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R Wróblewski

Publications and source records attributed to R Wróblewski.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1984

Freeze drying and freeze substitution combined with low temperature-embedding. Preparation techniques for microprobe analysis of biological soft tissues.

An X-ray microanalytical and morphological investigation was 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 which had been infiltrated, embedded and polymerised by ultraviolet irradiation at low temperatures with Lowicryl-HM20. The method of freeze drying, followed by embedding and polymerisation at low temperatures in vacuo was found to give satisfactory results, comparable with more tedious and hazardous freeze substitution technique.

Animals↗
PubMed1984

Sodium, phosphorus, sulphur, chlorine and potassium shifts in rat brain during embryonic development.

Concentrations of sodium (Na), phosphorus (P), sulphur (S), chlorine (Cl) and potassium (K) and their variations during brain development were measured in freeze-dried thick sections from rat brain (16-20 micron). Sprague-Dawley rats were bred and the day of finding vaginal spermatozoa was considered as day zero of pregnancy. On days 12E, 13E, 14E, 16E, 19E, 21E (embryonic) and postnatal day one whole embryos or fetal heads were rapidly frozen in liquid Freon 22 cooled with liquid nitrogen (-180 degrees C), sectioned in a cryostat (-20 to -40 degrees C), and processed for X-ray microanalysis on pure carbon plates. Concentrations of Na and Cl differed in the cells of the cerebral cortex, ependyma, choroid plexus and cerebral spinal fluid (CSF). During cerebral development, Na and Cl concentrations appeared to be correlated, while K was more related to P. S was low and unchanged in all compartments during development and was thus considered as an internal control. K was inversely related to Na and Cl fluctuations within the choroid plexus epithelia. Sharp phase changes of elemental composition appeared in all tissues at specific growth stages, e.g. days 14E and 19E. These results demonstrate rhythmic changes in the inorganic components of developing rat brain cells and fluid environment presumably reflecting physiological fluctuations and cell cycle phenomena. Such changes may also be related directly or indirectly to known 'growth phase changes' in the developing rat.

Animals↗
PubMed1984

Recent applications of X-ray microanalysis in muscle pathology.

X-ray microanalysis of single muscle fibres visualized in the scanning- and scanning-transmission mode of electron microscopy has been applied to human muscle biopsies to quantify changes of intracellular elements in different muscle disorders. To detect elements representing diffusible ions, cryofixation and cryosectioning was performed and analyses were conducted on freeze-dried cryosections 6 micron thick. Changes in the concentration of elements were found to differentiate certain muscular disorders. A large increase in sodium (Na) and chlorine (Cl), and a decrease in potassium (K) was typical of myotubular myopathy, while a moderate increase in Na and Cl was found in central core disease and nemaline myopathy. The normal elemental spectrum was found in multicore myopathy and facio- scapulo -humeral muscle dystrophy. In dystrophia myotonica there was constantly a decrease in K whereas in myotonia congenita an increase in K was a common finding. In myotonic dystrophy an increase in Na and Cl seemed to be related to the increase in the ring fibre formation. Experimental tenotomy of the soleus muscle of the rat is characterized by plasma membrane changes and the formation of core fibres. A marked increase in Na and Cl and a decrease in K was found to be a prominent elemental change in such core fibres. We conclude that changes in the concentration of certain intracellular elements demonstrable by X-ray microanalysis on cryosectioned freeze-dried muscle biopsies seem to be an indication of muscle fibre membrane disturbances.

Adolescent↗
PubMed1984

A unique striated muscle: further morphological and x-ray microanalytical investigation of the stapedius muscle of the guinea pig using thin and thick cryosections.

Thin (130-200 nm) and thick (10-16 micrometers) cryosections of stapedius muscle of guinea pig were examined by X-ray microanalysis in the scanning- and scanning-transmission mode of electron microscopy. To further explore the unusual elemental composition of stapedius muscle and to evaluate previously used method of analysis of thick cryosections cut at -30 degrees C (Wróblewski et al., 1981), thin cryosections cut at -100 degrees C were investigated. The ultrastructural organisation of stapedius muscle with the centrally located nuclei and the mitochondria could be easily recognized in unstained thin cryosections. X-ray microanalytical investigations of thin sections revealed nearly the same elemental composition of single organelles as results obtained on defined areas in thick cryosections. Analysis of mitochondrial matrix. The elements appeared, however, at the same ratio (1:1). The P/Ca ratio was approximately 6:1 in the nuclei. Myofibrils presented low K and higher Ca concentrations. The levels of other elements (P. S. Cl) were close to those found in the quadriceps muscle of the guinea pig, rat, or human.

Animals↗
PubMed1984

The freeze fracture technique in inner ear research.

Freeze-fracture studies on the inner ear have been focused mainly on the normal structure of junctions sealing the endolymphatic compartment, the compartmentalization of the stria vascularis and the junctional stability of the hair cells towards adjacent supporting cells. The hair cells have a very tight type of zonulae occludenetes as compared with other non-sensory epithelia in the inner ear. In contrast to other epithelial cells, the mature hair cells are in most species lacking gap junctions. During embryonic development a loss of gap junctions is an early and significant feature of cells differentiating into future hair cells. The tight junctions in the secretory epithelia (stria vascularis in the cochlea and dark cells around vestibular organs) are morphologically mature before the onset of the ionic maturation of endolymph. Freeze-fracture studies on inner ear pathology are few. The structural alterations of tight junctions in the diseased inner ear are minimal. The functional significance of such small morphological derangements is not known.

Animals↗
PubMed1983

Changes in elemental composition of single muscle fibers following tenotomy of the rat soleus muscle.

Cores were produced in type 1 muscle fibers by tenotomy of rat soleus muscle. The morphology and histochemistry of the muscle fibers was established by light microscopy on cryostat sections stained for hematoxylin-eosin or myofibrillar ATPase and on semithin plastic sections. The ultrastructure was visualized on thin plastic sections. On 6 micrometers of freeze-dried cryosections, energy dispersive X-ray microanalysis was performed on muscle fibers visualized in the scanning-transmission mode of electron microscopy. This procedure permitted quantification of different intracellular elements such as sodium (Na), chlorine (Cl), potassium (K), magnesium (Mg), sulphur (S), and phosphorus (P). Spectra from core fibers could easily be compared with those of normal fibers. A conspicuous finding was an increased Na and Cl content and a decreased K content in core fibers compared to normal fibers. It is known that core fibers produced after tenotomy exhibit distinct changes in plasma membrane morphology similar to that found in Duchenne muscular dystrophy (DMD). The results in this study point to a change in normal intracellular ion composition which could be a result of a deficiency of mechanisms maintaining normal membrane ion gradients.

Animals↗