Calcium and sulfur location in human nail.

Analytical electron microscopy was performed in order to elucidate the importance of calcium and sulfur to the hardness of nails. The free surfaces adsorb a considerable quantity of calcium from the environment. It is suggested that such adsorption occurs due to ion exchange. This concept is supported by the fact that the distribution of sulfur does not vary over the nail cross-section. Analytical electron microscopy offers unique possibilities of elemental distribution analysis in tissue fragments or sections from keratinized tissues.

The sensory epithelium and its innervation in the mole rat cochlea.

The mole rat (Spalax ehrenbergi) burrows throughout its life in subterranean tunnels. Several structural and functional features which adapt the mole rat to its habitat have been discerned. The goal of this study was to elucidate the structural basis for adaptation of the auditory end-organ to an environment where low-frequency acoustical signals prevail. For this purpose, cochleae of adult mole rats were studied with light and electron microscopy. Inner hair cells throughout the cochlea, and outer hair cells in the basal (high-frequency) portion of the cochlea, were similar in structure to those seen in other mammals. In contrast, outer hair cells in the apical (low-frequency) portion displayed unique structural features. These features resembled the structure of inner hair cells or immature outer hair cells. The innervation of outer hair cells was most uncommon, in that classical medial efferent innervation was not found throughout the cochlear spiral.

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.

Is copper hepatotoxic in primary biliary cirrhosis?

In primary biliary cirrhosis (PBC) liver copper retention occurs as a complication of cholestasis. By analogy with Wilson's disease, it has been suggested that copper retention is hepatotoxic in PBC, and this has been the rationale for the use of D-penicillamine in this disease. The hypothesis that copper is hepatotoxic in PBC has not been tested and in this study we have evaluated the role of liver copper retention in the pathogenesis of PBC. Sixty-four patients with PBC have been studied. Fifty-four had increased liver copper concentrations. Liver cell synthetic function was well preserved. All the patients had normal prothrombin times, and only two had subnormal serum albumin concentrations. There was no correlation between liver copper concentrations and the degree of liver cell damage assessed biochemically (aspartate transaminase), and histologically. Electron microscopy was performed on liver biopsies from five patients with markedly increased liver copper concentrations. The liver cell ultrastructure was compatible with cholestasis. Liver cells contained electron dense lysosomes, which were shown to contain copper and sulphur by x-ray probe microanalysis. The characteristic organelle changes associated with copper toxicity in Wilson's disease were not observed. The biochemical, histological, and histochemical differences between PBC complicated by liver copper retention, and Wilson's disease, indicates that there are differences in the handling of copper in these disease. In this study we could find no evidence to suggest that copper plays an important role in the pathogenesis of liver dysfunction in PBC.

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.

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.

Sarcoplasmic bodies in distal myopathy compared with nemaline rods.

Sarcoplasmic bodies in a late onset distal myopathy and rods in nemaline myopathy have been investigated by electron probe X-ray microanalysis correlated to light microscopy. Sarcoplasmic bodies were glassy, drop-shaped structures up to 10 microns of length and easily distinguished in the scanning mode of electron microscopy performed on thick freeze-dried cryosections. They were found to be source of X-ray spectra characterized by a high sulfur peak. On semithin epoxy sections the sarcoplasmic bodies were observed in the scanning transmission mode as electron-dense structures which, according to X-ray microanalysis, exhibited a high sulphur and osmium content, possibly indicating the presence of sulphydryl groups. The nemaline rods were of 3 different types as judged by their light-microscopical appearance. They were, however, not visible in the scanning mode of electron microscopy performed on freeze-dried unstained cryosections. There were small differences in the elemental composition between different rods within the same biopsy but these were not systematically related to the differences in morphological appearance.

Sulphur and phosphorus content in relation to fibre composition and atrophy of skeletal muscle in patients with Parkinson's disease.

Seventeen patients with Parkinson's disease have been compared with 8 normal individuals by biopsy of either the biceps brachii or quadriceps femoris muscles. All biopsies were investigated by enzyme histochemistry. With 13 patients, as well as all controls, scanning electron microscopy with X-ray microanalysis was employed on cryo-sections adjacent to those prepared for light microscopy. Thus, the elemental composition of single muscle fibres was obtained and could be related to histochemical fibre types. Fibre type analysis on the diseased material, based on differential stainability for alkali- and acid-stable ATPase, showed a normal type I and type IIA fibre frequency. A mild type IIB dominance at the expense of type IIA fibres was regarded as a significant deviation from normal. A slight to moderate muscle atrophy affected type IIB fibres almost exclusively. Normal content of sulphur and phosphorus was detected in type I and type IIA Fibres but a lowered sulphur content was obvious in type IIB fibres, especially in the atrophic ones, which also exhibited an increase in phosphorus content. The shift in fibre composition from IIA to IIB, the type IIB fibre atrophy and the change in sulphur and phosphorus content of type IIB fibres are interpreted as signs of a disuse which preferentially affects fast twitch type IIB motor units. These presumably have the highest threshold for activation under pathological conditions characterized by increased muscular tone and difficulties in the performance of rapid and strong voluntary movements.

Type distribution of muscle fibres and their ultrastructure related to intracellular elemental composition as revealed by energy dispersive X-ray microanalysis. A study of multicore myopathy.

Four cases of congenital myopathy, two children and two adults, are described whose light-microscopical and ultrastructural findings are consistent with a multicore myopathy. In all cases a hereditary etiology to the disorders is obvious presumably by an autosomal recessive trait. Fibres with central nuclei and multiple minicores was a prominent finding in all cases. Energy dispersive X-ray microanalysis of single muscle fibres revealed a normal intracellular content of elements (sodium, potassium and chlorine). This is in contrast to the findings in some cases of s.c. myotubular myopathy earlier described from our group where content of sodium and chlorine is markedly increased while that of potassium is decreased. It is suggested that in myotubular myopathy membrane dysfunction causing decreased ionic gradients is an important feature of the pathophysiology while in multicore myopathy other mechanisms, as a suggestion related to mitochondrial and myofibrillar function play a more prominent part. From a diagnostic point of view it seems that X-ray microanalysis can be used to differentiate the two conditions.

Measuring outcomes of hospital care using multiple risk-adjusted indexes.

Using existing data sources, we developed three risk-adjusted measures of hospital quality: the risk-adjusted mortality index (RAMI), the risk-adjusted readmissions index (RARI), and the risk-adjusted complication index (RACI). We describe the construction and validation of each of these indexes. After these measures were developed, we tested the relationships among the three indexes using a sample of 300 hospitals. Actual numbers of adverse events were observed for each hospital and compared to the number predicted by the RAMI, RARI, and RACI models. Then each hospital was ranked on each index. Our results showed that no relationship existed between a hospital's ranking on any one of these indexes and its ranking on the other two indexes. This result provides some evidence that no measure of quality should be used by itself to represent different aspects of the quality of hospital care. Adequate overall measures of hospital quality will need to include multiple measures in order to be credible and to reflect the complexity of hospital care. The findings suggest that consumers, payers, and policymakers cannot simply choose one hospitalwide measure, such as the mortality rate, to validly represent a hospital's performance: those hospitals with high rankings on their mortality rates do not necessarily rank high on their readmission rates or complication rates.

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.

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.

Elemental composition of the developing inner ear.

The elemental composition of the inner ear fluid-filled compartments has been analyzed using the x-ray energy dispersive technique (CBA mouse). Special attention has been focused on the maturation of endolymph. A few days before and after birth the relative peak intensity of potassium (RK) in the vestibular endolymphatic compartment was slightly surpassing that in the cochlear part of the labyrinth. From the fourth day after birth (DAB) a rapid increase occurred concerning the RK. The highest RK was found in the endolymphatic space in the basal part of the cochlea followed by that in the vestibular endolymph. The lowest RK was measured in endolymph of the apical part of the cochlea. These obvious differences were abandoned already on the sixth DAB. A mature composition of endolymph was reached on the eighth DAB. The present technique does not allow analyses of differences between cochlear and vestibular endolymph with regard to minimal shifts.

Xenogeneic neoplastic transplants in adult rats pretreated with thymic foetal extracts.

The paper deals with the effect of thymic extracts from young foetal pigs and calves on the development of tumour xenografts, i.e. mouse ascites Ehrlich carcinoma and hamster malignant melanoma, in rats. The results of experiments showed that Ehrlich carcinoma developed in 4 out of 72 animals, i.e., in those animals which had received thymic extracts from young foetal pigs, Ehrlich carcinoma led to their death. Ehrlich carcinoma assumed a solid form and the cells changed in their shape. It developed normally, when retransplanted to mice. Hamster melanoma, when transplanted to rats given thymic extracts from young foetal calves, developed and survived for long periods of time (in one animal up to 190 days, and in the remaining animals much longer than in the controls and the animals injected with liver extracts from young foetal calves). The melanoma xenograft was not morphologically changed. A normal development of the malignant process occurred when melanoma was retransplanted to hamsters. Animals injected with thymic extracts from young foetal calves exhibited lymphopoenia and a fall in gamma globulin levels before tumour transplantation.

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.

Elemental composition of the mature inner ear.

An X-ray energy dispersive analysis was performed on the mature serial sectioned freeze-dried mammalian labyrinth (CBA/CBA mouse). The elemental distribution in the endolymphatic and perilymphatic spaces revealed similar results as have been reported in the literature when withdrawing fluid for microanalysis. The cupulae of the vestibular organs showed only minimal relative peak intensities of chlorine and potassium in contrast to the tectorial membrane in the cochlea with elemental identification also of sodium, sulphur and calcium. A difference in structural composition is therefore likely between these two tissues.

Microprobe analysis of human otoconia.

Utricular and saccular otoconia (N = 510) from 1 fetus (39th gestational week; Trisomy-18), 2 babies (1 1/2 and 2 months) and 3 adults (58, 61 and 77 years) were analysed with an energy dispersive X-ray microprobe. In all otoconia the elemental composition showed an extremely high concentration of Ca but also Na, Mg, P, S, Cl and K were identified. The presence of non-Ca elements was interpreted as being due to a protein nucleus of the otoconia. Non-Ca elements are more prominent during fetal age and early post-natally than later in life. Old age otoconia contained high concentrations of P which indicates that a ' phosphatization ' of aging otoconia occurs, i.e. calcite otoconia are transformed in situ into apatite by reaction with phosphate ions. The elemental composition of otoconia in Trisomy-18 was similar to that in early postnatal inner ears.

Elemental composition of individual cells and tissues in the cochlea.

Localization of elements at the cellular and sub-cellular levels was performed with the energy dispersive X-ray microanalysis technique, using shock-frozen, freeze-dried and araldite-embedded mouse (CBA/CBA) cochleae sectioned dry. Anatomical identification occurred in the STEM (scanning transmission electron microscopy) mode. In inner hair cell stereocilia the K/Na ratio was 70:1 but only 20:1 in the cytoplasm. In outer hair cell cytoplasm the K/Na ratio was 11:1 while the ratio in stereocilia was similar to that in inner hair cells. Ca was identified in stereocilia and the upper part of the cytoplasm of both outer and inner hair cells. The elemental composition in the subtectorial space is endolymph-like and that in the inner tunnel of the organ of Corti is similar to extracellular fluid. Considerable regional differences in elemental composition occur in the tectorial membrane with regard to P, K and Ca. The highest concentration of Ca occurs in the basal part of the tectorial membrane towards the sensory hairs. The highest concentration of K occurs in the basal and outer parts whereas the middle part of the tectorial membrane contains low levels of both K and Ca. The elemental composition changes in two main directions: 1) from the limbal (growing) region to the tip of the tectorial membrane, and, 2) from upper to lower surfaces. The three cell types of the stria vascularis differ considerably in elemental composition. The highest concentration of K occurs in marginal cells. The basal cells contain more K than do the intermediate cells. A significantly higher concentration of Ca, Cl and Na occurs in marginal cell cytoplasm than in any other cell type in the stria vascularis.

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.

Microanalytic and light microscopic studies on the developing otic capsule.

The structure of the middle layer of the bone of the otic capsule is histologically unique: it is of endochondral origin, has no Haversian canals and the ossification develops from several centres which fuse to form the hardest bone in the body. Our study has been concentrated on the development of the otic capsule in the CBA/CBA mouse, followed from the 13th gestational day to early postnatal age. In the 14th gestational day inner ear, a condensation of mesenchyme is detected around the membranous labyrinth. A cartilaginous capsule is present on the 15th-16th gestational day. Prior to birth, ossification centres occur close to the stapedial footplate. Serial cryosectioning of the newborn inner ear reveals very few regions containing high levels of calcium (microprobe analysis) although by light microscopy, several ossification centres can be identified.