PubMed Health⌕ Search

Biomedical subjects

R Wróblewski

Publications and source records attributed to R Wróblewski.

At least 19 recordsLinked to original sources
PubMed1992

Characteristic ionic composition of endolymph is maintained in cultured inner ear.

Inner ear anlagen from mouse were explanted on the 16th gestational day (gd) and cultured for 5 days, i.e. corresponding to the time of birth. By using energy dispersive X-ray technique an elemental composition characteristic for endolymph was found within the membranous labyrinth of the explants. The sodium to potassium ratio in the endolymphatic space of the cultured inner ears corresponded to endolymph of the 16th gd fetus in vivo. There was no difference in the endolymph compartment between the cochlear and vestibular halves of the in vitro specimens. Differences in Na to K ratio between endolymph of the inner ears and the surrounding medium were statistically significant. Thus, endolymph-regulating mechanisms are active also under organ culture conditions, although not fully optimal.

Animals↗
PubMed1992

Correction for extraneous background in X-ray microanalysis of cell cultures.

Some practical aspects of the X-ray microanalysis of cell cultures have been investigated. Cells were cultured on titanium grids covered with Formvar films and analyzed at 100 kV either in the scanning transmission (STEM) or transmission mode (TEM) of the electron microscope. Different holders, grids and configurations were compared with respect to the relative contribution of different factors to the extraneous background in the X-ray spectrum. When low atomic number holders are used, the contribution to the spectrum of electrons scattered through high angles, may be negligible. In practice this may result in negative values for the contribution of these scattered electrons to the background. Computer programs for correction of the extraneous background should ignore these negative values and replace them by zero. When a brass holder is used, the contribution to the spectrum from electrons scattered through high angles becomes more important than that of the uncollimated radiation. The position of the analyzed cell relative to the grid bars is more important than the choice of grid or holder type. The data show that for the specimens used in the present study the correction for extraneous background is of little importance and can be neglected.

Brain Neoplasms↗
PubMed1991

Distribution of sodium, magnesium, phosphorus, sulphur, chlorine, potassium, calcium and iodine in mammalian thyroid gland.

Anhydrous specimen preparation techniques and X-ray microanalysis were used to study the elemental composition of the rat thyroid follicular cells and colloid in the follicular lumen. Analysis were performed in morphologically defined follicular cells and adjacent colloid. Use of semithin cryosections allowed us to selectively analyze cell nucleus and dense vesicles in the cytoplasm of follicular cells. The analytical results indicate that the basolateral and apical cell membrane of follicular cells act differently in the transport of iodine and other ions, thus creating ionic gradient in the follicular lumen as compared to that found in the follicular cells. High potassium levels were found in the colloid. This finding indicates an active secretion of potassium from the apical plasma membrane into the follicular lumen. High sulphur and calcium levels were also found in the colloid. Dense vesicles found in the cytoplasm of follicular cells varied in size and had different elemental composition.

Animals↗
PubMed1991

Elemental analysis and fine structure of mitochondrial granules in growth plate chondrocytes studied by electron energy loss spectroscopy and energy dispersive X-ray microanalysis.

Electron energy loss spectrometry--EELS, and energy dispersive X-ray microanalysis--XRMA, were used to study the elemental composition of mitochondrial dense granules-mdg. The study was performed on dry cut thin sections (80-200 nm) of freeze-dried and low temperature embedded cartilage. Results obtained by means of XRMA clearly showed high phosphorus and calcium content in the mdg. Using EELS at 100 kV primary voltage we found that small concentrations of elements (i.e. below typically 1% atomic weight) are difficult to analyze and map, this especially in sections thicker than 50-60 nm. Surprisingly, analysis of calcium can be successfully performed on thicker sections though the edge lies above the carbon K edge while this is not possible for the phosphorus edge which is located at lower energies. This is likely due to the edge shapes (sharp for calcium and delayed for phosphorus), and to the more intense contribution of multiple low loss scattering in the background for phosphorus between 100 and 130 eV. By means of EELS elemental mapping a centrally located core was found in numerous mdg. In the calcium map the signal was strongest in the middle of mdg which corresponds to the area of reduced carbon signal. We found that carbon maps might be used for high resolution structural studies of chemically unfixed and anhydrously processed biological tissues. As carbon is the main constituent of Lowicryl resin its distribution is reversed to the distribution of biological tissue in which the proportion of carbon is lower, but is proportional to water content in the specimen in vivo. Use of EELS in combination with electron microscope with accelerating voltages in range of 140-200 kV together with anhydrous techniques of the tissue preparation will provide a new type of information which might lead to better understanding of the etiology and function of small structures in the cell.

Animals↗
PubMed1989

In situ elemental analysis and visualization in cryofixed nervous tissues. X-ray microanalytical investigations of embryological and mature brain, inner ear, photoreceptors, muscle and muscle spindles. Comparison of preparation methods for analysis and visualization at cellular and subcellular levels.

For meaningful X-ray microanalysis (XRMA) in biology and medicine, the development of preparative and quantitative methods has been necessary. The methods need to preserve close to in vivo distribution of diffusible ions with at the same time reasonable morphological preservation of the tissue. Analyses at low and middle microanalytical resolution are useful at the initial stages of an investigation or when data from large populations of samples have to be acquired. Cryomicrotomy, which makes it possible for the single cells within semi-thin and thick cryosections examined by X-ray microanalysis to be further characterized histochemically (enzyme and substrate content), has been adopted for several pathophysiological studies. The method is particularly suitable for the analysis of complex morphological tissues with many cell types as in the brain or sensory organs of the internal ear. For microanalysis at the subcellular level, we developed a preparative procedure based on the frozen fixed preparation which is freeze-dried in vacuo at -80 degrees C and then at the same temperature, without breaking the vacuum, impregnated with a low-temperature Lowicryl-type resin. The resin is polymerized by u.v. light. This method prevents redistribution of the ions in the tissue and retains the antigenicity of the tissue. A considerable number of cells can be analysed simultaneously and the elemental composition in different cell compartments can be compared due to the similar analytical conditions within the section. An alternative to thin plastic sections of freeze-dried material is thin cryosections cut at -150 degrees C and analysed at low temperatures. Although some methodological problems still exist in preparation of cryosections, this type of section is potentially the most useful in analysis of diffusible ions, especially calcium which in most biological systems is present in very low concentrations. New preparative techniques for XRMA brought severe problems in visualization of the specimens prepared by cryomethods. Charging, low contrast, mass loss and contamination, which are often negligible in conventional electron microscopy, have still to be solved in XRMA of cryoprepared specimens. However, the methods of semi-thin and thick cryosectioning and low-temperature embedding were successfully used for analysis of cells and organelles and for the study of fluids in restricted biological spaces such as the inner ear, muscle spindles and ventricles of the brain in rats. Accordingly, examinations which were impossible by micropuncture and ion selective techniques could be carried out by XRMA.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Microprobe analysis of cryofixed, chemically untreated freeze-dried cochlear hair cells.

Elemental (energy-dispersive X-ray) microanalysis was performed of cryofixed, chemically untreated freeze-dried cochlear hair cells of 1-month-old CBA/CBA mice. This technique is very difficult to carry out and time consuming, requiring a large number of specimens to be sectioned, quite at random, in order to obtain absolutely perpendicular section planes. Microprobe determination was performed in different parts of outer and inner hair cells. In addition to the normal intracellular elemental content a high calcium concentration was found at the base of all hair cells. Lower calcium levels were evident in the entire cytoplasm, including the cuticular plate and stereocilia. Technically it was feasible to analyze individual stereocilia.

Animals↗
PubMed1989

Energy dispersive X-ray microanalysis, neutron activation analysis and atomic absorption spectrometry--comparison using biological specimens.

X-ray microanalysis, neutron activation analysis and atomic absorption spectrometry were performed on normal and injured skeletal muscle. X-ray microanalysis of tenotomized rat soleus muscle showed significantly elevated levels of sodium and chlorine and lower potassium compared with normal muscle. Similar ion shifts could be demonstrated by neutron activation analysis and atomic absorption spectrometry. The concentrations of sodium and chlorine obtained by these techniques were somewhat higher and that of potassium lower than the values obtained by X-ray microanalysis. This can probably be attributed to the fact that in atomic absorption spectrometry and in neutron activation analysis the entire muscle biopsy contents are measured while in X-ray microanalysis only the content of muscle cells unaffected by extracellular, non-muscular components are determined. It can be concluded that X-ray microanalysis is a reliable technique to study the elemental content of biological tissue, especially tissue undergoing pathological changes affecting the extracellular spaces. Other types of analysis should be used when elements not detectable by X-ray microanalysis are of interest.

Activation Analysis↗
PubMed1988

Low temperature techniques for X-ray microanalysis in pathology: alternatives to cryoultramicrotomy.

Many diseases are associated with a change in the distribution of diffusible ions at the cell or tissue level. These diseases can profitably be studied by X-ray microanalysis. This technique for the study of ion distribution requires the use of cryoprepared specimens. Analysis at low or medium resolution can be carried out on thick or semi-thick cryosections, or on frozen-hydrated or freeze-dried embedded bulk samples. Such analyses are particularly useful in the initial stages of an investigation or when data from a large number of samples have to be acquired. Also X-ray microanalysis of cultured or single cells prepared by freeze-drying can be used to rapidly collect information on a large number of cells. Analysis at high resolution has to be carried out on thin sections: Cryosections or sections of freeze-substituted or freeze-dried embedded tissue. For the latter type of specimens, the use of low-temperature embedding methods may have important advantages.

Animals↗
PubMed1988

Simultaneous investigations of elemental changes in individual cells of the stria vascularis and in endolymph.

Using the microprobe for energy dispersive X-ray microanalysis, the elemental compositions of both the individual cells of the stria vascularis and of the endolymph were followed simultaneously under normal conditions and after the administration of 120 mg/kg ethacrynic acid (EA). Marginal cells and intermediate cells showed reversible increases in potassium and decreases in sodium concentrations. Shifts in the ionic composition of endolymph occurred later than after elemental changes in the strial cells. The present results indicate that the marginal and the intermediate cells are the primary target for EA-induced ototoxicity. However, generalized toxic effects of EA are also indicated, with a general leakage of different elements occurring during the 30-60 min period after EA administration.

Animals↗
PubMed1988

Elemental microanalysis of biological specimens.

Although X-ray microanalysis in the electron microscope is the most common method for microanalysis of biological specimens, other methods of elemental microanalysis (electron energy loss spectroscopy, scanning Auger microanalysis, and proton, ion, and laser microprobe analysis) may provide important complementary information and help overcome some of the limitations of electron probe X-ray microanalysis. Despite differences in physical principles and instrumentation, the various microanalytical methods have much in common with regard to specimen preparation, quantitative analysis, and interpretation of analytical data. A common approach to microanalytical problems in the biological sciences, irrespective of the analytical techniques used, seems therefore indicated.

Electron Probe Microanalysis↗
PubMed1987

Low temperature techniques in biomedical microanalysis.

Many diseases are associated with a change in the distribution of diffusible ions at the cell or tissue level. These diseases can profitably be studied by X-ray microanalysis. This technique for the study of ion distribution requires the use of cryoprepared specimens. Analysis at low or medium resolution can be carried out on thick or semi-thick cryosections, or on frozen-hydrated or freeze-dried embedded bulk samples. Such analyses are particularly useful in the initial stages of an investigation, or when many data from a large number of samples have to be acquired. Quantitative analysis is then usually carried out with the peak-to-local background method: by taking the background in the same energy range as the characteristic peak, an intrinsic correction for absorption of X-rays within the sample is made. X-ray microanalysis of cultured cells prepared by freeze-drying can also be carried out in this way. Analysis at high resolution has to be carried out on thin sections: cryosections, or sections of freeze-substituted or freeze-dried embedded tissue. For the latter type of specimens, the use of low-temperature embedding methods may have important advantages. Quantitative analysis of thin sections is carried out by the established continuum method.

Animals↗
PubMed1986

Ionic environment of cochlear hair cells.

The scala media of the adult cochlea in mammals comprises a morphologically closed compartment sealed with tight junctions of the intermediate to tight types. The unique ionic composition of endolymph is maintained by the stria vascularis through active reabsorption of sodium and active secretion of potassium against ionic gradients. The subtectorial space is only a partially closed compartment which communicates with the endolymph via holes in the tectorial membrane at its outer insertion to the organ of Corti. Hardesty's membrane divides the subtectorial space into two compartments: one facing the surfaces of inner hair cells and one facing the surfaces of outer hair cells. In the study of comparative anatomy, hair cells, e.g. in the lizard, basilar papilla are of two types: those covered with a tectorial membrane and those being free-standing lacking the tectorial membrane. The ionic environment of the hair cell surface seems to be the same, independent of whether covered with a tectorial membrane or not. The tectorial membrane itself is semipermeable to ions in the endolymphatic space. Only the surface structures of the hair cell with the sensory hairs facing the subtectorial space are exposed to the high concentration of potassium, whereas the remaining parts of the hair cell are surrounded by a fluid having a more normal extracellular type of ionic composition (cortilymph/perilymph). During embryonic development the ionic composition of endolymph develops in parallel with the morphologic maturation of the stria vascularis. A completely mature composition of endolymph is reached before any electrophysiological potentials in the cochlea can be elicited. The sensory hair surface of hair cells has reached a mature morphology prior to the maturation of endolymph. In several species the tectorial membrane is morphologically only partially mature when the increase of the potassium concentration of endolymph starts. Drugs primarily affecting the stria vascularis causing a transient change of the ionic composition of endolymph result in a transient dysfunction of inner ear potentials. If the ionic changes persist for longer time, morphological changes can occur in both the stria vascularis and the hair cells of the organ of Corti. Whether such changes are primarily caused by the ototoxic drug itself or by changes in the ionic composition of endolymph has to be explored further.

Animals↗
PubMed1986

Why low temperature embedding for X-ray microanalytical investigations? A comparison of recently used preparation methods.

Freeze-drying followed by infiltration with resin and polymerization by UV light at low temperatures and under constant vacuum conditions is an alternative tissue preparation technique for microprobe analysis. Embedding is carried out with the nonpolar low-temperature embedding resin (Lowicryl HM20) which allows infiltration and polymerization at temperatures down to -50 degrees C. Sections of low temperature embedded material can be cut dry at -60 degrees C or at room temperature. Sectioning at low temperatures is an alternative for preparations that are difficult to cut at room temperature. The morphological preservation is adequate for the identification of structures such as mitochondria, lysosomes and different types of endoplasmic reticulum in liver cells. Some physical properties of Lowicryl resins, such as mass loss under the electron beam and high contrast, are positive characteristics for the analysis of semi-thick sections. No significant differences in the elemental composition could be detected between tissue which was freeze-dried or freeze-substituted prior to embedding. Freeze-drying is less time consuming. By avoiding contact with organic solvents the risks of ion loss and redistribution are diminished. In contrast to freeze-dried thin cryosections, low temperature embedded material can be sectioned for light microscopy and areas of interest chosen for further thin sectioning. This is of great importance in work with tissues with complicated morphology and heterogeneous cell populations. The initial preparative step--the cryofixation--determines to a high degree the morphological preservation of freeze-dried and embedded tissue.

Animals↗
PubMed1986

Linkage of sub-membrane-cisterns with the cytoskeleton and the plasma membrane in cochlear outer hair cells.

The fine structure of the organ of Corti in the mole-rat (Spalax sp.) was studied. All outer hair cells possessed a single layer of membrane bound vesicles which lined the plasma membrane along the lateral aspect of the cell, except in the cuticular plate and in synapse areas. This organization of vesicles was not observed in inner hair cells. Most of the vesicles were laterally linked to the cell membrane by pairs of 25 nm long 'arms' while medially they were connected to microtubules. The three-dimensional organization and the possible role of the sub-membrane vesicles in the process of auditory transduction are discussed.

Animals↗
PubMed1985

Energy dispersive X-ray microanalysis of individual vestibular hair cells.

Microprobe analysis was performed at the cellular and subcellular levels of type I and type II vestibular hair cells. In principle the same types of elemental histograms were found in the two types of hair cells studied. High concentrations of Cl and K were detected in stereocilia, whereas calcium was found when analyzing stereocilia and the supranuclear cytoplasm.

Animals↗
PubMed1985

Applications of scanning electron microscopy and X-ray microanalysis in inner ear pathology.

Surface pathology of inner ear structures so far described in detail concern cochlear and vestibular hair cells and the stria vascularis. In man, surgical intervention into the inner ear is very uncommon and when performed is in general with the primary objective of destroying the diseased peripheral end organs. The vast majority of inner ear tissue available for use with scanning electron microscopy (SEM) is therefore obtained from animals. The present paper reviews the progression of surface pathology caused by aminoglycoside antibiotics, acoustic overstimulation and in a guinea pig strain with genetic inner ear disease. The primary site of onset of surface pathology differs, depending on the underlying cause. Advanced surface pathology shows a similar type of morphological degeneration independent of cause. The combination of SEM and energy dispersive X-ray microanalysis (XRMA) of inner ear pathology has as yet been reported in only three studies, all concerning inner ear fluids or otoconia.

Aminoglycosides↗
PubMed1985

Freeze-drying and related preparation techniques for biological microprobe analysis.

An X-ray microanalytical and morphological investigation has been carried out on rapidly frozen, freeze-dried or freeze-substituted tissues. A comparison was made between different embedding and polymerisation procedures following freeze-substitution and freeze-drying. The investigation also included an analysis of specimens infiltrated, embedded and polymerised by ultraviolet irradiation at low temperatures with Lowicryl HM20. The morphological preservation of Lowicryl embedded tissue was adequate for the identification of different cell structures like nuclei, mitochondria, lysosomes and different types of endoplasmic reticulum. X-ray microanalytical investigation of low temperature embedded material displayed an elemental composition of cells and organelles similar to that found in freeze-dried cyosections. Compared with freeze-dried cryosections, low temperature embedded material could be sectioned for light microscopy and area of interest chosen for further thin sectioning. This is of great importance in work with tissues with complicated morphology and heterogenous cell populations.

Animals↗