Stereologic analysis of vacuolization of the T-system of frog muscle fibers, detected using confocal fluorescence microscopy

The confocal fluorescence microscopy has been used for quantitative evaluation of the T-system reversible vacuoles produced by efflux of 80-120 mM glycerol from frog skeletal muscle fibers. The fibers were stained by membrane probe RH414 and by water-soluble dye fluorescein dextran that marks the vacuolar lumen. Using morphometrical and stereological methods the volume and surface densities of vacuoles were measured on single optical sections and Z-series during a 30 min glycerol efflux. Various methods of measurements (three-dimensional reconstruction of vacuoles, computer morphometry, point counting method) applied to the same Z-series provide similar results. The vacuolar membranes stained by RH414 look like bright rings 0.3-0.4 micron in width. It is concluded that the real position of vacuolar membrane corresponds to the middle of the vacuolar envelope. The measurements of the external dimensions of the envelope overestimate the stereological parameters up to 50%. The volume density of vacuoles reaches 10% within 20-30 min of glycerol efflux. It means that the volume of the T-system may increase by 25-30 times compared to the control value (0.3-0.4%). The surface density of vacuoles during reversible vacuolation is equal to 0.20-0.35 micron-1 and does not exceed the surface density of normal T-system. The sufficiency of membrane material for the T-system reversible vacuolation is discussed in addition to the role of geometrical factor in this phenomenon.     

Stereological study of the early ultrastructural differentiation of chick embryo neuroepithelial cells during neurulation

The neuroectodermal cells of chick embryos have been analyzed during neurulation by stereological and morphometrical ultrastructural methods in an attempt to describe their cytometric evolution. A profound change of cellular form coefficient was observed which is related to the typical process of columnarization of these cells. At stages 7 and 8, the nucleus appeared round in shape, probably due to a loss of pressure of the vitelline inclusions. In this sense, the volume density of these inclusions falls during this period. There was also a significant increase of the nuclear surface density, the significance of which is discussed on the basis of the nucleo-cytoplasmic interchanges and the differentiation process. At the same time, an increase in the number of mitochondria was observed, which is related to the neural folding process. Simultaneously, the amount of rough endoplasmic reticulum increases, presumably related to the remarkable changes of the embryonic extracellular matrix.     

State of the endothelium of the aorta during chronic stress

In 50 white male rats (18-control and 32-experimental) using morphometrical and stereological methods for film preparations of endothelium, the state of the internal aortal lining at various stages of chronic stress developing during muscular activity has been studied. The data obtained are statistically treated by the sliding averages method, reliability of the differences is estimated according to the value of nonparametrical criterion of Wilcoxon-Mann-Whitney. For objective periodization of the endothelial reaction to stress, centered moving averages curves are performed with subsequent calculation of the coefficient of variation for the aligned ordinates of the approximated values in every temporal point. The aortal endothelium reaction to the stress is of phasic character. During the alarm reaction, destructive changes predominate; they are accompanied with an increased mitotic activity of endotheliocytes up to 2.2%. The resistance stage increases compensatory-adaptive rearrangements, which are manifested not only as cellular hyperplasia, but as hyperthrophy of nuclei, increasing number of strangulated nuclei, nucleoli, as well as binuclear and trinucler cells. To the exhaustion period the increase of alternative and restorative transformations of the tissue corresponds.     

On the Determination of Numerical Density of Cell Organelles Using Mitochondria as an Example

Methods of determining the numerical density of cell organelles described in literature were critically reviewed in a morphometrical and stereological study of muscle cell mitochondria (heart muscle cells, diaphragm cells, sceletal muscle cells). A review of the method described by WEIBEL and GOMEZ showed that the numerical density of the mitochondria depends to a great extent on their shape and not so much on their size distribution. For this reason serial sections should be used to determine the shape factor in biological objects of unknown geometric shape. Generally, the numerical densities of mitochondria determined by using the method proposed by DEHOFF and RHINES were higher than those obtained with the method described by WEIBEL and GOMEZ. This is attributed to certain corrections used in the former method. Elaborate computations are generally involved and the geometric shapes of the object examined must be known in order to determine the numerical density of cell organelles or of other biological structures. The numerous sources of error involved in these methods give this parameter the character of an objective estimate. For this reason it is recommended that the value obtained should be checked by determining a two-dimensional parameter. Our examinations of heart muscle mitochondria showed good agreement between the two parameters.     

Quantitative Measurements in the Human Hippocampus and Related Areas: Correspondence between Ex-Vivo MRI and Histological Preparations

The decrease of volume estimates in different structures of the medial temporal lobe related to memory correlate with the decline of cognitive functions in neurodegenerative diseases. This study presents data on the association between MRI quantitative parameters of medial temporal lobe structures and their quantitative estimate in microscopic examination. Twelve control cases had ex-vivo MRI, and thereafter, the temporal lobe of both hemispheres was sectioned from the pole as far as the level of the splenium of the corpus callosum. Nissl stain was used to establish anatomical boundaries between structures in the medial temporal lobe. The study included morphometrical and stereological estimates of the amygdaloid complex, hippocampus, and temporal horn of the lateral ventricle, as well as different regions of grey and white matter in the temporal lobe. Data showed a close association between morphometric MRI images values and those based on the histological determination of boundaries. Only values in perimeter and circularity of the piamater were different. This correspondence is also revealed by the stereological study, although irregular compartments resulted in a lesser agreement. Neither age (< 65 yr and > 65yr) nor hemisphere had any effect. Our results indicate that ex-vivo MRI is highly associated with quantitative information gathered by histological examination, and these data could be used as structural MRI biomarker in neurodegenerative diseases.     

Ultrastructural mechanisms of myocardial atrophy in white rats during starvation

By means of complex morphological, morphometrical and stereological analyses the myocardium of Wistar rats was studied in full alimentary starvation during 6 days. The availability of two mechanisms of myocardial atrophy in adaptation to full starvation was revealed, these are the diminution of dimensions of parenchymatous elements and the decrease of muscle cell number. By ultrastructural investigation of cardiomyocytes the signs of structural protein synthesis decline were revealed, this is a simple myocardial atrophy. In quantitative analysis of cardiomyocytes the decrease of their number without changes in proportions of cells with different nuclei number was observed, that indicated systematic character of muscle cell elimination out of myocardium by apoptosis mechanism involvement, this is a numerical myocardial atrophy. Stereological analysis of myocardial atrophy development in conditions of full starvation determined the main regulated indices, these are the absolute total myofibril mass and mitochondrial mass and surface area.     

Stereological evaluation of the soleus muscle isografted into fast extensor digitorum longus (EDL) muscle in rats with different thyroid status

The 2-D stereology can be used advantageously in the case of muscle cross sections stained by routine histochemical and immunocytochemical methods, such as mATPase reaction, when the quality of the image is often not sufficient for using image analysis techniques without considerable individual intervention. Other advantages of stereological methods in muscle morphometry are that measurements are made directly on specimens under the microscope and in their simplest arrangement they do not require sophisticated and expensive technical equipment. Furthermore, unbiased results are obtained, no segmentation and edge effect problems arise and the quantity of work invested in stereological estimation is reasonable. Therefore, we have used the stereological methods as our standard technique for assessment of fibre type composition in regenerated soleus muscles grafted from 21- to 28-day-old rats into fast EDL muscles of adult inbred recipients with different plasma levels of thyroid hormones.     

Stereological Implications of the Geometry of Cones

Given a cone around a curve β in IR³ it is shown that there is no relation between the curvature of β and the volume of the cone but, in contrast to the volume, the surface area of the cone depends, albeit rather weakly, on the curvature of β. The geometric meaning of the stereological formulae when applied to cones is considered and it is noted that stereological methods to estimate the surface area of a cone need to be extremely precise to provide information concerning curvature or tortuosity.     

Effects of dosed sympathectomy on the development of adaptation-compensation reaction of C-cells of the thyroid gland and chromaffin cells of the adrenal glands in young rats

Using radioimmunological, morphometrical, electron microscopic and luminescent methods, comparative analysis of thyroid C-cells and adrenal chromaffin cells has been carried out at guanethidine sympathectomy in young rats. Significant decrease of functional activity of C-cells with concomitant hyperplasia of C-cells population under blockade of sympathetic influences has been revealed. Compared to C-cells, adrenal chromaffinocytes of sympathectomized rats possess higher degree of structural-functional mobilization and are characterized by intensive secretion of catecholamines directed at restoration of tissue neurotransmitter deficit.     

Use of fractal dimensions to quantify coral shape

A morphometrical method to quantify and characterize coral corallites using Richardson Plots and Kaye’s notion of fractal dimensions is presented. A Jurassic coral species (Aplosmilia spinosa) and five Recent coral species were compared using the Box-Counting Method. This method enables the characterization of their morphologies at calicular and septal levels by their fractal dimensions (structural and textural). Moreover, it is possible to determine differences between species of Montastraea and to tackle the high phenotypic plasticity of Montastraea annularis. The use of fractal dimensions versus conventional methods (e.g., measurements of linear dimensions with a calliper, landmarks, Fourier analyses) to explore a rugged boundary object is discussed. It appears that fractal methods have the potential to considerably simplify the morphometrical and statistical approaches, and be a valuable addition to methods based on Euclidian geometry.     

The morphofunctional characteristics of the thymus in rat fetuses and progeny in venous occlusion]

Using histological and morphometrical methods, morphofunctional state of the thymus was studied in fetuses and offspring of rats in physiological pregnancy and under conditions of phlebo-occlusion. Phlebo-occlusion resulted in hypertrophy of the fetus, thymus and offspring, changes in structural zones and cellular reactions.     

Confocal stereology and image analysis: methods for estimating geometrical characteristics of cells and tissues from three-dimensional confocal images

A short review of confocal stereology and three-dimensional image analysis is presented, pointing out the achievements accomplished in this field by the Department of Biomathematics (Institute of Physiology, Prague). One of the methods of confocal stereology, the fakir method for surface area estimation, developed by this laboratory, is described. Methods for automatic measurement of geometrical characteristics of microscopical structures, based on 3-D image processing or surface triangulation, are discussed and compared with interactive stereological methods. Three-dimensional reconstruction programs and software implementation of stereological and digital methods as well as their practical applications are presented. The future trends are discussed.     

Identification of the elastic symmetry of bone and other materials

A simplified classification scheme for the elastic symmetries of a solid is applied to the identification of the elastic symmetry of a material by three different methods--visual, stereological and numerical algorithm. Each method is illustrated with an application to bone tissues, but the methods apply to all materials.     

Histologic organization of the myocardium of the rat during hypokinesia (stereologic study)

For 30 days Wistar rats have been kept under conditions of hypokinesia. Using stereological methods, the volume and surface density of muscle fibers, microcirculatory bed and connective tissue components have been defined, and volumetric and surface--volume ratios of parenchymal and stromal structures have been calculated. Intracellular stereological parameters of the cardiac myocytes have been studied. Three-dimensional parameters in the parenchymatous-stromal interrelations of the atrophying myocardium accompanied with a deep rearrangement of the intracellular architectonics have been determined, as well as certain destructive changes of the cardiac myocyte ultrastructures. Such a treatment as a decreased functional loading is demonstrated to be the cause of cardiomyocytic lesions.     

Topography of cell populations of the solitary tract nuclei of rats

At the light level by means of some morphometrical methods topography and morphological peculiarities of certain cell populations in the solitary tract nuclei (STN) of rats have been investigated. Basing on dimensions, form and tinctorial properties studied, as well as on arrangement density of the cells in the STN, 15 cell groups and 3 acellular areas have been distinguished. For designation of the subpopulations revealed a unified nomenclature, integrating the literature data is suggested.     

Structure of the mucous membrane of the distal part of the large intestine in the monkey

By means of histological, histochemical, immunohistochemical and morphometrical methods, according to 24 objective morphological parameters, bioptates of endoscopically unchanged mucosal membrane of the large intestine, obtained from 22 clinically healthy macaque rhesus males, have been studied. The data on vital structure and cellular interconnections in the mucous membrane of the distal part of the large intestine have been received. The data can be used for estimating certain pathological processes, when modelling the large intestine diseases in monkeys.     

Evaluation of the Cell Population of the Seminiferous Epithelium and Spermatic Indexes of the Bat Sturnira lilium (Chiroptera: Phyllostomidae)

Due to the scarcity of information about patterns of spermatogenesis in bats, this study aimed to provide information on the testicular activity of the bat Sturnira lilium along the annual seasons. Thus, a series of morphometrical and stereological analyses were made using the testes of adult S. lilium in order to achieve a better understanding of the sperm production dynamics. Light and transmission electron microscopy analyses were performed in testicular fragments of animals captured during dry and rainy seasons. The testes followed the pattern of organization described for other mammals, and there were no morphological differences between organs collected either in dry or in rainy seasons. Each tubular cross-section in stage 1 was made of 0.5 type-A spermatogonia, 4.4 primary spermatocytes in preleptotene/leptotene, 3.7 in zygotene, 11.9 in pachytene, 35.6 round spermatids and 8.5 Sertoli cells. The mitotic and meiotic indexes were 15.4 and 2.9 cells, respectively, while the spermatogenesis yield was 68.7 cells. The testicular sperm reserves was 37.61×10⁶ cells, and daily sperm production per gram of testis averaged 209.68×10⁶ cells, both highest averages occurring in the rainy season. S. lilium male bats have a continuous reproductive pattern, high spermatogenesis yield and low support capacity by the Sertoli cells.     

A stereological method for estimating fish fecundity

This paper describes the application and evaluation of a stereological method for counting fish oocytes. The method has been applied to herring, Clupea harengus L., Dover sole, Solea solea L., and mackerel, Scomber scombrus L., and compared with existing volumetric and/or automated particle counter methods. The stereological method has been shown to given similar results and to have a number of distinct advantages over the other methods.     

Changes in the structure and cell composition of the thymus as a result of dosed physical loads

By means of histological and morphometrical methods normal age involution of the thymus has been studied, as well as its changes under conditions of dosed physical loadings. The experiment has been performed in 92 non-inbred white male rats. At adaptation of the organism to the loadings, involution of the gland decelerates, and at an insufficient adaptation--accelerates. This is, probably, the cause of decreasing protective forces of the organism.     

The morphofunctional characteristics of the cecum in experimental escherichiosis]

Using the model of experimental escherichiosis in mice by means of morphological, immunomorphological, morphometrical and electron microscopy methods, the authors give morphofunctional characteristics of caecum 15 minutes to 2 weeks after inoculation. The authors show the dynamics of infectious process, characterized by changes of microcirculation, increasing lymphoplasmocellular infiltration, dystrophic changes in cells of neuroplexes and degranulation of mast and endocrine cells. The data obtained show that pathological process in caecum during experimental escherichiosis has an immune character, that the above portion of the intestine is a part of endocrine system.