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To maintain its functional abilities, the mature brain obtains energy from glucose produced in carbohydrate metabolism. When carbohydrates are eliminated from the diet, the energy comes from the oxidation of fatty acids. In this metabolic state called ketosis, ketone bodies are formed: β-hydroxybutyric acid (bHb), acetone, and acetoacetate as alternative source of energy passing through the blood–brain barrier easily. The ketosis state can be achieved through various strategies like caloric restriction, supplementation with medium-chain triglycerides, intense physical training, or ketogenic diet (KD). Using KD, drug-resistant epilepsy has been successfully treated in children and adults. It can also exert neuroprotective influences in cases of brain damage, glioblastoma multiforme, and Alzheimer's or Parkinson's diseases. Although many possible mechanisms of KD activity have been proposed, newer hypotheses appear with the research progress, mostly characterizing the brain under pathological but not normal conditions. Since different pathological conditions may affect the mechanism of KD action differently, additional research on the normal brain appears reasonable. For this purpose, young adult rats were treated with 4-month-lasting KD. Then, MRI structural measurements, spectroscopy, and tractography were performed. The procedures revealed significant increases in the concentration of glutamine, glutamate, glutathione and NAA, accompanied by changes in the pattern of neuronal connections of the striatum and hippocampal formation. This implies a possible involvement of these structures in the functional changes occurring in the brain after KD application. Thus, the investigations on the normal brain add important details concerning mechanisms underlying KD effects without their possible modification by a pathological status.

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.

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 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 main goal of this study was to evaluate the elemental changes occurring in the main rat organs (kidneys, spleen, heart, brain) as a result of polyethylene glycol-coated magnetic iron oxide nanoparticles (PEG-IONPs) administration. For this purpose, 24 animals were divided into four equinumerous groups, and the three of them were intravenously injected with PEG-IONPs dispersed in 15% solution of mannitol in dose of 0.03 mg of Fe per 1 kg of body weight. The organs were collected 2 h, 24 h and 7 days passing from NPs administration, respectively, for the 2H, 24H, and 7D experimental groups. The forth group of animals, namely control group, was injected with 1 mL of physiological saline solution. For the analysis of subtle elemental changes occurring in the organs after nanoparticles injection, highly sensitive method of total reflection X-ray fluorescence spectroscopy was used. Obtained results showed that administration of even such low doses of PEG-IONPs may lead to statistically significant changes in the accumulation of selected elements within kidneys and heart. Two hours and 7 days from NPs injection, the Fe level in kidneys was higher compared to that of control rats. Elevated levels of Cu, possibly associated with systemic action of ceruloplasmine enzyme, were found within kidneys in 24H and 7D groups, while in heart the similar observation was done only for 24H group. The levels of Ca and Zn increased in kidneys and heart during the first 2 h from the injection and were again elevated in these organs 7 days later. The abnormalities in Ca and Zn accumulations occurring exactly in the same manner may suggest that these elements may interplay either in the mechanisms responsible for the detoxification of the PEG-IONPs or pathological processes occurring as a result of their action.

The main purpose of this study was the determination and comparison of anomalies in creatine (Cr) accumulation occurring within CA3 and DG areas of hippocampal formation as a result of two high-fat, carbohydrate-restricted ketogenic diets (KD) with different ketogenic ratio (KR). To reach this goal, Fourier transformed infrared microspectroscopy with synchrotron radiation source (SRFTIR microspectroscopy) was applied for chemical mapping of creatine absorption bands, occurring around 1304, 1398 and 2800 cm-1. The samples were taken from three groups of experimental animals: control group (N) fed with standard laboratory diet, KD1 and KD2 groups fed with high-fat diets with KR 5:1 and 9:1 respectively. Additionally, the possible influence on the phosphocreatine (PhCr, the high energetic form of creatine) content was evaluated by comparative analysis of chemical maps obtained for creatine and for compounds containing phosphate groups which manifest in the spectra at the wavenumbers of around 1240 and 1080 cm-1. Our results showed that KD2 strongly modifies the frequency of Cr inclusions in both analyzed hippocampal areas. Statistical analysis, performed with Mann-Whitney U test revealed increased accumulation of Cr within CA3 and DG areas of KD2 fed rats compared to both normal rats and KD1 experimental group. Moreover, KD2 diet may modify the frequency of PhCr deposits as well as the PhCr to Cr ratio.

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.

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.

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.

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 Ammon's 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.

The main goal of this study was to evaluate in vivo effects of low dose of PEG-coated magnetic iron oxide nanoparticles (IONPs) on the rat liver. The IONPs was intravenously injected into rats at a dose equaled to 0.03 mg of Fe per 1 kg of an animal body weight. The elemental composition of liver tissue in rats subjected to IONPs action and controls were compared. Moreover, in order to determine the dynamics of nanoparticles (NPs) induced elemental changes, the tissues taken from animals 2 hours, 24 hours, and 7 days from IONPs injection were examined. The analysis of subtle elemental anomalies occurring as a result of IONPs action required application of highly sensitive analytical method. The total reflection X-ray fluorescence spectroscopy perfectly meets such requirements and therefore it was used in this study. The obtained results showed increasing trend of Fe level within liver occurring 2 hours from IONPs injection. One day after NPs administration, the liver Fe content presented the baseline level what suggests only the short-term accumulation of nanoparticles in the organ. The Ca, Cu, and Zn levels changed significantly as a result of NPs action. Moreover, the anomalies in their accumulation were still observed 7 days after IONPs injection. The level of Cu decreased while those of Ca and Zn increased in the liver of NPs-treated animals. The reduced liver Cu, followed by elevated serum level of this element, might be related in triggering the mechanisms responsible for Fe metabolism in the organism.

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.

In the present work, synchrotron radiation Fourier-transform infrared (SRFTIR) and Raman microspectroscopies were used to evaluate a possible role of creatine in the pathogenesis and progress of pilocarpine-evoked seizures and seizure-induced neurodegenerative changes in the rat hippocampal tissue. The main goal of this study was to identify creatine deposits within the examined brain area, to analyze their frequency in epileptic animals and naive controls and to examine correlations between the number of inclusions in the hippocampal formation of epileptic rats and the quantitative parameters describing animal behavior during 6-h observation period after pilocarpine injection. The presence of creatine in the brain tissue was confirmed based on the vibrational bands specific for this compound in the infrared and Raman spectra. These were the bands occurring at the wavenumbers around 2800, 1621, 1398, and 1304 cm−1 in IR spectra and around 1056, 908 and 834 cm−1 in the Raman spectra. Creatine was detected in eight of ten analyzed epileptic samples and in only one of six controls under the study. The number of deposits in epileptic animals varied from 1 to 100 and a relative majority of inclusions were detected in the area of the Dentate Gyrus and in the multiform hippocampal layer. Moreover, the number of creatine inclusions was positively correlated with the total time of seizure activity. Electronic supplementary material The online version of this article (doi:10.1007/s00216-011-5488-z) contains supplementary material, which is available to authorized users.

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 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.

A growing body of evidence demonstrates that dietary therapies, mainly the ketogenic diet, may be highly effective in the reduction of epileptic seizures. All of them share the common characteristic of restricting carbohydrate intake to shift the predominant caloric source of the diet to fat. Catabolism of fats results in the production of ketone bodies which become alternate energy substrates to glucose. Although many mechanisms by which ketone bodies yield its anticonvulsant effect are proposed, the relationships between the brain metabolism of the ketone bodies and their neuroprotective and antiepileptogenic action still remain to be discerned. In the study, X-ray fluorescence microscopy and FTIR microspectroscopy were used to follow ketogenic diet-induced changes in the elemental and biochemical compositions of rat hippocampal formation tissue. The use of synchrotron sources of X-rays and infrared allowed us to examine changes in the accumulation and distribution of selected elements (P, S, K, Ca, Fe, Cu, Zn, and Se) and biomolecules (proteins, lipids, ketone bodies, etc.) with the micrometer spatial resolution. The comparison of rats fed with the ketogenic diet and rats fed with the standard laboratory diet showed changes in the hippocampal accumulation of P, K, Ca, and Zn. The relations obtained for Ca (increased level in CA3, DG, and its internal area) and Zn (decreased areal density in CA3 and DG) were analogous to those that we previously observed for rats in the acute phase of pilocarpine-induced seizures. Biochemical analysis of tissues taken from ketogenic diet-fed rats demonstrated increased intensity of absorption band occurring at 1740 cm(-1), which was probably the result of elevated accumulation of ketone bodies. Moreover, higher absolute and relative (3012 cm(-1)/2924 cm(-1), 3012 cm(-1)/lipid massif, and 3012 cm(-1)/amide I) intensity of the 3012-cm(-1) band resulting from increased unsaturated fatty acids content was found after the treatment with the high-fat diet. This article is part of a Special Issue entitled "Status Epilepticus".

Gliosis, axonal sprouting and remodelling of nerve connections in the injured brain have been regarded as epileptogenic processes dependent on the age when the injury was inflicted. The present study examines whether brains injured at different developmental stages may acquire different susceptibility to experimental status epilepticus induced in adulthood. In 6- and 30-day-old Wistar rats (P6s and P30s, respectively), a mechanical injury was performed in the left cerebral hemisphere. On postnatal day 60, all the animals and controls received single injections of kainic acid to evoke status epilepticus. During a 6-h period following the injection, the animals were observed continuously and motor symptoms of seizure activity were recorded and rated. P6s showed significantly lower intensity of kainic acid-induced epileptic symptoms and significantly longer survival than controls or P30s. In P30s, no significant change was detected. The data provide evidence that the developmental stage when the brain is injured determines epileptogenecity of the lesion. However, a considerable discrepancy between these data and those obtained previously following pilocarpine administration in the same experimental models of brain injury shows that each of the two models of epilepsy may display different aspects of the same age-dependent process triggered by the brain injury.

In this study, ketogenic diet-induced biochemical changes occurring in normal and epileptic hippocampal formations were compared. Four groups of rats were analyzed, namely seizure experiencing animals and normal rats previously fed with ketogenic (KSE and K groups respectively) or standard laboratory diet (NSE and N groups respectively). Synchrotron radiation based Fourier-transform infrared microspectroscopy was used for the analysis of distributions of the main organic components (proteins, lipids, compounds containing phosphate group(s)) and their structural modifications as well as anomalies in creatine accumulation with micrometer spatial resolution. Infrared spectra recorded in the molecular layers of the dentate gyrus (DG) areas of normal rats on a ketogenic diet (K) presented increased intensity of the 1740 cm(-1) absorption band. This originates from the stretching vibrations of carbonyl groups and probably reflects increased accumulation of ketone bodies occurring in animals on a high fat diet compared to those fed with a standard laboratory diet (N). The comparison of K and N groups showed, moreover, elevated ratios of absorbance at 1634 and 1658 cm(-1) for DG internal layers and increased accumulation of creatine deposits in sector 3 of the Ammon's horn (CA3) hippocampal area of ketogenic diet fed rats. In multiform and internal layers of CA3, seizure experiencing animals on ketogenic diet (KSE) presented a lower ratio of absorbance at 1634 and 1658 cm(-1) compared to rats on standard laboratory diet (NSE). Moreover, in some of the examined cellular layers, the increased intensity of the 2924 cm(-1) lipid band as well as the massifs of 2800-3000 cm(-1) and 1360-1480 cm(-1), was found in KSE compared to NSE animals. The intensity of the 1740 cm(-1) band was diminished in DG molecular layers of KSE rats. The ketogenic diet did not modify the seizure induced anomalies in the unsaturation level of lipids or the number of creatine deposits.

Background Obesity is a worldwide epidemic with more than 600 million affected individuals. Human studies have demonstrated some alterations in brains of otherwise healthy obese individuals and elevated risk of neurodegenerative disease of old age; these studies have also pointed to slightly diminished memory and executive functions among healthy obese individuals. Similar findings were obtained in animal models of obesity induced by high fat diet. On the other hand, low carbohydrate high fat diets are currently promoted for losing weight (e.g., Atkin’s style diets). However, the long-term effects of such diets are not known. Additionally, high fat diets leading to (mild) ketonemia were shown to improve brain function in elderly humans and in some animal models. Aim To evaluate the hypothesis that long-term use of a high fat diet was associated with decreases in spatial memory, smaller hippocampi and hippocampi metabolite concentrations in Wistar rats. Methods Twenty five male Wistar rats were put on high fat diet (HFD; 60% calories from fat, 30% from carbohydrates) on their 55th day of life, while 25 control male rats (CONs) remained on chow. Adequate levels of essential nutrients were provided. Both groups underwent memory tests in 8-arm radial maze at 3rd, 6th, 9th, and 12th month. 1H magnetic resonance spectroscopy was employed to measure concentrations of tNAA (marker of neuronal integrity) at one month and one year, whereas MRI was used to evaluate hippocampal volumes. Results Obese rats (OBRs) consumed similar amount of calories as CONs, but less proteins. However, their protein intake was within recommended amounts. Throughout the experiment OBRs had statistically higher concentrations of blood ketone bodies than CONs, but still within normal values. At post-mortem assessment, OBRs had 38% larger fat deposits than CONs (p

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.

Influence of interferon gamma (IFNγ) on astrocytes proliferating in response to unilateral injury of the cerebral hemisphere was investigated in 30-day-old rats. The brain injury was followed by an immediate injection of IFNγ into the lesion cavity. On 1st or 2nd day following injury the animals were injected with [3H]thymidine and killed 4 h after the injection. The proliferating astrocytes were visualised by combination of immunocytochemical staining for glial fibrillary acidic protein (GFAP) and autoradiography. Thereafter, numbers of GFAP-immunopositive and autoradiographically-labeled astrocytes located within the region of injury were counted. On the day 1 after injury no effect of IFNγ administration was seen. However, on day 2 the average 43% reduction of the number of proliferating astrocytes was recorded. The reduction showed no dose-dependent changes. This in vivo evidence of IFNγ-induced suppression of astrocyte proliferation was considered in relation to other determinants of astrocyte reactivity existing in the injured brain.

Summary Rats with brain dysplasia evoked by interruption of different stages of prenatal neurogenesis show characteristic variations in susceptibility to seizures depending on the neurochemical specificity of pharmacological agents used to evoke seizures. To verify a discrepancy between the data obtained using different pharmacological models, neurochemically neutral electroshocks were applied here. To produce brain dysplasia of different degrees, pregnant Wistar rats were exposed to a single 1.0 Gy dose of gamma rays on gestation days 13, 15, 17 or 19. From the postnatal day 60, their male offspring (E13s, E15s, E17s and E19s, respectively) were subjected to 21 daily electrical stimulations to evoke seizures. Profiles of tonic and clonic reactivity to electrical stimulation significantly differed from those observed following pilocarpine or kainic acid administration. E17s showed minimal intensity of tonic but maximal of clonic responses. On the contrary, very high tonic and low clonic reactivity was observed in E13s and E15s. Periventricular nodular heterotopias (PNHs) were observed exclusively in E15s and E17s. Generally, the size of PNHs was correlated positively with susceptibility to tonic seizures but negatively with susceptibility to clonic seizures. Analogous correlations with the size of the neocortex were opposite. E13s and E19s had brains devoid PNHs but showed high tonic seizure susceptibility similar to that in E15s. It can therefore be concluded that PNHs modified the type of seizure reactivity from tonic to clonic, depending of their size, but the presence of PNHs was not necessary for the development of seizure susceptibility itself.

In the present paper, X-ray fluorescence microscopy was applied to follow the processes occurring in rat hippocampal formation during the post-seizure period. In the study, one of the status epilepticus animal models of epilepsy was used, namely the model of temporal lobe epilepsy with pilocarpine-induced seizures. In order to analyze the dynamics of seizure-induced elemental changes, the samples taken from seizure-experiencing animals 3 h and 1, 4, and 7 days after proconvulsive agent administration were analyzed. The obtained results confirmed the utility of X-ray fluorescence microscopy in the research of mechanisms involved in the pathogenesis and progress of epilepsy. The topographic and quantitative elemental analysis of hippocampal formations from different periods of epileptogenesis showed that excitotoxicity, mossy fibers sprouting, and iron-induced oxidative stress may be the processes responsible for seizure-induced neurodegenerative changes and spontaneous recurrent seizures occurring in the chronic phase of the pilocarpine model. The analysis of correlations between the recorded elemental anomalies and quantitative parameters describing animal behavior in the acute period of pilocarpine-induced status epilepticus showed that the areal densities of selected elements measured in the latent period strongly depend on the progress of the acute phase. Especially important seem to be the observations done for Ca and Zn levels which suggest that the intensity of the pathological processes such as excitotoxicity and mossy fibers sprouting depend on the total time of seizure activity. These results as well as dependencies found between the levels of S, K, and Cu and the intensity of maximal seizures clearly confirm how important it is to control the duration and intensity of seizures in clinical practice.

There is growing experimental evidence that tracing the elements involved in brain hyperexcitability, excitotoxicity, and/or subsequent neurodegeneration could be a valuable source of data on the molecular mechanisms triggering or promoting further development of epilepsy. The most frequently used experimental model of the temporal lobe epilepsy observed in clinical practice is the one based on pilocarpine-induced seizures. In the frame of this study, the elemental anomalies occurring for the rat hippocampal tissue in acute and silent periods after injection of pilocarpine in rats were compared. X-ray fluorescence microscopy was applied for the topographic and quantitative elemental analysis. The differences in the levels of elements such as P, S, K, Ca, Fe, Cu, and Zn between the rats 3 days (SE72) and 6 h (SE6) after pilocarpine injection as well as naive controls were examined. Comparison of SE72 and control groups showed, for specific areas of the hippocampal formation, lower levels of P, K, Cu, and Zn, and an increase in Ca accumulation. These results as well as further analysis of the differences between the SE72 and SE6 groups confirmed that seizure-induced excitotoxicity as well as mossy fiber sprouting are the mechanisms involved in the neurodegenerative processes which may finally lead to spontaneous seizures in the chronic period of the pilocarpine model. Moreover, in the light of the results obtained, Cu seems to play a very important role in the pathogenesis of epilepsy in this animal model. For all areas analyzed, the levels of this element recorded in the latent period were not only lower than those for controls but were even lower than the levels found in the acute period. The decreased hippocampal accumulation of Cu in the phase of behavior and EEG stabilization, a possible inhibitory effect of this element on excitatory amino acid receptors, and enhanced seizure susceptibility in Menkes disease (an inherited Cu transport disorder leading to Cu deficiency in the brain) suggest a neuroprotective role rather than neurodegenerative and proconvulsive roles of Cu in pilocarpine-induced epilepsy.

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 I 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.

Objective: Tacrolimus (FK506) and cyclosporin A (CsA), immunosuppressants widely used in post-transplantional therapy, have been reported to protect neurons in the injured brain. This effect can be exerted directly and indirectly via inflammatory cells. Since the data come exclusively from studies on the adult brain, we examined effects of the drugs on the macrophage recruitment in the brain injured at early developmental stages. Methods: Following the brain injury, 1- and 6-day-old Wistar rats (P1s and P6s, respectively) were treated with FK506 or CsA and injected with [3 H] thymidine. Brain sections were processed for BSI-B4 isolectin histochemistry and subjected to autoradiography to visualize proliferating and non-proliferating macrophages. Results: In P1s (n = 33), FK506 evoked a dose-dependent reduction in the number of macrophages. P6s (n = 30) presented greater decreases in macrophage numbers and their proliferative activity than the newborns. CsA application in P1s (n = 27) affected neither recruitment of macrophages to the region of injury nor their proliferation. In CsA-treated P6s (n = 28), reduction of the macrophage population and its proliferative activity was also seen but was much smaller than that following FK506 administration. Discussion: High effectiveness of FK506 in regulation of the inflammatory response and neuroprotection observed in the adult brain can also be considered as a possible indirect determinant of neuronal survival following the brain injury at very early developmental stages.

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.

Interruption of neurogenesis and/or neuronal migration produces brain dysplasia modifying susceptibility to epileptic seizures in adulthood. The course of neurogenesis has a strictly defined time-table. Consequently, the developmental stage at which the interruption occurs determines what functional subsystem potentially involved in epileptogenesis will suffer from irreversible neuronal deficit. The present study attempts to verify a hypothesis that brain dysplasias of different genesis should also lead to different susceptibility to seizures evoked by receptor agonists of different functional specificity, like kainic acid or pilocarpine, a cholinergic or glutaminergic agonist, respectively. Pregnant Wistar rats were exposed to gamma-rays on gestation days 13, 15, 17 or 19 (E13, E15, E17 or E19). Sixty-day-old offsprings of the females were injected with kainic acid or pilocarpine to evoke status epilepticus. During a 6-h period following the injection, motor manifestations of seizure activity were recorded. Generally, the intensity of pilocarpine-induced symptoms was relatively low in rats irradiated on E13 or E15 but high in rats irradiated on E17 or E19. In rats treated with kainic acid, the trend was opposite, viz. the later the prenatal irradiation was performed, the less intense epileptic symptoms were induced in adulthood. The data provide evidence that dysplasias acquired during prenatal development may significantly amplify or reduce the brain susceptibility to seizures. However, this relation depends not only on the developmental stage at which the dysplasias were produced but also on the functional specificity of epileptogenic stimuli used in the experimental model of epilepsy.

Newborn, 2-, 4-, 8-, 16- and 30-day-old Wistar rats were injected with 3 H -thymidine and sacrificed following 4 h. survival time. Brain sections containing the dorsal part of hippocampal formation were immunostained for S-100 β protein and subjected to autoradiography to visualize proliferating astrocytes. Microscopical observations revealed age-dependent changes in the number and distribution of proliferating astrocytes. The changes were considered as being related to the neurogenetic gradient characteristic to the hippocampal formation.

The present study examines the influence of interferon-gamma (IFN-gamma) on the astrocyte proliferation in the rat brain injured within the early period of postnatal development. Six-day-old male rats received a lesion in the left cerebral hemisphere and a single injection of recombinant rat IFN-gamma into the lesion cavity. One or 2 days after the injury the rats were injected with 3H-thymidine. Brain sections were immunostained for glial fibrillary acidic protein (GFAP), subjected to autoradiography, and examined microscopically to record proliferating GFAP-immunopositive astrocytes labeled with 3H-thymidine. In the IFN-gamma-injected rats, a statistically significant decrease in the intensity of reactive astrocyte proliferation was revealed. On day 1 after injury the intensity of astrocyte proliferation showed dose-dependent changes. Relations between the astrocyte reactivity and multiple factors existing in the injured and IFN-gamma-injected brain are discussed. The results represent the first in vivo evidence of a dose-dependent action of IFN-gamma on the astrocyte proliferation in response to injury.

Pregnant Wistar rats were exposed to a single 1.0 Gy dose of gamma-irradiation on gestational day 13, 15, 17 or 19. Thirty-day-old male offspring received a mechanical lesion in the left cerebral hemisphere. One, 2 or 4 days after the injury the rats were injected with [3H]thymidine and sacrificed 4 h after the injection. Thereafter, brain sections were immunostained for GFAP or S100 beta protein, subjected to autoradiography and examined microscopically to record immunopositive astrocytes labelled with [3H]thymidine. Statistically significant elevation of the reactive astrocyte proliferation was revealed on the 2nd day following injury in brains irradiated on gestational day 15. The results represent the first in vivo evidence that a low-dose prenatal gamma-irradiation can induce a long-term increase in the ability of astroglia to proliferate in response to injury.

A mechanical injury was inflicted to the left cerebral hemisphere in rats of four age groups: newborns, 6, 14 and 30 days old. The injury was followed by [3H]thymidine injections at different time intervals. Brain sections were immunostained for S-100 protein and subjected to autoradiography. During microscopic observations of the injury region, locations and numbers of the autoradiographically labeled astrocytes expressing S-100 protein were recorded. On the basis of the observations, injury-induced changes in the total number of proliferating astrocytes, as well as in their distribution, were analysed quantitatively. In rats injured neonatally, as well as those injured on postnatal days 6 and 14, the reactive increase in the number of proliferating astrocytes began on the first post-traumatic day. In 30-day-old rats the increase was slower and appeared on day 2. The maximal increase in the astrocyte proliferative activity occurred in 6-day-old rats as early as day 1 after injury and was about eight times higher than that recorded in newborns, and nearly twice as high as that recorded in brains of 30-day-old rats. The results suggest that the intensity of astrocyte proliferative response to injury cannot be regarded as simply being proportional to the developmental progress of the brain tissue. Rather, these results indicate that changes in glial proliferative responses to injury follow a developmental time course, with a peak around the end of the first postnatal week.

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. Figure The comparison of baseline corrected IR spectra recorded in selected creatine inclusion and nervous tissue (A). The microscopic view of the DG hippocampal area from the animal representing the SE72H group; creatine deposits are visible as darker points (B). The distributions of selected creatine bands: 2,800 cm−1 (C), 1,398 cm−1 (D), and 1,304 cm−1 (E) in the rectangular tissue area visible in part (B)

This paper describes the results of the application of X-ray fluorescence microscopy to the qualitative, topographic and quantitative elemental analysis of nervous tissue from rats with neocortical brain injury. The tissue samples were analyzed with a 15 mu m beam defined by the size of the polycapillary focus. Raster scanning of the samples generated 2D cartographies, revealing the distributions of elements such as P, S, Cl, K, Ca, Fe, Cu, and Zn. Special emphasis was placed on the analysis of the areas neighboring the lesion site and the hippocampal formation tissue. The results obtained for rats with mechanical brain injuries were compared with those recorded for controls and animals with pilocarpine-induced seizures. There were no significant differences in the elemental compositions of gray and white matter between injured and uninjured brain hemispheres. A higher level of Ca was observed in the gray matter of both of the hemispheres in brains with neocortical injuries. A similar relation was noticed for Fe in the white matter. A comparative study of hippocampal formation tissue showed a statistically significant decrease in the mass per unit area of P in the dentate gyrus (DG) and the hilus (H) of DG for animals with brain lesions in comparison with the control group. Analogous relations were found for Cu in the DG and Zn in sector 3 of Ammon's horn (CA3) and the DG. It is important to note that identical changes in the same areas were observed for animals with pilocarpine-induced seizures in our previous 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.

Proliferative response of astrocytes to unilateral injury of the cerebral hemisphere was investigated in 30-day-old rats using a combination of [^{3}H]thymidine autoradiography and immunocytochemical staining for glial fibrillary acidic protein. At different intervals following injury the animals were injected with [^{3}H]thymidine and the distribution of double-labeled cells was recorded 4 h (short-term experiment) or 40-days (long-term experiment) after each injection. Within the region of the lesion a strong reactive proliferation of astrocytes began as early as 2 h after injury, although, at that time all dividing neuroglial cells were located at a relatively long distance from the lesion margin. Forty days later, however, autoradiographically labeled astrocytes were observed in the center of the lesion. This was regarded as evidence for the ability of astrocytes to migrate towards the site of injury. Maximal reactive proliferation of astroglial cells was observed at day 2 after injury and then reached the postmitotic plateau at about day 8. The cells, while dividing, were mostly GFAP-negative. Therefore, the number of astrocytes produced at particular days after injury, and their contribution to post-traumatic scar formation could be assessed only in the long-term experiment, after the newly formed cells became GFAP-positive.