Clinical Hemorheology and Microcirculation - Volume 61, issue 2

Authors: Manz, Patrick | Cadeddu, Ron-Patrick | Wilk, Matthias | Fischer, Johannes C. | Fritz, Birgit | Haas, Rainer | Wenzel, Folker

Article Type: Research Article

Abstract: INTRODUCTION: Phthalates are a group of synthetic plasticizers that are ubiquitous environmental pollutants with toxic and endocrine disrupting characteristics. DEHP is the most commonly used plasticizer in the world and is still applied to stem cell transfusion bags used for storage of hematopoietic stem and progenitor cells (CD34+ HSPC), which are transferred during stem cell transplantation. Here we examined the effect of DEHP on vitality of CD34+ HSPC as well as stem cell specific properties like migration and differentiation capacity – both important for successful stem cell transplantations. MATERIAL AND METHODs: CD34+ HSPC were incubated …for 24 h and 72 h with DEHP concentrations ranging from 1 μg/ml to 250 μg/ml. DEHP was diluted in DMSO. Migration rate was analyzed along an SDF-1α gradient using Transwell migration inserts. Differentiation of CD34+ HSPC was investigated after two weeks in methylcellulose with colony stimulating factors. Apoptosis rate was measured via Annexin V and 7-AAD staining. RESULTS: 24 h of incubation with 10 μg/ml DEHP led to a significant (p  <  0.01) decrease in migration rate of CD34+ HSPC (70.70% ± 7.53% ) with a minimum migration rate of 48.33% ± 6.72% in relation to control after incubation with 100 μg/ml DEHP for 72 h. Incubation with the highest tested DEHP concentrations (50 and 100 μg/ml) significantly (p  <  0.05) altered colony formation rate and cell type distribution. Apoptosis rate of CD34+ HSPC significantly (p  <  0.05) increased after incubation with concentrations of 10 μg/ml DEHP for 24 h (1.46 ± 0.19) with a maximum apoptosis rate of 2.71 ± 0.66 after 24 h incubation with the highest DEHP concentration (250 μg/ml) in relation to control. CONCLUSIONS: As shown, DEHP takes impact on migration rate, apoptosis rate, and differentiation of CD34+ HSPC. As these are functions with an important role in stem cell transplantations, the usage of DEHP-free stem cell transfusion bags should be considered. Show more

Keywords: Phthalates, DEHP, hematopoietic stem cells, CD34+ , vitality, migration, differentiation, apoptosis, apheresis, stem cell transplantation

DOI: 10.3233/CH-151984

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 111-118, 2015

Authors: Vijaya Bhaskar, Thanga Bhuvanesh | Ma, Nan | Lendlein, Andreas | Roch, Toralf

Article Type: Research Article

Abstract: Silicones are widely used as biomaterials for medical devices such as extracorporeal equipments. However, there is often conflicting evidence about their supposed cell- and histocompatibility. Macrophages could mediate silicone-induced adverse responses such as foreign body reaction and fibrous encapsulation. The polarization behaviour of macrophages could determine the clinical outcome after implantation of biomaterials. Induction of classically activated macrophages (CAM) may induce and support uncontrolled inflammatory responses and undesired material degradation. In contrast, polarization into alternatively activated macrophages (AAM) is assumed to support healing processes and implant integration. This study compared the interaction of non-polarized macrophages (M0), CAM, and AAM …with commercially available tissue culture polystyrene (TCP) and a medical grade silicone-based biomaterial, regarding the secretion of inflammatory mediators such as cytokines and chemokines. Firstly, by using the Limulus amoebocyte lysate (LAL) test the silicone films were shown to be free of soluble endotoxins, which is the prerequisite to investigate their interaction with primary immune cells. Primary human monocyte-derived macrophages (M0) were polarized into CAM and AAM by addition of suitable differentiation factors. These macrophage subsets were incubated on the materials for 24 hours and their viability and cytokine secretion was assessed. In comparison to TCP, cell adhesion was lower on silicone after 24 hours for all three macrophage subsets. However, compared to TCP, silicone induced higher levels of certain inflammatory and chemotactic cytokines in M0, CAM, and AAM macrophage subsets. Conclusively, it was shown that silicone has the ability to induce a pro-inflammatory state to different magnitudes dependent on the macrophage subsets. This priming of the macrophage phenotype by silicone could explain the incidence of severe foreign body complications observed in vivo . Show more

Keywords: Biomaterials, silicone, macrophage subsets, cytokines/chemokines

DOI: 10.3233/CH-151991

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 119-133, 2015

Authors: Niessen, C. | Jung, E.M. | Beyer, L. | Pregler, B. | Dollinger, M. | Haimerl, M. | Scheer, F. | Stroszczynski, C. | Wiggermann, P.

Article Type: Research Article

Abstract: PURPOSE: To evaluate the use of contrast-enhanced ultrasound (CEUS) after irreversible electroporation (IRE) of prostatic cancer tissue to assess the ablation status by depicting microvascularisation in the ablation area. MATERIALS AND METHODS: Retrospective evaluation of CEUS of 13 patients (mean age: 61.4 ± 7.5 years) with histologically confirmed prostatic cancer who underwent percutaneous IRE. In the course of clinical routine, the tumor lesions were documented before, immediately after, and 1 day after the ablationusing color-coded transabdominal and transrectal CEUS. The obtained image data (DICOM loops and images) were subsequently evaluated by 2 experienced radiologists and assessed with regard …to micro vascularisation by means of a 5-point scale. RESULTS: CEUS images showed significantly reduced microcirculation of the lesions (mean 0.9 ± 0.6 cm (0.5–1.5 cm) after IRE. Microcirculation was reduced from 2.15 ± 0.56 prior to ablation to 0.65 ± 0.63 (p  <  0.001) immediately after the ablation and to 0.27 ± 0.44 one day after IRE (p  <  0.001). CONCLUSION: This study showed rapid and significant reduction of the microcirculation in the ablation area afterpercutaneous IRE of prostatic cancer tissue. Show more

Keywords: Irreversible electroporation, prostate cancer, percutaneous ablation, contrast-enhanced ultrasound

DOI: 10.3233/CH-151985

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 135-141, 2015

Authors: Paprottka, P.M. | Zengel, P. | Cyran, C.C. | Paprottka, K.J. | Ingrisch, M. | Nikolaou, K. | Reiser, M.F. | Clevert, D.A.

Article Type: Research Article

Abstract: PURPOSE: To evaluate the diagnostic benefits of multimodality imaging using image fusion with magnetic-resonance-imaging (MRI) and contrast-enhanced-ultrasound (CEUS) in an experimental small-animal-squamous-cell-carcinoma-model for the assessment of tissue hemodynamics and morphology. MATERIAL AND METHODS: Human hypopharynx-carcinoma-cells were injected subcutaneously into the left flank of 15 female athymic nude rats. After 10 daysof subcutaneous tumor growth, CEUS and MRI measurements were performed using a high-end-ultrasound-system and 3-T-MRI. After successful point-to-point or plan registration, the registered MR-images were simultaneously shown with the respective ultrasound sectional plane. Data evaluation was performed using the digitally stored video sequence data sets by two experienced …radiologists using a subjective 5-point scale. RESULTS: CEUS and MRI are well-known techniques for the assessment of tissue hemodynamics (score: mean 3.8 ± 0.4 SD and score 3.8 ± 0.4 SD). Real-time image fusion of MRI and CEUS yielded a significant (p  <  0.001) improvement in score (score 4.8 ± 0.4 SD). Reliable detection of small necrotic areas was possible in all animals with necrotic tumors. No significant intraobserver and interobserver variability was detected (kappa coefficient = +1). CONCLUSION: Image fusion of MRI and CEUS gives a significant improvement for reliable differentiation between different tumor tissue areas and simplifies investigations by showing the morphology as well as surrounding macro-/microvascularization. Show more

Keywords: Image fusion, multimodality imaging, magnetic resonance imaging (MRI), contrast-enhanced ultrasound (CEUS), experimental animal model

DOI: 10.3233/CH-151986

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 143-150, 2015

Authors: Krüger-Genge, A. | Fuhrmann, R. | Jung, F. | Franke, R.P.

Article Type: Research Article

Abstract: BACKGROUND: The evaluation of the interaction of human, venous endothelial cells (HUVEC) with body foreign materials on the cellular level cannot be performed in vivo , but is investigated in vitro under standard culture conditions. To maintain the vitality, proliferation and morphology of HUVEC seeded on body foreign substrates over days, the cell culture medium is usually exchanged every second day. It is well known, that alterations in the microenvironment of cells bear the risk of influencing cell morphology and function. In the current study the influence of cell culture medium exchange on HUVEC cytoskeletal microfilament structure and function …was investigated. MATERIAL AND METHODS: HUVEC in the third passage were seeded on extracellular matrix (ECM) - which was secreted from bovine corneal endothelial cells on glass- until functional confluence was reached. The experiment started 11 days after HUVEC seeding with an exchange of the cell culture medium followed by a staining of the actin microfilaments with phalloidin-rhodamin 1.5 and 5 minutes after medium exchange. The microfilaments were documented by use of an Olympus microscope (IMT-2) equipped with a UV lamp and online connected to a TV chain (Sony XC 50 ST/monochrome) implying an OPTIMAS – Image analysis system. Prostacyclin was analysed in the cell culture supernatant. RESULTS: 1.5 min after culture medium exchange in the functionally confluent cultures a slight disturbance of the actin microfilament structure with a broadening of the marginal filament band, a partial disconnection of cell-cell contacts and the appearance of intercellular fenestrations were observed. 5 minutes after medium exchange a redevelopment of the slightly disturbed microfilament structure with a condensation and narrowing of the marginal filament band was seen. 12 h later a further consolidation of the microfilament structure occurred. In addition, a perturbation of the cultured HUVEC occurred after cell culture medium exchange. The prostacyclin concentration in the supernatant increased significantly after 1.5 min to 466 ± 543 pg·mL–1 (p  <  0.001) and after 5 min to 408 ± 458 pg·mL–1 (p  <  0.001), while in control cells the prostacyclin concentration did not change remaining in the range of 50 ± 48.9 pg·mL–1 . CONCLUSION: This study revealed that the exchange of the cell culture medium led to a rapid disturbance of the HUVEC with stress fiber formation, disconnection of cell-cell contacts and an altered prostacyclin secretion, which had regressed nearly completely after 12 hours. Therefore, the evaluation of HUVEC on body foreign materials should be performed not earlier than 12 hours after cell culture medium exchange to avoid a misinterpretation of the endothelial cell morphological state. This procedure minimizes the risk of a misinterpretation of the endothelial cell morphology – caused by the culture medium exchange and not by the interaction between biomaterials and HUVEC. Show more

Keywords: HUVEC, morphology, microfilaments, adherence, culture medium

DOI: 10.3233/CH-151992

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 151-156, 2015

Authors: Khiao In, M. | Wallmeyer, L. | Hedtrich, S. | Richardson, K.C. | Plendl, J. | Kaessmeyer, S.

Article Type: Research Article

Abstract: INTRODUCTION: Inducing vascularization in three-dimensional skin constructs continues to be difficult. In this study, two variations of human full-thickness skin constructs were examined. Type KCFB consists of keratinocytes (epidermal equivalent) and fibroblasts that were embedded in a collagen matrix (dermal equivalent). Type KCFB-EC consists of keratinocytes as well as fibroblasts and vascular endothelial cells. The epidermal equivalent of KCFB-EC constructs underwent cellular alterations in their differentiation possibly induced by the presence of endothelial cells. The objective of the study was to assess the effect of endothelial cells, i.e., endothelialization of the dermal equivalent on the differentiation of keratinocytes by comparing …the morphology and ultrastructure of the two types of skin constructs, as well as to excised normal human skin. Hypothesis: The differentiation of keratinocytes is influenced by the presence of endothelial cells. METHODS, PATIENTS, MATERIAL: KCFB constructs (keratinocytes, fibroblasts) and KCFB-EC skin constructs(kera-tinocytes, fibroblasts, endothelial cells) were prepared according to Küchler et al. [25]. After two weeks, the skin constructs were processed for analysis by light microscopy (LM) and electron microscopy (TEM), followed by quantitative, semi-quantitative as well as qualitative assessment. For comparison, analysis by LM and TEM of excised normal human skin was also performed. RESULTS: Both KCFB and KCFB-EC skin constructs and the human skin had all strata of stratified soft-cornified epidermis present. The comparison of the respective layers of the skin constructs brought the following characteristics to light: The KCFB-EC constructs had significantly more mitotic cells in the stratum spinosum, more cell layers in the stratum granulosum and more keratohyalin granules compared to KCFB skin constructs. Additionally, the epidermal architecture was unorganized in the endothelialized constructs and features of excessive epidermal differentiation appeared in KCFB-EC skin constructs. CONCLUSION: The endothelialization of the dermal equivalent caused changes in the differentiation of the epidermis of KCFB-EC skin constructs that may be interpreted as an unbalanced, i.e., uncontrolled or enhanced maturation process. Show more

Keywords: Full-thickness skin constructs, human skin, epidermis, keratinocyte, fibroblast, endothelial cell

DOI: 10.3233/CH-151988

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 157-174, 2015

Authors: Riebe, Helene | Konschake, Wolfgang | Haase, Hermann | Jünger, Michael

Article Type: Research Article

Abstract: Standard of care in the therapy of chronic venous insufficiency (CVI) is the use of graduated elastic stockings (GECS). This paper is based on a prospective, mono-centric, open randomised, controlled and cross over study and discusses the hemodynamic effects of GECS and inverse graduated compression stockings (PECS) in 32 healthy volunteers and 32 patients with CVI and the consecutive impact on comfort. The application of stockings was performed sequentially, the allocation of the first stocking was randomized and double blind, wearing period for each stocking type about 7 days with one week of break between wearing periods. Measurements of the …interface pressure were carried out by Picopress® (Microlab Elettronica, Italy) and the venous drainage were registered by strain gauge plethysmography. Mean interface pressure: GECS (level B1): 27.3 mmHg; GECS (level C): 19,6 mmHg; PECS (level B1): 17,8 mmHg; PECS (level C): 24,7 mmHg. Significant increase of EF and decrease of VFI by wearing both types of compression stockings, comparatively GESC resulted in a significantly greater improvement of EF than the PECS. PECS were significantly easier to don and put on compared to the GECS, the PECS tended to slip towards the foot more often. Show more

Keywords: Chronic venous insufficiency, compression stockings, graduated elastic stockings, GECS, progressive pressure, PECS, interface pressure profile

DOI: 10.3233/CH-151989

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 175-183, 2015

Authors: Helling, H. | Stephan, B. | Pindur, G.

Article Type: Research Article

Abstract: Activation of coagulation and inflammatory response including the complement system play a major role in the pathogenesis of critical illness. However, only limited data are available addressing the relationship of both pathways and its assessment of a predictive value for the clinical outcome in intense care medicine. Therefore, parameters of the coagulation and complement system were studied in patients with septicaemia and multiple trauma regarded as being exemplary for critical illness. 34 patients (mean age: 51.38 years (±16.57), 15 females, 19 males) were investigated at day 1 of admittance to the intensive care unit (ICU). Leukocytes, complement factors C3a and …C5a were significantly (p  <  0.0500) higher in sepsis than in trauma, whereas platelet count and plasma fibrinogen were significantly lower in multiple trauma. Activation markers of coagulation were elevated in both groups, however, thrombin-antithrombin-complex was significantly higher in multiple trauma. DIC scores of 5 were not exceeded in any of the two groups. Analysing the influences on mortality (11/34; 32.35% ), which was not different in both groups, non-survivors were significantly older, had significantly higher multiple organ failure (MOF) scores, lactate, abnormal prothrombin times and lower C1-inhibitor activities, even more pronounced in early deaths, than survivors. In septic non-survivors protein C was significantly lower than in trauma. We conclude from these data that activation of the complement system as part of the inflammatory response is a significant mechanism in septicaemia, whereas loss and consumption of blood components including parts of the coagulation and complement system is more characteristic for multiple trauma. Protein C in case of severe reduction might be of special concern for surviving in sepsis. Activation of haemostasis was occurring in both diseases, however, overt DIC was not confirmed in this study to be a leading mechanism in critically ill patients. MOF score, lactate, C1-inhibitor and prothrombin time have been the only statistically significant predictors for lethal outcome suggesting that organ function, microcirculation, haemostasis and inflammatory response are essential elements of the pathomechanism and clinical course of diseases among critically ill patients. Show more

Keywords: Septicemia, multiple trauma, coagulation, complement, critical illness

DOI: 10.3233/CH-151993

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 185-193, 2015

Authors: Haimerl, M. | Jung, E.-M. | Beyer, L.P. | Pregler, B. | Dollinger, M. | Sieroń, D. | Niessen, C. | Stroszczynski, C. | Wiggermann, P.

Article Type: Research Article

Abstract: PURPOSE: To evaluate whether changes of dynamic contrast-enhanced ultrasound (CEUS)-based liver microcirculation during chronic liver disease are correlated with the extent of impaired liver function, expressed by the indocyanine green (ICG) clearance. MATERIALS AND METHODS: 19 patients underwent CEUS examinations with previous or consecutive ICG clearance test. The ICG plasma disappearance rate (ICG-PDR) was determined using the noninvasive pulse-densitometric LiMON system. Quantification of peak enhancement (PE), rise time (RT) and the wash-in rate (WiR) was performed in the liver parenchyma (ROIliver ) as well as in the portal vein (ROIportal vein ) using a novel quantification software (VueBoxTM ). …To compare quantification parameters, patients were classified in patients representing a healthy population (ICG-PDR >16, n  = 8) and those representing patients with liver disease (ICG-PDR ≤16, n  = 11). RESULTS: ROIportal vein showed significant differences comparing healthy patients and patients with liver disease for all perfusion parameters: PE and WiR were significantly higher in patients without liver disease (p  = 0.048; p  = 0.039). RT was significantly lower in healthy population (p  = 0.039). Comparing perfusion parameters for ROIliver , PE was significantly higher in patients without liver disease (p  = 0.039). There was no significant difference for RT (p  = 0.804) and WiR (p  = 0.058), respectively. CONCLUSION: Within the framework of this study CEUS derived estimation of microcirculation did not prove to be a reliable estimator of liver function. RT, PE and WiR measured in the portal vein were significant perfusion factors in predicting liver function. Show more

Keywords: Chronic liver disease, CEUS, ICG-test, microcirculation, liver function

DOI: 10.3233/CH-151990

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 195-204, 2015

Authors: Hiebl, Bernhard | Nennig, Ernst | Schiestel, Stefanie | Kovacs, Adalbert | Jung, Friedrich | Fischer, Harald

Article Type: Research Article

Abstract: Biomaterials made of zinc have been widely described to be antioxidative, hypothrombogenic, antiinflammatory and antiproliferative. Additionally in vivo zinc is toxic only in high concentrations and can completely be metabolized in vivo . Due to these properties zinc based vascular stents might be able to reduce the rate of restenosis in comparison to bare metal stents and zinc stents might be also able to limit the foreign body reaction. In the presented study we tested the biocompatibility and degradability of a stent made of zinc and characterized by a closed-cell-design to achieve high opening force and to increase stent …stiffness. After 100 days of enzymatic and hydrolytic degradation in 15 ml blood serum (fetal calf serum) a significant loss of weight (1.72 wt% ) was measured. Zinc was compared to other metals in terms of degradation rates. After six weeks of incubation in physiologic sodium chloride solution zinc showed the slowest degradation time, 6 times less than stainless steel and 4 times less than magnesium. In the tests for cytotoxic effects the degraded zinc stent caused no changes in the LDH-release and cell membrane integrity (3T3 cells, mouse fibroblasts) respectively, in the cell activity/proliferation (MTS assay) and in the morphological characteristics of the cells and cell layers in comparison to the control material (polystyrene). Based on these results the tested zinc stent proved to be non-cytotoxic and to be characterized by degradation characteristics which might be advantageous in comparison to magnesium and stainless steel. Show more

Keywords: zinc, stent, biocompatibility, degradation rate

DOI: 10.3233/CH-151983

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 205-211, 2015

Authors: Jung, Christian | Kelm, Malte

Article Type: Research Article

Abstract: Adequate monitoring of patients on intensive care units is of highest priority to provide optimal treatment and to detect patients at risk. Within recent years the microcirculation became more and more attention due to its central importance for the outcome of patients. Microcirculatory disorders may include capillary flow disturbances as well as changes in the density of perfused vessels. In the clinical setting, the most often used parameter to detect alterations in the microcirculation is serum lactate. Since this parameter is characterized by major limitations, other strategies including non-invasive methods to quantify microvascular perfusion have been developed. A successful surveillance …of the microcirculation in the individual patient may guide diagnostic and treatment strategies in order to optimize organ perfusion and oxygenation, subsequently leading to an individualized therapy. Intravital microscopy has been used to stratify patients at risk and to predict patients’ outcome. The aim of this review is to evaluate clinical correlates of microcirculatory disorders as well as giving an overview of newer diagnostic devices that may directly or indirectly evaluate the microcirculation and are available for use in critically ill patients. Show more

Keywords: Microcirculation, critically ill, intensive care, intravital microscopy

DOI: 10.3233/CH-151994

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 213-224, 2015

Authors: Braune, S. | Zhou, S. | Groth, B. | Jung, F.

Article Type: Research Article

Abstract: BACKGROUND: Platelet adhesion to artificial surfaces is one of the most important indicators for the thrombogenicity of implant materials. Currently, a variety of enzyme activity-based colorimetric assays or microscopy-based techniques are commonly in use to assess this characteristic. Studies about how data of colorimetric assays correlate with the image-based quantification of adherent platelets are scarce. To address this question, the present study compared two colorimetric assays (lactate dehydrogenase (LDH) and acid phosphatase (ACP)) with an image-based quantification of the density of platelets adhering on polymer-based biomaterial surfaces. MATERIALS AND METHODS: Tri-sodium citrated whole blood was collected from apparently …healthy subjects and platelet rich plasma (PRP) was prepared according to a standardized protocol. An in vitro static thrombogenicity test was applied to study platelet adhesion from PRP adjusted to 50,000 platelets per μL on three different polymers: medical grade polytetrafluoroethylene (PTFE), silicone and polyethylene terephthalate (PET). For the direct image-based approach, surface adherent platelets were fixed, fluorescently labelled and microscopically visualized. The image-based determination of platelet densities provided reference values for the comparison with data of the colorimetric assays. Correlation between standard platelet concentrations and ACP/LDH absorbance measurements were analysed to estimate accuracy and association of both parameters. ACP and LDH release from resting and ADP-stimulated platelets was studied to estimate how platelet activation influences colorimetric assay results. RESULTS: The density of adherent platelets ranged from 15,693 ± 2,487 platelets·mm–2 (PTFE) to 423 ± 99 platelets·mm–2 (silicone) and 4,621 ± 1,427 platelets·mm–2 (PET) and differed significantly between the three polymers (ANOVA: p  <  0.05). Correlation coefficients between microscopic and colorimetric determination of platelet densities ranged between r  = 0.93 (LDH, p  <  0.001) and r  = 0.94 (ACP, p  <  0.001). ACP absorbance measurements of platelet standards with different concentrations corresponded well to an ideal linear regression, while LDH data either deceeded or exceeded the expected values. The LDH release during ADP-induced platelet activation was significantly higher compared to the release of ACP. CONCLUSION: For an adjusted platelet concentration of 50,000 platelets·μL−1 , both colorimetric assays (ACP and LDH) allowed a similar accurate quantification of the mean platelet density compared to the microscopic evaluation. Better linearity of the assay standards, less variability of the results and a lower influence of platelet activation on the measurements mark the ACP assay as more suitable for the assessment of material surface adherent platelets compared to the LDH assay, particularly, if near physiological platelet concentrations are applied. Show more

Keywords: Platelet, biomaterial, thrombogenicity, lactate dehydrogenase, acid phosphatase, colorimetric assay, microscopy

DOI: 10.3233/CH-151995

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 225-236, 2015

Authors: Toguri, J.T. | Moxsom, R. | Szczesniak, A.M. | Zhou, J. | Kelly, M.E.M. | Lehmann, C.

Article Type: Research Article

Abstract: BACKGROUND: Leukocyte adhesion to the endothelium and decreased microvascular blood flow causing microcirculatory dysfunction are hallmarks of systemic inflammation. We studied the impact of cannabinoid receptor activation on the iridial microcirculation, which is accessible non-invasively in vivo , in systemic inflammation induced by endotoxin challenge. METHODS: 40 Lewis rats were used in the experiments. Endotoxemia was induced by 2 mg/kg i.v. lipopolysaccharide (LPS). Cannabinoid receptors (CBRs) were stimulated by i.v. administration of WIN 55212-2 (WIN; 1 mg/kg). CB1 R antagonist (AM281; 2.5 mg/kg i.v.) or CB2 R antagonist (AM630; 2.5 mg/kg i.v.) treatment prior to WIN was applied to identify the anti-inflammatory …effects underlying each CBR subtype. Leukocyte-endothelial interactions were examined in rat iridial microvas culature by intravital microscopy at baseline and 1 and 2 h post-LPS. Additionally, systemic (mean arterial pressure, heart rate) and local (laser Doppler flow) hemodynamic variables were measured prior to and during cannabinoid treatments. RESULTS: Endotoxemia resulted in severe inflammation as shown by significantly increased numbers of adherent leukocytes at 1 and 2 h observation time post-LPS challenge and decreased microcirculatory blood flow at 2 h within the iridial microcirculation. WIN treatment significantly reduced leukocyte adhesion in iridial microvessels with a diameter greater and less than 25 μm during endotoxemia (p  <  0.05). Pre-treatment of animals by CB1 R antagonist, AM281, did not affect WIN effects on LPS-induced leukocyte adhesion. When pre-treated with the CB2 R antagonist, AM630, a reversal of the WIN-induced reduction in leukocyte adhesion was noticed in vessels with a diameter of less than 25 μm (p  <  0.05). Cannabinoid treatment significantly increased the local iridial microcirculatory blood flow 2 hours after systemic LPS administration (p  <  0.05). CONCLUSIONS: Systemic administration of the CBR agonist, WIN, decreased leukocyte-adhesion and improved iridial microvascular blood flow. This effect is most likely mediated by CB2 R activation. Our findings indicate that the iris microvasculature can serve as a model to study the microcirculation during systemic inflammation and help to identify potential therapies to treat microcirculatory dysfunction in diseases such as sepsis. Show more

Keywords: Endotoxemia, microcirculation, cannabinoid receptor, WIN 55212-2

DOI: 10.3233/CH-151996

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 237-249, 2015

Authors: Thalhammer, Christoph | Alvarez, Elena | Husmann, Marc | Staub, Daniel | Kaspar, Mathias | Aschwanden, Markus | Amann-Vesti, Beatrice R.

Article Type: Research Article

Abstract: To perform a long term follow-up after endovascular brachytherapy (EVBT) and balloon angioplasty (PTA) regarding vessel patency and diameter. EVBT had been successfully used to decrease restenosis in short term, but long term data are lacking. Participants of a randomized study comparing EVBT and balloon angioplasty alone were invited for follow-up examination ten years after intervention. Using a standardized protocol measurement of the patency and vessel diameter was performed of femoral and popliteal arteries. 44 patients were included, 21 had been treated with EVBT and 23 had received PTA alone. Target lesion patency was similar between the two groups (90.5% …vs. 87.0%). Vessel diameter of the target lesion was significantly greater in the EVBT group (6.4 mm, range 3.9–9.9) compared to the controls (5.0 mm, range 3.1–7.4; p  = 0.002). Ten years after EVBT of femoro-popliteal arteries vessel diameter is significantly increased whereas patency rate is not different compared to angioplasty alone. Show more

Keywords: Endovascular brachytherapy, femoro-popliteal artery, long term follow-up, duplex sonography, peripheral artery disease, aneurysm

DOI: 10.3233/CH-151997

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 251-257, 2015

Authors: Matuszak, Jasmin | Dörfler, Philipp | Zaloga, Jan | Unterweger, Harald | Lyer, Stefan | Dietel, Barbara | Alexiou, Christoph | Cicha, Iwona

Article Type: Research Article

Abstract: Superparamagnetic iron oxide nanoparticles (SPIONs) are versatile and easily functionalized agents with high potential for diagnostic and therapeutic intravascular applications. In this study, we analyzed the responses of endothelial (ECs) and monocytic cells to three different types of SPIONs, in order to assess the influence of physico-chemical properties on the biological reactions to SPIONs. The following formulations were used: (1) Lauric acid-coated and BSA-stabilized SPION-1,(2) Lauric acid/BSA-coated SPION-2 and (3) dextran-coated SPION-3. SPION-1 were strongly internalized by ECs and reduced their viability in static conditions. Additionally, they had a dose-dependent inhibitory effect on monocytic cell chemotaxis to MCP-1, but did …not affect monocytic cell recruitment by ECs. SPION-2 uptake was less pronounced, both in ECs and monocytic cells, and these particles were better tolerated by the vascular cells. Not being internalized by endothelial or monocytic cells, SPION-3 did not induce relevant effects on cell viability, motility or endothelial-monocytic cell interactions. Taken together, localized accumulation of circulating SPION under physiologic-like flow conditions and their cellular uptake depends on the physicochemical characteristics. Our findings suggest that SPION-2 are suitable for magnetic targeting of atherosclerotic plaques. Due to their excellent biocompatibility and low internalization, SPION-3 may represent a suitable imaging agent for intravascular applications. Show more

Keywords: Atherosclerosis, magnetic nanoparticles, SPION uptake, endothelial-monocytic cell interactions, endothelial migration, monocytic cell chemotaxis, live-cell analysis

DOI: 10.3233/CH-151998

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 259-277, 2015

Authors: Haubner, F. | Muschter, D. | Schuster, N. | Pohl, F. | Ahrens, N. | Prantl, L. | Gassner, H.G.

Article Type: Research Article

Abstract: BACKGROUND: Platelet-rich plasma (PRP) products are currently suggested in the treatment of chronic wounds due to possible pro-angiogenic effects. Microvascular compromise represents the major component in radiogenic wound healing complications. The effects of PRP on irradiated cells of the cutaneous wound healing process are still poorly understood. MATERIAL AND METHODS: Human dermal microvascular endothelial cells (HDMEC) and human adipose derived stem cells (hASC) were cultured and irradiated with doses of 2 to 12 Gy. PRP was activated, characterized and added to the incubation media in different concentrations after external radiation. Cell count was determined 48 h after radiation using a …semi-automated cell counting system. Levels of interleukin-6 (IL-6), basic fibroblast growth factor (bFGF) and soluble intercellular adhesion molecule-1 (sICAM-1) in the supernatants of HDMEC and hASC co-cultures were determined by enzyme-linked immunosorbent assay (ELISA). Non-irradiated hASC and HDMEC served as controls. RESULTS: The employed PRP preparations were characterized and contained platelet derived growth factor (PDGF-AB), vascular endothelial growth factor (VEGF), bFGF and high levels of sICAM-1. Addition of PRP to irradiated cultures of HDMEC and hASC prevented profound radiation-induced decline in cell numbers. 10% PRP restored cell numbers to levels of untreated, non-irradiated cultures. Basic FGF expression was decreased significantly in hASC monocultures treated with 10% PRP without external radiation and after irradiation with 6 and 12 Gy. These inhibitory effects of PRP were also observed in HDMEC. In contrast, co-cultures of HDMEC-ASC showed a dose-dependent increase in bFGF expression when treated with 5 or 10% PRP. Doses of 6 and 12 Gy increased IL-6 expression in cultures stimulated with 5% PRP. CONCLUSIONS: Use of PRP in co-cultures of hASC and HDMEC restores proliferative defects caused by external radiation probably by induction of bFGF. Under irradiated conditions, PRP might induce pro-inflammatory stimuli which could be beneficial in treatment of chronic wounds where healing processes are defective. Combined use of hASC and PRP products might be helpful in the treatment of radiogenic wounds. Show more

Keywords: Microvascular endothelial cells, human adipose-derived stem cells, endothelial dysfunction, cytokines, adhesion molecules, growth factors, radiation therapy

DOI: 10.3233/CH-151982

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 279-290, 2015

Authors: Neuber, Christin | Pufe, Johanna | Pietzsch, Jens

Article Type: Research Article

Abstract: Survivors of Hodgkin’s disease as well as of breast and lung cancer are at risk of radiation-associated cardiovascular disease. Recent studies demonstrated a correlation between cardiovascular risk factors and circulating endothelial microparticles (EMP) and thereby suggest increased EMP levels in circulation to be an early biomarker of endothelial dysfunction and cardiovascular risk. This prompted us to analyze the amount of EMP released by human aortic endothelial cells (HAEC) after exposure to different doses of X-ray (0.4, 2, 4, 6, and 20 Gy) using antibodies against the endothelial cell markers CD31, CD144, and CD146 by flow cytometry. In this pilot experiment only …CD146 proved appropriate for quantification of HAEC-derived EMP. Exposure of HAEC to different doses of X-ray did not significantly influence formation of CD146-positive EMP. However, low doses (0.4 Gy) tended to decrease EMP formation, whereas higher doses (2 or 4 Gy) slightly increased release of CD146-positive EMP. By contrast, inflammatory activation of HAEC by TPA significantly increased EMP release about 15-fold (P  <  0.01). In conclusion, under the present experimental conditions EMP did not prove a suitable biomarker for radiation-induced endothelial dysfunction in vitro . Show more

Keywords: Radiation-associated cardiovascular disease, radiation therapy, endothelial dysfunction, human aortic endothelial cells (HAEC), flow cytometry, CD146

DOI: 10.3233/CH-151987

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 291-299, 2015

Authors: Wang, Weiwei | Kratz, Karl | Behl, Marc | Yan, Wan | Liu, Yue | Xu, Xun | Baudis, Stefan | Li, Zhengdong | Kurtz, Andreas | Lendlein, Andreas | Ma, Nan

Article Type: Research Article

Abstract: Polyether ether ketone (PEEK) as a high-performance, thermoplastic implant material entered the field of medical applications due to its structural function and commercial availability. In bone tissue engineering, the combination of mesenchymal stem cells (MSCs) with PEEK implants may accelerate the bone formation and promote the osseointegration between the implant and the adjacent bone tissue. In this concept the question how PEEK influences the behaviour and functions of MSCs is of great interest. Here the cellular response of human adipose-derived MSCs to PEEK was evaluated and compared to tissue culture plate (TCP) as the reference material. Viability and morphology of …cells were not altered when cultured on the PEEK film. The cells on PEEK presented a high proliferation activity in spite of a relatively lower initial cell adhesion rate. There was no significant difference on cell apoptosis and senescence between the cells on PEEK and TCP. The inflammatory cytokines and VEGF secreted by the cells on these two surfaces were at similar levels. The cells on PEEK showed up-regulated BMP2 and down-regulated BMP4 and BMP6 gene expression, whereas no conspicuous differences were observed in the committed osteoblast markers (BGLAP, COL1A1 and Runx2). With osteoinduction the cells on PEEK and TCP exhibited a similar osteogenic differentiation potential. Our results demonstrate the biofunctionality of PEEK for human MSC cultivation and differentiation. Its clinical benefits in bone tissue engineering may be achieved by combining MSCs with PEEK implants. These data may also provide useful information for further modification of PEEK with chemical or physical methods to regulate the cellular processes of MSCs and to consequently improve the efficacy of MSC-PEEK based therapies. Show more

Keywords: Polyether ether ketone, mesenchymal stem cells, biocompatibility, cell-material interaction, osteogenic differentiation

DOI: 10.3233/CH-152001

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 301-321, 2015

Authors: Thieme, Stefan F. | Vahldiek, Janis L. | Tummler, Katja | Poch, Franz | Gemeinhardt, Ole | Hiebl, Bernhard | Lehmann, Kai S. | Hamm, B. | Niehues, Stefan M.

Article Type: Research Article

Abstract: BACKGROUND: Radiofrequency ablation (RFA) is an evolving technique in treatment of hepatic malignant tumors. By heating local tissue it leads to coagulative necrotic areas around the ablation probe. Temperature falls with increasing distance to the probe, risking incomplete necrosis at the margins of the RFA-induced lesion. Therefore, immediate non-invasive and precise detection of incomplete ablation is necessary for early enlargement of the ablation if needed. OBJECTIVES: This in vivo pig study compares early experiences of immediate post-interventional computed tomography (CT) perfusion volume analysis to macroscopic and CT image evaluation in healthy pig liver. MATERIAL AND …METHODS: RFA was performed in vivo in healthy pig livers. Different CT perfusion algorithms (Maximum slope analysis and Patlak plot) were used to quantify three different perfusion parameters. Data points were acquired from rectangular grids. These grids were semiautomatically overlayed to macroscopic images documented after liver explantation. Each data point was visually assigned to zones defined as “inner” and “outer necrotic zone”, “margin” or “vital tissue”. RESULTS: Significant differences between necrotic zones and vital tissue are shown for equivalent blood volume (p  <  0.0001), arterial flow (p  <  0.01) and flow extraction product (p  <  0.001). Looking at equivalent blood volume and flow extraction product, there were also significant differences (EquivBV: p  <  0.0001, FE: p  <  0.001) between margins, necrotic and vital areas. CONCLUSIONS: In a porcine model these early results could show that all of the used CT perfusion parameters allowed discrimination of necrosis from vital tissue after RFA at high levels of significance. In addition, the parameters EquivBV and FE that give an estimate of the tissue blood volume and the permeability, were able to precisely discern different zones also seen macroscopically. From this data CT perfusion analysis could be precise tool for measurement and visualization of ablated liver lesions and for immediate detection of incomplete ablation areas. Show more

Keywords: Liver, animal model, radiofrequency ablation (RFA), interventional radiology, multidetector computed tomography (CT), perfusion CT

DOI: 10.3233/CH-152000

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 323-331, 2015

Authors: Pschierer, K. | Grothues, D. | Rennert, J. | da Silva, N. Platz Batista | Schreyer, A.G. | Melter, M. | Stroszczysnski, C. | Jung, E.M.

Article Type: Research Article

Abstract: OBJECTIVE: Comparison of the diagnostic findings of MRI, CT and CEUS in children with benign and malignant and portal venous anomalies of the liver. MATERIALS/METHODS: Retrospective analysis of the diagnostic findings of CEUS, MRI and CT scans in 56 children (age 0–17 years) with a total of 60 benign and malignant liver lesions and anomalies of the portal vein/perfusion. All patients underwent CEUS using sulphur hexafluoride microbubbles and a multi-frequency probe (1–5 MHz, 6–9 MHz). Cine-loops were stored up to 3 minutes. MRI was performed in 38 lesions. CT was performed in 8 lesions. RESULTS: Out of the …56 patients 49 liver lesions (48 benign, 1 malignant), 9 anomalies of the portal vein/perfusion and 2 of the biliary system were detected. 16/49 lesions were analyzed histopathologically. Using CEUS, the characterization of the lesions was possible in 45 out of 49 cases. In 32 cases, CEUS provided the exact diagnosis. Only two benign lesions were falsely categorized as malignant. Findings of MRI and CEUS were concordant in 84% of cases (n  = 32/38). CEUS considered 1 benign lesion to be malignant. 2 lesions were not detectable and in 3 lesions no definite diagnosis was established using MRI. Findings of CT and CEUS were concordant in 5 of 8 cases. In 21 lesions CEUS as the only imaging modality was found to be sufficient for diagnostics. CONCLUSION: Despite the restricted indications for using CEUS in children, it offers a high diagnostic detection rate (93%) for characterization of liver lesions and portal vein anomalies. Show more

Keywords: Contrast enhanced ultrasound (CEUS), children, liver, diagnostic value, safety

DOI: 10.3233/CH-152003

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 333-345, 2015

Authors: Roch, Toralf | Kratz, Karl | Ma, Nan | Lendlein, Andreas

Article Type: Research Article

Abstract: Dendritic cells (DC) contribute to immunity by presenting antigens to T cells and shape the immune response by the secretion of cytokines. Due to their immune stimulatory potential DC-based therapies are promising approaches to overcome tolerance e.g. against tumors. In order to enforce the immunogenicity of DCs, they have to be matured and activated in vitro , which requires an appropriate cell culture substrate, supporting their survival expansion and activation. Since most cell culture devices are not optimized for DC growth, it is hypothesized that polymers with certain physicochemical properties can positively influence the DC cultures. With the aim …to evaluate the effects that polymers with different chemical compositions have on the survival, the activation status, and the cytokine/chemokine secretion profile of DC, their interaction with polystyrene (PS), polycarbonate (PC), poly(ether imide) (PEI), and poly(styrene-co -acrylonitrile) (PSAN)-based cell culture inserts was investigated. By using this insert system, which fits exactly into 24 well cell culture plates, effects induced from the culture dish material can be excluded. The viability of untreated DC after incubation with the different inserts was not influenced by the different inserts, whereas LPS-activated DC showed an increased survival after cultivation on PC, PS, and PSAN compared to tissue culture polystyrene (TCP). The activation status of DC estimated by the expression of CD40, CD80, CD83, CD86 and HLA-DR expression was not altered by the different inserts in untreated DC but slightly reduced when LPS-activated DC were cultivated on PC, PS, PSAN, and PEI compared to TCP. For each polymeric cell culture insert a distinct cytokine profile could be observed. Since inserts with different chemical compositions of the inserts did not substantially alter the behavior of DC all insert systems could be considered as alternative substrate. The observed increased survival on some polymers, which showed in contrast to TCP a hydrophobic surface, could be beneficial for certain applications such as T cell expansion and activation. Show more

Keywords: Biomaterials, dendritic cells, cell culture device, amorphous polymers

DOI: 10.3233/CH-152004

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 347-357, 2015

Authors: Prantl, Lukas | Geis, Sebastian | Lamby, Philipp | Jung, Ernst Michael

Article Type: Research Article

Abstract: Complications rates after free flap transplantation still amount up to 5% . Consequently a reliable monitoring system is of high importance in plastic and reconstructive surgery. The following guidelines provide an overview of the current opportunities for free flap planning and monitoring with ultrasound and in particular with contrast enhanced ultrasound.

Keywords: Flap monitoring, ultrasound, CEUS, contrast enhanced ultrasound, microvascularization, microperfusion

DOI: 10.3233/CH-152005

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 359-365, 2015

Authors: Sievers, Henrieke | Hirschberg, Ruth M. | Hiebl, Bernhard | Hünigen, Hana | Plendl, Johanna

Article Type: Research Article

Abstract: Human microvascular ECs from the neonatal foreskin of two donors purchased from one distributor were used in an angiogenesis assay under the same culture conditions. Different angiogenic potency was apparent in these two batches (ECang and ECnon-ang ). During the cultivation period of three weeks, ECang ran through all stages of angiogenesis starting from proliferation to migration up to the formation of three-dimensional capillary-like structures. Despite of expression of endothelial markers, ECnon-ang showed excessive intracellular storage of lipids in form of multilamellar bodies and decreased angiogenic potency in contrast to its counterpart, ECang . Results indicate that …lipid metabolism differs in ECang versus ECnon-ang . This study points up that these differences are based on the different donors and presents a novel and valuable model for the study of mechanisms of atherosclerosis in endothelial cells in vitro . Show more

Keywords: Foreskin, fetal bovine serum, multilamellar bodies, atherosclerosis

DOI: 10.3233/CH-152002

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 367-383, 2015

Authors: George, S. | Noack, M. | Vanek, M. | Rentzsch, J. | Röber, N. | Conrad, K. | Roggenbuck, D. | Küpper, J.-H.

Article Type: Research Article

Abstract: Myasthenia gravis (MG) is an autoimmune disease characterized by the formation of pathogenic autoantibodies mostly targeting the nicotinic acetylcholine receptor (AChR). The AChR is composed of two alpha subunits and one subunit of each beta, delta and gamma (fetal AChR), or epsilon (adult AChR), respectively. Serological diagnostics is commonly done by radioimmunoassay (RIA). Here we used an indirect immunofluorescence assay with MG patient sera on transiently transfected HEp-2 cells expressing selected components of the AChR. Our data show that already 12 out of 13 MG patient sera showed autoantibody binding to HEp-2 cells transfected to express the alpha subunit solely. …Interestingly, 11 out of 13 patient sera reacted positive with cells transfected to reconstitute the complete fetal AChR, but only 6 out of 13 sera showed positive signals with cells expressing the components of adult AChR. Moreover, there was no strict correlation of the serum concentration required to obtain clear-cut fluorescence signals to the antibody titer as measured by RIA. It will be an interesting topic to further investigate if the optimal serum dilution for indirect immunofluorescence as well as the autoantibody binding preferences to defined AChR subunits and to the adult versus the fetal receptor variant could provide additional predictive value in MG diagnostics. Show more

Keywords: Acetylcholine receptor, autoantibodies, cell-based assay, HEp-2 cells, indirect immunofluorescence, multiplex diagnostics, Myasthenia gravis, radioimmunoassay, serological diagnostics

DOI: 10.3233/CH-151999

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 385-396, 2015

Authors: Kuss, N. | Bauknecht, E. | Felbinger, C. | Gehm, J. | Gehm, L. | Pöschl, J. | Ruef, P.

Article Type: Research Article

Abstract: Determination of shear stresses at given shear rates with the LS 300 allows approximation of flow curves by mathematical models and to calculate viscosities of non-Newtonian fluids across the entire physiological shear range. Particular in preterm neonates, which in comparison to term neonates and adults have a substantially lower mean arterial blood pressure (MAP), rheological blood properties play a decisive role to maintain the perfusion. Whole blood viscosity was measured in umbilical cord blood taken from 34 preterm neonates using the LS 300 viscometer. In addition aggregation index, plasma viscosity, hematocrit and red blood cell (RBC) deformability was determined. The …highest quality of approximation of the flow curve of whole blood was achieved by the method of Ostwald. Shear stresses of whole blood of preterm neonates were significantly lower compared to term neonates at similar shear rates (velocity range 6.16 s–1 –50 s–1 ). With hematocrit calculated to 0.40 the related exponent (n) of the viscosity of preterm neonates (by Ostwald) showed a significant lower exponent (n) (0.71 ± 0.07) than term neonates (0.76 ± 0.06). The highest quality of approximation of the flow curve of plasma was achieved with the model of Ostwald in preterm neonates as well as in term neonates. The viscosity of plasma determined by Newton was lower for preterm neonates (0.89 ± 0.19) than for term neonates (1.04 ± 0.16). Concurrent to term neonates the whole blood of preterm neonates showed a very low aggregation index (term neonates 2.97 ± 2.10; preterm neonates 2.37 ± 1.32) and preterm neonates showed higher RBC deformability than term neonates. Because of the physiologically lower MAP and the particular viscous properties of neonatal blood special attention should be given when treating neonates with conventional blood products derived from adult donors. Show more

Keywords: Ostwald, Casson, preterm, viscosity, flow curve, yield shear stress

DOI: 10.3233/CH-152007

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 397-405, 2015