The use of SR-FTIR microspectroscopy for a preliminary biochemical study of the rat hippocampal formation tissue in case of pilocarpine induced epilepsy and neutroprotection with FK-506

The main aim of the work was the biochemical analysis of the hippocampal formation tissue in the case of epileptic rats treated with the neuroprotective agent FK-506. Three groups of animals were compared: rats with pilocarpine induced seizures treated and non-treated with tacrolimus as well as naive controls. Synchrotron radiation Fourier transform infrared (SR-FTIR) microspectroscopy was used for the biomolecular analysis of studied samples. The measurements were carried out at SISSI beamline of ELETTRA. A Bruker IFS 66v/S interferometer coupled to a Bruker Hyperion 2000 microscope was used. The tissue samples were analyzed in transmission mode with a beam defined by a small aperture and spatial resolution steps of 10 mu m which allowed us to probe the selected cross-line of the sample at cellular resolution. The obtained results enabled to compare the distributions of proteins and lipids in the three hippocampal cellular layers, i.e. in molecular, multiform and granular layers. For epileptic animals both treated and non-treated with FK-506, the tendency for increase of the ratio of the absorbance at around 1548 and 1658 cm(-1) (amide II/amide I ratio) was observed, however only for the multiform layer these changes were statistically significant. Similar relation was noticed in case of the ratio of the absorbance at around 1631 and 1658 cm(-1). The mentioned results may suggest conformational changes of proteins in the direction of beta-sheet secondary structure. Additionally, a statistically significant increase in the lipid massif and a decrease of the ratio of absorbance at around 2921 and 2958 cm(-1) were observed for epileptic animals treated with tacrolimus comparing to the control group

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

The 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

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

Altered Elemental Distribution in Male Rat Brain Tissue as a Predictor of Glioblastoma Multiforme Growth : Studies Using SR-XRF Microscopy

Glioblastoma multiforme (GBM) is a particularly malignant primary brain tumor. Despite enormous advances in the surgical treatment of cancer, radio- and chemotherapy, the average survival of patients suffering from this cancer does not usually exceed several months. For obvious ethical reasons, the search and testing of the new drugs and therapies of GBM cannot be carried out on humans, and for this purpose, animal models of the disease are most often used. However, to assess the efficacy and safety of the therapy basing on these models, a deep knowledge of the pathological changes associated with tumor development in the animal brain is necessary. Therefore, as part of our study, the synchrotron radiation-based X-ray fluorescence microscopy was applied for multi-elemental micro-imaging of the rat brain in which glioblastoma develops. Elemental changes occurring in animals after the implantation of two human glioma cell lines as well as the cells taken directly from a patient suffering from GBM were compared. Both the extent and intensity of elemental changes strongly correlated with the regions of glioma growth. The obtained results showed that the observation of elemental anomalies accompanying tumor development within an animal’s brain might facilitate our understanding of the pathogenesis and progress of GBM and also determine potential biomarkers of its extension. The tumors appearing in a rat’s brain were characterized by an increased accumulation of Fe and Se, whilst the tissue directly surrounding the tumor presented a higher accumulation of Cu. Furthermore, the results of the study allow us to consider Se as a potential elemental marker of GBM progression

Promieniowanie synchrotonowe w badaniach składu pierwiastkowego oraz zwiazków organicznych w komórkach nerwowych dla wybranych schorzeń neurodegeneracyjnych : rozprawa doktorska [...] wykonana w Zakładzie Metod Jądrowych pod kierunkiem Marka Lankosza /

Recenzenci pracy: Krzysztof Janeczko, Květoslava Burda.Praca doktorska. Akademia Górniczo-Hutnicza im. Stanisława Staszica (Kraków), 2007.Bibliogr. k. 138-150.Charakterystyka badanych schorzeń neurodegeneracyjnych, procesów związanych z ich patogenezą, choroba Parkinsona, stwardnienie boczne zanikowe, stres oksydacyjny, ekscytotoksyczność, dysfunkcja mitochondriów, agregacja białek, mutacja genu SOD1 kodującego miedziowo-cynkową dysmutazę nadtlenkową, dotychczasowy stan badań w zakresie analizy pierwiastkowej tkanki OUN w przypadku badanych schorzeń, podstawy fizyczne, krótka charakterystyka stosowanych metod pomiarowych, własności promieniowania synchrotronowego, rentgenowska mikroskopia fluorescencyjna, spektroskopia absorpcyjna promieniowania X w pobliżu krawędzi absorpcji, absorpcja promieniowania X, zjawisko fluorescencji rentgenowskiej, wymagania pomiarowe techniki μ-SRXRF, spektroskopia absorpcyjna promieni X w pobliżu krawędzi absorpcji, charakterystyka widma absorpcyjnego promieniowania X w pobliżu krawędzi absorpcji, wymagania pomiarowe metody XANES, spektroskopia absorpcyjna w zakresie IR, absorpcja promieniowania podczerwonego, rodzaje drgań cząsteczkowych, pasma absorpcji najważniejszych makromolekuł biologicznych, aparatura pomiarowa stosowana w mikrospektroskopii promieniowania podczerwonego, metody obrazowania składu chemicznego próbki z wykorzystaniem spektroskopii IR, wielowymiarowa analiza wariancji, analiza klastrowa, analiza dyskryminacyjna, materiał badawczy, preparatyka próbek, aparatura pomiarowa, linia pomiarowa L, HASYLAB, ID22, ESRF, MIRAGE (SuperACO, SA5), LURE, badania tkanek OUN z wykorzystaniem rentgenowskiej mikroskopii fluorescencyjnej, dobór metody preparatyki próbek tkanki dla potrzeb techniki μ-SRXRF, przygotowanie próbek tkanki, warunki pomiarowe, analiza danych pomiarowych, wykorzystanie rentgenowskiej mikroskopii fluorescencyjnej w badaniach dotyczących choroby Parkinsona, wykorzystanie rentgenowskiej mikroskopii fluorescencyjnej w badaniach dotyczących stwardnienia bocznego zanikowego, analiza statystyczna wyników badań uzyskanych metodą rentgenowskiej mikroskopii fluorescencyjnej, zastosowanie technik WAW w badaniach dotyczących PD, SBZ, wykorzystanie spektroskopii absorpcyjnej promieniowania X w badaniach stopnia utlenienia metali w tkance OUN, badanie stopnia utlenienia żelaza w neuronach istoty czarnej, badanie stopnia utlenienia miedzi w neuronach istoty czarnej, badania rozkładów związków organicznych w tkance OUN przy wykorzystaniu mikrospektroskopii promieniowania IR z transformatą Fouriera, zastosowanie mikrospektroskopii IR w badaniach dotyczących choroby Parkinsona, stwardnienia bocznego zanikowego, próba interpretacji, podsumowanie uzyskanych wynikó

Differences in the hippocampal frequency of creatine inclusions between the acute and latent phases of pilocarpine model defined using synchrotron radiation-based FTIR microspectroscopy

Temporal lobe epilepsy (TLE) is the most common type of epilepsy in adults. Of the animal models developed to investigate the pathogenesis of TLE, the one with pilocarpine-induced seizures is most often used. After pilocarpine administration in animals, three distinct periods-acute, latent, and chronic-can be distinguished according to their behavior. The present paper is the continuation of our previous study which has shown an increased occurrence of creatine inclusions in rat hippocampal formations from the acute phase of pilocarpine-induced status epilepticus (SE) and positive correlation between their quantity and the total time of seizure activity within the observation period. In this paper, we tried to verify if anomalies in hippocampal creatine accumulation were the temporary or permanent effect of pilocarpine-evoked seizures. To realize this purpose, male Wistar rats in the latent phase (3 days after pilocarpine administration) were examined. The results obtained for the period when stabilization of animal behavior and EEG occurs were afterwards compared with ones obtained for the acute phase of pilocarpine-induced SE and for naive controls. To investigate the frequency of creatine inclusions within the hippocampal formation as well as in its selected areas (sectors 1-3 of Ammon's horn (CA1-CA3), dentate gyrus (DG), and hilus of DG) and cellular layers (pyramidal, molecular, multiform, and granular cell layers), synchrotron radiation-based Fourier-transform infrared microspectroscopy was used. The applied technique, being a combination of light microscopy and infrared spectroscopy, allowed us to localize microscopic details in the analyzed samples and provided information concerning their chemical composition. Moreover, the use of a synchrotron source of IR radiation allowed us to carry out the research at the diffraction-limited spatial resolution which, because of the typical size of creatine inclusions (from a few to dozens of micrometers), was necessary for our study. The comparison of epileptic animals in the latent phase with controls showed statistically significant increase in the number of creatine inclusions for most of the analyzed hippocampal regions, all examined cellular layers, as well as the whole hippocampal formation. Moreover, for the hilus of the DG and CA3 area, the number of creatine deposits was higher in the latent than in the acute phase after pilocarpine injection. In light of the obtained results, an anomaly in the hippocampal accumulation of creatine is the long-term effect of pilocarpine-evoked seizures, and the intensity of this phenomenon may increase with time passing from the primary injury

The use of fourier transform infrared microspectroscopy for the determination of biochemical anomalies of the hippocampal formation characteristic for the kindling model of seizures

[Image: see text] The animal models of seizures and/or epilepsy are widely used to identify the pathomechanisms of the disease as well as to look for and test the new antiseizure therapies. The understanding of the mechanisms of action of new drugs and evaluation of their safety in animals require previous knowledge concerning the biomolecular anomalies characteristic for the particular model. Among different models of seizures, one of the most widely used is the kindling model that was also applied in our study. To examine the influence of multiple transauricular electroshocks on the biochemical composition of rat hippocampal formation, Fourier transform infrared (FT-IR) microspectrosopy was utilized. The chemical mapping of the main absorption bands and their ratios allowed us to detect significant anomalies in both the distribution and structure of main biomolecules for electrically stimulated rats. They included an increased relative content of proteins with β-sheet conformation (an increased ratio of the absorbance at the wavenumbers of 1635 and 1658 cm(–1)), a decreased level of cholesterol and/or its esters and compounds containing phosphate groups (a diminished intensity of the massif of 1360–1480 cm(–1) and the band at 1240 cm(–1)), as well as increased accumulation of carbohydrates and the compounds containing carbonyl groups (increased intensity of the bands at 1080 and 1740 cm(–1), respectively). The observed biomolecular abnormalities seem to be the consequence of lipid peroxidation promoted by reactive oxygen species as well as the mobilization of glucose that resulted from the increased demand to energy during postelectroshock seizures

The methods of vibrational microspectroscopy reveals long-term biochemical anomalies within the region of mechanical injury within the rat brain

Traumatic brain injury (TBI), meaning functional or structural brain damage which appear as a result of the application of the external physical force, constitutes the main cause of death and disability of individuals and a great socioeconomic problem. To search for the new therapeutic strategies for TBI, better knowledge about posttraumatic pathological changes occurring in the brain is necessary. Therefore in the present paper the Fourier transform infrared microspectroscopy and Raman microscopy were used to examine local and remote biochemical changes occurring in the rat brain as a result of focal cortex injury. The site of the injury and the dorsal part of the hippocampal formation together with the above situated cortex and white matter were the subject of the study. The topographic and quantitative biochemical analysis followed with the statistical study using principal component analysis showed significant biomolecular anomalies within the lesion site but not in the area of the dorsal hippocampal formation and in the above situated white matter and cortex. The observed intralesional anomalies included significantly decreased accumulation of lipids and their structural changes within the place of injury. Also the levels of compounds containing phosphate and carbonyl groups were lower within the lesion site comparing to the surrounding cortex. The opposite relation was, in turn, found for the bands characteristic to proteins and cholesterol/cholesterol esters

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 mu m 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 Ammon's 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