Glioblastoma multiforme influence on the elemental homeostasis of the distant organs: the results of inter-comparison study carried out with TXRF method.

Glioblastoma (GBM) is a fast-growing and aggressive brain tumor which invades the nearby brain tissue but generally does not spread to the distant organs. Nonetheless, if untreated, GBM can result in patient death in time even less than few months from the diagnosis. The influence of the tumor progress on organs other than brain is obvious but still not well described. Therefore, we examined the elemental abnormalities appearing in selected body organs (kidney, heart, spleen, lung) in two rat models of GBM. The animals used for the study were subjected to the implantation of human GBM cell lines (U87MG and T98G) characterized by different levels of invasiveness. The elemental analysis of digested organ samples was carried out using the total reflection X-ray fluorescence (TXRF) method, independently, in three European laboratories utilizing various commercially available TXRF spectrometers. The comparison of the data obtained for animals subjected to T98G and U87MG cells implantation showed a number of elemental anomalies in the examined organs. What is more, the abnormalities were found for rats even if neoplastic tumor did not develop in their brains. The most of alterations for both experimental groups were noted in the spleen and lungs, with the direction of the found element changes in these organs being the opposite. The observed disorders of element homeostasis may result from many processes occurring in the animal body as a result of implantation of cancer cells or the development of GBM, including inflammation, anemia of chronic disease or changes in iron metabolism. Tumor induced changes in organ elemental composition detected in cooperating laboratories were usually in a good agreement. In case of elements with higher atomic numbers (Fe, Cu, Zn and Se), 88% of the results were classified as fully compliant. Some discrepancies between the laboratories were found for lighter elements (P, S, K and Ca). However, also in this case, the obtained results fulfilled the requirements of full (the results from three laboratories were in agreement) or partial agreement (the results from two laboratories were in agreement).

Surface-enhanced Raman scattering (SERS) and tip-enhanced Raman scattering (TERS) in label-free characterization of erythrocyte membranes and extracellular vesicles at the nano-scale and molecular level.

The manuscript presents the potential of surface-enhanced Raman spectroscopy (SERS) and tip-enhanced Raman spectroscopy (TERS) for label-free characterization of extracellular microvesicles (EVs) and their isolated membranes derived from red blood cells (RBCs) at the nanoscale and at the single-molecule level, providing detection of a few individual amino acids, protein and lipid membrane compartments. The study shows future directions for research, such as investigating the use of the mentioned techniques for the detection and diagnosis of diseases. We demonstrate that SERS and TERS are powerful techniques for identifying the biochemical composition of EVs and their membranes, allowing the detection of small molecules, lipids, and proteins. Furthermore, extracellular vesicles released from red blood cells (REVs) can be broadly classified into exosomes, microvesicles, and apoptotic bodies, based on their size and biogenesis pathways. Our study specifically focuses on microvesicles that range from 100 to 1000 nanometres in diameter, as presented in AFM images. Using SERS and TERS spectra obtained for REVs and their membranes, we were able to characterize the chemical and structural properties of microvesicle membranes with high sensitivity and specificity. This information may help better distinguish and categorize different types of EVs, leading to a better understanding of their functions and potential biomedical applications.

Ketogenic diet influence on the elemental homeostasis of internal organs is gender dependent.

The ketogenic diet (KD) is a low-carbohydrate and high-fat diet that gains increasing popularity in the treatment of numerous diseases, including epilepsy, brain cancers, type 2 diabetes and various metabolic syndromes. Although KD is effective in the treatment of mentioned medical conditions, it is unfortunately not without side effects. The most frequently occurring undesired outcomes of this diet are nutrient deficiencies, the formation of kidney stones, loss of bone mineral density, increased LDL (low-density lipoprotein) cholesterol levels and hormonal disturbances. Both the diet itself and the mentioned adverse effects can influence the elemental composition and homeostasis of internal organs. Therefore, the objective of this study was to determine the elemental abnormalities that appear in the liver, kidney, and spleen of rats subjected to long-term KD treatment. The investigation was conducted separately on males and females to determine if observed changes in the elemental composition of organs are gender-dependent. To measure the concentration of P, S, K, Ca, Fe, Cu, Zn and Se in the tissues the method of the total reflection X-ray fluorescence (TXRF) was utilized. The obtained results revealed numerous elemental abnormalities in the organs of animals fed a high-fat diet. Only some of them can be explained by the differences in the composition and intake of the ketogenic and standard diets. Furthermore, in many cases, the observed anomalies differed between male and female rats.

Vibrational spectroscopy methods for investigation of the animal models of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and devastating primary brain tumor among adults. It is highly lethal disease, as only 25% of patients survive longer than 1 year and only 5% more than 5 years from the diagnosis. To search for the new, more effective methods of treatment, the understanding of mechanisms underlying the process of tumorigenesis is needed. The new light on this problem may be shed by the analysis of biochemical anomalies of tissues affected by tumor growth. Therefore, in the present work, we applied the Fourier transform infrared (FTIR) and Raman microspectroscopy to evaluate changes in the distribution and structure of biomolecules appearing in the rat brain as a result of glioblastoma development. In turn, synchrotron X-ray fluorescence microscopy was utilized to determine the elemental anomalies appearing in the nervous tissue. To achieve the assumed goals of the study animal models of GBM were used. The rats were subjected to the intracranial implantation of glioma cells with different degree of invasiveness. For spectroscopic investigation brain slices taken from the area of cancer cells administration were used. The obtained results revealed, among others, the decrease content of lipids and compounds containing carbonyl groups, compositional and structural changes of proteins as well as abnormalities in the distribution of low atomic number elements within the region of tumor.

Does Ketogenic Diet Used in Pregnancy Affect the Nervous System Development in Offspring?─FTIR Microspectroscopy Study.

Anti-seizure medications used during pregnancy may have transient or long-lasting impact on the nervous system of the offspring. Therefore, there is a great need to search for alternative therapies for pregnant women suffering from seizures. One of the solutions may be the use of the ketogenic diet (KD), which has been successfully applied as a treatment of drug-resistant epilepsy in children and adults. However, the risks associated with the use of this dietary therapy during pregnancy are unknown and more investigation in this area is needed. To shed some light on this problem, we attempted to determine the potential abnormalities in brain biomolecular composition that may occur in the offspring after the prenatal exposure to KD. To achieve this, the female Wistar rats were, during pregnancy, fed with either ketogenic or standard laboratory diet, and for further studies, their male offspring at 2, 6, or 14 days of age were used. Fourier transform infrared microspectroscopy was applied for topographic and quantitative analysis of main biological macromolecules (proteins, lipids, compounds containing phosphate and carbonyl groups, and cholesterol) in brain samples. Performed chemical mapping and further semi-quantitative and statistical analysis showed that the use of the KD during pregnancy, in general, does not lead to the brain biochemical anomalies in 2 and 6 days old rats. The exception from this rule was increased relative (comparing to proteins) content of compounds containing phosphate groups in white matter and cortex of 2 days old rats exposed prenatally to KD. Greater number of abnormalities was found in brains of the 14 days old offspring of KD-fed mothers. They included the increase of the relative level of compounds containing carbonyl groups (in cortex as well as multiform and molecular cells of the hippocampal formation) as well as the decrease of the relative content of lipids and their structural changes (in white matter). What is more, the surface of the internal capsule (structure of the white matter) determined for this age group was smaller in animals subjected to prenatal KD exposure. The observed changes seem to arise from the elevated exposition to ketone bodies during a fetus life and the disturbance of lipid metabolism after prenatal exposure to the KD. These changes may be also associated with the processes of compensation of mother organism, which slowly began to make up for the deficiencies in carbohydrates postpartum.

Influence of measurement mode on the results of glioblastoma multiforme analysis with the FTIR microspectroscopy.

Fourier Transform Infrared (FTIR) microspectroscopy is well known for its effectiveness in spectral and biochemical analyses of various materials. It enables to determine the sample biochemical composition by assigning detected frequencies, characteristic for functional groups of main biological macromolecules. In analysis of tissue sections one of two measurement modes, namely transmission and transflection, is usually applied. The first one has relatively straightforward geometry, hence it is considered to be more precise and accurate. However, IR-transparent media are very fragile and expensive. Transflection does not require expensive substrates, but is more prone to disruptive influence of Mie scattering as well as electric field standing wave effect. The excessive comparison of spectra' characteristics, obtained via both measurement modes, was performed in this paper. By the means of Mann-Whitney non-parametrical U test and PCA, the comparison of results obtained with both modes and assessment of usefulness of IR spectra obtained with transmission and transflection modes to differentiate between healthy and GBM-affected tissue, were performed. The main objective of the presented research is to compare the results of FTIR analysis of unfixed biological samples performed with transflection and transmission mode. In frame of the study we demonstrated the discrepancies between results of biochemical analysis performed based on data obtained with transmission and transflection. Such observation suggests that caution should be taken in drawing conclusions from the results obtained with transflection geometry, as its more prone to disruptive effects. Despite that, IR spectra developed with both modes allowed to distinguish GBM area from healthy tissue, which proves their diagnostic potential. Especially, application of the ME-EMSC correction of spectra before PCA enhances the performance of both methods to distinguish the analysed tissue areas.

Biochemical changes of macrophages and U87MG cells occurring as a result of the exposure to iron oxide nanoparticles detected with the Raman microspectroscopy.

The core size of iron oxide nanoparticles (IONPs) is a crucial factor defining not only their magnetic properties but also toxicological profile and biocompatibility. On the other hand, particular IONPs may induce different biological response depending on the dose, exposure time, but mainly depending on the examined system. New light on this problem may be shed by the information concerning biomolecular anomalies appearing in various cell lines in response to the action of IONPs with different core diameters and this was accomplished in the present study. Using Raman microscopy we studied the abnormalities in the accumulation of proteins, lipids and organic matter within the nucleus, cytoplasm and cellular membrane of macrophages, HEK293T and U87MG cell line occurring as a result of 24-hour long exposure to PEG-coated magnetite IONPs. The examined nanoparticles had 5, 10 and 30 nm cores and were administered in doses 5 and 25 µg Fe/ml. The obtained results showed significant anomalies in biochemical composition of macrophages and the U87MG cells, but not the HEK293T cells, occurring as a result of exposure to all of the examined nanoparticles. However, IONPs with 10 nm core diminished the accumulation of biomolecules in cells only when they were administered at a larger dose. The Raman spectra recorded for the macrophages subjected to 30 nm IONPs and for the U87MG cells exposed to 5 and 10 nm showed the presence of additional bands in the wavenumber range 1700-2400 cm-1, probably resulting from the appearance of Fe adducts within cells. Our results indicate, moreover, that smaller IONPs may be effectively internalized into the U87MG cells, which points at their diagnostic/therapeutic potential in the case of glioblastoma multiforme.

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.

Ketogenic diet impairs neurological development of neonatal rats and affects biochemical composition of maternal brains: evidence of functional recovery in pups.

The ketogenic diet (KD) is a type of diet in which the intake of fats significantly increases at the cost of carbohydrates while maintaining an adequate amount of proteins. This kind of diet has been successfully used in clinical therapies of drug-resistant epilepsy, but there is still insufficient evidence on its safety when used in pregnancy. To assess KD effects on the course of gestation and fetal development, pregnant females were fed with: (i) KD during pregnancy and lactation periods (KD group), (ii) KD during pregnancy replaced with ND from the day 2 postpartum (KDND group) and (iii) normal diet alone (ND group). The body mass, ketone and glucose blood levels, and food intake were monitored. In brains of KD-fed females, FTIR biochemical analyses revealed increased concentrations of lipids and ketone groups containing molecules. In offspring of these females, significant reduction of the body mass and delays in neurological development were detected. However, replacement of KD with ND in these females at the beginning of lactation period led to regainment of the body mass in their pups as early as on the postnatal day 14. Moreover, the vast majority of our neurological tests detected functional recovery up to the normal level. It could be concluded that the ketogenic diet undoubtedly affects the brain of pregnant females and impairs the somatic and neurological development of their offspring. However, early postnatal withdrawal of this diet may initiate compensatory processes and considerable functional restitution of the nervous system based on still unrecognized mechanisms.

The Use of Fourier Transform Infrared Microspectroscopy for the Determination of Biochemical Anomalies of the Hippocampal Formation Characteristic for the Kindling Model of Seizures.

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 ß-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 influence of IONPs core size on their biocompatibility and activity in in vitro cellular models.

Although the key factor affecting the biocompatibility of IONPs is the core size, there is a lack of regular investigation concerning the impact of the parameter on the toxicity of these nanomaterials. Therefore, such studies were carried out in this paper. Their purpose was to compare the influence of PEG-coated-magnetite NPs with the core of 5, 10 and 30 nm on six carefully selected cell lines. The proliferation rate, viability, metabolic activity, migration activity, ROS levels and cytoskeleton architecture of cells have been evaluated for specified incubation periods. These were 24 and 72-h long incubations with IONPs administered in two doses: 5 and 25 µg Fe/ml. A decrease in viability was observed after exposure to the tested NPs for all the analyzed cell lines. This effect was not connected with core diameter but depended on the exposure time to the nanomaterials. IONPs increased not only the proliferation rate of macrophages-being phagocytic cells-but also, under certain conditions stimulated tumor cell divisions. Most likely, the increase in proliferation rate of macrophages contributed to the changes in the architecture of their cytoskeleton. The growth in the level of ROS in cells had been induced mainly by the smallest NPs. This effect was observed for HEK293T cells and two cancerous lines: U87MG (at both doses tested) and T98G (only for the higher dose). This requires further study concerning both potential toxicity of such IONPs to the kidneys and assessing their therapeutic potential in the treatment of glioblastoma multiforme.

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.

The assessment of the usability of selected instrumental techniques for the elemental analysis of biomedical samples.

The fundamental role of major, minor and trace elements in different physiological and pathological processes occurring in living organism makes that elemental analysis of biomedical samples becomes more and more popular issue. The most often used tools for analysis of the elemental composition of biological samples include Flame and Graphite Furnace Atomic Absorption Spectroscopy (F-AAS and GF-AAS), Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Each of these techniques has many advantages and limitations that should be considered in the first stage of planning the measurement procedure. Their reliability can be checked in the validation process and the precision, trueness and detection limits of elements belong to the most frequently determined validation parameters. The main purpose of this paper was the discussion of selected instrumental techniques (F-AAS, GF-AAS, ICP-OES and ICP-MS) in term of the achieved validation parameters and the usefulness in the analysis of biological samples. The focus in the detailed literature studies was also put on the methods of preparation of the biomedical samples. What is more based on the own data the usefulness of the total reflection X-ray fluorescence spectroscopy for the elemental analysis of animal tissues was examined. The detection limits of elements, precision and trueness for the technique were determined and compared with the literature data concerning other of the discussed techniques of elemental analysis. Reassuming, the following paper is to serve as a guide and comprehensive source of information concerning the validation parameters achievable in different instrumental techniques used for the elemental analysis of biomedical samples.

Comparison of Elemental Anomalies Following Implantation of Different Cell Lines of Glioblastoma Multiforme in the Rat Brain: A Total Reflection X-ray Fluorescence Spectroscopy Study.

Glioblastoma multiforme (GBM) is a primary brain tumor with a very high degree of malignancy and is classified by WHO as a glioma IV. At present, the treatment of patients suffering from GBM is based on surgical resection of the tumor with maximal protection of surrounding tissues followed by radio- and pharmacological therapy using temozolomide as the most frequently recommended drug. This strategy, however, does not guarantee success and has devastating consequences. Testing of new substances or therapies having potential in the treatment of GBM as well as detection of their side effects cannot be done on humans. Animal models of the disease are usually used for these purposes, and one possibility is the implantation of human tumor cells into rodent brains. Such a solution was used in the present study the purpose of which was comparison of elemental anomalies appearing in the brain as a result of implantation of different glioblastoma cell lines. These were two commercially available cell lines (U87MG and T98G), as well as tumor cells taken directly from a patient diagnosed with GBM. Using total reflection X-ray fluorescence we determined the contents of P, S, K, Ca, Fe, Cu, Zn, and Se in implanted-left and intact-right brain hemispheres. The number of elemental anomalies registered for both hemispheres was positively correlated with the invasiveness of GBM cells and was the highest for animals subjected to U87MG cell implantation, which presented significant decrease of P, K, and Cu levels and an increase of Se concentration within the left hemisphere. The abnormality common for all three groups of animals subjected to glioma cell implantation was increased Fe level in the brain, which may result from higher blood supply or the presence of hemorrhaging regions. In the case of the intact hemisphere, elevated Fe concentration may also indicate higher neuronal activity caused by taking over some functions of the left hemisphere impaired as a result of tumor growth.

Intravenously administered d-mannitol-coated maghemite nanoparticles cause elemental anomalies in selected rat organs.

In this study novel d-mannitol coated maghemite nanoparticles (MIONPs) are presented in terms of their influence on elemental homeostasis of living organisms and for this purpose highly sensitive total reflection X-ray fluorescence was used. Because of the biological indifference of d-mannitol and presumed lower toxicity of maghemite, compared to the most commonly used magnetite in nanomedicine, such nanoparticles seem to be promising candidates for biomedical applications. The examined dose of MIONPs was comparable with one of the lowest doses used in medical diagnostics. However, it should be emphasized that the amount of iron injected in this form is still significant compared to its total content in organs, especially in kidneys or the heart, and may easily disrupt their elemental homeostasis. The aim of the present study was to evaluate the elemental changes occurring in selected rat organs after injecting a low dose of MIONPs. The results were compared with those obtained for previously examined PEG-coated nanoparticles with magnetite cores. In the light of our findings the elemental changes observed after exposure to MIONPs were less extensive than those following PEG-coated magnetite nanoparticle administration.

Comparison of ultrasmall IONPs and Fe salts biocompatibility and activity in multi-cellular in vitro models.

In the paper, the results of the first regular studies of ultra-small iron oxide nanoparticles (IONPs) toxicity in vitro were presented. The influence of PEG-coated NPs with 5 nm magnetite core on six different cell lines was examined. These were: human bronchial fibroblasts, human embryonic kidney cells (HEK293T), two glioblastoma multiforme (GBM) cell lines as well as GBM cells isolated from a brain tumor of patient. Additionally, mouse macrophages were included in the study. The influence of IONPs in three different doses (1, 5 and 25 µg Fe/ml) on the viability, proliferation and migration activity of cells was assessed. Moreover, quantifying the intracellular ROS production, we determined the level of oxidative stress in cells exposed to IONPs. In the paper, for the first time, the effect of Fe in the form of IONPs was compared with the analogical data obtained for iron salts solutions containing the same amount of Fe, on the similar oxidation state. Our results clearly showed that the influence of iron on the living cells strongly depends not only on the used cell line, dose and exposure time but also on the form in which this element was administered to the culture. Notably, nanoparticles can stimulate the proliferation of some cell lines, including glioblastoma multiforme. Compared to Fe salts, they have a stronger negative impact on the viability of the cells tested. Ultra-small NPs, also, more often positively affect cell motility which seem to differ them from the NPs with larger core diameters.

FTIR microspectroscopy revealed biochemical changes in liver and kidneys as a result of exposure to low dose of iron oxide nanoparticles.

Iron oxide nanoparticles (IONPs) have biomedical and biotechnological applications in magnetic imaging, drug-delivery, magnetic separation and purification. The biocompatibility of such particles may be improved by covering them with coating. In presented paper the biochemical anomalies of liver and kidney occurring in animals exposed to d-mannitol-coated iron(III) oxide nanoparticles (M-IONPs) were examined with Fourier transform infrared (FTIR) microspectroscopy. The dose of IONPs used in the study was significantly lower than those used so far in other research. Liver and kidney tissue sections were analysed by chemical mapping of infrared absorption bands originating from proteins, lipids, compounds containing phosphate groups, cholesterol and cholesterol esters. Changes in content and/or structure of the selected biomolecules were evaluated by comparison of the results obtained for animals treated with M-IONPs with those from control group. Biochemical analysis of liver samples demonstrated a few M-IONPs induced anomalies in the organ, mostly concerning the relative content of the selected compounds. The biomolecular changes, following exposition to nanoparticles, were much more intense within the kidney tissue. Biochemical aberrations found in the organ samples indicated at increase of tissue density, anomalies in fatty acids structure as well as changes in relative content of lipids and proteins. The simultaneous accumulation of lipids, phosphate groups as well as cholesterol and cholesterol esters in kidneys of rats exposed to IONPs may indicate that the particles stimulated formation of lipid droplets within the organ.

Organ Metallome Processed with Chemometric Methods Enable the Determination of Elements that May Serve as Markers of Exposure to Iron Oxide Nanoparticles in Male Rats.

The systemic influence of iron oxide nanoparticles on the elemental homeostasis of key organs was examined in male rats. In tissues taken at different intervals from nanoparticles injection, the dynamics of elemental changes was analyzed. The organ metallome was studied using total reflection X-ray fluorescence. The obtained data were processed with advanced cluster and discriminant analyses-to classify the tissues according to their organs of origin and to distinguish accurately the nanoparticle-treated and normal rats. Additionally, in the case of liver and heart, it was possible to determine the elements of highest significance for different treatments, which may serve as markers of exposure to iron oxide nanoparticles.

Biochemical Changes Indicate Developmental Stage in the Hippocampal Formation.

The literature showing how age of humans or animals influences the IR absorption spectra recorded in different brain regions is very poor. A very limited number of studies used FTIR microspectroscopy for analysis of the aging process, however there is lack of data concerning the biomolecular changes occurring in the course of postnatal development of the central nervous system. Therefore, in this paper the topographic and semiquantitative biochemical changes occurring within the rat hippocampus during postnatal development were examined. To achieve the goal of the study, three groups of normal male rats differing in age were investigated. These were 6, 30, and 60 day old animals, and the chosen ages correspond to the neonatal period, childhood, and early adulthood in humans, respectively. Already, preliminary topographic analysis identified a number of significant changes in the accumulation of biomolecules within the hippocampal formation occurring during brain development. Such observation was confirmed by further semiquantitative analysis of intensities of selected absorption bands or ratios of their intensities. The detailed examinations were done for four hippocampal cellular layers (multiform, molecular, pyramidal, and granular layers), and the results showed that the accumulation of most biomolecules, including both saturated and unsaturated lipids as well as compounds containing phosphate and carbonyl groups, was significantly higher in adulthood comparing to the neonatal period. What is more, the increases in their levels were observed mostly between 6th and 30th days of animals' life. The unsaturation level of lipids did not change during postnatal development, although the differences in unsaturated and saturated lipids contents were noticed between examined animal groups. Significant differences in relative secondary structure of proteins were found between young adult rats and animals in neonatal period for which the relative level of proteins with ß-type secondary structure was the highest.