Post-mortem distribution of Iodinated Contrast Media (ICM) (iodixanol versus iopromide) in the porcine kidney after multiple bolus injections in vivo into the supra-renal aorta1.

 Iodinated contrast media (ICM) are widely used for diagnostic and interventional procedures in radiology and cardiology. Ideally, they should not interact with blood cells or vascular wall cells to avoid deteriorations of the blood circulation. However, it is well known that ICM can affect erythrocytes as well as endothelial cells which consequently might perturb especially the microcirculation. In former studies the influence of two ICM (iodixanol versus iopromide) on the vascular system, the development of blood stasis, on changes in renal resistive index (RRI) and vascular diameters, and on the post-mortem distribution of iodine as marker for ICM in the explanted kidneys was examined. The modus of ICM application into the supra-renal aorta followed the regime in interventional cardiology, so that 10 bolus injections were administered at steady intervals (iopromide 4,32 ml / iodixanol 5 ml) accompanied by infusion of 500 ml isotonic NaCl-solution.In the present study, the post-mortem X-ray analysis revealed that there were no differences in iodine content in the regions of the mid-cortex and the medullo-pelvic transition zone of the kidneys after application of both ICM. Remarkable differences, however, were found in the region of the capsule-near cortex, where the application of iopromide led to a significantly lower iodine content in the microcirculation. This is in good agreement with former studies, in which a maldistribution in this area, presumably due to a decrease in arteriolar inflow as a result of stasis/occlusion was shown.

Influence of polymeric microspheres on the myocardial oxygen partial pressure in the beating heart of pigs.

Injection of labeled microspheres is an established method in animal models to analyze the capillary organ blood flow at different time points. However, the microspheres can lead to stenoses of the capillary lumen, which might affect tissue oxygen supply. Our study aimed to investigate the influence of repeated injections of microspheres into the left coronary artery on the tissue oxygen partial pressure (pO(2)) in the downstream supplied myocardium of Göttingen minipigs. Tests (n=6 pigs each) were performed with two differently sized microspheres (ø=10 ± 0.1 µm (M10) or ø=15 ± 0.15 µm (M15)) from polystyrene. The pO(2) was measured in the midmyocardium of the left and right ventricle for 6 min continuously after each of five injections (1 × 10(6) microspheres each). There was a time laps of 12 min between each injection. In addition, the influence of the carrier solution was analyzed solely in the identical time frame. pO(2) decreased significantly in the myocardial area supplied by the ramus interventricularis paraconalis after injection of M15 microspheres. In contrast, the application of the M10 microspheres did not change the myocardial pO(2). This finding suggests to use microspheres with diameters not exceeding 10 µm for the coronary blood flow assessment.

COVID-19 and the endothelium.

There is growing evidence that COVID-19 not only affects the lungs but beyond that the endothelial system. Recent studies showed that this can lead to microcirculatory impairments and in consequence to functional disorders of all inner organs. The combination of endothelial dysfunction with a generalized inflammatory state and complement elements may together contribute to the overall pro-coagulative state described in COVID-19 patients leading to venular as well as to arteriolar occlusions.

Extreme reduction of the capillary lumen in segments of the venular legs of human cutaneous capillaries.

While structure and function of precapillary sphincter cells were assured in skin capillaries it is unclear whether segmental reduction of capillary lumina can occur in human capillaries. It has been shown that endothelial cells are able to exert dynamical reactions. Since the first description of the vascular endothelium a great variety of findings were described concerning the active role of capillary endothelial cells in regulation of the capillary lumen applying intravital microscopy. The intravital microscopy was performed in the framework of an observational study to document the long-term stability of capillaries in healthy subjects over many years. In the second year one of the participants showed remarkable changes in capillaries compared to recent recordings. Control recordings were performed 1, 3, 4, 5 and 20 h after the initial examination - until a complete normalization of the capillaries occurred. This case report is documenting for the first time clearly that extreme luminal narrowing of long segments of cutaneous capillaries can also appear in humans, in this case restricted exclusively to the venular leg of the capillaries. Different from the reductions of the capillary lumen induced by electrical irritation in frogs which lasted only for seconds, the capillary lumen narrowing in this case lasted considerably longer, almost over a whole day. It is important to note that the demonstrated findings did not occur in all capillaries and it remains unclear whether such findings are restricted to skin capillaries or might occur also in other regions of the body or even systemically. It could be demonstrated clearly, however, that segmental narrowing of capillary lumina can occur in humans possibly leading to a temporary stillstand of perfusion.

Detection of osteoarthritis using acoustic emission analysis.

Osteoarthritis (OA) of the knee is a widespread disease, often resulting in pain, restricted mobility and a reduction of activities and participation. Initial studies gave hints that Acoustic Emission Analysis (AEA) is capable of detecting early changes in cartilage structure. However, up to date no in vivo validation studies have been conducted. A prospective pilot study was conducted to investigate this diagnostic capability and the accuracy of the AEA, using magnetic resonance imaging (MRI) as a reference standard. Additionally, potential factors influencing false positive or negative results were studied. Twenty-eight patients, receiving MRI due to discomfort of the knee, were examined with AEA. Sensitivity was 0.92 for the whole knee and 0.86 to 1 for different parts of the knee. The specificity was 0.7 and 0.59 to 0.78, respectively. Confidence intervals varied between 0 and 0.33 for sensitivity and 0.1 and 0.24 for specificity. The diagnostic accuracy of the AEA was shown to be good to very good. However, because of the relatively small number of patients involved, interpretation of the data should be handled with care. Future studies with greater sample sizes have to be conducted to confirm the results of this investigation.

Evaluation of Laser-Doppler-Fluxmetry for the diagnosis of microcirculatory disorders.

BACKGROUND: The laser Doppler fluxmetry (LDF) is a non-invasive method to assess skin blood perfusion, measuring the flow of blood cells inside a tissue volume without harming the tissue. In the diagnosis of skin circulation disorders, the results of the LDF measurement are generally used in such a way that "normal" (or non-ill) or "pathological" values are achieved by comparison with a reference sample, for example of apparently healthy subjects. MATERIAL AND METHODS: In this study, the values of LDF for the diagnosis of microcirculatory disorders in patients with coronary artery disease (n = 20) or in patients with microcirculatory disorders, already diagnosed by capillary microscopy (n = 46), were examined. RESULTS: The mean values of LD amplitudes in the four frequency windows for patients with coronary artery disease were in the reference range. However, some of the patients showed reduced LD values: in eleven of the twenty patients, one or more mean LD amplitudes were below the reference range. Four of the eleven patients had pathologically decreased capillary erythrocyte velocities of very = 0.09-0.21 [mm/s], while the other seven patients had normal blood circulation at rest.For all patients with a proven cutaneous microcirculatory disorder, the mean LD amplitude in at least one of the frequency windows FF2 to FF4 was pathologically reduced. CONCLUSION: The Laser-Doppler fluxmetry method used in the study allows the reliable diagnosis of cutaneous microcirculatory disorders.

Effect of iodinated contrast media on the oxygen tension in the renal cortico-medullary region of pigs.

Repeated injections of iodinated contrast media (CM) can lead to a deterioration of the renal blood flow, can redistribute blood from the renal cortex to other parts of the kidney and can cause small decreases of the blood flow in cortical capillaries, a significant reduction in blood flow in peritubular capillaries and a significant reduction in blood flow in the vasa recta. Therefore, a study in pigs was designed, to show whether the repeated injection of CM boli, alone, can cause a reduction of oxygenation in the cortico-medullar renal tissue - the region with the highest oxygen demand in the kidney - of pigs.While the mean pO2-value had only decreased by 0.3 mmHg from 29.9±4.3 mmHg to 29.6±4.3 mmHg (p = 0.8799) after the tenth Iodixanol bolus, it decreased by 5.9 mmHg from 34.0±4.3 mmHg to 28.1±4.3 mmHg after the tenth Iopromide bolus (p = 0.044). This revealed a remarkable difference in the influence of these CM on the oxygen partial pressure in the kidney.Repeated applications of CM had a significant influence on the renal oxygen partial pressure. In line with earlier studies showing a redistribution of blood from the cortex to other renal areas, this study revealed that Iodixanol - in contrast to Iopromide - induced no changes in the pO2 in the cortico-medullar region which confirms that Iodixanol did not hinder the flow of blood through the renal micro-vessels. These results are in favor of a hypothesis from Brezis that a microcirculatory disorder might be the basis for the development of CI-AKI.

Characterization of cryopreserved CD14+-human monocytes after differentiation towards macrophages and stimulation with VEGF-A(165).

Monocytes are broadly discussed in the literature as cells, which can get properties of endothelial progenitor cells after angiogenic stimulation. Angiogenically stimulated monocytes can be used to promote implant vascularisation. A necessity therefore is that these cells can be stored and used after storage without a loose of their characteristic phenotype. In this study we tested, if freshly thawed cryopreserved human monocytes are positive for the mo/macrophage markers CD14 and CD68 and the endothelial marker CD31 after thawing and following angiogenic stimulation in a VEGF-A(165) enriched (10 ng/ml) angiogenic medium. Thereby the monocytes were tested before and after differentiation towards macrophages. The results revealed that freshly thawed human CD14 positive monocytes are positive for CD14, CD68 and CD31 after angiogenic stimulation. This CD specification was much more intense in the differentiated cells. The differentiation step also resulted in an increased cell count. Both results can be attributed to the method of differentiation, were cell culture bags were used instead of common cell culture dishes. Additionally the differentiation medium (X-VIVO 10+10% FCS) was specifically adapted to the requirements of monocytes/macrophages. The study showed that human CD14 positive monocytes can be thawed after cryopreservation without loss of their monocytes/macrophage phenotype and without loss of their ability to get angiogenically stimulated. To enhance the efficiency of both steps (thawing, angiogenic stimulation) it can be useful to differentiate the thawed cells in cell culture bags by the use of X-VIVO 10 (+10% FCS) before angiogenic stimulation.

Stimulation of monocytes and macrophages: possible influence of surface roughness.

The aim of this study was to understand the mechanisms of interaction of monocytes/macrophages and foreign body giant cell (FBGC) with implant materials, with respect to the roughness and the solubility of calcium phosphate based coatings. Anderson et al. (Bone Engineering, J.E. Davies, ed., Toronto, 2000, pp. 81-93) showed that the presence of FBGC's and monocytes/macrophages influenced the strength of the implant-tissue integration and that more monocytes/macrophages rested on smooth surfaces compared to rough surfaces. We seeded human bone marrow cells on uncoated ultrasmooth polished TiAl6V4 samples as well as on coated TiAl6V4 discs of the same diameter with two different calcium phosphates coatings, monetite (DCP) and hydroxyapatite (OHAp), both with rougher surfaces. On uncoated ultrasmooth polished TiAl6V4 discs (UUTi, diameter 16 mm, thickness 2 mm) and on TiAl6V4 discs of same diameter coated with OHAP or DCPA, human bone marrow cells (HMBC) were seeded and cultivated under standard culture conditions for 90 days without addition of inducing substances like ascorbic acid, Na-beta-glycerophosphate or dexamethasone. The roughnesses of the virgin samples were assessed with atomic force microscopy and light profilometry. After 90 days of cultivation a fraction of the samples, with cells and extracellular matrix, were stained with hematoxylin eosin (HE) and examined in light microscopy. R(a) roughness values of virgin uncoated TiAl6V4 samples were 0.001 microm, of DCP coated discs 4 microm and of OHAp coated discs 3 microm. The examination of HE stained samples showed a high number of FBGC and monocytes/macrophages on the UUTi samples. On the DCP coated samples there were less FBGC and monocytes/macrophages and on the OHAp coated samples we could not find any FBGC and monocytes/macrophages. The extracellular matrix (ECM) we found on the UUTi samples was finer and thinner than on the coated samples. The ECM was vastly spread and not dense on the UUTi samples in contrast to the calcium phosphate coated samples, where the ECM was much thicker and stronger. The ultrasmooth surface of the uncoated TiAl6V4 samples, a material which is accepted to be biocompatible, evidently induced the differentiation of cells of the monocytic lineage and the formation of FBGC out of the cell populations present in the human bone marrow.

Reversibility of echinocyte formation after contact of erythrocytes with various radiographic contrast media.

Various radiographic contrast media (RCM) significantly influence the morphology of erythrocytes, especially the formation of echinocytes [Scand. J. Clin. Lab. Invest. 35 (1975), 1-43; Microvasc. Res. 60 (2000), 193-200; Herz 23 (2003), 35-41]. Microscopic studies, however, have shown that these changes of erythrocyte morphology are possibly reversible [Acta Radiol. 37 (1996), 214-217]. The aim of this study was to proof if the RCM-induced echinocyte formation can be reversed by a resuspension in autologous plasma. In this study four RCMs were tested (Iodixanol, Iohexol, Iomeprol and Iopromide). These RCM induced echinocyte formation (after suspension of erythrocytes in plasma/RCM mixtures for 10 min at 37 degrees C), which was reversible after resuspension in autologous RCM-free plasma (resuspension time 5 min at 37 degrees C). Especially for Iomeprol and Iopromide - the RCMs which induced the strongest echinocyte formation - an echinocyte reduction from 94.2% to 44.5% and for Iopromide from 80.6% to 50.4% occurred. The echinocyte formation was influenced by the type of RCM as well as by the RCM concentration. The same was true for the reversibility of echinocyte formation due to resuspension in autologous plasma (type of RCM: p

The effect of radiographic contrast media on the morphology of human erythrocytes.

Echinocyte formation is associated with a rigidification of the cells that possibly affects capillary diffusion and, consequently, the tissue's oxygen supply. This study examines how many echinocytes appeared after the addition of various concentrations of radiographic contrast media (RCM) (Iodixanol 320, Iohexol 350, Iopromide 370, Iomeprol 350 and Iomeprol 400 mg Iodine/ml) compared to red blood cells in isotonic saline solution as well as in autologous plasma. Isotonic saline solution, Iodixanol, Iohexol, Iomeprol 350, Iomeprol 400 and Iopromide in concentrations of 10%, 20% or 40% were added to the plasma of six healthy subjects. Subsequently, the erythrocytes were resuspended in these RCM/plasma mixtures, incubated for 5 minutes at 37 degrees C and then examined under the microscope.The various mixtures and concentrations of the RCM in the mixture all had a significant effect on the number of discocytes (p<0.0001). The percentage of discocytes for all concentrations significantly depended on the RCM/plasma mixture (p=0.0097). Of all the RCM/plasma mixtures used as well as of the NaCl/plasma mixtures, the Iodixanol/plasma mixture showed the most similar discocyte fraction compared to red blood cells in the autologous plasma. At the same time, while Iodixanol in this respect differed from all other RCMs, the other RCMs only differed little from one another with respect to the discocyte fraction.

In vitro 3D assay to test angiogenic effects of human CD14+ monocytes seeded on macroporous PLGA/CaP polymers with a CaP nanostructured surface.

In this study we present a three-dimensional angiogenesis assay in vitro that allows the evaluation of the influence of Poly(lactic-co-glycolic acid) based implants seeded with VEGF-A165 stimulated/activated human CD14+ monocytes on the attraction and migration of human micro vascular endothelial cells (HMVEC-L). Primary HMEC of the capillary bed were cultured on an extracellular matrix generated by bovine corneal endothelial cells (BCEC). The HMEC layer was covered by an agarose gel, upon which a Poly(lactic-co-glycolic acid)/CaP polymer with a Calcium-Phosphate (CaP) nanostructured surface was placed. This scaffold has already been shown to interact with endothelial cells and endothelial progenitor cells respectively in vivo. It was seeded with angiogenically stimulated (VEGF-A165) human CD14+ monocytes, to get a monocyte/macrophage fraction, which can promote angiogenesis, tissue remodelling and tissue repair due to the secretion of growth factors, cytokines, chemokines and enzymes. The study demonstrated that this assay is suitable to test angiogenic effects by stimulated human CD14+ monocytes on human microvascular endothelial cells influenced by Poly(lactic-co-glycolic acid)/CaP scaffolds with a nanostructured CaP surface. The assay can exclude effects on migration caused by gravity and also allows testing in a physiological environment on an extracellular matrix secreted by endothelial cells.

Acute effects of Iodixanol on renal function after intra-arterial administration in patients with end-stage kidney disease.

BACKGROUND: Contrast-induced acute kidney injury (CI-AKI), a potentially life-threatening complication of iodinated contrast media in patients with impaired renal function, has attracted increasing attention in recent years. There is overwhelming evidence that the most important pre-disposing factor for a contrast-medium induced nephropathy is the pre-existence of a renal impairment. METHODS: The registry was performed as a part of a quality management project in the Dresden-Friedrichstadt heart catheter laboratory. In compliance with the Declaration of Helsinki/Somerset West, 9,026 patients were included between 2010 and 2015. 100 patients of these were participants in a chronic dialysis program. All patients were dialyzed on the day before angiography. In all patients a coronary angiography, in 28 patients a stent implantation and in 12 patients a surgical reconstruction had to be performed. Prior to the intervention and one, two and three days thereafter the serum creatinine was measured. RESULTS: Up to the third day after application of the iodinated contrast medium no significant changes of the serum creatinine (baseline value: 423.3±42.6µmol/l) occurred (ANOVA for repeated measures: p = 0.507). On average, a slight decrease of the serum creatinine was found.All patients remained in their routine dialysis-program. 18 out of 100 died during the next three months after the procedure. CONCLUSION: The study revealed that the coronary angiography using Iodixanol as iodinated contrast medium did not result in an increase of serum creatinine, which was drastically elevated in these patients before application of the iodinated contrast medium.

Albumin solder covalently bound to a polymer membrane: New approach to improve binding strength in laser tissue soldering in-vitro.

Laser tissue soldering (LTS) based on indocyanine green (ICG)-mediated heat-denaturation of proteins might be a promising alternative technique for micro-suturing, but up to now the problem of too weak shear strength of the solder welds in comparison to sutures is not solved. Earlier reports gave promising results showing that solder supported by carrier materials can enhance the cohesive strength of the liquid solder. In these studies, the solder was applied to the carriers by dip coating. Higher reliability of the connection between the solder and the carrier material is expected when the solder is bound covalently to the carrier material. In the present study a poly(ether imide) (PEI) membrane served as carrier material and ICG-supplemented albumin as solder substrate. The latter was covalently coupled to the carrier membrane under physiological conditions to prevent structural protein changes. As laser source a diode continuous-wave laser emitting at 808 nm with intensities between 250 mW and 1500 mW was utilized. The albumin functionalized carrier membrane was placed onto the tunica media of explanted pig thoracic aortae forming an overlapping area of approximately 0.5×0.5 cm2. All tests were performed in a dry state to prevent laser light absorption by water. Infrared spectroscopy, spectro-photometrical determination of the secondary and primary amine groups after acid orange II staining, contact angle measurements, and atomic force microscopy proved the successful functionalization of the PEI membrane with albumin. A laser power of 450 mW LTS could generate a membrane-blood vessel connection which was characterized by a shear strength of 0.08±0.002 MPa, corresponding to 15% of the tensile strength of the native blood vessel. Theoretically, an overlapping zone of 4.1 mm around the entire circumference of the blood vessel could have provided shear strength of the PEI membrane-blood vessel compound identical to the tensile strength of the native blood vessel. These in-vitro results confirmed the beneficial effects of solder reinforcement by carrier membranes, and suggest LTS with covalently bound solders on PEI substrates for further studies in animal models.

Influence of the surface structure of a multiblock copolymer on the cellular behavior of primary cell cultures of the upper aerodigestive tract in vitro.

The influence of the surface topography of a biodegradable copolymer on adhesion, proliferation, and cellular activity of primary cell cultures of the upper aerodigestive tract (ADT) was investigated. On the basis of the important functions of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitor of MMPs (TIMPs) in regulating extracellular matrix remodeling, cellular adhesion and growth, the appearance and kinetics of these enzymes were investigated in primary cells of the upper ADT seeded on different surfaces of a polymeric biomaterial. Primary cell cultures of the upper ADT of Sprague-Dawley rats were seeded on different surfaces (smooth versus rough surface) of a biodegradable multiblock copolymer and on polystyrene surface as control. Conditioned media of the primary cells were analyzed for MMPs and TIMPs by both zymography and radiometric enzyme assay. Cell adhesion and proliferation as well as the kinetics of appearance and activity level of MMP-1, MMP-2, and TIMPs were significantly different depending on the cell type and the surface structure of the multiblock copolymer. In this study, the data obtained indicated that surface topography governed the biological response to biomaterials. Knowledge as to how cells interact with the interface of biomaterials will be necessary in order to eventually design the "ideal" surface of biomaterials, which will be both tissue and organ-optimized in order to best provide clinicians with specific and viable novel therapeutical options in medicine.

Establishment and biochemical characterization of primary cells of the upper aerodigestive tract.

The upper aerodigestive tract, composed of the oral cavity, the pharynx and the esophagus, is a complex system whose components function in both organ-specific ways as well in serving as a protective barrier against the enzymes which initiate digestion as well as against the mechanical functions which serve to ensure movement of food through the upper aerodigestive tract. Given these diverse functional requirements, the study of the anatomy and physiology of this region are uniquely complex and significantly understudied. The goal of the current study was to develop a simple and reproducible method for the isolation, growth, and maintenance of primary epithelial cells from the oral cavity, the pharynx and the esophagus. In addition, given the increased interest in diseases characterized by a loss of mucosal integrity in these areas which is often accompanied by a diminished wound healing capability, these cells were biochemically characterized with a focus on the components of the extracellular matrix remodeling axis including the activity and inhibition of the matrix metalloproteinases (MMPs).

The effect of radiographic contrast media on the morphology of human venous endothelial cells.

Radiographic contrast media (RCM) can affect the morphology of red blood cells in very different ways but research on how they affect endothelial cell morphology is rudimentary. The effect of two conventional RCMs on human umbilical venous cells over the short term was studied in vitro under static conditions. Cell circumference length, the number of dissolved cell contacts and the number of denuded subendothelial matrix areas were interactively quantified by a computer imaging system after histochemical processing. 1.5 minutes after RCM exposure a significant effect of both RCMs on cell circumference length (CCL) compared to the control cells was evident (p=0.0001 each). The increase after iodixanol was larger than after iomeprol (p=0.0087). After five minutes of exposure, the CCL of exposed cells were significantly larger than those of control cells (p<0.0001 each). The CCL after exposure hardly differed anymore at that time (iomeprol/iodixanol: p=0.0547), though cells exposed to iomeprol tended to be bigger. After both iomeprol (p<0.0001) and iodixanol (p=0.0018), the number of dissolved cell contacts (DCC) increased compared to the control cells. The increases after either RCM were similar (p=0.9633). After five minutes of RCM exposure, the number of DCC was significantly higher than for the control cells (control/iomeprol: p<0.0001; control/iodixanol: p=0.0012). After exposure to iodixanol, significantly fewer DCC were recorded than after iomeprol (p=0.0018). At 1.5 minutes after RCM exposure, the number of denuded subendothelial matrix areas (DSMA) in the cell layer increased both after iomeprol (p<0.0002) and after iodixanol (p=0.0002) compared to the control cells. The increases with the two RCMs were similar (p=0.8618). After five minutes of exposure, the number of DSMA in the cell layer was significantly higher than for the control cells (control/iomeprol: p<0.0001; control/iodixanol: p=0.0015). However, after iodixanol significantly fewer DSMA were recorded than after iomeprol (iomeprol/iodixanol: p=0.0353). The number of dissolved cell/cell contacts and the number of denuded subendothelial matrix areas in the confluent endothelial layer were significantly greater after exposing the endothelial cells for five minutes to iomeprol than after iodixanol.

Reference range and variability of Laser-Doppler-Fluxmetry.

The Laser Doppler technique (Laser-Doppler-Fluxmetry, LDF), a noninvasive method to estimate skin blood flow (LDF), is frequently used in research and clinical routine [1]. Here, the measurements were carried out with a new Laser Doppler system, the DOP-system, which allows to measure frequency spectra in four different frequency windows according to the velocities in venules (low velocity), capillaries (low to medium velocities), and in arteries (with high and very high velocities). However, the diagnostic reliability or the effectiveness of the LDF has not yet been evaluated sufficiently, which is indispensable, where medical diagnostics and therapy controls are concerned. For a valid interpretation of LDF values of individual patients, the knowledge of the reference range and the variability of the measured parameters is required.In four successive studies the reference range (62 apparently healthy subjects), the circadian variability (8 subjects), the variability from day-to-day (6 subjects) and over one year with monthly measurements (6 subjects) were evaluated.With the knowledge of the reference range, microcirculatory disorders can now be diagnosed using the DOP method. Following a standard measurement procedure there was no dependence of the measured data on the day or season of measurement.

Effects of Tacrolimus or Sirolimus on the adhesion of vascular wall cells: Controlled in-vitro comparison study.

In drug eluting stents the cytostatic drugs Sirolimus or Tacrolimus are used to inhibit blood vessel restenosis by limiting the proliferation of smooth muscle cells. However, the cytostatic activity of both drugs was shown to be not cell specific and could also affect the stent endothelialisation, respectively. Currently, only limited in vitro data are available about the impact of Sirolimus and Tacrolimus on endothelial cell proliferation over a broad concentration range. To answer this question the following study was performed.Commercially obtained HUVEC were expanded with DMEM cell culture medium (GIBCO, Germany) supplemented with 5 vol% fetal calf serum on non-coated regular polystyrene-based 24-multiwell plates. For drug testings 2×104 cells/cm2 were seeded and grown for 24 h until 30-40% of the multiwell surfaces were covered and then exposed to Sirolimus (1.0×10-11 - 1.0×10-5 mol/l) or Tacrolimus (2.0×10-8 - 6.2×10-5 mol/l), both dissolved in DMSO. 12, 24 and 48 h after adding the drugs cell numbers per area were quantified by counting the cells in six wells with four fields of view per well, representing 0.6 mm2, using a confocal laser microscope.After 48 h of cell growth in the drug-free cell culture medium, the HUVEC number increased from 2.0×104 to 3.55×104 cells/cm2 (mean cell doubling time: 53.6 h, n = 6). At lower concentrations (≤2.0×10-6 mol/l) Tacrolimus reduced the number of adherent HUVEC significantly less than Sirolimus (p < 0.05). However, at higher concentrations (≥2.07×10-5 mol/l) the effect of Tacrolimus on the number of adherent endothelial cells was significantly greater than that of Sirolimus (p < 0.05). At the highest concentration applied (6.22×10-5 mol/l), Tacrolimus induced detachment of all HUVECs within 12 h after drug application. The number of adherent HUVEC decreased only slightly (about 9%) after Sirolimus application at the highest concentration (1.09×10-5 mol/l).These data show that in a non-flow model the cytostatic drug Tacrolimus reduced the number of adherent endothelial cells less than Sirolimus, as long as the drug concentration did not surpass 10-6 mol/l. At the limits of solubility, Sirolimus (1×10-5 mol/l) reduced the number of adherent endothelial cells less than Tacrolimus (6×10-5 mol/l), which induced detachment of endothelial cells.

Influence of Ultrasound Microbubbles on kidney oxygen tension.

Ultrasound contrast agents (USCA) allows the dynamic detection of blood flow of both the macro and microvasculature. An obvious prerequisite for USCAs is the unhindered passage of clinically relevant dose levels through the microcirculation especially of the lungue, where they have to pass capillaries with diameters of around 4 µm. While smaller microbubbles rapidly passed through the microcirculation along with the red blood cells, larger microbubbles, however, were observed to coalesce and interrupt the blood flow. Whether this might influence the tissue oxygen tension is unclear up to now.To examine this question a bolus of 2.4 ml SonoVue™ was injected into the suprarenal aorta at a flow rate of 10 ml/s (a dosage usually applied in the clinic). The pO2 in the outer medulla of the kidney was continuously measured using a flexible pO2 microcatheter. In addition, the SonoVue™ injection and its passage through the renal vasculature were documented by the CEUS technology to assess whether the microbubbles passed the kidney.The study revealed that SonoVue™ induced no changes of the mean oxygen partial pressure in the outer medulla which confirms that these microbubbles on their way through the medullar capillaries did not hinder the co-flow of blood through the renal microvessels in a big animal model with a renal morphology and function comparable to human kidneys. These results demonstrate that the CEUS diagnostic itself did not influence the system to be examined which is a most important prerequisite for any diagnostic method.