of clinical symptoms. Erythrocyte filtration by Nuclepore filter increased significantly over the 6-month examination period (initial value: ; after 6 months medication: 0.246±0.030). In addition, in the single-pore erythrocyte rigidometer (SER) a significantly improved passage time of individual erythrocytes could be demonstrated (initial value: 62.43±15.72 ms; after 4 weeks medication: 28.13±7.0 ms). The hitherto high number of rheological occlusions in the SER (48±30.9 from 200 individual passages) almost completely disappeared after treatment (1.7±1.2).

Endothelial cells of the arterial vascular system and the heart contain straight actin filament bundles, of which there are few, if any, in the venous endothelium1–4. Since stress fibre-containing endothelial cells within the vascular system tend to be located at sites exposed to particularly high shear stress of blood flow, we have investigated, in an experimental rheological system (Fig. 1), the response of the endothelial actin filament skeleton to controlled levels of fluid shear stress. Here we report that endothelial stress fibres can be induced by a 3-h exposure of confluent monolayer cultures of human vascular endothelium to a fluid shear stress of 2 dynes cm−2, approximately the stress occurring in human arteries in vivo. Fourfold lower levels of shear stress that normally occur only in veins, had no significant effect on the endothelial actin filament system. The formation of endothelial stress fibres in response to critical levels of fluid shear stress is probably a functionally important mechanism that protects the endothelium from hydrodynamic injury and detachment.

Summary In the present investigation the effect of pentoxifylline on single red cell deformability was determined. The deformability of human red blood cells were measured in a Singlepore Erythrocytes Rigidometer (SER). The new method determines optoelectronically the passage of each individual cell through a singlepore membrane (6 µm diameter and 50 µm length) under a driving pressure gradient (?p=100 Pa, t=3 Pa). Venous human blood samples were taken, prepared and divided into three samples. Sample A served as a physiological control, while to sample B and C CaCl2-solution was added. After Ca2+ stress sample C was incubated with 0.36 mmol/l pentoxifylline1. The medium passage time (MPT) for sample A was 43.3 ± 6.5 ms and for sample B 87.5 ± 14.7 ms. The addition of pentoxifylline to the Ca2+-treated red cell suspension (sample C) reduced the MPT to 51.6 ± 11.3 ms.