Petr Karen

Topological Properties and Spatial Organization of Villous Capillaries in Normal and Diabetic Placentas

Spatial arrangement and complexity of the capillary bed of placental terminal villi were analyzed in 9 normal and 11 diabetic placentas. Specimens were taken by systematic random sampling, fixed and stained in toto, and embedded in paraffin. Fifteen fields of view were sampled systematically from 120-µm-thick sections of specimens and examined using a confocal laser scanning microscope. Series of thin optical sections of terminal villi and their developmental forms were recorded by the confocal microscope and used as initial data for three-dimensional visualization of the spatial arrangement of villous capillaries. Vascular topology and branching were studied by focusing through the villus, making a schematic drawing of the villous capillary bed and counting redundant capillary connections. It was found that the basic arrangement of villous capillaries is similar in both normal and diabetic placentas. Nevertheless, the proportion of simple forms of the capillary bed without redundant connections is significantly higher in normal placentas and the mean number of redundant connections per villus is significantly higher in diabetic placentas. It is concluded that both the longitudinal growth and branching of capillaries contribute to the increase in the placental capillary bed in late gestation and that the capillary bed of diabetic villi is more complicated due to more intense capillary branching.

Volume reconstruction of large tissue specimens from serial physical sections using confocal microscopy and correction of cutting deformations by elastic registration

A set of methods leading to volume reconstruction of biological specimens larger than the field of view of a confocal laser scanning microscope (CLSM) is presented. Large tissue specimens are cut into thin physical slices and volume data sets are captured from all studied physical slices by CLSM. Overlapping spatial tiles of the same physical slice are stitched in horizontal direction. Image volumes of successive physical slices are linked in axial direction by applying an elastic registration algorithm to compensate for deformations because of cutting the specimen. We present a method enabling us to keep true object morphology using a priori information about the shape and size of the specimen, available from images of the cutting planes captured by a USB light microscope immediately before cutting the specimen by a microtome. The errors introduced by elastic registration are evaluated using a stereological point counting method and the Procrustes distance. Finally, the images are enhanced to compensate for the effect of the light attenuation with depth and visualized by a hardware accelerated volume rendering. Algorithmic steps of the reconstruction, namely elastic registration, object morphology preservation, image enhancement, and volume visualization, are implemented in a new Rapid3D software package. Because confocal microscopes get more and more frequently used in scientific laboratories, the described volume reconstruction may become an easy‐to‐apply tool to study large biological objects, tissues, and organs in histology, embryology, evolution biology, and developmental biology. In this work, we demonstrate the reconstruction using a postcranial part of a 17‐day‐old laboratory Wistar rat embryo. Microsc. Res. Tech., 2009.

Role of nifedipine-sensitive sympathetic vasoconstriction in maintenance of high blood pressure in spontaneously hypertensive rats: effect of Gi-protein inactivation by pertussis toxin.

Background High blood pressure (BP) in spontaneously hypertensive rats (SHRs) is attributed to excessive activity of sympathetic nervous system (SNS) and relative nitric oxide deficiency. An important part of SNS hypertensive action is exerted by calcium influx through L-type of voltage-dependent calcium channels (L-VDCC). The overexpression of pertussis toxin (PTX)-sensitive inhibitory G-proteins (Gi) participating in the development and maintenance of high BP in SHRs suggested us to study Gi-protein involvement in the pathway through which noradrenergic vasoconstriction and calcium influx can be coupled. Method The participation of main vasoactive systems (angiotensin II, norepinephrine, nitric oxide) in BP maintenance was investigated in conscious SHR and WKY rats (half of them being pretreated with PTX, 10 μg/kg i.v., 48 h before the experiment). To evaluate the contribution of Gi-proteins and L-VDCC to vasoconstriction induced by exogenous norepinephrine, dose–response curves were determined before and after acute nifedipine administration. Results PTX pretreatment of SHRs significantly decreased BP and reduced sympathetic vasoconstriction, which was partially substituted by enhanced angiotensin II-dependent vasoconstriction. PTX pretreatment also reduced nitric oxide-dependent vasodilation in both rat strains. PTX pretreatment of SHRs decreased BP component sensitive to acute blockade of calcium entry by nifedipine. In both strains, PTX pretreatment as well as acute nifedipine administration caused substantial rightward shift of norepinephrine dose–response curves (without additive effects of both treatments). Conclusion The enhanced contribution of SNS to hypertension maintenance in SHRs is mediated by Gi-protein-coupled pathway controlling calcium influx through L-VDCC.

Three-dimensional reconstructions from non-deparaffinized tissue sections.

Three-dimensional (3-D) reconstruction from microscopic images represents a useful tool for the study of biological structures in embryology and developmental biology. However, it is usually necessary to cope with many difficulties connected with the preparation of specimens. In order to minimize mutual displacement of structures in successive sections, the applicability of non-deparaffinized tissue sections for 3-D reconstruction was tested. Chicken embryos were fixed and stained in toto with eosin and then embedded in paraffin. About 30-μm-thick non-deparaffinized serial sections were used for obtaining initial data for 3-D reconstruction of larger stacks of embryonic bodies using either fluorescence or confocal microscope. The same sections served for both collecting optical serial sections of mesonephros as source images for its 3-D reconstruction, and immunohistochemical detection of fibronectin, laminin and vimentin. It was found that sections with retained paraffin preserve the mutual spatial relationships of tissue components as well as provide an excellent differentiation of structure. It makes the process of 3-D reconstruction easier. The localization of the products of immunohistochemical reactions demonstrated the co-localization of fibronectin and laminin in basal laminas and the presence of vimentin in glomeruli and mesenchymal tissue. The use of non-deparaffinized sections represents a less time consuming and more effective alternative to thin histological sections for the purpose of 3-D reconstruction, and enables further application of material.

Ca2+ sensitization and Ca2+ entry in the control of blood pressure and adrenergic vasoconstriction in conscious Wistar-Kyoto and spontaneously hypertensive rats.

Background: Calcium entry through nifedipine-sensitive L-type voltage-dependent calcium channels (L-VDCC) is augmented in spontaneously hypertensive rats (SHR) characterized by enhanced sympathetic vasoconstriction. However, the changes of calcium sensitization mediated by RhoA/Rho kinase pathway are less understood. Methods and results: The participation of calcium entry and calcium sensitization in the control of blood pressure (BP) and vascular contraction was studied in SHR and normotensive Wistar–Kyoto (WKY) rats. The acute administration of fasudil (Rho kinase inhibitor) caused BP decrease which lasted longer in SHR. Fasudil also attenuated adrenergic contraction in femoral or mesenteric arteries of WKY and SHR. BP reduction elicited by fasudil in WKY was more pronounced than that induced by L-VDCC blocker nifedipine (−33 ± 2 vs. −15 ± 3% of baseline BP, P < 0.001), whereas both inhibitors were similarly effective in SHR (−36 ± 4 vs. −41 ± 2%). Fasudil pretreatment also attenuated BP elevation elicited by L-VDCC agonist BAY K8644 more in WKY than in SHR (−63 ± 4 vs. −42 ± 5%, P < 0.001), indicating reduced calcium sensitization in SHR. Moreover, fasudil pretreatment shifted norepinephrine dose–response curves to the right more in WKY than in SHR. The additional nifedipine pretreatment shifted these curves further to the right but this shift was more pronounced in SHR than in WKY. Thus adrenergic vasoconstriction is more dependent on L-VDCC in SHR and on RhoA/Rho kinase pathway in WKY rats. Conclusion: Ca2+ sensitization mediated by RhoA/Rho kinase pathway is attenuated in SHR compared with normotensive WKY rats. This might be a part of the compensation for enhanced Ca2+ entry through L-VDCC in genetic hypertension.

Influence of calcium-dependent potassium channel blockade and nitric oxide inhibition on norepinephrine-induced contractions in two forms of genetic hypertension

The activation of Ca(2+)-dependent K(+) channels (BK(Ca)) leads to the attenuation of vascular contraction. Our study aimed to evaluate BK(Ca) influence on norepinephrine (NE)-induced femoral artery contraction in two forms of genetic hypertension. NE dose-response curves were studied before and after BK(Ca) blockade or after combined blockade of BK(Ca) and NO synthase (NOS) in femoral arteries with intact endothelium from normotensive Wistar (WIS), hypertensive hereditary hypertriglyceridemic (HTG), or spontaneously hypertensive rats (SHR). NE-induced contractions of femoral arteries were augmented in both hypertensive strains compared with Wistar rats, but acetylcholine-induced relaxation was impaired in HTG only. The increase of basal vascular tone of isolated arteries after BK(Ca) blockade was similar in all rat strains, but subsequent NOS inhibition increased basal vascular tone more in vessels from both hypertensive rat strains. NOS inhibition increased sensitivity to NE in all strains, but BK(Ca) blockade in SHR only. Neither treatment enhanced maximal NE-induced contraction. NO-dependent attenuation of NE-induced contractions was greater in SHR than HTG or Wistar vessels, whereas large conductance Ca(2+)-dependent K(+) channels may play a greater role in modulating vascular contraction in the severe form of hypertension.

Contribution of Ca²⁺-dependent Cl⁻ channels to norepinephrine-induced contraction of femoral artery is replaced by increasing EDCF contribution during ageing.

The activation of Ca2+-dependent Cl− channels during norepinephrine-induced contraction of vascular smooth muscle was suggested to depolarize cell membrane and to increase Ca2+ entry. Hypertension and ageing are associated with altered Ca2+ handling including possible activation of Ca2+-dependent Cl− channels. Our study was aimed to determine Ca2+-dependent Cl− channels contribution to norepinephrine-induced contraction during hypertension and ageing. Norepinephrine-induced concentration-response curves of femoral arteries from 6- and 12-month-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were recorded using wire myograph. Pretreatment with Ca2+-dependent Cl- channel inhibitor indanyloxyacetic acid 94 [R(+)-IAA-94](IAA) attenuated norepinephrine-induced contraction in all groups, but relatively more in WKY than SHR arteries. The attenuation of norepinephrine-induced contraction after Ca2+-dependent Cl− channels blockade was partially reduced in 12-month-old WKY rats, but substantially diminished in 12-month-old SHR. IAA effect was enhanced after NO synthase inhibition but decreased by ageing. In 20-month-old WKY rats norepinephrine-induced contraction was not affected by IAA but was almost abolished after cyclooxygenase inhibition by indomethacin or niflumic acid. In conclusion, contribution of Ca2+-dependent Cl− channels to norepinephrine-induced contraction diminished with age, hypertension development, and/or NO synthesis inhibition. Ca2+-dependent Cl− channels are important for maintenance of normal vascular tone while their inactivation/closing might be a pathological mechanism.

Stereological and Digital Methods for Estimating Geometrical Characteristics of Biological Structures Using Confocal Microscopy

Stereological methods and automatic digital methods for the quantitative evaluation of the structure of three-dimensional (3D) objects are introduced. Classical stereological methods based on observations made on two-dimensional (2D) sections, applying point, linear or planar test probes are presented and their application for estimating geometrical characteristics such as volume, surface area, and length of microscopical structures is demonstrated together with two practical trainings presented in a “do it yourself, can do approach”. Further, stereological methods based on evaluation of 3D image data obtained by confocal microscopy from thick specimens and using computer-generated virtual test probes are presented. Such methods, allowing arbitrary orientation of the thick slice, can be used for estimating number, volume, surface area, and length in a very efficient way. The methods using spatial grid of points, disector, fakir and slicer probes are described and compared with automatic digital methods. The applications of presented methods are shown on practical examples. A special attention is paid to the sampling design and problems connected with practical implementation of the methods.

Erratum to “Contribution of Ca2

[This corrects the article DOI: 10.1155/2014/289361.].

APPLICATION OF CONFOCAL MICROSCOPY TO 3-D RECONSTRUCTION AND MORPHOMETRICAL ANALYSIS OF CAPILLARIES

The main advantage of a confocal laser scanning microscope (CLSM) consists in its possibility to capture images of thin, perfectly registered serial optical sections of the studied specimen. Such stacks of sections are useful in 3-D rendering of objects contained in the specimen as well as in implementing some of the recent efficient stereological methods. However, when using CLSM, a suitable fluorescence staining of the components of interest must be found.