Continuous activation of the immune system inside a tissue can lead to remodelling of the tissue structure and creation of a specific microenvironment, such as during the tumour development. Chronic inflammation is a central player in stimulating changes that alter the tissue stroma and can lead to fibrotic evolution. In the colon mucosa, regulatory mechanisms, including TGF-β1, avoid damaging inflammation in front of the continuous challenge by the intestinal microbiome. Inducing either DSS colitis or AOM colorectal carcinogenesis in AVN-Wistar rats, we evaluated at one month after the end of each treatment whether immunological changes and remodelling of the collagen scaffold were already in development. At this time point, we found in both models a general downregulation of pro-inflammatory cytokines and even of TGF-β1, but not of IL-6. Moreover, we demonstrated by multi-photon microscopy the simultaneously presence of pro-fibrotic remodelling of the collagen scaffold, with measurable changes in comparison to the control mucosa. The scaffold was significantly modified depending on the type of induced stimulation. These results suggest that at one month after the end of the DSS or AOM inductions, a smouldering inflammation is present in both induced conditions, since the pro-inflammatory cytokines still exceed, in proportion, the local homeostatic regulation of which TGF-β1 is a part (inflammatory threshold). Such an inflammation appears sufficient to sustain remodelling of the collagen scaffold that may be taken as a possible pathological marker for revealing pre-neoplastic inflammation.
- Klíčová slova
- AOM, DSS-induced colitis, IL-6, chronic inflammation, collagen, colorectal cancer, tissue scaffold, tumour niche,
- Publikační typ
- časopisecké články MeSH
Remodeling of nanoscopic structures is not just crucial for cell biology, but it is also at the core of bioinspired materials. While the microtubule cytoskeleton in cells undergoes fast adaptation, adaptive materials still face this remodeling challenge. Moreover, the guided reorganization of the microtubule network and the correction of its abnormalities is still a major aim. This work reports new findings for externally triggered microtubule network remodeling by nanosecond electropulses (nsEPs). At first, a wide range of nsEP parameters, applied in a low conductivity buffer, is explored to find out the minimal nsEP dosage needed to disturb microtubules in various cell types. The time course of apoptosis and microtubule recovery in the culture medium is thereafter assessed. Application of nsEPs to cells in culture media result in modulation of microtubule binding properties to end-binding (EB1) protein, quantified by newly developed image processing techniques. The microtubules in nsEP-treated cells in the culture medium have longer EB1 comets but their density is lower than that of the control. The nsEP treatment represents a strategy for microtubule remodeling-based nano-biotechnological applications, such as engineering of self-healing materials, and as a manipulation tool for the evaluation of microtubule remodeling mechanisms during various biological processes in health and disease.
- Klíčová slova
- bioinspired materials, cytoskeleton remodeling, end-binding proteins, microtubules, nanosecond pulsed electric fields,
- MeSH
- elektřina * MeSH
- lidé MeSH
- mikrotubuly metabolismus MeSH
- nádorové buněčné linie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In obesity, the skeletal muscle capillary network regresses and the insulin-mediated capillary recruitment is impaired. However, it has been shown that in the early stage of advanced obesity, an increased functional vascular response can partially compensate for other mechanisms of insulin resistance. The present study aimed to investigate the changes in the capillary network around individual muscle fibres during the early stage of obesity and insulin resistance in mice using 3D analysis. Capillaries and muscle fibres of the gluteus maximus muscles of seven high-fat-diet-induced obese and insulin-resistant mice and seven age-matched lean healthy mice were immunofluorescently labelled in thick transverse muscle sections. Stacks of images were acquired using confocal microscope. Capillary network characteristics were estimated by methods of quantitative image analysis. Muscle fibre typing was performed by histochemical analysis of myosin heavy chain isoforms on thin serial sections of skeletal muscle. Capillary length per muscle fibre length and capillary length per muscle fibre surface were increased by 27% and 23%, respectively, around small muscle fibres in obese mice, while there were no significant comparative differences around large fibres of obese and lean mice. Furthermore, the capillarization was larger around small compared to large fibres and there was a shift toward fast type myosin heavy chain isoforms, with no significant changes in muscle fibre diameters, tortuosity and anisotropy in obese mice. Overall, the results show that obese insulin-resistant mice have selective increase in capillarization around small predominantly intermediate muscle fibres, which is most likely related to the impaired glucose metabolism characteristic of type 2 diabetes.
- Klíčová slova
- 3D analysis, Capillary network, Fibre type, Insulin resistance, Obesity, Skeletal muscle,
- MeSH
- inzulinová rezistence MeSH
- kapiláry chemie metabolismus MeSH
- kosterní svaly chemie metabolismus MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- obezita metabolismus patologie MeSH
- těžké řetězce myosinu analýza metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- těžké řetězce myosinu MeSH
Tubulin self-assembly into microtubules is a fascinating natural phenomenon. Its importance is not just crucial for functional and structural biological processes, but it also serves as an inspiration for synthetic nanomaterial innovations. The modulation of the tubulin self-assembly process without introducing additional chemical inhibitors/promoters or stabilizers has remained an elusive process. This work reports a versatile and vigorous strategy for controlling tubulin self-assembly by nanosecond electropulses (nsEPs). The polymerization assessed by turbidimetry is dependent on nsEPs dosage. The kinetics of microtubules formation is tightly linked to the nsEPs effects on structural properties of tubulin, and tubulin-solvent interface, assessed by autofluorescence, and the zeta potential. Moreover, the overall size of tubulin assessed by dynamic light scattering is affected as well. Additionally, atomic force microscopy imaging reveals the formation of different assemblies reflecting applied nsEPs. It is suggested that changes in C-terminal modification states alter tubulin polymerization-competent conformations. Although the assembled tubulin preserve their integral structure, they might exhibit a broad range of new properties important for their functions. Thus, these transient conformation changes of tubulin and their collective properties can result in new applications.
- Klíčová slova
- microtubules, nanosecond pulsed electric field, self-assembly, tubulin,
- MeSH
- elektřina * MeSH
- hydrodynamika MeSH
- kinetika MeSH
- kvarterní struktura proteinů MeSH
- mikrotubuly metabolismus MeSH
- molekulární modely MeSH
- multimerizace proteinu * MeSH
- tubulin chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- tubulin MeSH
Capillary network characteristics are invaluable for diagnostics of muscle diseases. Biopsy material is limited in size and mostly not accessible for intensive research. Therefore, especially in human tissue, studies are performed on autopsy material. To approach the problem whether it is reliable to deduce hypotheses from autopsy material to explain physiological and pathological processes, we studied capillarity in pig soleus muscle 1 and 24 hr after death. Capillaries and muscle fibers were immunofluorescently marked, and images were acquired with a confocal microscope. Characteristics of the capillary network were estimated by image analysis methods using several plugins of the Ellipse program. Twenty-four hours after death, the measured characteristics of the capillary network differ by up to 50% when compared with samples excised 1 hr after death. Muscle fiber diameter, the measured capillary length, and tortuosity were reduced, and capillary network became more anisotropic. The main postmortem change that affects capillaries is evidently geometric deformation of muscle tissue. In conclusion, when comparing results from biopsy samples with those from autopsy samples, the effect of postmortem changes on the measured parameters must be carefully considered.
- Klíčová slova
- capillaries, confocal microscopy, image analysis, pig, postmortem changes, skeletal muscle,
- MeSH
- kapiláry patologie ultrastruktura MeSH
- konfokální mikroskopie metody MeSH
- kosterní svaly krevní zásobení patologie MeSH
- počítačové zpracování obrazu metody MeSH
- posmrtné změny MeSH
- prasata MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
During pregnancy, oxygen diffuses from maternal to fetal blood through villous trees in the placenta. In this paper, we simulate blood flow and oxygen transfer in feto-placental capillaries by converting three-dimensional representations of villous and capillary surfaces, reconstructed from confocal laser scanning microscopy, to finite-element meshes, and calculating values of vascular flow resistance and total oxygen transfer. The relationship between the total oxygen transfer rate and the pressure drop through the capillary is shown to be captured across a wide range of pressure drops by physical scaling laws and an upper bound on the oxygen transfer rate. A regression equation is introduced that can be used to estimate the oxygen transfer in a capillary using the vascular resistance. Two techniques for quantifying the effects of statistical variability, experimental uncertainty and pathological placental structure on the calculated properties are then introduced. First, scaling arguments are used to quantify the sensitivity of the model to uncertainties in the geometry and the parameters. Second, the effects of localized dilations in fetal capillaries are investigated using an idealized axisymmetric model, to quantify the possible effect of pathological placental structure on oxygen transfer. The model predicts how, for a fixed pressure drop through a capillary, oxygen transfer is maximized by an optimal width of the dilation. The results could explain the prevalence of fetal hypoxia in cases of delayed villous maturation, a pathology characterized by a lack of the vasculo-syncytial membranes often seen in conjunction with localized capillary dilations.
- MeSH
- biologické modely * MeSH
- choriové klky embryologie MeSH
- difuze MeSH
- kapiláry metabolismus fyziologie MeSH
- krevní oběh * MeSH
- kyslík metabolismus MeSH
- lidé MeSH
- placenta krevní zásobení MeSH
- plod krevní zásobení MeSH
- těhotenství MeSH
- zobrazování trojrozměrné * MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- kyslík MeSH
Quantitative measurements of geometric forms or counting of objects in microscopic specimens is an essential tool in studies of microstructure. Confocal stereology represents a contemporary approach to the evaluation of microscopic structures by using a combination of stereological methods and confocal microscopy. 3-D images acquired by confocal microscopy can be used for the estimation of geometrical characteristics of microscopic structures by stereological methods, based on the evaluation of optical sections within a thick slice and using computer-generated virtual test probes. Such methods can be used for estimating volume, number, surface area and length using relevant spatial probes, which are generated by specific software. The interactions of the probes with the structure under study are interactively evaluated. An overview of the methods of confocal stereology developed during the past 30 years is presented. Their advantages and pitfalls in comparison with other methods for measurement of geometrical characteristics of microscopic structures are discussed.
Chloroplast number per cell is a frequently examined quantitative anatomical parameter, often estimated by counting chloroplast profiles in two-dimensional (2D) sections of mesophyll cells. However, a mesophyll cell is a three-dimensional (3D) structure and this has to be taken into account when quantifying its internal structure. We compared 2D and 3D approaches to chloroplast counting from different points of view: (i) in practical measurements of mesophyll cells of Norway spruce needles, (ii) in a 3D model of a mesophyll cell with chloroplasts, and (iii) using a theoretical analysis. We applied, for the first time, the stereological method of an optical disector based on counting chloroplasts in stacks of spruce needle optical cross-sections acquired by confocal laser-scanning microscopy. This estimate was compared with counting chloroplast profiles in 2D sections from the same stacks of sections. Comparing practical measurements of mesophyll cells, calculations performed in a 3D model of a cell with chloroplasts as well as a theoretical analysis showed that the 2D approach yielded biased results, while the underestimation could be up to 10-fold. We proved that the frequently used method for counting chloroplasts in a mesophyll cell by counting their profiles in 2D sections did not give correct results. We concluded that the present disector method can be efficiently used for unbiased estimation of chloroplast number per mesophyll cell. This should be the method of choice, especially in coniferous needles and leaves with mesophyll cells with lignified cell walls where maceration methods are difficult or impossible to use.
- Klíčová slova
- Chloroplast counting, Norway spruce (Picea abies L. Karst.), confocal microscopy, coniferous needle structure, disector method, mesophyll, profile counting, stereology.,
- MeSH
- biologické modely MeSH
- chloroplasty metabolismus MeSH
- mezofylové buňky cytologie metabolismus MeSH
- smrk MeSH
- zobrazování trojrozměrné metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Studies of the capillary bed characterized by its length or length density are relevant in many biomedical studies. A reliable assessment of capillary length from two-dimensional (2D), thin histological sections is a rather difficult task as it requires physical cutting of such sections in randomized directions. This is often technically demanding, inefficient, or outright impossible. However, if 3D image data of the microscopic structure under investigation are available, methods of length estimation that do not require randomized physical cutting of sections may be applied. Two different rat brain regions were optically sliced by confocal microscopy and resulting 3D images processed by three types of capillary length estimation methods: (1) stereological methods based on a computer generation of isotropic uniform random virtual test probes in 3D, either in the form of spatial grids of virtual "slicer" planes or spherical probes; (2) automatic method employing a digital version of the Crofton relations using the Euler characteristic of planar sections of the binary image; and (3) interactive "tracer" method for length measurement based on a manual delineation in 3D of the axes of capillary segments. The presented methods were compared in terms of their practical applicability, efficiency, and precision.
- MeSH
- biometrie metody MeSH
- kapiláry anatomie a histologie MeSH
- konfokální mikroskopie metody MeSH
- krysa rodu Rattus MeSH
- laboratorní automatizace metody MeSH
- mozek anatomie a histologie MeSH
- zobrazování trojrozměrné metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Testate amoeba (TA) assemblages were collected in 2005 from four ponds in Komořany (Prague, Czech Republic). An analysis of seasonal taxonomic variability of TA populations and its correlation with the limnological characteristics of the area (temperature, pH, total organic carbon, nitrogen, phosphorus, heavy metals, etc.) was performed. The predominant genera were Difflugia, Arcella, and Centropyxis. The most significant changes in the TA community occurred between March and July. Arcella genus dominated in March and April; in May, Arcella and Centropyxis genera were present in the same amount; in June, Arcella genus disappeared, and Difflugia genus started to dominate the community. A multivariate redundancy analysis showed statistically significant correlations between the environmental parameters and the composition of the TA community. The results indicate a negative correlation between TA quantities and Ni, Cd, PAH, Mn, As, and Pb. TA were also affected by concentrations of NH4(+), NO3(-), and P, as well as by temperature variations. The observed correlations between the species composition and environmental parameters can be used in paleoecological interpretations of fossil TA communities. Our results also prove the suitability of TA as water quality indicators in urban areas.
- MeSH
- Amoeba klasifikace izolace a purifikace MeSH
- biodiverzita MeSH
- ekosystém * MeSH
- fylogeneze MeSH
- roční období MeSH
- rybníky chemie parazitologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
