Crespo Pv

Changes in intracellular sodium, chlorine, and potassium concentrations in staurosporine-induced apoptosis

Ion gradients across the plasma membrane, fundamentally K+, play a pivotal role in the execution phase of apoptosis. However, little is known about other monovalent anions (Cl−) or cations (Na+) in apoptosis. In addition, the relationship between changes in total ion composition and morphological and biochemical events are poorly understood. We investigated simultaneous changes in sodium (Na), chlorine (Cl), and potassium (K) concentrations in stauroporine‐induced apoptosis by quantitative electron probe X‐ray microanalysis (EPXMA) in single cells. Apoptotic cells identified unequivocally from the presence of chromatin condensation in backscattered electron images were characterized by an increase in intracellular Na, a decrease in intracellular Cl and K concentrations, and a decrease in K/Na ratio. The ouabain‐sensitive Rb‐uptake assay demonstrated a net decrease in Na+/K+‐ATPase activity, suggesting that increases in Na and decreases in K and the K/Na ratio in apoptotic cells were related with inhibition of the Na+/K+‐ATPase pump. These changes in diffusible elements were associated with externalization of phosphatidyl serine and oligonucleosomal fragmentation of DNA. This alteration in ion homeostasis and morphological hallmarks of apoptosis occur in cells that have lost their inner mitochondrial transmembrane potential and before the plasma membrane becomes permeable.

Evaluation of the viability of cultured corneal endothelial cells by quantitative electron probe X-ray microanalysis

Construction of artificial organs and tissues by tissue engineering is strongly dependent on the availability of viable cells. For that reason, the viability and the physiological status of cells kept in culture must be evaluated before the cells can be used for clinical purposes. In this work, we determined the viability of isolated rabbit corneal endothelial cells by trypan blue staining and quantitative electron probe X‐ray microanalysis. Our results showed that the ionic content of potassium in cultured corneal endothelial cells tended to rise initially, but significantly decreased in cells in the fifth (and final) subculture, especially in comparison to cells in the fourth subculture (P < 0.001). However, the concentration of sulfur was higher in the fifth subculture than in the fourth subculture (P < 0.001), with a nonsignificant increase in sodium in the fifth subculture (P = 0.031). These data imply a remarkable decrease in the K/Na ratio from the fourth to the fifth subculture. Our microanalytical results, along with the morphological differences between cells in the last two subcultures, are compatible with an early phase of the preapoptotic process in the fifth subculture, and suggest that cells of the first four subcultures would be better candidates for tissue engineering. J. Cell. Physiol. 211: 692–698, 2007.

X-ray microanalytical histochemistry of human circumpulpar and mantle dentine.

An X-ray microanalytical histochemistry study was carried out, on thick sections observed under scanning electron microscopy, of five freshly extracted human premolars and five molars. In particular the circumpulpar and mantle dentine were examined to determine levels of calcium, phosphorus and sulphur, the basic elements involved in the process of biomineralization. Semiquantitative analysis was subsequently performed after the appropriate ZAF (Z, atomic number; A, absorption; F, secondary fluorescence) correction. Ca/P mass ratio was found to be similar for both regions in molars and slightly higher in circumpulpar dentine in premolars implying that this parameter is independent of the processes of biomineralization in the two dentinal regions. In contrast, statistically significant differences were recorded in sulphur content upon comparing mantle and circumpulpar dentine between premolars and molars (P less than 0.02). If sulphur, which has been associated with sulphated glycosaminoglycans (GAGs) decreases as mineralization progresses it would therefore seem reasonable to posit sulphur-rich areas in both topographical regions of the matrix--more significant in premolars than molars--able to undergo subsequent mineralization if required. The possible application of these findings in remineralization therapies is suggested.

Pantothenic Acid and Coenzyme A in Experimental Cisplatin-Induced Ototoxia

We have observed that pantothenic acid (PA) prevents deafness induced by cisplatin (CP) in the guinea pig if both drugs are administered jointly. When deafness was previously produced, recovery was sometimes obtained after the administration of PA; so, we studied the effects of PA on cisplatian-induced ototoxia in guinea pigs, both as a prophylactic agent in healthy animals, and as a therapeutic agent in animals previously made deaf by the drug. To elucidate why PA protects the ear from the toxic effects of CP, we used coenzyme A (CoA) instead of PA-since PA is a component of CoA-to test the hypothesis that the action of PA is due to CoA. The results were practically the same in both experiences, the compound action potential of the auditory nerve (CAP) was tested and cochleas were examined by scanning electron microscopy (SEM). Our results suggest that the protective effect of PA takes place through CoA. Both substances had the same effect on CP ototoxicity, but CoA appears to be much more active, since the dose tested here was much lower than that of PA.

Gentamicin Ototoxicity in Otoconia: Quantitative Electron Probe X-ray Microanalysis

Chronic gentamicin ototoxicity was evaluated in the otolithic membrane of adult OF1 mice at the otoconial layer of the saccule and utricle by quantitative electron probe X-ray microanalysis of Ca and K. The otolithic membranes were plunge-frozen and freeze-dried. The analysis was carried out with an energy dispersive detector using the peak-to-back-ground ratio method and different inorganic salts of Ca and K as standards to calibrate the microprobe. Ca and K in the otoconia are related via a linear function in both the saccule and the utricle. This association is not maintained after exposure to gentamicin, which suggests that this aminoglycoside antibiotic interferes with the Ca-K equilibrium in the otoconia. A dose of 200 mg/kg gentamicin twice a day for 5 days did not affect Ca in the mineral phase of the otoconia, but did increase K in both saccular (p < 0.05) and utricular (p < 0.01) otoconia. These increases in K may reflect a modification in the composition of the endolymph, resulting from cellular damage at the plasma membrane.

The crystalline pattern of calcium in different topographical regions of the otoconial membrane. An electron probe and spectroscopic diffraction study.

Quantitative electron probe microanalysis and electron spectroscopic diffraction analysis was used to determine the gradient of distribution of calcium and its crystalline pattern at different levels (lower gelatinous membrane, upper gelatinous membrane and otoliths) in the otoconial membrane of adult OF1 mice. Our quantitative electron probe microanalytical data, obtained with scanning-transmission electron microscopy, indicated that there was a gradient in calcium concentration which increased from the vestibular surface towards the otoliths. Differences between the three regions of the otoconial membrane were statistically significant in both the utricle and saccule. Our results with electron spectroscopic diffraction revealed an increasing crystalline development from the lower gelatinous membrane towards the otoliths. Our findings with both techniques suggest that the gelatinous membrane is involved in the maturation and crystallization of the otoliths.

X-ray microanalytical determination of P, S and K concentrations in the gelatinous membrane of the utricle

Electron probe X-ray microanalysis was used to determine the concentrations of P, S and K (Cp, CS, CK) in the gelatinous membrane of the mouse utricle. The otolithic membranes were plunge-frozen in liquid N2, freeze-dried and carbon-coated. Quantitative analysis was carried out with an energy dispersive detector using the peak-to-background ratio method and different concentrations of KH2PO4 and K2SO4 salts dissolved in dextran solutions to calibrate the microprobe. P, S and K were measured and their concentrations plotted as bar graphs to study the frequency distributions. Regression analysis revealed a dependence between the concentrations of P and K (CK = 1454.10 - 2.83 CP, r = -0.68745, p < 0.05), and P and S (CS = 43.18 + 0.23 CP, r = 0.66949, p < 0.05); however, no correlation was found between CK and CP (r = -0.25424). The findings obtained in the present study show an inverse relationship between P and K ions, and direct relationship between P and S in the gelatinous membrane of the utricle.

Standards for quantification of elements in the otolithic membrane by electron probe X-ray microanalysis: Calibration curves and electron beam sensitivity

An absolute quantitative standardization technique has been developed to measure Ca and K weight fractions (WF) in the otolithic membrane of the saccule and utricle by scanning electron microscopy and electron probe X‐ray analysis using the peak‐to‐background (P/B) ratio method. Microcrystalline salt standards were used to calibrate Ca and K Kα P/B or Y = (P/B) · Z2/A (Z = atomic number; A = atomic weight) against WF at 10, 15, 20 and 25 kV accelerating voltage. The effect of voltage on the calibration, plotting the coefficient of correlation (r) as a function of voltage, was not dependent on the voltage in the range 10–25 kV for Ca standards. K standards were also independent when P/B was corrected for Z2/A. Background counts in the otoconia (Bo) were obtained at 5, 25, 50, 100, 200 and 500 s and used to test the electron beam sensitivity of saccular and utricular otoconia. Bo was not dependent on the spectra acquisition time, with the exception of Bo under Kα K peak in the saccule at 10 kV. Ca and K WF were determined at 10, 15, 20 and 25 kV in the saccule and utricle, showing similar values regardless of the voltage used. This method of calibration offers several advantages, such as stability, homogeneity, known composition of the standards, high reproducibility at different voltages even without Z2/A correction and the similarity between the otoconia and crystal standards. We recommend the application of this method for other elements and biomineral systems.

Mineralization in the Newborn Rat Statoconia: An EDAX Study

Several types of statoconia, differing in size and shape, have been observed in macula utriculi, the smaller ones in the macular periphery. The larger statoconia seem to be formed by the association of smaller units and calcium presence is comparatively higher: In some parts of the marginal zone there exists a granular material in which the presence of calcium is significant.

Bone mineral density aspects in the femoral neck of hip fracture patients

Elderly people, due to neurological conditions and muscular atrophy, present a greater propensity to falls and thus are very susceptible to hip fractures. Other variables, such as osteoporosis, may also be related to the etiopathogenesis of hip fractures, although osteoporosis is in fact a concurrent disease, and merely a coadjutant cause. Nonetheless, osteoporosis can make fracture patterns more severe and interfere with osteosynthesis. Osteoporosis is the radiological image of osteopenia, a pathological concept meaning a smaller quantity of bone per unit of volume. The radiological expression of osteopenia is therefore that of bone tissue with a lower radiological density than normal. In the context of hip fractures, bone mineral density and bone architecture of the femoral neck together with protein expression profiles and cross-links of this anatomical area are of special interest which is reviewed in the current paper. Spatial variations in bone mineral density in the femoral neck were found in the literature with increased porosity from the periosteal to the endosteal region and also from the distal to the proximal part of the femoral neck. Furthermore, increased crystal size, increased cortical porosity, reduced osteocyte lacunar density and an increased Ca/P ratio associated with higher concentrations of Ca and P were described in hip fracture patients compared to control patients. Osteocalcin/collagen type 1 expression ratio and enzymatic cross-link content in high-density bone was found to be significantly lower in hip fractures compared to controls. In conclusion, further research in bone mineral density and associated parameters are of interest to deepen the understanding of osteoporotic hip fractures.