J. Chwiej

Synchrotron FTIR micro-spectroscopy study of the rat hippocampal formation after pilocarpine-evoked seizures

In the present work, synchrotron radiation Fourier transform infrared (SRFTIR) micro-spectroscopy and imaging were used for topographic and semi-quantitative biochemical analysis of rat brain tissue in cases of pilocarpine-induced epilepsy. The tissue samples were analyzed with a beam defined by small apertures and spatial resolution steps of 10 microm which allowed us to probe the selected cellular layers of hippocampal formation. Raster scanning of the samples has generated 2D chemical cartographies revealing the distribution of proteins, lipids and nucleic acids. Spectral analysis has shown changes in the saturation level of phospholipids and relative secondary structure of proteins. Special interest was put in the analysis of two areas of the hippocampal formation (sector 3 of the Ammons horn, CA3 and dentate gyrus, DG) in which elemental abnormalities were observed during our previous studies. Statistically significant increase in the saturation level of phospholipids (increased ratio of the absorption intensities at around 2921 and 2958 cm(-1)) as well as conformational changes of proteins (beta-type structure discrepancies as shown by the increased ratio of the absorbance intensities at around 1631 and 1657 cm(-1) as well as the ratio of the absorbance at 1548 and 1657 cm(-1)) were detected in pyramidal cells of CA3 area as well as in the multiform and molecular layers of DG. The findings presented here suggest that abnormalities in the protein secondary structure and increases in the level of phospholipid saturation could be involved in mechanisms of neurodegenerative changes following the oxidative stress evoked in brain areas affected by pilocarpine-induced seizures.

Investigations of differences in iron oxidation state inside single neurons from substantia nigra of Parkinson’s disease and control patients using the micro-XANES technique

X-ray absorption near edge structure spectroscopy was applied in order to investigate differences in iron chemical state between the nerve cells of substantia nigra (SN) representing Parkinson’s disease (PD) and those of control cases. Autopsy samples were cut using a cryotome, and were not fixed and not embedded in paraffin. The comparison of the absorption spectra near the iron K-edge measured in melanized neurons from SN of PD and control samples did not show significant differences in iron oxidation state. Measurements of inorganic reference materials containing iron in the second and third oxidation states indicate that most of the iron in all the nerve cell bodies examined was oxidized and occurred as trivalent ferric iron (Fe3+).

The role of trace elements in the pathogenesis and progress of pilocarpine-induced epileptic seizures

X-ray fluorescence microscopy was applied for topographic and quantitative elemental analysis within the areas of the rat brain that undergo neurodegenerative changes in consequence of pilocarpine-induced seizures. Significant changes in levels of selected elements were observed in epileptic animals. They included an increased tissue content of Ca in the CA1 and CA3 regions of the hippocampus and in the cerebral cortex. The opposite relation was observed for the Cu level in the dentate gyrus and for Zn in the CA3 region of the hippocampus and in the dentate gyrus.

Study of Cu chemical state inside single neurons from Parkinson's disease and control substantia nigra using the micro-XANES technique.

Parkinsons disease (PD) is referred to as idiopathic disorder, which means that its causes have not been found yet. However, a few processes such as oxidative stress, protein aggregation and mitochondrial dysfunction are suspected to lead to the atrophy and death of substantia nigra (SN) neurons in case of this neurodegenerative disorder. Cu is a trace element whose role in the pathogenesis of PD is widely discussed. The investigation of Cu oxidation state inside single nerve cells from SN of PD and control cases may shed some new light on the role of this element in PD. The differences in Cu chemical state were investigated with the use of X-ray absorption near edge structure (XANES) spectroscopy. The least-square fitting method was applied for the analysis of XANES spectra. The comparison of the positions of white line, multiple scattering and pre-edge peak maximum at the energy scale did not reveal the existence of differences in Cu chemical state between PD and control samples. However, it was found that most of the Cu inside SN neurons occurs in tetrahedral environment and probably as Cu(II).

Neuroprotective action of FK-506 (tacrolimus) after seizures induced with pilocarpine: quantitative and topographic elemental analysis of brain tissue

In the present work, X-ray fluorescence microscopy with a synchrotron source for the exciting radiation was applied for topographic and quantitative elemental analysis of rat brain tissue in pilocarpine-induced epilepsy and neuroprotection with FK-506. The mass per unit area of the elements P, S, Cl, K, Ca, Fe, Cu, Zn, Se, Br, and Rb was determined in four fields of the hippocampal formation (sectors 1 and 3 of Ammon’s horn–CA1, CA3; dentate gyrus; hilus of dentate gyrus) and the parietal cortex. The results obtained for epileptic rats treated with FK-506 (SNF) were compared with data obtained previously for epileptic rats (SNS) and a control group. Many statistically significant differences in elemental composition were observed between the SNF and SNS groups. Higher mass per unit area of P was noticed in CA1 and CA3 regions of the hippocampus of SNF rats in comparison with SNS rats. A similar relation was observed for K in all five brain areas analyzed. Also, Fe in CA3 and dentate gyrus, Cu in the parietal cortex, and Zn in CA3 and in the cortex were present at a higher level in the SNF group in comparison with the SNS group. The findings obtained in the present study suggest that the neuroprotective action of FK-506 in epileptic rat brain may involve not only the inhibition of calcineurin but also blockade of the K+ channels.

The use of cluster and discriminant analysis in the investigations of the role of trace metals in the pathogenesis of Parkinson's disease.

X-ray fluorescence microscopy was applied for two-dimensional elemental analysis of substantia nigra (SN) tissue. The samples representing Parkinsons disease (PD) and control cases were examined at HASYLAB beamline L and at ESRF beamline ID22. Two-dimensional mapping of P, S, Cl, K, Ca, Fe, Cu, Zn, Se and Br was done with the spatial resolution of 15 and 5 microm. The masses per unit area of elements in neuromelanin reach nerve cells of SN were determined. The elemental data were processed using two multivariate techniques, namely cluster and discriminant analysis. The statistical methods were used for data reduction, both unsupervised and supervised classification as well as for the creation of a model that would simplify case identification based on the elemental analysis of SN tissue. The results of cluster analysis confirmed the statistical significance of the differences in elemental composition of PD and control SN nerve cells. Based on the results of discriminant analysis, the elements (P, Cl, Fe, Cu and Zn) that played the greatest role in the process of differentiation between neurons from examined groups were determined.

Elemental anomalies in the hippocampal formation after repetitive electrical stimulation: an X-ray fluorescence microscopy study

Our previous studies carried out on the pilocarpine model of seizures showed that highly resolved elemental analysis might be very helpful in the investigation of processes involved in the pathogenesis of epilepsy, such as excitotoxicity or mossy fiber sprouting. In this study, the changes in elemental composition that occurred in the hippocampal formation in the electrical kindling model of seizures were examined to determine the mechanisms responsible for the phenomenon of kindling and spontaneous seizure activity that may occur in this animal model. X-ray fluorescence microscopy was applied for topographic and quantitative analysis of selected elements in tissues taken from rats subjected to repetitive transauricular electroshocks (ES) and controls (N). The detailed comparisons were carried out for sectors 1 and 3 of the Ammon’s horn (CA1 and CA3, respectively), the dentate gyrus (DG) and hilus of DG. The obtained results showed only one statistically significant difference between ES and N groups, namely a higher level of Fe was noticed in CA3 region in the kindled animals. However, further analysis of correlations between the elemental levels and quantitative parameters describing electroshock-induced tonic and clonic seizures showed that the areal densities of some elements (Ca, Cu, Zn) strongly depended on the progress of kindling process. The areal density of Cu in CA1 decreased with the cumulative (totaled over 21 stimulation days) intensity and duration of electroshock-induced tonic seizures while Zn level in the hilus of DG was positively correlated with the duration and intensity of both tonic and clonic seizures.

Elemental changes in the hippocampal formation following two different formulas of ketogenic diet: an X-ray fluorescence microscopy study.

AbstractThe main purpose of the following study was the determination of elemental changes occurring within hippocampal formation as a result of high-fat and carbohydrate-restricted ketogenic diet (KD). To realize it, X-ray fluorescence microscopy was applied for topographic and quantitative analysis of P, S, K, Ca, Fe, Cu, Zn and Se in hippocampal formations taken from rats fed with two different KDs and naive controls. The detailed comparisons were done for sectors 1 and 3 of the Ammon’s, the dentate gyrus and hilus of dentate gyrus. The results of elemental analysis showed that the KDs induced statistically significant changes in the accumulation of P, K, Ca, Zn and Se in particular areas of hippocampal formation and these alterations strongly depended on the composition of the diets. Much greater influence on the hippocampal areal densities of examined elements was found for the KD which was characterized by a lower content of carbohydrates, higher content of fats and increased proportion of unsaturated fatty acids. The levels of P, K and Zn decreased whilst those of Ca and Se increased as a result of the treatment with the KDs.

Various ketogenic diets can differently support brain resistance against experimentally evoked seizures and seizure-induced elemental anomalies of hippocampal formation

In this paper the influence of two different ketogenic diets (KDs) on the seizure-evoked elemental anomalies of hippocampal formation was examined. To achieve this purpose normal and pilocarpine treated rats previously fed with one of the two high fat and carbohydrate restricted diets were compared with animals on standard laboratory diet. The ketogenic ratios of the examined KDs were equal to 5:1 (KD1) and 9:1 (KD2). KD1 and standard diet fed animals presented similar patterns of seizure-evoked elemental changes in hippocampal formation. Also the analysis of behavioral data recorded after pilocarpine injection did not show any significant differences in intensity and duration of seizures between KD1 and standard diet fed animals. Higher ketogenic ratio KD2 introduced in the normal hippocampal formation prolonged changes in the accumulation of P, K, Zn and Ca. Despite this, both the intensity and duration of seizures were significantly reduced in rats fed with KD2 which suggests that its saving action on the nerve tissue may protect brain from seizure propagation. Also seizure-evoked elemental anomalies in KD2 animals were different than those observed for rats both on KD1 and standard diets. The comparison of seizure experiencing and normal rats on KD2, did not show any statistically significant differences in elemental composition of CA1 and H hippocampal areas whilst in CA3 area only Zn level changed as a result of seizures. DG was the area mostly affected by seizures in KD2 fed rats but areal densities of all examined elements increased in this hippocampal region.

Elemental changes of hippocampal formation occurring during postnatal brain development

In this paper the elemental changes of rat hippocampal formation occurring during the postnatal development were examined. Three groups of animals were used in the study. These were naive Wistar rats at the age of 6-, 30- and 60-days and the chosen life periods corresponded to the neonatal period, childhood and early adulthood in humans, respectively. For the topographic and quantitative elemental analysis X-ray fluorescence microscopy was applied and the measurements were done at the FLUO beamline of ANKA. The detailed quantitative and statistical analysis was done for four areas of hippocampal formation, namely sectors 1 and 3 of the Ammons horn (CA1 and CA3, respectively), dentate gyrus (DG) and its internal area (hilus of DG, H). The obtained results showed that among the all examined elements (P, S, K, Ca, Fe, Cu, Zn and Se), only the levels of Fe and Zn changed significantly during postnatal development of the hippocampal formation and both the elements were significantly higher in young adults comparing to the rats in neonatal period. The increased Fe areal density was found in all examined hippocampal areas whilst Zn was elevated in CA3, DG and H. In order to follow the dynamics of age-dependent elemental changes, the statistical significance of differences in their accumulation between subsequent moments of time was examined. The obtained results showed statistically relevant increase of Zn level only in the first observation period (between 6th and 30th day of life). Afterwards the areal density of the element did not change significantly. The increase of Fe areal density took place in both examined periods, however the observed changes were small and usually not statistically relevant.