A ssessment of the cerebral microcirculation by on-line visualization has been impossible for a long time. Sidestream dark-field (SDF) imaging is a relatively new method allowing direct visualization of cerebral surface layer microcirculation using hand-held probe for direct contact with target tissue. The aim of this study was to elucidate the feasibility of studying the cerebral microcirculation in situ by SDF imaging and to assess the basic cerebral microcirculatory parameters in mechanically ventilated rabbits. Images were obtained using SDF imaging from the surface of the brain via cranio tomy. Clear high contrast SDF images were successfully obtained. Total small-vessel density was 14.6±1.8 mm/mm 2 , total all-vessel density was 17.9±1.7 mm/mm2, DeBacker score was 12.0±1.6 mm-1 and microvascular flow index was 3.0±0.0. This method seems to be applicable in animal studies with possibility to use SDF imaging also intraoperatively, providing unique opportunity to study cerebral microcirculation during various ex perimental and clinical settings., M. Šitina ... [et al.]., and Obsahuje bibliografii a bibliografické odkazy
Assessment of hepatic microcirculation by on-line visualization has been impossible for a long time. Sidestream dark-field (SDF) imaging is a relatively new method allowing direct visualization of both mucosal microcirculation and surface layers microcirculation of solid organs using hand-held probe for direct contact with target tissue. The aim of this study was to evaluate the feasibility of studying the rat hepatic microcirculation in situ by SDF imaging. The liver lobes were left in situ, and images were obtained using SDF imaging on the surface of the liver via upper midline laparotomy. Images were captured intermittently during 10-sec apnoea and recorded. The microvascular parameters were compared with previous validation studies. Clear high contrast SDF images were successfully obtained. Quantitative analysis revealed a mean FSD (functional sinusoidal density) of 402±15 cm/cm2, a sinusoidal diameter of 10.2±0.5 μm and postsinusoidal venular diameter of 33.9±13 μm. SDF imaging is a suitable noninvasive method for accurate quantification of the basic microcirculatory parameters of the liver in situ without a need to exteriorize the liver lobes. This method seems to be applicable in animal studies with possibility to use SDF imaging also intraoperatively, providing unique opportunity to study liver microcirculation during various experimental and clinical settings., V. Černý, Z. Turek, R. Pařízková., and Obsahuje seznam literatury
Flavin7 (F7) is a nutritional supplement often taken by cancer patients in Central Europe during chemo- and radiation therapy. In this study, investigation of the antiproliferative and antiangiogenic activities of this supplement were performed. Flavin7 showed antiprolif erative activity in Jurkat as well as in HeLa cells. It significantly reduced the growth of both cancer cell lines at the doses of 200 μg/ml to 20 μg/ml (p<0.001 and p<0.01, respectively). In F7-treated Jurkat cells we found a significant increase in the fraction of cells with sub-G0/G1 DNA content, which is considered to be a marker of apoptotic cell death. Apoptosis was also confirmed by annexin V staining and DNA fragmentation. Furthermore, F7 at the doses of 100 μg/ml to 4 μg/ml inhibited endothelial cell migration and capillary tube formation what indicates its potential antiangiogenic properties. Flavin7 also inhibited the activity of matrix metalloproteinases (MMPs), preferentially MM P-9, at the doses of 100 μg/ml to 4 μg/ml. Our data suggest that F7 possesses marked antiproliferative and antiangiogenic properties in vitro. Further research is needed to elucidate also its in vivo activities., J. Mojžiš, M. Šarišský, M. Pilátová, V. Voharová, L. Varinská, G. Mojžišová, A. Ostro, P. Urdzík, R. Dankovčik, L. Mirossay., and Obsahuje bibliografii a bibliografické odkazy
t would be desirable to expand the existing general knowledge concerning direct action of metals on the ovary. Nevertheless, the results of testing of iron compound on porcine ovarian cells should be interpreted carefully because iron is an essential element which could also induce changes in cellular processes. The aim of this in vitro study was 1) to examine dose-dependent effects of iron on the secretory activity of porcine ovarian granulosa cells, and 2) to outline the potential intracellular mediators mediating these effects. Specifically, we evaluated the effect of iron sulphate on the release of insulin-like growth factor I (IGF-I) and progesterone, as well as the expression of markers of proliferation (cyclin B1) and apoptosis (caspase-3) in porcine ovarian granulosa cells. Concentrations of IGF-I and progesterone were determined by RIA, cyclin B1 and caspase-3 expression by immunocytochemistry (ICC). Our results show a significantly decreased IGF-I secretion by ovarian granulosa cells after iron sulphate addition at the doses 0.5 and 1.0 mg/ml. The iron sulphate additions at do ses 0.17 and 1.0 mg/ml had no effect on progesterone secretion. In contrast, iron sulphate addition at doses 0.17-1.0 mg/ml resulted in stimulation of cyclin B1 and caspase-3 expression. In conclusion, the present results indicate a direct effect of iron on 1) secretion of growth factor IGF-I but not steroid hormone progesterone, 2) expression of markers of proliferation (cyclin B1), or 3) apoptosis (caspase-3) of porcine ovarian granulosa cells. These results support an idea that iron could play a regulatory role in porcine ovarian function: hormone release, prolif eration and apoptosis., A. Kolesarova ... [et al.]., and Obsahuje bibliografii a bibliografické odkazy
Nickel is a ubiquitous environmental pollutant, which has various effects on reproductive endocrinology. In this study, human adrenocortical carcinoma (NCI-H295R) cell line was used as an in vitro biological model to study the effect of nickel chloride (NiCl2) on the viability and steroidogenesis. The cells were exposed to different concentrations (3.90; 7.80; 15.60; 31.20; 62.50; 125; 250 and 500 μM) of NiCl2 and compared with control group (culture medium without NiCl2). The cell viability was measured by the metabolic activity assay. Production of sexual steroid hormones was quantified by enzyme linked immunosorbent assay. Following 48 h culture of the cells in the presence of NiCl2 a dose-dependent depletion of progesterone release was observed even at the lower concentrations. In fact, lower levels of progesterone were detected in groups with higher doses (≥125 μM) of NiCl2 (P<0.01), which also elicited cytotoxic action. A more prominent decrease in testosterone production (P<0.01) was also noted in comparison to that of progesterone. On the other hand, the release of 17β-estradiol was substantially increased at low concentrations (3.90 to 62.50 μM) of NiCl2. The cell viability remained relatively unaltered up to 125 μM (P>0.05) and slightly decreased from 250 μM of NiCl2 (P<0.05). Our results indicate endocrine disruptive effect of NiCl2 on the release of progesterone and testosterone in the NCI-H295R cell line. Although no detrimental effect of NiCl2 (≤62.50 μM) could be found on 17β-estradiol production, its toxicity may reflect at other points of the steroidogenic pathway., Norbert Lukac, Zsolt Forgacs, Hana Duranova, Tomas Jambor, Jirina Zemanova, Peter Massanyi, Barbara Tombarkiewicz, Shubhadeep Roychoudhury, Zuzana Knazicka., and Obsahuje bibliografii
The aim of this in vitro study was to examine the secretion activity (progesterone, 17β-estradiol and insulin-like growth factor-I) of rat ovarian fragments after molybdenum (Mo) addition. Rat ovarian fragments were incubated with ammonium molybdate (NH4)6Mo7O24.4H2O at the doses 90, 170, 330 and 500 μg.ml-1 for 24 h and compared with control group without Mo addition. Release of progesterone (P4), estradiol (17β-estradiol) and insulin-like growth factor I (IGF-I) by ovarian fragments was assessed by radioimmunoassay (RIA). Data show that P4 release by ovarian fragments was not affected by (NH4)6.Mo7O24.4H2O addition at all the doses used (90-500 μg.ml-1). However, addition of ammonium molybdate was found to cause a significant (P<0.05) dose-dependent decrease (at the doses 90, 170 and 500 μg.ml-1) in release of 17β-estradiol by ovarian fragments in comparison to control. Also, addition of ammonium molybdate significantly (P<0.05) inhibited IGF-I release at all the doses (90-500 μg.ml-1) used in the study. Results suggest ammonium molybdate induced inhibition in the release of growth factor IGF-I and its dosedependent effect on secretion of steroid hormone 17β-estradiol but not progesterone. These data contribute to new insights regarding the mechanism of action of Mo on rat ovarian functions., S. Roychoudhury, L. Detvanova, A. V. Sirotkin, R. Toman, A. Kolesarova., and Obsahuje bibliografii
Skin healing process is postnatally always associated with scarring of various extent. Based on the clinical experience of plastic surgeons, the healing after lip cleft reconstruction is surprisingly almost scar-less when it is carried out within a few first days after birth. This phenomenon is not seen in delayed cases. In order to decipher causative mechanism, we have isolated and studied principal cell populations, keratinocytes and fibroblast, from residual tissue samples after reconstructive operation (N=39) performed at various age (0-9 years). These cells play the pivotal role in the healing and that is why we focused on description of their phenotype and also functionality with respect to age. We have identified a population of remarkably small cells in explants from newborns (day 0-10). These small cells were strongly positive for markers of low differentiated keratinocytes, keratin-8 and -19, and moreover also for vimentin. In the explants cultures from older babies this population was missing. Fibroblasts from newborns and older patients differed namely in terms of nestin expression and also in the production of extracellular matrix components. We conclude that in vitro described properties of keratinocytes and fibroblasts in newborns could participate on the almost scar-less wound healing in earliest neonatal period., E. Krejčí, O. Kodet, P. Szabo, J. Borský, K. Smetana Jr., M. Grim, B. Dvořánková., and Obsahuje bibliografii
Interspecies differences in glycosidation potential in mammalian tissues represent a factor contributing to ambiguity when endobiotic and/or xenobiotic metabolic pathways are extrapolated from animals to man. Using the TLC/autoradiographic technique, we conducted an in vitro investigation involving mouse, rat, monkey, as well as human liver and kidney microsomes to evaluate their glycoconjugation potential towards 3H-labeled, purine-derived selective inhibitors of cyclin-dependent kinases such as olomoucine, bohemine, roscovitine, 6-(2-hydroxybenzyl)amino-2-(1-hydroxymethyl-2-methylpropyl)amino-9-isopropylpurine (compound A-4), and 6-(3-hydroxybenzyl)amino-2-[(1(R/S)-hydroxymethyl)propyl]amino-9-isopropylpurine (compound A-5) as aglycones. Principally, this study confirmed the aliphatic hydroxyl group of olomoucine-type inhibitors as a relatively suitable target for glucuronide, glucoside, xyloside, galactoside, and/or N-acetylaminoglucoside conjugation. Of the tissues examined, only the mouse microsomes were able to perform glucosidation and galactosidation reactions with the aglycones. On the other hand, monkey microsomes were superior to the mouse microsomes in a variety of glucuronide conjugates produced with compounds A-4 and A-5., K. Červenková, M. Belejová, Z. Chmela, M. Rypka, D. Riegrová, K. Michnová, K. Michalíková, I. Šúrová, A. Brejcha, J. Hanuš, B. Černý, K. Fuksová, L. Havlíček, J. Veselý., and Obsahuje bibliografii
Several recent studies bring evidence of cell death enhancement in photodynamic compound loaded cells by ultrasonic treatment. There are a number of hypotheses suggesting the mechanism of the harmful ultrasonic effect. One of them considers a process in the activation of photosensitizers by ultrasonic energy. Because the basis of the photodynamic damaging effect on cells consists in the production of reactive oxygen species (ROS), we focused our study on whether the ultrasound can increase ROS production within cancer cells. Particularly, we studied ROS formation in ultrasound pretreated breast adenocarcinoma cells during photodynamic therapy in the presence of chloroaluminum phthalocyanine disulfonate (ClAlPcS2). Production of ROS was investigated by the molecular probe CM-H2DCFDA. Our results show that ClAlPcS2 induces higher ROS production in the ultrasound pretreated cell lines at a concentration of 100 μM and light intensity of 2 mW/cm2. We also observed a dependence of ROS production on photosensitizer concentration and light dose. These results demonstrate that the photodynamic effect on breast cancer cells can be enhanced by ultrasound pretreatment., H. Kolářová, R. Bajgar, K. Tománková, E. Krestýn, L. Doležal, J. Hálek., and Obsahuje bibliografii
Electrophysiological investigations in mice, particularly with altered myelination, require reference data of the nerve conduction velocity (CV). CVs of different fibre groups were determined in the hindlimb of anaesthetized adult mice. Differentiation between afferent and efferent fibres was performed by recording at dorsal roots and stimulating at ventral roots, respectively. Correspondingly, recording or stimulation was performed at peripheral hindlimb nerves. Stimulation was performed with graded strength to differentiate between fibre groups. CVs of the same fibre groups were different in different nerves of the hindlimb. CVs for motor fibres were for the tibial nerve (Tib) 38.5±4.0 m/s (Aγ: 16.7±3.0 m/s), the sural nerve (Sur) 39.3±3.1 m/s (12.0±0.8 m/s) and the common peroneal nerve (Per) 46.7±4.7 m/s (22.2±4.4 m/s). CVs for group I afferents were 47.4±3.1 m/s (Tib), 43.8±3.8 m/s (Sur), 55.2±6.1 m/s (Per) and 42.9±4.3 m/s for the posterior biceps (PB). CVs of higher threshold afferents, presumably muscle and cutaneous, cover a broad range and do not really exhibit nerve specific differences. Ranges are for group II 22-38 m/s, for group III 9-19 m/s, and for group IV 0.8-0.9 m/s. Incontrovertible evidence was found for the presence of motor fibres in the sural nerve. The results are useful as references for further electrophysiological investigations particularly in genetically modified mice with myelination changes., H. Steffens, P. Dibaj, E. D. Schomburg., and Obsahuje seznam literatury