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.

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.

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.

Effects of dorsal root transection on morphology and chemical composition of degenerating nerve fibers and reactive astrocytes in the dorsal funiculus.

The morphology and chemical (elemental) composition of the dorsal funiculus of the rat spinal cord were examined 1 and 7 days after unilateral transection (rhizotomy) of the L4 and L5 dorsal roots, using light and electron microscopy as well as X-ray microanalysis. Changes were observed only in the dorsal funiculus on the side of injury and included disintegration of the axonal cytoskeleton, enlargement of axonal mitochondria, and widening of the myelin lamellae of the injured axons. X-ray microanalysis demonstrated a significant increase in intraaxonal sodium at 1 day after injury. This increase was abolished at 7 days, but at this stage there was a significant lowering of potassium in axons and myelin sheaths and of phosphorus in myelin as well as a marked increase in calcium in the axoplasm of the degenerating axons. The nonneuronal cell compartment, largely composed of astrocytes, showed elevated sodium, chlorine, and calcium and lowered potassium levels. The changes in chemical composition paralleled an increase in immunoreactivity for the calcium-binding Mts1 (S100A4) protein, which is exclusively expressed by white matter astrocytes. The influx of calcium is likely to play a crucial role in the loss of axonal integrity after rhizotomy, while the alterations in potassium, and perhaps also phosphorus, may contribute to activation of the nonneuronal cells, including the up-regulation of Mts1 expression in astrocytes.

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.

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.

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.

Elemental composition of muscle at rest and potassium levels in muscle, plasma and sweat of horses exercising at 20 degrees C and 35 degrees C.

In this study, 4 Standardbred geldings were exercised at 20 and 35 degrees C. The exercise test (ET) consisted of 2 exercise bouts separated by 2 h of rest in their boxes. Blood samples were taken before, during and after the second exercise bout and muscle (m. gluteus medius) biopsies were taken before the first exercise bout and after an intensive trot over 2600 m in the second exercise bout. The blood samples were analysed for plasma potassium and total plasma protein concentration (TPP) and the muscle fibres were analysed for elemental composition by x-ray microanalysis. The intracellular content was as follows: sodium (Na) = 40 +/- 7; magnesium (Mg) = 32 +/- 4; phosphorus (P) = 282 +/- 15; sulphur (S) = 222 +/- 13; chloride (Cl) = 119 +/- 31; potassium (K) = 304 +/- 21 and calcium (Ca) = 8 +/- 2 mmol/kg dry weight under resting conditions. Intracellular potassium content increased after exercise compared to resting values. There was a good correlation between exercise intensity, plasma potassium concentration and shifts in plasma volume, indicated by alterations in TPP. This probably reflects the very fast shift of potassium and fluid between muscle and plasma. Plasma potassium concentrations decreased below resting values post exercise. The higher dehydration degree and potassium sweat loss after exercise at 35 degrees C was not reflected in lower muscular potassium content, but by a lower plasma potassium/total plasma protein ratio after exercise, indicating less circulating potassium.

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.

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.

X-ray microanalytical and morphological observations of the interface region between ceramic implant and bone.

In 8 monkeys both the maxillary lateral incisors were extracted. In the 16 extraction sites an Al2O3-ceramic dental implant (Frialit) was immediately inserted. Eight implants were submerged under the oral mucosa and the other 8 were allowed to penetrate through the mucosa during the observation period. None of the implants was loaded during the observation periods. After experimental periods of 1, 2, 4 and 8 months, the interface region between the implant and bone was examined using scanning-electron microscopy, X-ray microanalysis, back scatter, microradiography and light microscopy. The results showed that osseointegration of Al2O3-ceramic dental implant was obtained. The newly formed bone around the implant filled exactly the contours of the implant. Newly formed bone had a mineral content and a phosphorus-to-calcium ratio similar to that of the adjacent older mature jaw bone. There were no differences in the osseointegration rate between the submerged and the nonsubmerged implants. No leakage of aluminium from the implant could be detected in the bone.

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.

Concentrations of sodium, potassium, calcium, magnesium and chlorine in the muscle cells of downer cows and cows with parturient paresis.

The concentrations of sodium, potassium, calcium, magnesium, phosphorus, sulphur and chlorine, in the skeletal muscle fibres of cows with parturient paresis, downer cows, normally calving cows and unmated heifers were determined by means of an X-ray microanalysis technique. The most consistent finding was an increase in the concentration of chlorine in the muscle samples from the downer cows. There were no other significant differences between the concentrations of these elements in samples from the different groups of cows, but the calcium concentration was higher in the muscle samples collected one month after parturition than in the samples collected close to parturition.

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.

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.

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.

Intracellular and extracellular elemental composition of the endolymphatic sac studied by X-ray microanalysis.

X-ray microanalysis was performed along with light microscopy (LM) on rapidly frozen and cryo-sectioned endolymphatic sac tissues of adult guinea pigs, to determine the elemental composition of the different cell types in this tissue as well as the content of the sac lumen. The morphological preservation and spatial resolution of cryo-sectioned endolymphatic sac was found adequate for the identification of the different cell types of the sac in the transmission electron microscope. Further cell type identification was performed by comparing scanning transmission electron microscopy images with LM images on adjacent serial sections. X-ray microanalysis demonstrated differences between epithelial and sub-epithelial cells in the intracellular concentrations of sodium, chlorine, potassium, calcium, phosphorus and sulphur. Measurements performed in the lumen of the endolymphatic sac showed elevated sodium and decreased potassium levels as compared with the known levels of these elements in cochlear or vestibular endolymph. High phosphorus and sulphur levels were also found in the endolymph of the sac. Other morphological and analytical findings on the luminal content point out that otoconial destruction and cleaning of the endolymph from the cell debris and other products such as lipids and proteins take place in the endolymphatic sac. Our results suggest that the endolymphatic sac participates in fluid absorption (osmoregulation), ion transport and otoconial destruction. The data support the longitudinal flow theory of the endolymph.

X-ray microanalysis of chief cells in rat parathyroid gland.

X-ray microanalysis was used to study the elemental composition of the rat parathyroid gland. Analyses were performed in morphologically defined cells. Use of semi-thin cryosections allowed us to selectively analyze cell nucleus, cytoplasm and occasionally dense granules in the cytoplasm. The concentration of elements (mmol/kg dry weight) in chief cells of parathyroid gland were as follows: Na 83, Mg 48, P 853, S 224, Cl 164, K 551 and Ca 10. Significantly higher phosphorus and potassium concentrations were found in the dense granules as compared to cytoplasm of the parathyroid chief cells. Choosing rat parathyroid gland gave the possibility to exclusively analyze chief cells. Data obtained from such a homogenous cell population will be of interest when studying human parathyroid in which chief cells are mixed together with other types of cells.

Growth regulation of LLC-PK1 cells: lack of effect of Na(+)-loading.

To gain more information about the growth regulation of renal epithelial cells, we examined the growth stimulatory effect of serum and intracellular sodium in the renal epithelial cell line, LLC-PK1. In subconfluent LLC-PK1 cells serum-starved for 5 days and exposed to [3H]thymidine for 24 h, 22.9% of the cells synthesized DNA. Stimulation with 10% foetal calf serum (FCS) caused an almost three-fold increase in the fraction of labelled nuclei (62.2%). Serum-starved LLC-PK1 cells exposed to 10% FCS responded with an increased abundance of c-jun transcripts. The maximal expression of the c-jun transcripts occurred at 60 min and declined 120 min after serum stimulation. It has been suggested that an increase in Na+ influx plays a role in the growth regulation of renal epithelial cells. This prompted us to study the effect of intracellular Na+ loading on the growth response of LLC-PK1 cells. Serum-starved LLC-PK1 cells were incubated in a low K+ medium or exposed to Nystatin. Incubation in a low K+ medium or with Nystatin resulted in a marked increase in intracellular Na after only 5 min. A low K+ medium did not significantly influence the intracellular pH. No effect was observed on DNA synthesis or the abundance of c-jun transcripts in LLC-PK1 cells. Nor did Na+ loading enhance the growth stimulatory effect of serum. The results suggest that an increase in intracellular sodium does not directly regulate the growth of renal epithelial cells.

X-ray micro-analysis of cultured respiratory epithelial cells from patients with cystic fibrosis.

X-ray micro-analysis was carried out on cultured respiratory cells from polyps removed from individuals with and without cystic fibrosis (CF). In a first set of experiments, proper experimental conditions were established. Washing the cells with 300 mmol l-1 mannitol in distilled water was found to give the best removal of the culture medium. The elemental concentrations stabilized in about 10 min after the start of the preincubation. Intracellular [Na] and [Cl] increased slightly with increasing passage number, whereas intracellular [K] decreased. Under resting conditions there were no significant differences in elemental content between CF and control cells, and there were no indications for abnormally high total [Ca] in CF cells. In normal cells, stimulation with a cAMP-analogue resulted in a decrease of cellular [Cl], whereas in CF cells an increase was measured. Exposure of both normal and CF cells to ouabain resulted in decreased [K] and increased [Na] and [Cl] level. The calcium ionophore A23187 had a similar effect on normal cells but did not affect CF cells markedly. Application of amiloride to the apical side of the cells resulted in a decrease of cellular [Na] in CF cells, whereas [Na] in control cells was not affected. The results correspond with what is known about the defective cAMP-regulated transepithelial Cl-transport in CF cells. The effect of the calcium ionophore on cellular electrolyte content is more complicated and may be the result of two separate effects: efflux of Cl- via a Ca(2+)-dependent mechanism and inhibition of the Na(+)-K(+)-ATPase by intracellular Ca2+ ions causing an influx of Na+ and Cl- ions.