Serum C-peptide level correlates with the course of muscle tissue healing in the rabbit model of critical limb ischemia.

AIM: The therapeutic potential of adipose-derived stem cell conditioned medium (ASC-CM) was studied in the rabbit model of critical limb ischemia (CLI). METHODS: Rabbits received treatment with ASC-CM or placebo. Gastrocnemius muscle tissue was collected 35 days after ischemia induction. Ischemic changes were evaluated in hematoxylin-eosin stained tissues for early (necrotic lesions/granulation tissue) and late (fibrous scars) phases of tissue repair. The expression of proangiogenic miR-126 was also evaluated using in situ hybridization. The levels of cytokines, insulin, and C-peptide were measured in blood. RESULTS: Early repair phases were observed more often in placebo-treated samples (45.5%) than in ASC-CM-treated ones (22.2%). However, the difference was not statistically significant. We demonstrated a statistically significant positive correlation between the early healing phases in tissue samples and C-peptide levels in peripheral blood. The expression of proangiogenic miR-126 was also shown in a number of structures in all phases of ischemic tissue healing. CONCLUSION: Based on our results, we believe that treatment with ASC-CM has the potential to accelerate the healing process in ischemic tissues in the rabbit model of CLI. The whole healing process was accompanied by miR-126 tissue expression. C-peptide could be used to monitor the course of the tissue healing process.

The Role of miR-126 in Critical Limb Ischemia Treatment Using Adipose-Derived Stem Cell Therapeutic Factor Concentrate and Extracellular Matrix Microparticles.

BACKGROUND Paracrine factors secreted by adipose-derived stem cells can be captured, fractionated, and concentrated to produce therapeutic factor concentrate (TFC). The present study examined whether TFC effects could be enhanced by combining TFC with a biological matrix to provide sustained release of factors in the target region. MATERIAL AND METHODS Unilateral hind limb ischemia was induced in rabbits. Ischemic limbs were injected with either placebo control, TFC, micronized small intestinal submucosa tissue (SIS), or TFC absorbed to SIS. Blood flow in both limbs was assessed with laser Doppler perfusion imaging. Tissues harvested at Day 48 were assessed immunohistochemically for vessel density; in situ hybridization and quantitative real-time PCR were employed to determine miR-126 expression. RESULTS LDP ratios were significantly elevated, compared to placebo control, on day 28 in all treatment groups (p=0.0816, p=0.0543, p=0.0639, for groups 2-4, respectively) and on day 36 in the TFC group (p=0.0866). This effect correlated with capillary density in the SIS and TFC+SIS groups (p=0.0093 and p=0.0054, respectively, compared to placebo). A correlation was observed between miR-126 levels and LDP levels at 48 days in SIS and TFC+SIS groups. CONCLUSIONS A single bolus administration of TFC and SIS had early, transient effects on reperfusion and promotion of ischemia repair. The effects were not additive. We also discovered that TFC modulated miR-126 levels that were expressed in cell types other than endothelial cells. These data suggested that TFC, alone or in combination with SIS, may be a potent therapy for patients with CLI that are at risk of amputation.

Therapeutic Potential of Adipose-Derived Therapeutic Factor Concentrate for Treating Critical Limb Ischemia.

Transplantation of adipose-derived stem cells (ADSCs) is an emerging therapeutic option for addressing intractable diseases such as critical limb ischemia (CLI). Evidence suggests that therapeutic effects of ADSCs are primarily mediated through paracrine mechanisms rather than transdifferentiation. These secreted factors can be captured in conditioned medium (CM) and concentrated to prepare a therapeutic factor concentrate (TFC) composed of a cocktail of beneficial growth factors and cytokines that individually and in combination demonstrate disease-modifying effects. The ability of a TFC to promote reperfusion in a rabbit model of CLI was evaluated. A total of 27 adult female rabbits underwent surgery to induce ischemia in the left hindlimb. An additional five rabbits served as sham controls. One week after surgery, the ischemic limbs received intramuscular injections of either (1) placebo (control medium), (2) a low dose of TFC, or (3) a high dose of TFC. Limb perfusion was serially assessed with a Doppler probe. Blood samples were analyzed for growth factors and cytokines. Tissue was harvested postmortem on day 35 and assessed for capillary density by immunohistochemistry. At 1 month after treatment, tissue perfusion in ischemic limbs treated with a high dose of TFC was almost double (p < 0.05) that of the placebo group [58.8 ± 23 relative perfusion units (RPU) vs. 30.7 ± 13.6 RPU; mean ± SD]. This effect was correlated with greater capillary density in the affected tissues and with transiently higher serum levels of the angiogenic and prosurvival factors vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). The conclusions from this study are that a single bolus administration of TFC demonstrated robust effects for promoting tissue reperfusion in a rabbit model of CLI and that a possible mechanism of revascularization was promotion of angiogenesis by TFC. Results of this study demonstrate that TFC represents a potent therapeutic cocktail for patients with CLI, many of whom are at risk for amputation of the affected limb.

Colorectal cancer liver metastases: laparoscopic and open radiofrequency-assisted surgery.

INTRODUCTION: The liver is the most common site of colorectal metastases (colorectal liver metastases - CLM). Surgical treatment in combination with oncological therapy is the only potentially curative method. Unfortunately, only 10-25% of patients are suitable for surgery. Traditionally, open liver resection (OLR) is usually performed. However, laparoscopic liver resection (LLR) has become popular worldwide in the last two decades. AIM: To evaluate the effectiveness and benefits of radiofrequency minor LLR of CLM in comparison with OLR. MATERIAL AND METHODS: The indication for surgery was CLM and the possibility to perform minor laparoscopic or OLR not exceeding two hepatic segments according to Couinaud's classification. RESULTS: Sixty-six minor liver resections for CLM were performed. Twenty-five (37.9%) patients underwent a laparoscopic approach and 41 (62.1%) patients underwent OLR. The mean operative time was 166.4 min for LLR and 166.8 min for OLR. Average blood loss was 132.3 ±218.0 ml during LLR and 149.5 ±277.5 ml during OLR. Length of hospital stay was 8.4 ±2.0 days for LLR and 10.5 ±5.8 days for OLR. All resections were R0. There was no case of mortality. Postoperative complications were recognized in 9 (13.6%) patients: 8 in the group of OLR patients and 1 in the LLR group. The median survival time for LLR was 70.5 months and for OLR 61.9 months. The 5-year overall survival rate was higher for LLR vs. OLR - 82.1% vs. 69.8%. The average length of disease-free interval after LLR was greater (52.2 months) in comparison with OLR (49.4%). The 5-year disease-free interval was 63.2% for LLR and 58% for OLR. CONCLUSIONS: Outcomes and oncological radicality of minor laparoscopic liver resections of CLM are comparable to outcomes of OLR.

Technical development of a new semispherical radiofrequency bipolar device (RONJA): ex vivo and in vivo studies.

The aim of this study is to inform about the development of a new semispherical surgical instrument for the bipolar multielectrode radiofrequency liver ablation. Present tools are universal; however they have several disadvantages such as ablation of healthy tissue, numerous needle punctures, and, therefore, longer operating procedure. Our newly designed and tested semispherical surgical tool can solve some of these disadvantages. By conducting an in vivo study on a set of 12 pigs, randomly divided into two groups, we have compared efficiency of the newly developed instrument with the commonly used device. Statistical analysis showed that there were no significant differences between the groups. On average, the tested instrument RONJA had shorter ablation time in both liver lobes and reduced the total operating time. The depth of the thermal alteration was on average 4 mm larger using the newly tested instrument. The new radiofrequency method described in this study could be used in open liver surgery for the treatment of small liver malignancies (up to 2 cm) in a single application with the aim of saving healthy liver parenchyma. Further experimental studies are needed to confirm these results before clinical application of the method in the treatment of human liver malignancies.

In situ effects of urban river pollution on the mudsnail Potamopyrgus antipodarum as part of an integrated assessment.

The freshwater mudsnail (Potamopyrgus antipodarum) is sensitive to toxicity of both sediment and water and also to the endocrine disrupting compounds (EDC) at environmentally relevant concentrations. This study determined effects of in situ exposure of P. antipodarum as a part of a complex assessment of the impact of a city metropolitan area with large waste water treatment plant (WWTP) for 0.5 million population equivalents on two urban rivers. The study combined the in situ biotest with detailed chemical analyses and a battery of in vitro bioassays of both sediment and water. Passive sampling of river water was conducted during the course of exposure of the mudsnail. P. antipodarum was exposed for 8 weeks in cages permeable to sediment and water at localities up- and down-stream of the city of Brno, Czech Republic and downstream of the WWTP in two rivers. Greater mortality and significantly decreased embryo production of P. antipodarum were observed immediately downstream of the city of Brno. P. antipodarum exposed at locations downstream of the metropolitan area and WWTP exhibited greater mortality, while numbers of embryos produced by surviving individuals were comparable or slightly greater than for individuals held at the least polluted location. Comparisons with results of chemical analysis and in vitro assays indicate occurrence of groups of compounds contributing to observed effects. Differences in mortalities of mudsnails among sites corresponded well with in vitro cytotoxicity and concentrations of metals. The results of this study confirm the applicability of this novel field biotest with P. antipodarum for the evaluation of the effects of river pollution on metazoans, especially as suitable in situ part of integrative contamination assessment.

Radiofrequency-assisted liver resections: comparison of open and laparoscopic techniques.

BACKGROUND/AIMS: After the first reported laparoscopic liver resection (LLR) twenty years ago, liver surgery still remains one of the last areas of resistance to the offensive of laparoscopy. Radiofrequency assisted laparoscopic liver resection has been recently developed technique for treatment of primary and secondary liver tumors. METHODOLOGY: Over a 5-year period, a total of 134 laparoscopic and open radiofrequency assisted operations were performed in a single institution. LLR was done in 47 patients, and open liver resection (OLR) in 87 patients. RESULTS: The study selection criteria were fulfilled by 134 patients. The mean blood loss for LLR was 68.7 mL, the difference between the groups was significant with lower median of blood loss using laparoscopy (p=0.046). The mean of length of hospital stay in LLR was 7.5 days versus 8.7 days in OLR (p=0.071). The 5-year survival rate was 67.0% after LLR and 63.8% after OLR. The 5-year disease-free survival rate was 59.4% after LLR, and 62.2% after OLR. The difference between groups was not statistically significant. CONCLUSIONS: Laparoscopic liver resection is safe and feasible procedure. The hand-assisted laparoscopic radiofrequency technique can be applied effectively for selected patients. Preliminary oncological results suggest non-inferiority of laparoscopic to open procedures.

Occurrence of perfluoroalkyl substances in fish and water from the Svitava and Svratka rivers, Czech Republic.

Perfluoroalkyl substances (PFHxS, FHUEA, PFOA, PFOS, FOSA, N-methyl FOSA and PFNA) from seven sites on the Svitava and Svratka rivers in the Brno conurbation (Czech Republic) were determined in fish blood plasma and water. Concentrations of PFHxS, FHUEA, FOSA, and N-methyl FOSA were below detection limits. Major compound in fish blood was PFOS (38.9-57.8 ng mL(-1)), followed by PFNA and PFOA. In water, the major compound detected was PFOA (1.7-178.0 ng mL(-1)), followed by PFOS and PFNA. A significant (p < 0.05) correlation for PFOA concentration in blood plasma and water was found (r = 0.74).

Assessment of contamination of the Svitava and Svratka rivers in the Czech Republic using selected biochemical markers.

The aim of the present study is to assess aquatic ecosystem contamination using selected biochemical markers: cytochrome P450, ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), tripeptide glutathione, vitellogenin, and 11-ketotestosterone in chub (Leuciscus cephalus L.). Seven locations on the Svitava and Svratka rivers (in the Brno conurbation, Czech Republic) were assessed. The results were compared with the levels of the most important inductors of these biomarkers: organic pollutants hexachlorocyclohexane (HCH), hexachlorobenzene (HCB), DDT and its metabolites, polychlorinated biphenyls, and polycyclic aromatic hydrocarbons in bottom sediment, fish muscle, and semipermeable membrane devices (SPMDs) and metals in sediment. The highest levels of pollutants were observed at sites situated downstream from Brno, especially at Modrice and Rajhradice. Significant positive correlations (p < 0.05) were found between EROD activity and HCH concentration in SPMDs, and also between GST and EROD activity with HCB concentration in muscle, after adjusting for age.

Passive sampling methods for monitoring endocrine disruptors in the Svratka and Svitava rivers in the Czech Republic.

Endocrine-disrupting chemicals (EDC) are manmade or natural chemicals that have the ability to interfere with the endocrine system of animals. They have not been monitored systematically in the Czech Republic. The goal of the present study was the characterization of aquatic environmental pollution from the Brno (Czech Republic) city agglomeration focusing on EDC. Passive sampling devices, as well as semipermeable membrane devices (SPMD) and polar organic chemical integrative samplers (POCIS), were used for the pilot assessment of EDC. They were deployed for 21- to 28-d periods at nine locations in the Svratka and Svitava Rivers, Brno, Czech Republic, including at the inlet and outlet of Brno's wastewater treatment plant. The SPMDs were used to monitor nonpolar compounds such as polychlorinated biphenyls (PCBs), polynuclear aromatic hydrocarbons (PAHs), and organochlorinated pesticides (OCP). The POCIS were used to monitor polar compounds such as pesticides and their metabolites, perfluoro-organic compounds (PFOC), and pharmaceuticals. The passive samplers allowed very low detection limits for soluble (bioavailable) fractions of pollutants. The contribution of PAH, PCB, and HCB in sewage water to pollution of the Svratka River was low. The Brno wastewater treatment plant was identified as the main source of pharmaceuticals, triclosan, methyl triclosan, and some polar pesticides.

1-Hydroxypyrene--a biochemical marker for PAH pollution assessment of aquatic ecosystem.

The aim of the present study was to assess aquatic contamination by polycyclic aromatic hydrocarbons (PAH), using the 1-hydroxypyrene (1-OHP) content in fish bile as a biochemical marker. A total of 71 chub (Leuciscus cephalus L.) were collected from seven locations on the Svitava and Svratka rivers in and around the industrial city of Brno, Czech Republic. The levels of 1-OHP were determined by reverse phase HPLC with fluorescence detection after deconjugation. Normalising the molar concentration of the biliary 1-OHP to the biliary protein content reduced sample variation. The content of 1-OHP was correlated with the PAH level in bottom sediment and semi-permeable membrane devices (SPMD), which was analyzed by a combination of HPLC/FLD and GC/MS methods. The highest mean values of 1-OHP were found in fish caught at the Svratka River at locations Modrice (169.2 ± 99.7 ng · mg(-1) protein) and Rajhradice (152.2 ± 79.7 ng · mg(-1) protein), which are located downstream from Brno. These values were significantly (P < 0.05) higher than those obtained from localities Knínicky (98.4 ± 66.1 ng · mg(-1) protein) and Bílovice nad Svitavou (64.1 ± 31.4 ng · mg(-1) protein). The lowest contents of PAH in sediment and SPMD were found at location Knínicky (1.5 mg · kg(-1) dry mass and 19.4 ng · L(-1), respectively). The highest contents of PAH in sediment and SPMD were found in Rajhradice (26.0 mg · kg(-1) dry mass) and Svitava before junction (65.4 ng · L(-1)), respectively. A Spearman correlation test was applied to determine the relationship between biliary 1-OHP and the sum of PAH in sediment and SPMD. A positive, but no statistically significant correlation was found. The main impact sources of elevated level of PAHs in sites located downstream from Brno are most probably intensive industrial and agricultural activities and domestic waste.

Fish biochemical markers as a tool for pollution assessment on the Svitava and Svratka rivers, Czech Republic.

OBJECTIVES: The study was designed to assess the pollution of the Svitava and Svratka rivers in and around the industrial city of Brno (Czech Republic) by persistent organic pollutants using selected biochemical markers in chub. DESIGN: Levels of selected biochemical markers were measured in liver and plasma samples of chub. The concentrations of persistent organic pollutants (POPs) were determined in bottom sediment, semi-permeable membrane devices (SPMDs) and muscle samples, and consequently used for correlation with biochemical markers. RESULTS: Significant alterations (p < 0.05) in some biochemical markers were observed and associated with combined exposure to pollutants. The highest levels of pollutants were found at sites situated downstream from Brno. The most widespread changes were identified in the function of phase I detoxifying enzymes. Significant positive correlations were observed in cytochrome P450 content and DDT concentration in the semi-permeable membrane device (p = 0.019, rs = 0.886), and between ethoxyresorufin-O-deethylase activity and content of DDT (p = 0.041, rs = 0.352) and polychlorinated biphenyls (p = 0.034, rs = 0.365) in muscle tissues of indicator fish. CONCLUSION: The results presented in our study indicate the highest contamination of sites situated downstream from Brno, where the intensive industrial and agricultural activities as well as domestic waste and sewage most probably comprise the main impact sources of the enhanced level of pollutants and some biochemical markers in fish.

Biliary 1-hydroxypyrene as a biomarker of exposure to polycyclic aromatic hydrocarbons in fish.

OBJECTIVES: The aim of this study was the assessment of the Svitava and Svratka rivers contamination by polycyclic aromatic hydrocarbons (PAH) using 1-hydroxypyrene (1-OHP) concentrations in fish bile as a biomarker. DESIGN: Levels of 1-OHP were determined by reverse phase HPLC with fluorescence detection. For valid assessment of bile accumulation levels, the 1-OHP concentration was normalized to the biliary protein content. The content of 1-OHP was correlated with the PAH content of river bottom sediments. RESULTS: The highest level of 1-OHP in fish bile (136.1 ng.mg-1 protein) was found at the locality Rajhradice, which is situated downstream of Brno. Also the greatest level of sum of PAHs was found at this locality (17.1 microg.g-1 dry mass). Significant positive correlation (p< or =0.05) between the level of 1-OHP and sum of PAHs in sediment was found only in case of sediments collected in the same month as fish samples (in June). CONCLUSION: Our results document that 1-OHP in fish bile is a suitable biochemical marker for the assessment of aquatic ecosystem contamination by PAHs.