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The influence of electrical stimulation of vagus nerve on elemental composition of dopamine related brain structures in rats
Neurochemistry International 61 (2012) 156–165
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The influence of electrical stimulation of vagus nerve on elemental composition of dopamine related brain structures in rats Magdalena Szczerbowska-Boruchowska a,⇑, Anna Krygowska-Wajs b, Agata Ziomber c, Piotr Thor c, Pawel Wrobel a, Mateusz Bukowczan d, Ivo Zizak e a
AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, al. A. Mickiewicza 30, 30-059 Krakow, Poland Department of Neurology, Jagiellonian University Medical College, Krakow, Poland Department of Pathophysiology, Jagiellonian University Medical College, Krakow, Poland d Department of Neurosurgery, Jagiellonian University Medical College, Krakow, Poland e Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, Institute for Nanometre Optics and Technology, Berlin, Germany b c
a r t i c l e
i n f o
Article history: Received 23 September 2011 Received in revised form 14 April 2012 Accepted 18 April 2012 Available online 25 April 2012 Keywords: Synchrotron radiation X-ray fluorescence Electrical stimulation of vagus nerve Rat brain Elemental composition
a b s t r a c t Recent studies of Parkinson’s disease indicate that dorsal motor nucleus of nerve vagus is one of the earliest brain areas affected by alpha-synuclein and Lewy bodies pathology. The influence of electrical stimulation of vagus nerve on elemental composition of dopamine related brain structures in rats is investigated. Synchrotron radiation based X-ray fluorescence was applied to the elemental microimaging and quantification in thin tissue sections. It was found that elements such as P, S, Cl, K, Ca, Fe, Cu, Zn, Se, Br and Rb are present in motor cortex, corpus striatum, nucleus accumbens, substantia nigra, ventral tectal area, and dorsal motor nucleus of vagus. The topographic analysis shows that macroelements like P, S, Cl and K are highly concentrated within the fiber bundles of corpus striatum. In contrast the levels of trace elements like Fe and Zn are the lowest in these structures. It was found that statistically significant differences between the animals with electrical stimulation of vagus nerve and the control are observed in the left side of corpus striatum for P (p = 0.04), S (p = 0.02), Cl (p = 0.05), K (p = 0.02), Fe (p = 0.04) and Zn (p = 0.02). The mass fractions of these elements are increased in the group for which the electrical stimulation of vagus nerve was performed. Moreover, the contents of Ca (p = 0.02), Zn (p = 0.07) and Rb (p = 0.04) in substantia nigra of right hemisphere are found to be significantly lower in the group with stimulation of vagus nerve than in the control rats. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Sporadic Parkinson’s disease (PD) is the second most frequent neurodegenerative disorder characterized clinically by bradykinesia, rigidity, tremor and postural instability (de Lau and Breteler, 2006). The pathologic process in PD is progressive and takes years to reach its full extent. In PD the loss (depletion) of dopamine in nigro-striatal system as well as the presence of a-synuclein immunoreactive inclusions (Lewy neuritis, Lewy bodies) develop within specific types of projection neurons in substantia nigra, striatum and in other regions of the central nervous system (CNS). Autopsy based studies indicate that the a-synuclein and Lewy body pathology in sporadic PD does not evolve simultaneously at all of the susceptible nervous system sites but at predisposed locations. It is now appreciated that neurodegeneration with Lewy bodies is
⇑ Corresponding author. Tel.: +48 12 617 4424; fax: +48 12 634 0010. E-mail address: Magdalena.Boruchowska@fis.agh.edu.pl (M. SzczerbowskaBoruchowska). 0197-0186/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2012.04.018
widespread and can be seen in noradrenergic neurons in the locus coeruleus, cholinergic neurons in the nucleus basalis of Meynert, as well as in nerve cells in the dorsal motor nucleus of the vagus, olfactory regions and peripheral autonomic nervous system (Braak et al., 2003; Forno, 1966). The widely cited work of Braak and colleagues emphasizes the importance of early extranigral involvement in PD (Braak et al., 2003). Recently, it has become clearer that non-dopaminergic pathology may predate the classic dopaminergic pathology. A recent hypothesis about neuropathological stages of PD suggests that PD generally begins in the anterior olfactory nucleus and medulla including the dorsal motor nucleus of the vagal nerve, and nigrostriatal pathology develops after lower brainstem areas have become affected (Braak et al., 2003). Degeneration of substantia nigra is reached in temporal sequence of PD, with motor symptoms emerging in stage 3 or 4. Catecholaminergic neurons in the dorsal vagal area (corresponding to the A2 group in the rat) and those in the intermediate reticular zone (A1 group) that do not project to the periphery via the vagus nerve but, instead, generate ascending projections to higher levels of the central nervous system (Del Treduci et al., 2002).
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PD is diagnosed in biochemically and morphologically advanced stages of the disease when motor symptoms (bradykinesia, rigidity and tremor) are clinically developed (Kochman et al., 2006). Therefore, it is difficult to determine the exact cause of PD and mechanism of changes in early phase of this disorder. The recognition of extranigral aspects of PD will lead to earlier recognition of the onset of the disease and thus improve effectiveness and use of future neuroprotective therapies. Since the dorsal motor nucleus of the vagal nerve can be considered the true point of departure of the disease process we evaluated effect of stimulation of vagal nerve (VNS) on dopaminergic system as well as on elemental composition of dopamine related brain structures. We have found that chronic stimulation of the peripheral vagus nerve in rats causes a meaningful dopamine system inhibition in the different brain structures containing both the dopamine cell bodies and nerve endings. Moreover, we have observed that this effect is specific to dopamine neurons and does not affect other monoamine system for example, serotonin neurons (Ziomber et al., submitted for publication). As it is known, selected chemical elements are involved in biochemical processes that may lead to degeneration and atrophy of nerve cells in Parkinson’s disease. It has been reported that trace elements, mainly transition metals play a crucial role, acting as mediators of neurotoxicity either by favoring plaque formation or redox cycling in Parkinson’s disease (Gaeta and Hider, 2005). Moreover, some other biological processes such as excitotoxicity or mitochondrial dysfunction that involve metallochemical reactions are putative ways of neuronal degeneration and atrophy in PD (Hencheliffe et al., 2002). Both literature and our previous studies indicate presence of elemental abnormalities (mainly related to Fe, Zn, Ca) in human autopsy samples of substantia nigra in case of Parkinson’s disease (Szczerbowska-Boruchowska et al., 2005, 2012; Dexter et al., 1989, 1992). So, in short, two biochemical features are observed in PD, i.e. inhibition of dopamine system and elemental dyshomeostasis. In that light the question arises: whether the vagus nerve dysfunction may also affect elemental composition of dopamine related brain structures? Therefore, we have undertaken studies on the effect of electrical stimulation on vagus nerve on elemental composition of brain structures. Alterations in macro- and trace element-dependent processes as a result of VNS may occur. This is due to the fact that ions of K, Na, Mg, Ca, Cl are essential for physiological functions of neurons (Wöhrle et al., 2003). These and other macro-elements directly reflect the homeostasis of the cell. Moreover, trace elements like Fe, Zn, Cu serve many essential functions in the brain under normal conditions (Zecca et al., 2004; Lee and Koh, 2010). Since the alterations of chemical elements may occur only in finite anatomical structure of brain the analytical tool of high spatial resolution is desirable. Moreover, the level of trace elements in brain tissue is relatively low (mg/kg range) Boruchowska et al., 2001. Therefore, the technique of high detectability is necessary. In this study, the synchrotron radiation based X-ray fluorescence (SRXRF) was used. Synchrotron radiation is an excellent excitation beam to perform X-ray fluorescence analysis (XRF) Bertsch and Hunter, 2001. The technique is based on the detection of X-rays emitted from sample atoms irradiated with X-rays of higher energy (Ortega et al., 2009). The energy of the emitted X-rays is characteristic of the excited element thus enabling the identification of the composition of the atoms present in the sample. The analysis is multi-elemental and quantitative since the intensity of the fluorescence is proportional to the concentration of the elements within the sample (Ortega et al., 2009). The SRXRF technique has been used in a number of applications in biology and medicine. A comprehensive overview of recent applications of X-ray fluorescence microanalysis in biomedical field was presented by Paunesku et al. (2006). The main
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applications of SRXRF include research on physiology of trace elements, cancer chemotherapy of inorganic compounds, and metal neurotoxicity (Ilinski et al., 2003; Finney et al., 2007; Duong et al., 2009; Kehr et al., 2009). Because dysregulations of trace elements homeostasis, in particular Fe, Cu and Zn, may play an important role in selected neurodegenerative disorders, including Parkinson’s disease, Alzheimer’s disease and amyotrophic lateral sclerosis, the SRXRF technique was applied to visualize the metal ion distribution in brain tissue or nerve cells (Szczerbowska-Boruchowska et al., 2005; Miller et al., 2006; Ide-Ektessabi et al., 2002; Yoshida et al., 2003; Tomik et al., 2006; Ide-Ektessabi and Rabionet, 2005; Ortega et al., 2007). In the presented work the SRXRF technique is applied to topographic and quantitative analysis of macro- and trace elements in rat brains. Since multiple lines of evidence suggest an important role of chemical elements in neurodegenerative processes in PD in this experiment we have attempted to test the effect of chronic stimulation of the vagal nerve on the elemental composition of dopamine related brain structures in rats. 2. Materials and methods 2.1. Surgical procedures Twelve male Wistar rats were divided into two subgroups. Group 1 underwent surgical implantation of the microchip (Institute of Electron Technology, Cracow, Poland) in the abdominal region of the vagus. In group 2 (control) sham-laparotomy was performed. The 1 cm-diameter silicon-coated (RTV 3140, Dow Corning) and battery driven microchip (signal period: 20 s; signal duration: 0.1 s; amplitude 200 mV) was connected to the subdiaphragmatic part of the left vagus nerve. After 12 h of food deprivation and under general anesthesia with pentobarbital intraperitoneally (Vetbutal 25 mg/kg of body mass, Biowet, Pulawy, Poland), the subcutaneous pocket was formed by skin and underlining fascia where the microchip (MiC) was placed. Next, the left vagus nerve was localized in the subdiaphragmatic part of the esophagus and the MiC’s electrodes were brought into the abdominal cavity to connect with it. Un-isolated wires of the electrodes were wrapped around the nerve – cathode and anode were positioned 0.5 cm from each other (Ziomber et al., 2009; Bugajski et al., 2007). After closing the wounds, the rats were placed into the cages with free access to food and water. Control group underwent sham-laparatomy. After a 7 day-stimulation period of the left vagus nerve the animals were terminated. Jagiellonian University Bioethical Committee approved care and use of the animals (20/11/2009). 2.2. Tissue preparation The brain of each rat was removed immediately after decapitation and frozen in liquid nitrogen. Shortly before the measurements the specimens were cut into 20 lm-thick slices with the use of a cryomicrotome (at 20 °C). The coronal sections containing the following areas of rat brain: motor cortex (MCX), striatum (STR), nucleus accumbens (NA), substantia nigra (SN), ventral tectal area (VTA), and dorsal motor nucleus of vagus (DMV) were taken. The tissue slices designed for elemental analysis were mounted immediately onto ultralene (SPEX SAMPLE PREP) foil of 4 lm thickness suspended on a Plexiglas holder. Afterwards, samples prepared in such a way were freeze-dried at –80 °C. The samples destined for SRXRF measurements were neither fixed nor paraffinized. A visible image of the coronal section showing location of MCX, NA and STR is presented in Fig. 1.
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Fig. 1. The visible image of unstained section of rat brain showing loci of the areas analyzed using the SRXRF technique (MCX – motor cortex; STR – striatum, the medial part of caudate putamen; NA – nucleus accumbens). Scale bar: 1 mm.
Fig. 2. A typical spectrum excited in rat brain tissue (the area of nucleus accumbens) with the use of synchrotron radiation (beam energy: 17 keV; beam diameter: 12 lm; acquisition time: 10 s/point; XRF Ka lines of elements are shown).
each analyzed element i was calculated using the following expression: 2.3. Measurement conditions
Si ¼ Y S =C S
The SRXRF measurements were carried out at the mySpot beamline of the synchrotron light source facility BESSY II (HZB, Berlin, Germany) (Erko and Zizak, 2009). The exciting photon energy was set to 17 keV. Poly-capillary lens was used to focus the X-ray beam on the sample surface to spot sizes of about 12 lm in diameter. The X-ray beam from the capillary was incident at 45° to the sample surface. The detection of the fluorescence from the sample was performed in standard 90° geometry with respect to the incident exciting X-ray beam, providing the best fluorescence-scattering ratio. The sample was mounted on a translational stage with a precision of <1 lm. The areas of interest were selected by means of an on-line optical microscope, normal to the sample face. The acquisition tame was 10 s per one measurement point. The characteristic X-ray lines were measured by 7-element Si(Li) detector. The measurements were carried out in air. The size of the area of scanning was depending on the brain structure and it was 500 lm 500 lm for SN, VTA, NA, 1000 lm x 1000 lm for MCX, STR and 600 lm (horizontally) 300 lm (vertically) for NV. Spectrometer calibration was carried out based on the measurements of thin film standard samples of known concentration of elements. These homemade standards consisted of a mixture of nitrates of metals such as Cl, Ti, K, Fe, Sc, Zn, Mn, Rb, Cu, Y, Se, Sr in Tissue Freezing Medium (Jung Leica) were prepared in the same way as the tissue slices.
where Ys is the normalized net peak area of measured element for the standard sample, Cs the concentration of measured element i in the standard sample. For the calibration of the sensitivity versus atomic number, the fitting curve was constructed based on the concentration of elements in the standard materials. Based on the spectral data from each point of scanned areas of samples the concentrations of elements were determined according to the following formula:
2.4. Data analysis A synchrotron radiation X-ray fluorescence microprobe analysis technique was used to visualize two-dimensional distribution of elements in rat brain areas. A typical X-ray fluorescence spectrum excited in nucleus accumbens is illustrated in Fig. 2. Evaluation of the spectra was performed using the PyMCA software package v. 4.4.1 (Solé et al., 2007) freely distributed by European Synchrotron Radiation Facility. The spectral data collected for each measurement point was normalized to the detector live time as well as the beam flux before the sample. The intensities of elemental Ka lines, determined after background subtraction, were used for further topographic and quantitative analysis. For calculation of the concentrations of elements in tissue samples the external standard method was used. The sensitivity (Si) for
C i ¼ Y t =Si where Yt is the normalized net peak area of measured element i for the tissue sample, Si the sensitivity for the measured element i. 3. Results and discussion The anatomical structures of rat brains were imaged by synchrotron radiation X-ray fluorescence. It allowed finding P, S, Cl, K, Ca, Fe, Cu, Zn, Se, Br and Rb in the samples. By raster – scanning the sample and mapping the fluorescence yield, the distributions of the accessible chemical elements were determined. The examples of the X-ray fluorescence maps for striatum (the medial part of caudate nucleus), substantia nigra and dorsal motor nucleus of vagus are shown in Figs. 3–5. The corpus striatum consists of two masses of gray matter separated from each other by numerous stria of white fibers, which ascend from below upwards through its substance. In Fig. 3 the results of two-dimensional XRF analysis of corpus striatum are shown. To present elemental distribution at better spatial resolution only fragment of whole scanned area of this structure is shown. The synchrotron radiation X-ray fluorescence displays a prominent labeling of fiber bundles within the striatum (cf. Fig. 3). The topographic analysis shows that macro-elements like P, S, Cl and K are highly concentrated within the fiber bundles. In contrast the levels of trace elements like Fe and Zn are the lowest in these structures. They are located mainly in the areas occupied by striatal neurons. The domination of phosphorus contribution within the fiber bundles undoubtedly is related to high concentration of phospholipids in myelin. Strong accumulation of P, S, Cl was previously observed by Ducic´ et al. (2011) in single myelinated fibers isolated form sciatic nerve. The presence of K in the fiber bun-
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Fig. 3. Distribution of selected elements in corpus striatum obtained using the SRXRF technique. Arrows show fiber bundles.
Fig. 4. X-ray fluorescence maps showing distribution of P, S, Cl, K, Fe, and Zn in the scanned area including substantia nigra (SN) and midbrain reticular nucleus (MRN).
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Fig. 5. Multi-element imaging of coronal section of rat brain in the region of dorsal motor nucleus of vagus (DMV) and intermediate reticular nucleus (IRN).
dles is also not surprising given the role of this element in the physiological functions of nerve fibers, mainly in the nerve conduction. The X-ray fluorescence maps obtained for the substantia nigra are presented in Fig. 4. Apart from SN the raster-scanning analysis included also small adjoining area of midbrain reticular nucleus (MRN). It was found that the concentrations of elements like Fe, Zn, S, K and Cl are higher in substantia nigra than within MRN. Two-dimensional XRF maps for other elements such as P, Ca, Cu, Br and Rb display homogeneity of these components in both structures. The obtained result may suggest that high abundance of Fe, Zn, S, K and Cl is related to cell bodies of dopaminergic neurons. The spatial resolution achieved in the experiment was insufficient to image nerve cells separately. Therefore, we may only speculate on these findings based on the results presented previously by Szczerbowska-Boruchowska et al. (2005). It was reported that the mentioned elements are highly concentrated within nerve cell bodies of human substantia nigra. A mentioned above recent postmortem immunohistochemical studies of Parkinson’s disease have found that dorsal motor nucleus of vagus nerve is one of the earliest brain areas affected by alpha-synuclein and Lewy bodies pathology (Braak et al., 2003). Therefore this structure was included to the elemental investigation. The results of X-ray fluorescence mapping are presented in Fig. 5. It is easy to notice that phosphorus differ prominently between the DMV and intermediate reticular nucleus (IRN) located below. The concentration of P is about 2, three times higher in the IRN in respect to the DMV area. It allowed for the precise localization of the topography of the DMV in the scanned area of the samples. No other element distinguished so clearly between these two areas however concentrations of Ca, Fe, Cu and Zn showed an evident enhancement within the DMV. Moreover, the levels of S, K are slightly higher in the IRN area. The contributions of other chemical elements are comparable in both structures. The other studied brain areas i.e. motor cortex, core part of the nucleus accumbens, ventral tectal area are characterized by relatively homogenous distributions of chemical elements in whole structures at the applied spatial resolution of the SRXRF technique. For each animal the X-ray fluorescence maps were used for further quantitative and statistical analysis. For this purpose only data corresponding strictly with the structure of interest were extracted from a whole area of scanning. This procedure was applied especially to the cases of SN, DMV, VTA for which the elemental mapping included also parts of surrounding anatomical areas due to anc irregularity in brain structures anatomy. Additionally all artifacts, damages of the samples etc., were excluded from the quantitative analysis. Since electrical stimulation was carried out for left vagus nerve the brain areas from left end right hemispheres were analyzed
separately. For each brain structure the mean values of elemental mass fractions were calculated individually for each animal. To compare the MC animals with the control group the medians of the elemental mass fractions were evaluated for each brain area analyzed in the groups examined. Due to the small size of the examined populations the significance of any difference between median values was tested using the nonparametric Mann–Whitney U test. P value less than 0.1 (10% level) was considered as statistically significant. It was found that statistically significant differences between the MC and the control group are observed in the left side of corpus striatum for P (p = 0.04), S (p = 0.02), Cl (p = 0.05), K (p = 0.02), Fe (p = 0.04) and Zn (p = 0.02). The mass fractions of these elements are increased in the group for which the electrical stimulation of vagus nerve was performed. Moreover the contents of Ca (p = 0.02), Zn (p = 0.07) and Rb (p = 0.04) in substantia nigra of right hemisphere are found to be significantly lower in the MC group than in the control rats. No more statistically significant differences were noticed in the brain areas between the analyzed groups of animals. The selected results of the statistical analysis are graphically presented in Fig. 6. As an example, the mass fractions of S, K, Fe and Zn as well as Ca, Fe, Zn and Rb determined in STR (Fig. 6A) and SN (Fig. 6B) respectively are shown. The data related to left and right side areas are presented separately. Moreover, the comparison of elemental composition between the brain structures located in left and right hemispheres in the groups examined was carried out. The Mann– Whitney U test showed that there are no statistical differences (at p < 0.1) in the mass fractions of the elements determined for the given area between the left and right side of brains. Since the X-ray fluorescence analysis provides information of the multi-element content of the specimens simultaneously it seemed to be useful for comparative studies to keep all the data together. For this purpose we applied the multivariate statistical technique of hierarchical cluster analysis (HCA) to emphasize the structure of the data. Cluster analysis, which identifies groups of samples that behave similarly or show similar characteristics, was used to find natural groups in brain areas represent MC and control group, regarding brain hemispheres. Squared Euclidean was applied to calculate the distance matrix and Ward’s algorithm was used for hierarchical clustering. The experimental data were standardized before the analysis by subtracting their mean values and dividing them by their standard deviations. The reason of this procedure was that the range of mass fractions differed widely from element to element. The results of HCA analysis are presented in Fig. 7 as the dendrogram, showing in graphical form the similarity/dissimilarity of the elemental composition of the analyzed cases. As an example the dendrograms obtained for motor cortex, corpus striatum, substantia nigra and dorsal motor nucleus of vagus are shown. For all the analyzed brain areas excluding DMV
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Fig. 6. Median values of mean mass fractions of selected elements in corpus striatum (A) and substantia nigra (B) from the animals with the left vagus stimulation and the control group. CL – control, left brain hemisphere; CR – control, right brain hemisphere; MCL – with vagus nerve stimulation, left brain hemisphere; MCR – with vagus nerve stimulation, right brain hemisphere.
the dendrograms reveal a clear cluster separation between the MC group and the control. As one can noticed the similarity of mean
elemental composition between areas located in left and right hemispheres in each experimental group is significantly higher
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Fig. 7. Dendrograms showing the clustering of rat brain tissue samples from the animals with the left vagus stimulation (MC) and the control group (C) for the following anatomical brain structures: motor cortex (MCX), corpus striatum (STR), substantia nigra (SN), and dorsal motor nucleus of vagus (DMV). CL – control, left brain hemisphere; CR – control, right brain hemisphere; MCL – with vagus nerve stimulation, left brain hemisphere; MCR – with vagus nerve stimulation, right brain hemisphere.
than between the analyzed groups. However, the applied approach yielded only entire information about the analyzed groups. Therefore, to elucidate similarity in the elemental composition between the cases individually the hierarchical cluster analysis was carried out based on the mean values of elemental mass fractions in the given brain area for each animal separately. In Fig. 8, the dendrograms obtained on the basis of the elemental composition of motor cortex and corpus striatum are shown. As for MCX in all the analyzed cases the samples taken from left and right hemispheres of the same brain reveal strong similarity in the mean elemental content. They are grouped in separate clusters. As one can see the distances between the objects forming the clusters CR–CL, related to left and right side of motor cortex, for all control animals are significantly smaller in comparison to these obtained for MC group. Moreover a clear distinction between the control samples denoted as 3 and 10 and other objects can be observed. Generally, either for the presented here MCX or the other analyzed brain areas excluding STR the selected samples representing the control and MC group reveal similarity between each other and are joined to form clusters. Based on the shapes of the obtained dendrograms and squared Euclidean distances between objects it is not possible to confirm the existence of the differences in the elemental composition between control samples and these representing the MC group. Unlike to other brain areas, the mean elemental composition of STR samples differs significantly between the control and
the MC group. The results presented in Fig. 8 show a clear cluster separation in accordance with analyzed group. The two well separated major clusters indicate dissimilarity of elemental composition between electrically stimulated animals and these implanted with inactive microchip. Moreover, the different tendencies to clastering are observed for both groups. In the control objects, samples belonging to the antithetic hemispheres form the separate clusters (excluding sample denoted as 3). This result suggests higher similarity in elemental content between the samples taken from different brains but from the same hemisphere than between the left and right part of the same brain. Contrary, in the MC group the clusters of the smallest distances are formed by the elements representing both hemispheres of the same brain. In this case the corpus striatum probes of left and right side of each brain reveal strong similarity in elemental composition. The presented work shows changes in elemental composition of corpus striatum and substantia nigra of rat brains as a result of electrical stimulation of vagus nerve. In general, the mechanism of action of VNS is still poorly understood. Electrical stimulation of vagus nerve probably mediates neurochemical and electrical effects in brain areas through several mechanisms. It was previously reported that electrical stimulation of the central and peripheral nervous systems can affect both somatic and axonal activity in vivo, in vitro and in situ (Durand et al., 2010). These studies have showed the effect of electrical stimulation on activity at the cell
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Fig. 8. Dendrograms showing the clustering of the samples of motor cortex (MCX) and corpus striatum (STR) for all individuals analyzed by the SRXRF technique and hierarchical cluster analysis with Ward’s algorithm. CL – control, left brain hemisphere; CR – control, right brain hemisphere; MCL – with vagus nerve stimulation, left brain hemisphere; MCR – with vagus nerve stimulation, right brain hemisphere.
body, with suppression of somatic activity during stimulation. Moreover, it was emphasized that electrical stimulation also generates changes in axonal activity including a depression of excitatory synaptic currents, and conduction blockade in the peripheral and central nervous system (Durand et al., 2010). It is worth pointing out that the chemical elements, for which the anomalies were observed in our experiment in rat brain structures play important role in physiology of central nervous system. Therefore, it is highly probable that the mechanisms triggered by electrical stimulation of vagus nerve can result in changes of elemental composition of brain areas as observed in this work. Since the early stage of Parkinson’s disease is still poorly understood it is difficult to accurately relate the results obtained in our experiment to potential mechanisms initiating this disorder. However, the potential role of macro- and trace elements in biochemical processes in nervous system is extensively discussed. As mentioned above, striatal level of phosphorus is significantly increased in the MC group. The presence of phosphorus in striatum area is bound up mainly with the myelin sheath due to the contribution of phospholipids. They account for about 40% of the total lipid mass (Quarles et al., 2006). Since P was observed predominantly in the fiber bundles (cf. Fig. 3) there is a substantial likelihood that the increase of this chemical element in striatum of MC group results from anomalies in content of myelin phospholipids. On the other hand, one important function of phosphate groups of organic molecules within living organisms is energy storage. Adenosine triphosphate (ATP) is one of the possible forms of such compounds. The observed fact of higher concentration of P in left corpus striatum of MC rats may be related with alterations of energy metabolites such as ATP. Therefore, it can indicate a different metabolic state in this part of brain as a result of electrical stimulation of vagus nerve. Impaired energy metabolism, an obvious form of mitochondrial dysfunction is discussed as a mechanism resulting in nerve cell death in PD (Hencheliffe et al., 2002). Moreover, the regulated release of ATP is a fundamental process in cell-to-cell signaling. The sources of extracellular ATP have been discussed in view of stress-sensory transduction (Burnstock and Williams, 2000). Adenosine triphosphate can also participate in communication between neurons and glia as an activity-depend signaling molecule (Fields and Stevens, 2000). It is worth pointing out that accumulation of phosphorus in nervous tissue may also stem from cyclic adenosine monophosphate (cAMP). Bolton and Butt (Bolton and Butt, 2006) showed that cAMP signaling pathways
regulate myelin formation by oligodendrocytes. The numerous functions of phosphorus in central nervous system may reason the observed anomaly in accumulation of this chemical element in the area of left corpus striatum of MC rats. Similarly to phosphorus, sulfur level in left-side striatum was elevated for the group of the animals with active microchip implanted. Among non-metallic elements essential to life, sulfur stands out. This is due to its unique multifunctional role in chemical interactions including these occurring in central nervous system. Sulfur metabolism supplies cells with important reagents such as glutathione, S-adenosylmethionine or taurine (Stipanuk, 2004). Neurons and astrocytes are interdependent for their sulfur transactions, what is exhibited in both the glutathione and taurine biosynthetic branches of sulfur metabolism (Banerjee et al., 2008). Glutathione, the powerful antioxidant neutralizes neurotoxins that may cause Parkinson’s disease, and other neurotoxic events in the body (Zeevalk et al., 2007). The enhancement of chlorine accumulation was observed in corpus striatum of left hemisphere in case of the animals for which the electrical stimulation of vagus nerve was carried out. Chlorine ions are involved in an electrochemical impulse transduction. Apart from ions of sodium, potassium and calcium the chloride ions pass the neuronal membrane to process information, providing the fundamental basis for perception, learning and behavior (Rinke, 2010). The proper ionic concentration gradient is necessary to ensure electrical signaling and information processing between nerve cells. Moreover the intra-cellular concentration of chloride changes during development determining the neuronal response to the transmitter c-aminobutyric acid or glycine. It is known that altered chloride homeostasis can cause several human inherited diseases, such as cystic fibrosis (Quinton, 1983), myotonia (Koch et al., 1992), epilepsy (Cohen et al., 2002) or Dent’s disease (Lloyd et al., 1996). The quantitative analysis shows that the concentration of potassium is significantly higher in corpus striatum of MC group than in the control. As mentioned above potassium ions play important role in neuron communication (Rinke, 2010). The changes in potassium concentration can impact a wide variety of neuronal processes, such as synaptic transmission, maintenance of membrane potential, activation and inactivation of voltagegated channels, and electrogenic transport of neurotransmitters (Kofuji and Newman, 2004). It is known that local variations in extracellular potassium ions may occur following neuronal activity changes
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(Kofuji and Newman, 2004). The increase of extracellular K+ concentration can also be evoked by direct electrical stimulation of afferent pathways. Generally, increases of neuronal activity induce local increases of extracellular potassium ions level. In the case of uptake of potassium ions, these are temporarily sequestered into glial cells. The excess of K+ was found under pathophysiological conditions such as in anoxia (Vyskocil et al., 1972) or in spreading depression (Somjen, 2002). Moreover MPP+-induced increases in extracellular potassium ion activity in rat striatal slices was reported by Hollinden et al. (1988). The results of our research show increase of Fe accumulation in striatal area as an effect of electrical stimulation of vagus nerve. The increase in striatal iron level in aging was reported previously by Cass et al. (2007). Relation of changes in the nigrostriatal system, including alterations in dopamine release, regulation and transport in the striatum and substantia nigra, striatal atrophy and elevated iron levels in the basal ganglia with the motor abnormalities was studied. It was found that decreases in motor ability were correlated with significantly increased iron accumulation in the striatum. Therefore it was suggested that striatal iron level may be a biomarker of motor dysfunction in aging. The correlation between age and basal ganglia iron content was also research by Martin et al. (1998). A strong direct relationship between age and regional iron increase in striatum (putamen and caudate nucleus) was found. The Fe increase may increase the probability of free-radical formation in the striatum, leading to the development of neurodegenerative disorders such as Parkinson’s disease. In this case, nigrostriatal neurons may be affected by increased oxidative stress. In general, iron is critical to both neurodevelopment and normal function of the adult brain. Within the brain, the basal ganglia are particularly rich in iron therefore several diseases of the basal ganglia are related to iron dysregulation (Hallgren and Sourander, 1958). Under normal conditions Fe is present most abundantly in oligodendrocytes, but is also found in neurons, microglia, and astrocytes (Zecca et al., 2004). Furthermore oligodendrocytes are critical for axon myelination, and this process is iron-dependent (Badaracco et al., 2010). Mitochondria play a crucial role in regulating cellular iron homeostasis amongst other by synthesis of iron-sulfur clusters and heme prosthetic groups (Horowitz and Greenamyre, 2010). However, it is known that excessive cellular iron is harmful to cells (Stankiewicz et al., 2007). This may promote reactive oxygen species formation through Fenton chemistry. That is why any regional increase in brain iron level may increase the potential for local free-radical formation. Several neurodegenerative diseases are associated with accumulation of iron in the central nervous system. Excessive cellular iron has consequences for many of the well-established pathogenic mechanisms of Parkinson’s disease (Horowitz and Greenamyre, 2010). We also observed alteration in zinc concentration in both left STR and right SN between the groups analyzed. Excessive increase of intracellular zinc has been reported in degenerating neurons in the various brain areas including striatum after transient forebrain ischemia in rats (Koh et al., 1996). Moreover it was also indicated that zinc enters into post-synaptic neurons in toxic excess during seizures (Frederickson et al., 1989) and traumatic brain injury (Suh et al., 2000). Elevated Zn content was previously observed in dopaminergic neurons of substantia nigra and in motor cortex in human autopsy samples in case of Parkinson’s disease (Szczerbowska-Boruchowska et al., 2005). Zinc is necessary for brain normal function and maturation. The brain contains a high concentration of zinc ions (Lo et al., 2004). Most of them are bound to proteins (mainly metalloproteins) and has a variety of structural or catalytic roles. A small percentage (about 10%) of the zinc ions is present in the synaptic vesicles (Frederickson et al., 1989) and is histochemically reactive (Fre, 2004). The functions of many en-
zymes are zinc-dependent. Zinc serves many essential functions in the body under normal conditions, and is necessary for cellular development and survival (Lee and Koh, 2010). In general, a severe zinc deficiency leads to development of anomalies in humans and animals. On the other hand, increased free zinc content in a cell can be highly cytotoxic (Lee and Koh, 2010). Because cells are susceptible to severe changes in intracellular free zinc, they are equipped with a number of proteins that function to regulate zinc levels. Recent studies have revealed that free zinc levels change in various organelles in response to physiologic or pathological stimuli, and suggest important functional consequences of these zinc dynamics. Zn plays a crucial role in the metabolism of the brain therefore it has been linked to neurodegenerative disorders (Mocchegiani et al., 2005) such as Alzheimer’s disease, amyotrophic lateral sclerosis and Huntington disease as well as apoptosis, where it was found to occur in abnormal levels. Apart from Zn, calcium level was found as significantly lower in right-side SN of the MC group than in the control as well. This chemical element is supposed to play a significant role in apoptosis (Marcilhac, 2004). Calcium level must be kept at low levels because of its toxicity. Brain cells have the ability to control calcium through pumps and calcium-binding proteins that protect the cell from excess calcium. Most of the cellular calcium is stored in the endoplasmic reticulum (Demaurex and Distelhorst, 2003). Failure in the regulation of intracellular concentration of Ca may lead to cytotoxicity and neuronal death (Jiménez-Jiménez et al., 1996). Calcium is also required for the electrical signals of the nervous system (Braet et al., 2004). Changes in intracellular calcium ion concentration induce several events in dopaminergic cells, including spike afterhyperpolarizations and subthreshold oscillations underlying autonomous firing (Foehring et al., 2009). The decreased level of rubidium was also observed in right side of substantia nigra for MC group in comparison with the control. It is well-known that Rb increases synaptic neurotransmitter levels (Krachler and Wirnsberger, 2000). It was also showed that rubidium may replace potassium in the sodium–potassium pump (Krulík et al., 1977). As is supposed, potassium and rubidium are transported into brain and cerebrospinal fluid (CSF) across the same sites. Both ions may be moved out of CSF and brain into blood by a sodium–potassium pump sited at the choroid epithelium or the blood–brain barrier (Bradbury, 1970). It is not easy to speculate on the meaning of our findings from the biochemical perspective. However, the present work pointed out clearly the close relationship between the peripheral vagus nerve function and altered elemental composition in dopamine related brain structures (corpus striatum and substantia nigra). Since the inhibition of dopamine system was previously observed as a result of VNS (Ziomber et al., 2012), the abnormalities in elemental composition of dopamine related brain structures suggest possible role of selected macro- and trace elements, especially P, S, Ca, Fe, and Zn in mechanisms that may occur in the onset of Parkinson’s disease.
4. Conclusions The application of synchrotron radiation allowed an explicit identification of macro- and trace elements in nigrostriatal and mesolimbic dopaminergic system in rats. A relatively high-resolution imaging which is essential for tissue structure analysis enabled quantitative evaluation of elemental content in precisely selected brain areas. The comparison of cases representing the animals with stimulation of vagus nerve and the control group supported by statistical tests, helped to establish a relationship within the analyzed groups. The most prominent observation is that electrical stimulation of left vagus nerve causes significant
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increase of elemental concentration in left corpus striatum. It is related to either macro-elements like P, S, Cl, K or trace elements such us Fe and Zn. The dissimilarity in elemental composition of left corpus striatum between the MC and the control group was confirmed by cluster analysis as well. Moreover, chronic stimulation of vagus nerve results in significant decrease of Ca, Zn and Rb in substantia nigra of right hemisphere. Based on the results obtained we can conclude that the highest influence of electrical stimulation of left vagus nerve is observed in corpus striatum and substantia nigra. The other structures of nigrostriatal and mesolimbic system do not reveal any significant alterations in content of chemical elements. It may suggest that electrical stimulation of vagus nerve (at the applied parameters) do not cause any considerable disturbances in biochemical processes mediated by macro- or trace elements in these structures. However, the alterations observed for selected elements in striatum and substantia nigra point out clearly that impairment of the vagus nerve function is in close relationship with elemental dyshomeostasis. Taking into account the fact that dopamine system inhibition is also observed as a result of VNS the role of minor- and trace elements in pathological processes of early stage of PD is highly probable. Acknowledgments We acknowledge the Helmholtz-Zentrum Berlin – Electron storage ring BESSY II for provision of synchrotron radiation at beamline mySpot. We also thank Katarzyna Ciesielczyk and Andrzej Bugajski for their technical assistance. This work was supported by: the Polish Ministry of Science and Higher Education and its Grants for Scientific Research, European Community’s Seventh Framework Programme (FP7/2007-2013) under Grant agreement No. 226716. References Badaracco, M.E., Siri, M.V., Pasquini, J.M., 2010. Biofactors 36, 98–102. Banerjee, R., Vitvitsky, V., Garg, S.K., 2008. Trends Biochem. Sci. 33, 413–419. Bertsch, P.M., Hunter, D.B., 2001. Chem. Rev. 101, 1809–1842. Bolton, S., Butt, A., 2006. Exp. Neurol. 202, 36–43. Boruchowska, M., Lankosz, M., Adamek, D., Korman, A., 2001. X-ray Spectrom. 30, 174–179. Braak, H., Del Treduci, K., Rb, U., de Vos, R.A.I., Jansen Steur, E.N.H., Braak, E., 2003. Neurobiol. Aging 24, 197–211. Bradbury, M.W., 1970. Brain Res. 24, 311–321. Braet, K., Cabooter, L., Paemeleire, K., Leybaert, L., 2004. Biol. Cell 96, 79–91. Bugajski, A.J., Gil, K., Ziomber, A., Zurowski, D., Zaraska, W., Thor, P.J., 2007. J. Physiol. Pharmacol. 58 (Suppl. 1), 5–12. Burnstock, G., Williams, M., 2000. J. Pharmacol. Exp. Ther. 295, 862–869. Cass, W.A., Grondin, R., Andersen, A.H., Zhang, Z., Hardy, P.A., Hussey-Andersen, L.K., Rayens, W.S., Gerhardt, G.A., Gash, D.M., 2007. Neurobiol. Aging 28, 258–271. Cohen, I., Navarro, V., Clemenceau, S., Baulac, M., Miles, R., 2002. Science 298, 1418– 1421. de Lau, L.M.L., Breteler, N.M.B., 2006. Lancet Neurol. 5, 525–535. Del Treduci, K., Rub, U., de Vos, R.A.I., Bohl, J.R.E., Braak, H., 2002. J. Neuropathol. Exp. Neurol. 61, 413–423. Demaurex, N., Distelhorst, C., 2003. Science 300, 65–67. Dexter, D.T., Wells, F.R., Lee, A.J., Agid, F., Agid, Y., Jenner, P., Marsden, C.D., 1989. J. Neurochem. 52, 1830–1836. Dexter, D.T., Jenner, P., Schapira, A.H., Marsden, C.D., 1992. Ann. Neurol. 32, S94– 100. Ducic´, T., Quintes, S., Nave, K.A., Susini, J., Rak, M., Tucoulou, R., Alevra, M., Guttmann, P., Salditt, T., 2011. J. Struct. Biol. 173, 202–212. Duong, T.T., Witting, P.K., Antao, S.T., Parry, S.N., Kennerson, M., Lai, B., Vogt, S., Lay, P.A., Harris, H.H., 2009. J. Neurochem. 108, 1143–1154. Durand, D.M., Park, E.H., Jensen, A.L., 2010. Philos. Trans. R. Soc. Lond. B Biol. Sci. 365, 2347–2362. Erko, A., Zizak, I., 2009. Spectrochim. Acta, Part B 64, 833–848. Fields, R., Stevens, B., 2000. Trends Neurosci. 23, 625–633. Finney, L., Mandava, S., Ursos, L., Zhang, W., Rodi, D., Vogt, S., Legnini, D., Maser, J., Ikpatt, F., Olopade, O.I., Glesne, D., 2007. Proc. Natl. Acad. Sci. USA 104, 2247– 2252. Foehring, R.C., Zhang, X.F., Lee, J.C., Callaway, J.C., 2009. J. Neurophysiol. 102, 2326– 2333.
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The influence of electrical stimulation of vagus nerve on elemental composition of dopamine related brain structures in rats Magdalena Szczerbowska-Boruchowska a,⇑, Anna Krygowska-Wajs b, Agata Ziomber c, Piotr Thor c, Pawel Wrobel a, Mateusz Bukowczan d, Ivo Zizak e a
AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, al. A. Mickiewicza 30, 30-059 Krakow, Poland Department of Neurology, Jagiellonian University Medical College, Krakow, Poland Department of Pathophysiology, Jagiellonian University Medical College, Krakow, Poland d Department of Neurosurgery, Jagiellonian University Medical College, Krakow, Poland e Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, Institute for Nanometre Optics and Technology, Berlin, Germany b c
a r t i c l e
i n f o
Article history: Received 23 September 2011 Received in revised form 14 April 2012 Accepted 18 April 2012 Available online 25 April 2012 Keywords: Synchrotron radiation X-ray fluorescence Electrical stimulation of vagus nerve Rat brain Elemental composition
a b s t r a c t Recent studies of Parkinson’s disease indicate that dorsal motor nucleus of nerve vagus is one of the earliest brain areas affected by alpha-synuclein and Lewy bodies pathology. The influence of electrical stimulation of vagus nerve on elemental composition of dopamine related brain structures in rats is investigated. Synchrotron radiation based X-ray fluorescence was applied to the elemental microimaging and quantification in thin tissue sections. It was found that elements such as P, S, Cl, K, Ca, Fe, Cu, Zn, Se, Br and Rb are present in motor cortex, corpus striatum, nucleus accumbens, substantia nigra, ventral tectal area, and dorsal motor nucleus of vagus. The topographic analysis shows that macroelements like P, S, Cl and K are highly concentrated within the fiber bundles of corpus striatum. In contrast the levels of trace elements like Fe and Zn are the lowest in these structures. It was found that statistically significant differences between the animals with electrical stimulation of vagus nerve and the control are observed in the left side of corpus striatum for P (p = 0.04), S (p = 0.02), Cl (p = 0.05), K (p = 0.02), Fe (p = 0.04) and Zn (p = 0.02). The mass fractions of these elements are increased in the group for which the electrical stimulation of vagus nerve was performed. Moreover, the contents of Ca (p = 0.02), Zn (p = 0.07) and Rb (p = 0.04) in substantia nigra of right hemisphere are found to be significantly lower in the group with stimulation of vagus nerve than in the control rats. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Sporadic Parkinson’s disease (PD) is the second most frequent neurodegenerative disorder characterized clinically by bradykinesia, rigidity, tremor and postural instability (de Lau and Breteler, 2006). The pathologic process in PD is progressive and takes years to reach its full extent. In PD the loss (depletion) of dopamine in nigro-striatal system as well as the presence of a-synuclein immunoreactive inclusions (Lewy neuritis, Lewy bodies) develop within specific types of projection neurons in substantia nigra, striatum and in other regions of the central nervous system (CNS). Autopsy based studies indicate that the a-synuclein and Lewy body pathology in sporadic PD does not evolve simultaneously at all of the susceptible nervous system sites but at predisposed locations. It is now appreciated that neurodegeneration with Lewy bodies is
⇑ Corresponding author. Tel.: +48 12 617 4424; fax: +48 12 634 0010. E-mail address: Magdalena.Boruchowska@fis.agh.edu.pl (M. SzczerbowskaBoruchowska). 0197-0186/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2012.04.018
widespread and can be seen in noradrenergic neurons in the locus coeruleus, cholinergic neurons in the nucleus basalis of Meynert, as well as in nerve cells in the dorsal motor nucleus of the vagus, olfactory regions and peripheral autonomic nervous system (Braak et al., 2003; Forno, 1966). The widely cited work of Braak and colleagues emphasizes the importance of early extranigral involvement in PD (Braak et al., 2003). Recently, it has become clearer that non-dopaminergic pathology may predate the classic dopaminergic pathology. A recent hypothesis about neuropathological stages of PD suggests that PD generally begins in the anterior olfactory nucleus and medulla including the dorsal motor nucleus of the vagal nerve, and nigrostriatal pathology develops after lower brainstem areas have become affected (Braak et al., 2003). Degeneration of substantia nigra is reached in temporal sequence of PD, with motor symptoms emerging in stage 3 or 4. Catecholaminergic neurons in the dorsal vagal area (corresponding to the A2 group in the rat) and those in the intermediate reticular zone (A1 group) that do not project to the periphery via the vagus nerve but, instead, generate ascending projections to higher levels of the central nervous system (Del Treduci et al., 2002).
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PD is diagnosed in biochemically and morphologically advanced stages of the disease when motor symptoms (bradykinesia, rigidity and tremor) are clinically developed (Kochman et al., 2006). Therefore, it is difficult to determine the exact cause of PD and mechanism of changes in early phase of this disorder. The recognition of extranigral aspects of PD will lead to earlier recognition of the onset of the disease and thus improve effectiveness and use of future neuroprotective therapies. Since the dorsal motor nucleus of the vagal nerve can be considered the true point of departure of the disease process we evaluated effect of stimulation of vagal nerve (VNS) on dopaminergic system as well as on elemental composition of dopamine related brain structures. We have found that chronic stimulation of the peripheral vagus nerve in rats causes a meaningful dopamine system inhibition in the different brain structures containing both the dopamine cell bodies and nerve endings. Moreover, we have observed that this effect is specific to dopamine neurons and does not affect other monoamine system for example, serotonin neurons (Ziomber et al., submitted for publication). As it is known, selected chemical elements are involved in biochemical processes that may lead to degeneration and atrophy of nerve cells in Parkinson’s disease. It has been reported that trace elements, mainly transition metals play a crucial role, acting as mediators of neurotoxicity either by favoring plaque formation or redox cycling in Parkinson’s disease (Gaeta and Hider, 2005). Moreover, some other biological processes such as excitotoxicity or mitochondrial dysfunction that involve metallochemical reactions are putative ways of neuronal degeneration and atrophy in PD (Hencheliffe et al., 2002). Both literature and our previous studies indicate presence of elemental abnormalities (mainly related to Fe, Zn, Ca) in human autopsy samples of substantia nigra in case of Parkinson’s disease (Szczerbowska-Boruchowska et al., 2005, 2012; Dexter et al., 1989, 1992). So, in short, two biochemical features are observed in PD, i.e. inhibition of dopamine system and elemental dyshomeostasis. In that light the question arises: whether the vagus nerve dysfunction may also affect elemental composition of dopamine related brain structures? Therefore, we have undertaken studies on the effect of electrical stimulation on vagus nerve on elemental composition of brain structures. Alterations in macro- and trace element-dependent processes as a result of VNS may occur. This is due to the fact that ions of K, Na, Mg, Ca, Cl are essential for physiological functions of neurons (Wöhrle et al., 2003). These and other macro-elements directly reflect the homeostasis of the cell. Moreover, trace elements like Fe, Zn, Cu serve many essential functions in the brain under normal conditions (Zecca et al., 2004; Lee and Koh, 2010). Since the alterations of chemical elements may occur only in finite anatomical structure of brain the analytical tool of high spatial resolution is desirable. Moreover, the level of trace elements in brain tissue is relatively low (mg/kg range) Boruchowska et al., 2001. Therefore, the technique of high detectability is necessary. In this study, the synchrotron radiation based X-ray fluorescence (SRXRF) was used. Synchrotron radiation is an excellent excitation beam to perform X-ray fluorescence analysis (XRF) Bertsch and Hunter, 2001. The technique is based on the detection of X-rays emitted from sample atoms irradiated with X-rays of higher energy (Ortega et al., 2009). The energy of the emitted X-rays is characteristic of the excited element thus enabling the identification of the composition of the atoms present in the sample. The analysis is multi-elemental and quantitative since the intensity of the fluorescence is proportional to the concentration of the elements within the sample (Ortega et al., 2009). The SRXRF technique has been used in a number of applications in biology and medicine. A comprehensive overview of recent applications of X-ray fluorescence microanalysis in biomedical field was presented by Paunesku et al. (2006). The main
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applications of SRXRF include research on physiology of trace elements, cancer chemotherapy of inorganic compounds, and metal neurotoxicity (Ilinski et al., 2003; Finney et al., 2007; Duong et al., 2009; Kehr et al., 2009). Because dysregulations of trace elements homeostasis, in particular Fe, Cu and Zn, may play an important role in selected neurodegenerative disorders, including Parkinson’s disease, Alzheimer’s disease and amyotrophic lateral sclerosis, the SRXRF technique was applied to visualize the metal ion distribution in brain tissue or nerve cells (Szczerbowska-Boruchowska et al., 2005; Miller et al., 2006; Ide-Ektessabi et al., 2002; Yoshida et al., 2003; Tomik et al., 2006; Ide-Ektessabi and Rabionet, 2005; Ortega et al., 2007). In the presented work the SRXRF technique is applied to topographic and quantitative analysis of macro- and trace elements in rat brains. Since multiple lines of evidence suggest an important role of chemical elements in neurodegenerative processes in PD in this experiment we have attempted to test the effect of chronic stimulation of the vagal nerve on the elemental composition of dopamine related brain structures in rats. 2. Materials and methods 2.1. Surgical procedures Twelve male Wistar rats were divided into two subgroups. Group 1 underwent surgical implantation of the microchip (Institute of Electron Technology, Cracow, Poland) in the abdominal region of the vagus. In group 2 (control) sham-laparotomy was performed. The 1 cm-diameter silicon-coated (RTV 3140, Dow Corning) and battery driven microchip (signal period: 20 s; signal duration: 0.1 s; amplitude 200 mV) was connected to the subdiaphragmatic part of the left vagus nerve. After 12 h of food deprivation and under general anesthesia with pentobarbital intraperitoneally (Vetbutal 25 mg/kg of body mass, Biowet, Pulawy, Poland), the subcutaneous pocket was formed by skin and underlining fascia where the microchip (MiC) was placed. Next, the left vagus nerve was localized in the subdiaphragmatic part of the esophagus and the MiC’s electrodes were brought into the abdominal cavity to connect with it. Un-isolated wires of the electrodes were wrapped around the nerve – cathode and anode were positioned 0.5 cm from each other (Ziomber et al., 2009; Bugajski et al., 2007). After closing the wounds, the rats were placed into the cages with free access to food and water. Control group underwent sham-laparatomy. After a 7 day-stimulation period of the left vagus nerve the animals were terminated. Jagiellonian University Bioethical Committee approved care and use of the animals (20/11/2009). 2.2. Tissue preparation The brain of each rat was removed immediately after decapitation and frozen in liquid nitrogen. Shortly before the measurements the specimens were cut into 20 lm-thick slices with the use of a cryomicrotome (at 20 °C). The coronal sections containing the following areas of rat brain: motor cortex (MCX), striatum (STR), nucleus accumbens (NA), substantia nigra (SN), ventral tectal area (VTA), and dorsal motor nucleus of vagus (DMV) were taken. The tissue slices designed for elemental analysis were mounted immediately onto ultralene (SPEX SAMPLE PREP) foil of 4 lm thickness suspended on a Plexiglas holder. Afterwards, samples prepared in such a way were freeze-dried at –80 °C. The samples destined for SRXRF measurements were neither fixed nor paraffinized. A visible image of the coronal section showing location of MCX, NA and STR is presented in Fig. 1.
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Fig. 1. The visible image of unstained section of rat brain showing loci of the areas analyzed using the SRXRF technique (MCX – motor cortex; STR – striatum, the medial part of caudate putamen; NA – nucleus accumbens). Scale bar: 1 mm.
Fig. 2. A typical spectrum excited in rat brain tissue (the area of nucleus accumbens) with the use of synchrotron radiation (beam energy: 17 keV; beam diameter: 12 lm; acquisition time: 10 s/point; XRF Ka lines of elements are shown).
each analyzed element i was calculated using the following expression: 2.3. Measurement conditions
Si ¼ Y S =C S
The SRXRF measurements were carried out at the mySpot beamline of the synchrotron light source facility BESSY II (HZB, Berlin, Germany) (Erko and Zizak, 2009). The exciting photon energy was set to 17 keV. Poly-capillary lens was used to focus the X-ray beam on the sample surface to spot sizes of about 12 lm in diameter. The X-ray beam from the capillary was incident at 45° to the sample surface. The detection of the fluorescence from the sample was performed in standard 90° geometry with respect to the incident exciting X-ray beam, providing the best fluorescence-scattering ratio. The sample was mounted on a translational stage with a precision of <1 lm. The areas of interest were selected by means of an on-line optical microscope, normal to the sample face. The acquisition tame was 10 s per one measurement point. The characteristic X-ray lines were measured by 7-element Si(Li) detector. The measurements were carried out in air. The size of the area of scanning was depending on the brain structure and it was 500 lm 500 lm for SN, VTA, NA, 1000 lm x 1000 lm for MCX, STR and 600 lm (horizontally) 300 lm (vertically) for NV. Spectrometer calibration was carried out based on the measurements of thin film standard samples of known concentration of elements. These homemade standards consisted of a mixture of nitrates of metals such as Cl, Ti, K, Fe, Sc, Zn, Mn, Rb, Cu, Y, Se, Sr in Tissue Freezing Medium (Jung Leica) were prepared in the same way as the tissue slices.
where Ys is the normalized net peak area of measured element for the standard sample, Cs the concentration of measured element i in the standard sample. For the calibration of the sensitivity versus atomic number, the fitting curve was constructed based on the concentration of elements in the standard materials. Based on the spectral data from each point of scanned areas of samples the concentrations of elements were determined according to the following formula:
2.4. Data analysis A synchrotron radiation X-ray fluorescence microprobe analysis technique was used to visualize two-dimensional distribution of elements in rat brain areas. A typical X-ray fluorescence spectrum excited in nucleus accumbens is illustrated in Fig. 2. Evaluation of the spectra was performed using the PyMCA software package v. 4.4.1 (Solé et al., 2007) freely distributed by European Synchrotron Radiation Facility. The spectral data collected for each measurement point was normalized to the detector live time as well as the beam flux before the sample. The intensities of elemental Ka lines, determined after background subtraction, were used for further topographic and quantitative analysis. For calculation of the concentrations of elements in tissue samples the external standard method was used. The sensitivity (Si) for
C i ¼ Y t =Si where Yt is the normalized net peak area of measured element i for the tissue sample, Si the sensitivity for the measured element i. 3. Results and discussion The anatomical structures of rat brains were imaged by synchrotron radiation X-ray fluorescence. It allowed finding P, S, Cl, K, Ca, Fe, Cu, Zn, Se, Br and Rb in the samples. By raster – scanning the sample and mapping the fluorescence yield, the distributions of the accessible chemical elements were determined. The examples of the X-ray fluorescence maps for striatum (the medial part of caudate nucleus), substantia nigra and dorsal motor nucleus of vagus are shown in Figs. 3–5. The corpus striatum consists of two masses of gray matter separated from each other by numerous stria of white fibers, which ascend from below upwards through its substance. In Fig. 3 the results of two-dimensional XRF analysis of corpus striatum are shown. To present elemental distribution at better spatial resolution only fragment of whole scanned area of this structure is shown. The synchrotron radiation X-ray fluorescence displays a prominent labeling of fiber bundles within the striatum (cf. Fig. 3). The topographic analysis shows that macro-elements like P, S, Cl and K are highly concentrated within the fiber bundles. In contrast the levels of trace elements like Fe and Zn are the lowest in these structures. They are located mainly in the areas occupied by striatal neurons. The domination of phosphorus contribution within the fiber bundles undoubtedly is related to high concentration of phospholipids in myelin. Strong accumulation of P, S, Cl was previously observed by Ducic´ et al. (2011) in single myelinated fibers isolated form sciatic nerve. The presence of K in the fiber bun-
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Fig. 3. Distribution of selected elements in corpus striatum obtained using the SRXRF technique. Arrows show fiber bundles.
Fig. 4. X-ray fluorescence maps showing distribution of P, S, Cl, K, Fe, and Zn in the scanned area including substantia nigra (SN) and midbrain reticular nucleus (MRN).
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Fig. 5. Multi-element imaging of coronal section of rat brain in the region of dorsal motor nucleus of vagus (DMV) and intermediate reticular nucleus (IRN).
dles is also not surprising given the role of this element in the physiological functions of nerve fibers, mainly in the nerve conduction. The X-ray fluorescence maps obtained for the substantia nigra are presented in Fig. 4. Apart from SN the raster-scanning analysis included also small adjoining area of midbrain reticular nucleus (MRN). It was found that the concentrations of elements like Fe, Zn, S, K and Cl are higher in substantia nigra than within MRN. Two-dimensional XRF maps for other elements such as P, Ca, Cu, Br and Rb display homogeneity of these components in both structures. The obtained result may suggest that high abundance of Fe, Zn, S, K and Cl is related to cell bodies of dopaminergic neurons. The spatial resolution achieved in the experiment was insufficient to image nerve cells separately. Therefore, we may only speculate on these findings based on the results presented previously by Szczerbowska-Boruchowska et al. (2005). It was reported that the mentioned elements are highly concentrated within nerve cell bodies of human substantia nigra. A mentioned above recent postmortem immunohistochemical studies of Parkinson’s disease have found that dorsal motor nucleus of vagus nerve is one of the earliest brain areas affected by alpha-synuclein and Lewy bodies pathology (Braak et al., 2003). Therefore this structure was included to the elemental investigation. The results of X-ray fluorescence mapping are presented in Fig. 5. It is easy to notice that phosphorus differ prominently between the DMV and intermediate reticular nucleus (IRN) located below. The concentration of P is about 2, three times higher in the IRN in respect to the DMV area. It allowed for the precise localization of the topography of the DMV in the scanned area of the samples. No other element distinguished so clearly between these two areas however concentrations of Ca, Fe, Cu and Zn showed an evident enhancement within the DMV. Moreover, the levels of S, K are slightly higher in the IRN area. The contributions of other chemical elements are comparable in both structures. The other studied brain areas i.e. motor cortex, core part of the nucleus accumbens, ventral tectal area are characterized by relatively homogenous distributions of chemical elements in whole structures at the applied spatial resolution of the SRXRF technique. For each animal the X-ray fluorescence maps were used for further quantitative and statistical analysis. For this purpose only data corresponding strictly with the structure of interest were extracted from a whole area of scanning. This procedure was applied especially to the cases of SN, DMV, VTA for which the elemental mapping included also parts of surrounding anatomical areas due to anc irregularity in brain structures anatomy. Additionally all artifacts, damages of the samples etc., were excluded from the quantitative analysis. Since electrical stimulation was carried out for left vagus nerve the brain areas from left end right hemispheres were analyzed
separately. For each brain structure the mean values of elemental mass fractions were calculated individually for each animal. To compare the MC animals with the control group the medians of the elemental mass fractions were evaluated for each brain area analyzed in the groups examined. Due to the small size of the examined populations the significance of any difference between median values was tested using the nonparametric Mann–Whitney U test. P value less than 0.1 (10% level) was considered as statistically significant. It was found that statistically significant differences between the MC and the control group are observed in the left side of corpus striatum for P (p = 0.04), S (p = 0.02), Cl (p = 0.05), K (p = 0.02), Fe (p = 0.04) and Zn (p = 0.02). The mass fractions of these elements are increased in the group for which the electrical stimulation of vagus nerve was performed. Moreover the contents of Ca (p = 0.02), Zn (p = 0.07) and Rb (p = 0.04) in substantia nigra of right hemisphere are found to be significantly lower in the MC group than in the control rats. No more statistically significant differences were noticed in the brain areas between the analyzed groups of animals. The selected results of the statistical analysis are graphically presented in Fig. 6. As an example, the mass fractions of S, K, Fe and Zn as well as Ca, Fe, Zn and Rb determined in STR (Fig. 6A) and SN (Fig. 6B) respectively are shown. The data related to left and right side areas are presented separately. Moreover, the comparison of elemental composition between the brain structures located in left and right hemispheres in the groups examined was carried out. The Mann– Whitney U test showed that there are no statistical differences (at p < 0.1) in the mass fractions of the elements determined for the given area between the left and right side of brains. Since the X-ray fluorescence analysis provides information of the multi-element content of the specimens simultaneously it seemed to be useful for comparative studies to keep all the data together. For this purpose we applied the multivariate statistical technique of hierarchical cluster analysis (HCA) to emphasize the structure of the data. Cluster analysis, which identifies groups of samples that behave similarly or show similar characteristics, was used to find natural groups in brain areas represent MC and control group, regarding brain hemispheres. Squared Euclidean was applied to calculate the distance matrix and Ward’s algorithm was used for hierarchical clustering. The experimental data were standardized before the analysis by subtracting their mean values and dividing them by their standard deviations. The reason of this procedure was that the range of mass fractions differed widely from element to element. The results of HCA analysis are presented in Fig. 7 as the dendrogram, showing in graphical form the similarity/dissimilarity of the elemental composition of the analyzed cases. As an example the dendrograms obtained for motor cortex, corpus striatum, substantia nigra and dorsal motor nucleus of vagus are shown. For all the analyzed brain areas excluding DMV
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Fig. 6. Median values of mean mass fractions of selected elements in corpus striatum (A) and substantia nigra (B) from the animals with the left vagus stimulation and the control group. CL – control, left brain hemisphere; CR – control, right brain hemisphere; MCL – with vagus nerve stimulation, left brain hemisphere; MCR – with vagus nerve stimulation, right brain hemisphere.
the dendrograms reveal a clear cluster separation between the MC group and the control. As one can noticed the similarity of mean
elemental composition between areas located in left and right hemispheres in each experimental group is significantly higher
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Fig. 7. Dendrograms showing the clustering of rat brain tissue samples from the animals with the left vagus stimulation (MC) and the control group (C) for the following anatomical brain structures: motor cortex (MCX), corpus striatum (STR), substantia nigra (SN), and dorsal motor nucleus of vagus (DMV). CL – control, left brain hemisphere; CR – control, right brain hemisphere; MCL – with vagus nerve stimulation, left brain hemisphere; MCR – with vagus nerve stimulation, right brain hemisphere.
than between the analyzed groups. However, the applied approach yielded only entire information about the analyzed groups. Therefore, to elucidate similarity in the elemental composition between the cases individually the hierarchical cluster analysis was carried out based on the mean values of elemental mass fractions in the given brain area for each animal separately. In Fig. 8, the dendrograms obtained on the basis of the elemental composition of motor cortex and corpus striatum are shown. As for MCX in all the analyzed cases the samples taken from left and right hemispheres of the same brain reveal strong similarity in the mean elemental content. They are grouped in separate clusters. As one can see the distances between the objects forming the clusters CR–CL, related to left and right side of motor cortex, for all control animals are significantly smaller in comparison to these obtained for MC group. Moreover a clear distinction between the control samples denoted as 3 and 10 and other objects can be observed. Generally, either for the presented here MCX or the other analyzed brain areas excluding STR the selected samples representing the control and MC group reveal similarity between each other and are joined to form clusters. Based on the shapes of the obtained dendrograms and squared Euclidean distances between objects it is not possible to confirm the existence of the differences in the elemental composition between control samples and these representing the MC group. Unlike to other brain areas, the mean elemental composition of STR samples differs significantly between the control and
the MC group. The results presented in Fig. 8 show a clear cluster separation in accordance with analyzed group. The two well separated major clusters indicate dissimilarity of elemental composition between electrically stimulated animals and these implanted with inactive microchip. Moreover, the different tendencies to clastering are observed for both groups. In the control objects, samples belonging to the antithetic hemispheres form the separate clusters (excluding sample denoted as 3). This result suggests higher similarity in elemental content between the samples taken from different brains but from the same hemisphere than between the left and right part of the same brain. Contrary, in the MC group the clusters of the smallest distances are formed by the elements representing both hemispheres of the same brain. In this case the corpus striatum probes of left and right side of each brain reveal strong similarity in elemental composition. The presented work shows changes in elemental composition of corpus striatum and substantia nigra of rat brains as a result of electrical stimulation of vagus nerve. In general, the mechanism of action of VNS is still poorly understood. Electrical stimulation of vagus nerve probably mediates neurochemical and electrical effects in brain areas through several mechanisms. It was previously reported that electrical stimulation of the central and peripheral nervous systems can affect both somatic and axonal activity in vivo, in vitro and in situ (Durand et al., 2010). These studies have showed the effect of electrical stimulation on activity at the cell
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Fig. 8. Dendrograms showing the clustering of the samples of motor cortex (MCX) and corpus striatum (STR) for all individuals analyzed by the SRXRF technique and hierarchical cluster analysis with Ward’s algorithm. CL – control, left brain hemisphere; CR – control, right brain hemisphere; MCL – with vagus nerve stimulation, left brain hemisphere; MCR – with vagus nerve stimulation, right brain hemisphere.
body, with suppression of somatic activity during stimulation. Moreover, it was emphasized that electrical stimulation also generates changes in axonal activity including a depression of excitatory synaptic currents, and conduction blockade in the peripheral and central nervous system (Durand et al., 2010). It is worth pointing out that the chemical elements, for which the anomalies were observed in our experiment in rat brain structures play important role in physiology of central nervous system. Therefore, it is highly probable that the mechanisms triggered by electrical stimulation of vagus nerve can result in changes of elemental composition of brain areas as observed in this work. Since the early stage of Parkinson’s disease is still poorly understood it is difficult to accurately relate the results obtained in our experiment to potential mechanisms initiating this disorder. However, the potential role of macro- and trace elements in biochemical processes in nervous system is extensively discussed. As mentioned above, striatal level of phosphorus is significantly increased in the MC group. The presence of phosphorus in striatum area is bound up mainly with the myelin sheath due to the contribution of phospholipids. They account for about 40% of the total lipid mass (Quarles et al., 2006). Since P was observed predominantly in the fiber bundles (cf. Fig. 3) there is a substantial likelihood that the increase of this chemical element in striatum of MC group results from anomalies in content of myelin phospholipids. On the other hand, one important function of phosphate groups of organic molecules within living organisms is energy storage. Adenosine triphosphate (ATP) is one of the possible forms of such compounds. The observed fact of higher concentration of P in left corpus striatum of MC rats may be related with alterations of energy metabolites such as ATP. Therefore, it can indicate a different metabolic state in this part of brain as a result of electrical stimulation of vagus nerve. Impaired energy metabolism, an obvious form of mitochondrial dysfunction is discussed as a mechanism resulting in nerve cell death in PD (Hencheliffe et al., 2002). Moreover, the regulated release of ATP is a fundamental process in cell-to-cell signaling. The sources of extracellular ATP have been discussed in view of stress-sensory transduction (Burnstock and Williams, 2000). Adenosine triphosphate can also participate in communication between neurons and glia as an activity-depend signaling molecule (Fields and Stevens, 2000). It is worth pointing out that accumulation of phosphorus in nervous tissue may also stem from cyclic adenosine monophosphate (cAMP). Bolton and Butt (Bolton and Butt, 2006) showed that cAMP signaling pathways
regulate myelin formation by oligodendrocytes. The numerous functions of phosphorus in central nervous system may reason the observed anomaly in accumulation of this chemical element in the area of left corpus striatum of MC rats. Similarly to phosphorus, sulfur level in left-side striatum was elevated for the group of the animals with active microchip implanted. Among non-metallic elements essential to life, sulfur stands out. This is due to its unique multifunctional role in chemical interactions including these occurring in central nervous system. Sulfur metabolism supplies cells with important reagents such as glutathione, S-adenosylmethionine or taurine (Stipanuk, 2004). Neurons and astrocytes are interdependent for their sulfur transactions, what is exhibited in both the glutathione and taurine biosynthetic branches of sulfur metabolism (Banerjee et al., 2008). Glutathione, the powerful antioxidant neutralizes neurotoxins that may cause Parkinson’s disease, and other neurotoxic events in the body (Zeevalk et al., 2007). The enhancement of chlorine accumulation was observed in corpus striatum of left hemisphere in case of the animals for which the electrical stimulation of vagus nerve was carried out. Chlorine ions are involved in an electrochemical impulse transduction. Apart from ions of sodium, potassium and calcium the chloride ions pass the neuronal membrane to process information, providing the fundamental basis for perception, learning and behavior (Rinke, 2010). The proper ionic concentration gradient is necessary to ensure electrical signaling and information processing between nerve cells. Moreover the intra-cellular concentration of chloride changes during development determining the neuronal response to the transmitter c-aminobutyric acid or glycine. It is known that altered chloride homeostasis can cause several human inherited diseases, such as cystic fibrosis (Quinton, 1983), myotonia (Koch et al., 1992), epilepsy (Cohen et al., 2002) or Dent’s disease (Lloyd et al., 1996). The quantitative analysis shows that the concentration of potassium is significantly higher in corpus striatum of MC group than in the control. As mentioned above potassium ions play important role in neuron communication (Rinke, 2010). The changes in potassium concentration can impact a wide variety of neuronal processes, such as synaptic transmission, maintenance of membrane potential, activation and inactivation of voltagegated channels, and electrogenic transport of neurotransmitters (Kofuji and Newman, 2004). It is known that local variations in extracellular potassium ions may occur following neuronal activity changes
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(Kofuji and Newman, 2004). The increase of extracellular K+ concentration can also be evoked by direct electrical stimulation of afferent pathways. Generally, increases of neuronal activity induce local increases of extracellular potassium ions level. In the case of uptake of potassium ions, these are temporarily sequestered into glial cells. The excess of K+ was found under pathophysiological conditions such as in anoxia (Vyskocil et al., 1972) or in spreading depression (Somjen, 2002). Moreover MPP+-induced increases in extracellular potassium ion activity in rat striatal slices was reported by Hollinden et al. (1988). The results of our research show increase of Fe accumulation in striatal area as an effect of electrical stimulation of vagus nerve. The increase in striatal iron level in aging was reported previously by Cass et al. (2007). Relation of changes in the nigrostriatal system, including alterations in dopamine release, regulation and transport in the striatum and substantia nigra, striatal atrophy and elevated iron levels in the basal ganglia with the motor abnormalities was studied. It was found that decreases in motor ability were correlated with significantly increased iron accumulation in the striatum. Therefore it was suggested that striatal iron level may be a biomarker of motor dysfunction in aging. The correlation between age and basal ganglia iron content was also research by Martin et al. (1998). A strong direct relationship between age and regional iron increase in striatum (putamen and caudate nucleus) was found. The Fe increase may increase the probability of free-radical formation in the striatum, leading to the development of neurodegenerative disorders such as Parkinson’s disease. In this case, nigrostriatal neurons may be affected by increased oxidative stress. In general, iron is critical to both neurodevelopment and normal function of the adult brain. Within the brain, the basal ganglia are particularly rich in iron therefore several diseases of the basal ganglia are related to iron dysregulation (Hallgren and Sourander, 1958). Under normal conditions Fe is present most abundantly in oligodendrocytes, but is also found in neurons, microglia, and astrocytes (Zecca et al., 2004). Furthermore oligodendrocytes are critical for axon myelination, and this process is iron-dependent (Badaracco et al., 2010). Mitochondria play a crucial role in regulating cellular iron homeostasis amongst other by synthesis of iron-sulfur clusters and heme prosthetic groups (Horowitz and Greenamyre, 2010). However, it is known that excessive cellular iron is harmful to cells (Stankiewicz et al., 2007). This may promote reactive oxygen species formation through Fenton chemistry. That is why any regional increase in brain iron level may increase the potential for local free-radical formation. Several neurodegenerative diseases are associated with accumulation of iron in the central nervous system. Excessive cellular iron has consequences for many of the well-established pathogenic mechanisms of Parkinson’s disease (Horowitz and Greenamyre, 2010). We also observed alteration in zinc concentration in both left STR and right SN between the groups analyzed. Excessive increase of intracellular zinc has been reported in degenerating neurons in the various brain areas including striatum after transient forebrain ischemia in rats (Koh et al., 1996). Moreover it was also indicated that zinc enters into post-synaptic neurons in toxic excess during seizures (Frederickson et al., 1989) and traumatic brain injury (Suh et al., 2000). Elevated Zn content was previously observed in dopaminergic neurons of substantia nigra and in motor cortex in human autopsy samples in case of Parkinson’s disease (Szczerbowska-Boruchowska et al., 2005). Zinc is necessary for brain normal function and maturation. The brain contains a high concentration of zinc ions (Lo et al., 2004). Most of them are bound to proteins (mainly metalloproteins) and has a variety of structural or catalytic roles. A small percentage (about 10%) of the zinc ions is present in the synaptic vesicles (Frederickson et al., 1989) and is histochemically reactive (Fre, 2004). The functions of many en-
zymes are zinc-dependent. Zinc serves many essential functions in the body under normal conditions, and is necessary for cellular development and survival (Lee and Koh, 2010). In general, a severe zinc deficiency leads to development of anomalies in humans and animals. On the other hand, increased free zinc content in a cell can be highly cytotoxic (Lee and Koh, 2010). Because cells are susceptible to severe changes in intracellular free zinc, they are equipped with a number of proteins that function to regulate zinc levels. Recent studies have revealed that free zinc levels change in various organelles in response to physiologic or pathological stimuli, and suggest important functional consequences of these zinc dynamics. Zn plays a crucial role in the metabolism of the brain therefore it has been linked to neurodegenerative disorders (Mocchegiani et al., 2005) such as Alzheimer’s disease, amyotrophic lateral sclerosis and Huntington disease as well as apoptosis, where it was found to occur in abnormal levels. Apart from Zn, calcium level was found as significantly lower in right-side SN of the MC group than in the control as well. This chemical element is supposed to play a significant role in apoptosis (Marcilhac, 2004). Calcium level must be kept at low levels because of its toxicity. Brain cells have the ability to control calcium through pumps and calcium-binding proteins that protect the cell from excess calcium. Most of the cellular calcium is stored in the endoplasmic reticulum (Demaurex and Distelhorst, 2003). Failure in the regulation of intracellular concentration of Ca may lead to cytotoxicity and neuronal death (Jiménez-Jiménez et al., 1996). Calcium is also required for the electrical signals of the nervous system (Braet et al., 2004). Changes in intracellular calcium ion concentration induce several events in dopaminergic cells, including spike afterhyperpolarizations and subthreshold oscillations underlying autonomous firing (Foehring et al., 2009). The decreased level of rubidium was also observed in right side of substantia nigra for MC group in comparison with the control. It is well-known that Rb increases synaptic neurotransmitter levels (Krachler and Wirnsberger, 2000). It was also showed that rubidium may replace potassium in the sodium–potassium pump (Krulík et al., 1977). As is supposed, potassium and rubidium are transported into brain and cerebrospinal fluid (CSF) across the same sites. Both ions may be moved out of CSF and brain into blood by a sodium–potassium pump sited at the choroid epithelium or the blood–brain barrier (Bradbury, 1970). It is not easy to speculate on the meaning of our findings from the biochemical perspective. However, the present work pointed out clearly the close relationship between the peripheral vagus nerve function and altered elemental composition in dopamine related brain structures (corpus striatum and substantia nigra). Since the inhibition of dopamine system was previously observed as a result of VNS (Ziomber et al., 2012), the abnormalities in elemental composition of dopamine related brain structures suggest possible role of selected macro- and trace elements, especially P, S, Ca, Fe, and Zn in mechanisms that may occur in the onset of Parkinson’s disease.
4. Conclusions The application of synchrotron radiation allowed an explicit identification of macro- and trace elements in nigrostriatal and mesolimbic dopaminergic system in rats. A relatively high-resolution imaging which is essential for tissue structure analysis enabled quantitative evaluation of elemental content in precisely selected brain areas. The comparison of cases representing the animals with stimulation of vagus nerve and the control group supported by statistical tests, helped to establish a relationship within the analyzed groups. The most prominent observation is that electrical stimulation of left vagus nerve causes significant
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increase of elemental concentration in left corpus striatum. It is related to either macro-elements like P, S, Cl, K or trace elements such us Fe and Zn. The dissimilarity in elemental composition of left corpus striatum between the MC and the control group was confirmed by cluster analysis as well. Moreover, chronic stimulation of vagus nerve results in significant decrease of Ca, Zn and Rb in substantia nigra of right hemisphere. Based on the results obtained we can conclude that the highest influence of electrical stimulation of left vagus nerve is observed in corpus striatum and substantia nigra. The other structures of nigrostriatal and mesolimbic system do not reveal any significant alterations in content of chemical elements. It may suggest that electrical stimulation of vagus nerve (at the applied parameters) do not cause any considerable disturbances in biochemical processes mediated by macro- or trace elements in these structures. However, the alterations observed for selected elements in striatum and substantia nigra point out clearly that impairment of the vagus nerve function is in close relationship with elemental dyshomeostasis. Taking into account the fact that dopamine system inhibition is also observed as a result of VNS the role of minor- and trace elements in pathological processes of early stage of PD is highly probable. Acknowledgments We acknowledge the Helmholtz-Zentrum Berlin – Electron storage ring BESSY II for provision of synchrotron radiation at beamline mySpot. We also thank Katarzyna Ciesielczyk and Andrzej Bugajski for their technical assistance. This work was supported by: the Polish Ministry of Science and Higher Education and its Grants for Scientific Research, European Community’s Seventh Framework Programme (FP7/2007-2013) under Grant agreement No. 226716. References Badaracco, M.E., Siri, M.V., Pasquini, J.M., 2010. Biofactors 36, 98–102. Banerjee, R., Vitvitsky, V., Garg, S.K., 2008. Trends Biochem. Sci. 33, 413–419. Bertsch, P.M., Hunter, D.B., 2001. Chem. Rev. 101, 1809–1842. Bolton, S., Butt, A., 2006. Exp. Neurol. 202, 36–43. Boruchowska, M., Lankosz, M., Adamek, D., Korman, A., 2001. X-ray Spectrom. 30, 174–179. Braak, H., Del Treduci, K., Rb, U., de Vos, R.A.I., Jansen Steur, E.N.H., Braak, E., 2003. Neurobiol. Aging 24, 197–211. Bradbury, M.W., 1970. Brain Res. 24, 311–321. Braet, K., Cabooter, L., Paemeleire, K., Leybaert, L., 2004. Biol. Cell 96, 79–91. Bugajski, A.J., Gil, K., Ziomber, A., Zurowski, D., Zaraska, W., Thor, P.J., 2007. J. Physiol. Pharmacol. 58 (Suppl. 1), 5–12. Burnstock, G., Williams, M., 2000. J. Pharmacol. Exp. Ther. 295, 862–869. Cass, W.A., Grondin, R., Andersen, A.H., Zhang, Z., Hardy, P.A., Hussey-Andersen, L.K., Rayens, W.S., Gerhardt, G.A., Gash, D.M., 2007. Neurobiol. Aging 28, 258–271. Cohen, I., Navarro, V., Clemenceau, S., Baulac, M., Miles, R., 2002. Science 298, 1418– 1421. de Lau, L.M.L., Breteler, N.M.B., 2006. Lancet Neurol. 5, 525–535. Del Treduci, K., Rub, U., de Vos, R.A.I., Bohl, J.R.E., Braak, H., 2002. J. Neuropathol. Exp. Neurol. 61, 413–423. Demaurex, N., Distelhorst, C., 2003. Science 300, 65–67. Dexter, D.T., Wells, F.R., Lee, A.J., Agid, F., Agid, Y., Jenner, P., Marsden, C.D., 1989. J. Neurochem. 52, 1830–1836. Dexter, D.T., Jenner, P., Schapira, A.H., Marsden, C.D., 1992. Ann. Neurol. 32, S94– 100. Ducic´, T., Quintes, S., Nave, K.A., Susini, J., Rak, M., Tucoulou, R., Alevra, M., Guttmann, P., Salditt, T., 2011. J. Struct. Biol. 173, 202–212. Duong, T.T., Witting, P.K., Antao, S.T., Parry, S.N., Kennerson, M., Lai, B., Vogt, S., Lay, P.A., Harris, H.H., 2009. J. Neurochem. 108, 1143–1154. Durand, D.M., Park, E.H., Jensen, A.L., 2010. Philos. Trans. R. Soc. Lond. B Biol. Sci. 365, 2347–2362. Erko, A., Zizak, I., 2009. Spectrochim. Acta, Part B 64, 833–848. Fields, R., Stevens, B., 2000. Trends Neurosci. 23, 625–633. Finney, L., Mandava, S., Ursos, L., Zhang, W., Rodi, D., Vogt, S., Legnini, D., Maser, J., Ikpatt, F., Olopade, O.I., Glesne, D., 2007. Proc. Natl. Acad. Sci. USA 104, 2247– 2252. Foehring, R.C., Zhang, X.F., Lee, J.C., Callaway, J.C., 2009. J. Neurophysiol. 102, 2326– 2333.
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