Clinical Hemorheology and Microcirculation

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.

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.

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.

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.

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.

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.

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).

This study showed rapid and significant reduction of the microcirculation in the ablation area afterpercutaneous IRE of prostatic cancer tissue.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 (ROI_liver) as well as in the portal vein (ROI_{portal vein}) using a novel quantification software (VueBox^TM). 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).

ROI_{portal 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 ROI_liver, 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.

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.

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.

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.

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.

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.

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.

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). CB₁R antagonist (AM281; 2.5 mg/kg i.v.) or CB₂R 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.

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 CB₁R antagonist, AM281, did not affect WIN effects on LPS-induced leukocyte adhesion. When pre-treated with the CB₂R 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).

Systemic administration of the CBR agonist, WIN, decreased leukocyte-adhesion and improved iridial microvascular blood flow. This effect is most likely mediated by CB₂R 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.

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.

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.

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.

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.

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.

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.

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”.

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.

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.

Comparison of the diagnostic findings of MRI, CT and CEUS in children with benign and malignant and portal venous anomalies of the liver.

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.

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.

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.