Biomedical subjects

R Wróblewski

Publications and source records attributed to R Wróblewski.

At least 19 recordsLinked to original sources

PubMed2002

Effects of irradiation on intestinal cells in vivo and in vitro.

The effects of irradiation on intestinal epithelial cells were analyzed in vivo and in vitro. The in vivo study was carried out on the rat small intestine and for the in vitro study the intestinal crypt cell-line IEC-6 was used. Rat intestine and IEC-6 cells were irradiated with X-ray doses ranging between 1-16 Gy. Energy-dispersive X-ray microanalysis was used for detection of the elemental changes in the cells. Cell morphology was investigated in the scanning electron microscope, DNA-synthesis by autoradiography of 3H-thymidine incorporating nuclei and proliferation by cell counting. Our results indicate that in vivo, in the crypt cells, the increasing doses of irradiation led to increased sodium and lowered potassium and phosphorus concentrations. Corresponding ion shifts were found in the irradiated IEC-6 cells. Cells continued to proliferate up to the dose of 8 Gy, although the proliferation rate became lower with increasing dose of irradiation. The increasing dose of irradiation significantly reduced DNA-synthesis (16 Gy decreased DNA-synthesis by 50%) which resulted in a complete inhibition of cell proliferation. Analysis of goblet cells also showed characteristic radiation-dependent elemental changes. Scanning electron microscopical investigation of cells in culture revealed that most of the control cells were flat and had rather smooth cell membranes. Irradiation led to the appearance of numerous different membrane manifestations (microvilli of varying length and distribution, and blebs). Frequency of differences in the topology of the cells was related to the dose of irradiation. Our study clearly demonstrates that even low doses of irradiation cause changes in the ionic composition of the cells and inhibit DNA-synthesis and cell proliferation. The effects observed in the crypt cells in vivo were the same as in the intestinal cell line in vitro, which indicates that IEC-6 cells can be used for investigation of side effects of radiation to the abdomen.

Animals↗

PubMed2002

Effects of nicotine on intestinal and respiratory epithelium.

The effects of nicotine on intestinal and tracheal mucosa and epithelial cells were studied in vivo and in vitro. Rats received 108 microM nicotine in their drinking water for 10 days. Intestine and trachea were removed and prepared for X-ray microanalysis, transmission electron microscopy and immunocytochemistry. X-ray microanalysis of freeze-dried cryosections of intestine and trachea showed an increase in Na and Cl, and a decrease in K in the lamina propria of the intestine and the epithelial cells and the submucosal compartment of the trachea. Analysis of frozen-hydrated trachea, in order to determine ionic changes in the airway-surface liquid (ASL), indicated that the Na and Cl content in the ASL decreased after nicotine treatment. Immunocytochemistry showed upregulation of ICAM-1 in the submucosal connective tissue of the trachea and the lamina propria of the intestine. Transmission electron microscopy showed a somewhat increased number of eosinophils in the lamina propria of nicotine-treated rats, increased edema in the submucosal connective tissue, and a somewhat increased number of damaged basal cells in the trachea of nicotine-treated rats, compared to the controls. These data indicate that nicotine may evoke an inflammatory reaction, in particular in the trachea, that could cause cell damage and with that changes in the ionic relations of the epithelial cells. The in vitro experiments showed that nicotine could directly affect ion transport by inhibiting cAMP-stimulated (but not ATP-stimulated) chloride efflux from cultured respiratory epithelial cells. This indicates that apart from indirect effects via inflammation, nicotine can directly affect the ionic homeostasis of the cells and the composition of the airway-surface liquid.

Administration, Oral↗

PubMed2000

Electron probe X-ray microanalysis of cultured myogenic C2C12 cells with scanning and scanning transmission electron microscopy.

Heterogeneity of the elemental content of myogenic C2C12 cultured cells was studied by electron probe X-ray microanalysis (EPXMA) with scanning (SEM EPXMA) and scanning transmission electron microscopy (STEM EPXMA). The best plastic substrate for growing cells was Thermanox. For STEM EPXMA, a Formvar film coated with carbon was found to be suitable substrate. The cells examined by scanning transmission electron microscopy showed great heterogeneity in their elemental content in comparison with the cells examined in the scanning electron microscope despite of an almost identical preparation procedure for EPXMA. Nevertheless the K/Na ratios obtained from both methods of EPXMA were very close (4.1 and 4.3). We conclude that the observed discrepancy in the elemental content obtained by the two methods may be due to differences in instrumentation and this must be taken into account when planning a comparative study.

Animals↗

PubMed2000

Elemental changes in skin from patients with basal cell carcinoma.

Intracellular ionic shifts have been described in connection with normal and abnormal cell proliferation. This study was conducted to determine the elemental composition of a cell tumor classified as basal cell carcinoma in skin. The biopsies were taken from 10 patients. Control biopsies were taken from the non-affected skin from the same patients. Cryosections were cut and attached to carbon specimen holders. After freeze drying the sections were analyzed by X-ray microanalysis in a scanning microscope. The X-ray microanalytical results showed that the content of magnesium, phosphorus and potassium was significantly higher in the cancer cells than in the control cells (p < 0.05). The content of sodium, chlorine, calcium and sulfur was similar in both affected and unaffected cells. We conclude from these data that basal cell carcinoma has a characteristic elemental composition. It is possible that increased potassium and magnesium levels have a stimulatory effect on this tumor type, in contrast to other types of cancer cells where elevated sodium and chlorine and lowered potassium levels have frequently been found.

Adult↗

PubMed1999

X-ray microanalytical and morphological observations of aqueous humor from cataract human eyes with and without diabetes mellitus.

This study was conducted to determine the elemental composition of the aqueous humor in cataract human eyes, with and without diabetes mellitus. The aqueous humor was obtained during cataract surgery from 11 patients with diabetes mellitus and 11 controls. Droplets of aqueous humor, after air-drying on carbon plates, were analyzed by X-ray microanalysis. Before surgery, the protein concentration in the anterior chamber was measured with a laser flare meter. Flare intensity was significantly higher in eyes with diabetes mellitus than in controls (p < 0.05). The X-ray microanalytical results showed that the relative sulfur content in the aqueous humor was significantly higher in the diabetic eyes than in the controls (p < 0.05). The content of elements representing the mobile ions in the aqueous humor, i.e., Na, Cl, K, and Ca was similar in both studied groups. We conclude from these data that the high sulfur content in aqueous humor of diabetic eyes may be related to a higher level of proteins found in the humor in these eyes.

Aged↗

PubMed1998

X-ray microanalysis of in situ and isolated pancreatic islets.

The effect of stimulation of insulin secretion in pancreatic beta cells on the elemental composition of these cells was investigated by x-ray microanalysis. In vitro experiments on isolated islets of Langerhans from ob/ob mice were compared to in situ experiments. The only significant difference in the elemental composition of beta cells from ob/ob mice versus their lean counterparts is a lower Ca concentration in the ob/ob animals. The nucleus of the beta cells has a higher concentration of P, K, and Na than the cytoplasm, which has a higher concentration of S and Cl. No polarized ion distribution in the cytoplasm of the beta cells was observed. Isolated beta cells show a higher concentration of Na and Cl and a lower concentration of K than their in situ counterparts. Stimulation of insulin secretion with glucose both in situ and in vitro showed only very small effects on the elemental composition of the beta cells: a tendency to a decreased P content was noted. In vitro experiments using stimulation with high extracellular K+ showed, in addition, a small increase in the intracellular K concentration. In conclusion, while the elemental content of beta cells in vitro differs from that in situ, the response to glucose stimulation appears to be similar in both systems.

Animals↗

PubMed1996

X-ray microanalysis of elemental changes in human parathyroid glands in primary and secondary hyperparathyroidism.

The elemental composition of chief cells of parathyroid glands from patients with adenomatous primary hyperparathyroidism (HPT) and uremic secondary HPT was studied by X-ray microanalysis. Glands histologically deemed normal were used as controls. The analyses were also carried out on tissue specimens incubated in hypo-, normo- and hypercalcemic media (0.5, 1.25, and 3.0 mM calcium concentration). Analysis of chief cells from normal glands did not reveal any significant differences in ionic composition after exposure to the different calcium concentrations. In chief cells from adenomatous and uremic hyperplastic glands, elemental changes were noted. In comparison with specimens incubated in 1.25 mM calcium medium, cells in 0.5 mM calcium medium had a lower content of potassium and phosphorus. After stimulation with increasing extracellular concentration, an increase in the K/Na ratio was observed, due to a marked decrease of sodium and an increase of potassium; the calcium concentration was almost unchanged. Our findings indicate that in HPT an increase in serum calcium concentration might exert a stimulatory effect on the Na/K pump (sodium pump) and on the calcium-activated potassium channels. Either of these mechanisms might contribute to a lowering of cytoplasmic calcium. Our observations suggest that changes in ionic content of the parathyroid cells may be of importance for the stimulus secretion process in the cells.

Adult↗

PubMed1996

Morphological and X-ray microanalytical changes in mammalian tissue after overhydration with irrigating fluids.

We compared morphological changes in the heart, brain, liver and kidneys after giving 100 ml/kg of 5 irrigating fluids by intravenous infusion to 30 rabbits. Glycine 1.5% and 1.0%, both with ethanol 1% as a tracer for absorption, received the highest scores for tissue swelling and were sometimes followed by focal necrosis and an inflammatory cell reaction in subendocardial areas of the heart. Three rabbits died shortly after infusion of glycine 1.5% + ethanol 1%, the deaths being preceded by bradycardia and an irregular cardiac rhythm. This solution also lowered the intracellular potassium and chlorine content as shown by X-ray microanalysis of myocardial cells. Mannitol 3% + ethanol 1% produced the most favorable outcome in our evaluation, with the lowest scores for tissue swelling, while sorbitol 2% + mannitol 1% and normal saline took intermediate positions.

Animals↗

PubMed1995

Low concentrations of mercury induce changes in ion composition of cultured myoblasts.

The effects of low concentrations (1 pM, 1 nM, 1 microM) of mercuric chloride on ion distribution in cultured myoblasts were analysed by energy dispersive X-ray microanalysis. An increase in intracellular sodium concentration was observed five minutes after addition of HgCl2 to the culture medium. This increase was dose dependent and accompanied by a transient decrease in potassium concentration. Exposure to 1 nM and 1 microM HgCl2 led to a two-fold increase in the cytoplasmic chlorine concentration. The higher HgCl2 concentration (1 microM) induced morphological alterations in the form of cell membrane blebs, perforations and shrinkage or flattening of the myoblasts. It was concluded that even low concentrations of mercuric chloride cause elemental and morphological changes in cultured myoblasts, which may reflect effects of the metal on membrane permeability.

Animals↗

PubMed1994

Effect of delay in cryofixation on the elemental composition of biopsies and post mortem specimens of the thyroid gland.

The time between the excision and cryofixation of a biopsy is most important regarding its elemental composition as demonstrated by an investigation of the thyroid glands of rats and pigs. Biopsies taken and cryofixed immediately served as control specimens. Biopsies that were allowed to stand at room temperature for 20 min before cryofixation and specimens cryofixed at 1 h post mortem were also investigated. Significant changes in the ion concentration of the cells and colloid were apparent in biopsies in which cryofixation was delayed for 20 min and in thyroids cryofixed 1 h post mortem. It was demonstrated that redistribution of electrolytes occurs within 1h post mortem and that similar changes occur in biopsies allowed to stand for 20 min at room temperature before cryofixation. The results stress the importance of immediate cryofixation after surgical excision of a biopsy. This is especially important since numerous elemental changes due to delayed cryofixation resemble those which occur in pathological processes.

Animals↗

PubMed1994

X-ray microanalysis of endocrine, exocrine and intestinal cells and organs in culture: technical and physiological aspects.

In the present study methods for preparation of cultured cells and organ cultures for analytical electron microscopy are investigated. These methods allow qualitative and quantitative analysis of mobile ions in combination with biochemical or morphological studies. Cultured cells can be easily prepared for analytical microscopy and therefore use of in vitro systems for X-ray microanalysis has increased over the last few years. Two major, anhydrous preparation techniques, by which loss or redistribution of ions is minimized, were used: (1) Cells were cryosectioned and analysis carried out on freeze-dried sections obtained from frozen cell monolayers, pelleted cells or organ cultures. (2)Cells cultured on supports compatible with elemental analysis were frozen after removal of experimental media by rinsing, freeze-dried and analyzed. The first technique was applied to the studies of the elemental content of isolated Langerhans islets and thyroid follicles cultured in collagen gel. The second was used in studies of the ionic changes in enterocytes. Data obtained from organotypic cell cultures and cultures of single cells were compared with analytical data obtained from sections of corresponding tissues, where isolation, culturing and steps in processing such as removal of culture or experimental medium were omitted. It was shown that often culture systems fully acceptable to physiologists have an elemental composition different from that of tissue in situ and can not be regarded as fully normal tissue.

Animals↗

PubMed1994

Distribution of sodium, magnesium, chlorine, calcium, potassium, phosphorus and sulphur in Z-, I- and A-bands in mammalian striated muscle.

The regularity of the striation of skeletal muscle offers a unique possibility to analyze different bands of the sarcomere in longitudinally cut semithin cryosections. The aim of the present study was to investigate the elemental content of the Z-, I- and A-bands within the sarcomere which may be related to the affinity of an element to different contractile elements and the water content in different bands. The highest potassium levels were found in the Z-band (802 mmol/kg dry weight (d.w.)) as compared to the I-band (697 mmol/kg d.w.) and the A-band (731 mmol/kg d.w.). The difference between A-band and Z-band, but not between I-band and A-band, was significant. The highest phosphorus values were detected in the Z- and I- bands and the lowest within the A-bands (632, 615 and 540 mmol/kg d.w. respectively). No significant differences were found in the concentrations of Na, S, or Cl. Ca was significantly lower in the I-band as compared to A- and Z-band. The Mg concentration in the I- and A-band was significantly higher than in the Z-band. By means of computerized densitometry, relative densities (proportional to the dry mass content) of the Z-, I- and A-band were calculated (23.9, 11.6, and 19.2, respectively). The mean value of dry mass over several sarcomeres varied between 19.5-22.5 which corresponds well with dry weight mass concentrations obtained by traditional methods. The values for dry mass content allowed recalculation of the elemental concentrations as mmol/kg wet weight.

Animals↗

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↗