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Influence of a low dose of silver nanoparticles on cerebral myelin and behavior of adult rats
Accepted Manuscript Title: Influence of a low dose of silver nanoparticles on cerebral myelin and behavior of adult rats. Author: Beata D˛abrowska-Bouta Mateusz Zi˛eba Jolanta Orzelska-G´orka Joanna Skalska Grzegorz Sulkowski Małgorzata Frontczak-Baniewicz Sylwia Talarek Joanna Listos Lidia Stru˙zy´nska PII: DOI: Reference:
S0300-483X(16)30124-X http://dx.doi.org/doi:10.1016/j.tox.2016.07.007 TOX 51704
To appear in:
Toxicology
Received date: Revised date: Accepted date:
25-5-2016 11-7-2016 13-7-2016
Please cite this article as: D˛abrowska-Bouta, Beata, Zi˛eba, Mateusz, Orzelska-G´orka, Jolanta, Skalska, Joanna, Sulkowski, Grzegorz, Frontczak-Baniewicz, Małgorzata, Talarek, Sylwia, Listos, Joanna, Stru˙zy´nska, Lidia, Influence of a low dose of silver nanoparticles on cerebral myelin and behavior of adult rats.Toxicology http://dx.doi.org/10.1016/j.tox.2016.07.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Influence of a low dose of silver nanoparticles on cerebral myelin and behavior of adult rats.
Beata Dąbrowska-Bouta1a, Mateusz Zięba2b, Jolanta Orzelska-Górka2, Joanna Skalska1, Grzegorz Sulkowski1, Małgorzata Frontczak-Baniewicz3, Sylwia Talarek2, Joanna Listos2, Lidia Strużyńska1*
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Laboratory of Pathoneurochemistry, Department of Neurochemistry, Mossakowski Medical Research Centre, Polish Academy of Sciences,5 Pawińskiego str., 02-106 Warsaw, Poland 2 Department of Pharmacology and Pharmacodynamics, Medical University of Lublin, 4a Chodźki str., 20-093 Lublin, Poland 3 Electron Microscopy Platform, Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawińskiego str., 02-106 Warsaw, Poland
* Corresponding author: Lidia Strużyńska, PhD, Laboratory of Pathoneurochemistry, Department of Neurochemistry, Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawińskiego str., 02-106 Warsaw, Poland. E-mail: [email protected] a, b - Authors equally contributed to the manuscript
Abstract Nanoscale particles have large surface to volume ratio that significantly enhances their chemical and biological reactivity. Although general toxicity of nano silver (nanoAg) has been intensively studied in both in vitro and in vivo models, its neurotoxic effects are poorly known, especially those of low-dose exposure. In the present study we assess whether oral administration of nanoAg influences behavior of exposed rats and induces changes in cerebral myelin. We examine the effect of prolonged exposure of adult rats to small (10 nm) citrate-stabilized nanoAg particles at a low dose of 0.2 mg/kg b.w. (as opposed to the ionic silver) in a comprehensive behavioral analysis. Myelin ultrastructure and the expression of myelin-specific proteins are also investigated. The present study reveals slight differences with respect to behavioral effects of Ag+- but not nanoAg-treated rats. A weak depressive effect and hyperalgesia were observed after Ag+ exposure whereas administration of nanoAg was found to specifically increase body weight and body temperature of animals. Both nanoAg and Ag+ induce morphological disturbances in myelin sheaths and alter the expression of myelin-specific proteins CNP, MAG and MOG. These results suggest that the CNS may be a target of low-level toxicity of nanoAg.
Keywords: nanosilver; nanotoxicity; neurotoxicity; CNP; MAG; MOG; behavioral tests
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1. Introduction Nanosilver (nanoAg), one of the most commonly used metal-derived nanomaterials, has broad-spectrum antibacterial and antifungal properties. These qualities have promoted its use in many life domains. NanoAg is currently used in a wide array of commercial goods, biomedical products (such as medical implants, catheters and wound dressings), as well as in food production and bioengineering (Marambio-Jones and Hoek, 2010; Cushen et al., 2014). One of the main aims of nanomedicine is to enhance drug availability within the central nervous system (CNS) by providing a mechanism for delivery of drugs past the blood-brain-barrier (BBB) to increase therapeutic efficacy (Sharma et al. 2013; Chaloupka et al. 2010). NanoAg is regarded as a nanoparticulate drug-carrier system for delivery of drugs to CNS (Leite et al., 2015). The increasing production and use of engineered nanomaterials may inevitably lead to contamination of the environment and thus warrants risk assessment. On the other hand, medical applications also carry potential risks of toxic effects in humans. Hence, there is a need for research on low-level toxicity of nanoAg. A number of studies, both in vitro and in vivo, have indicated toxic properties of nanoAg in a wide range of concentrations and sizes. Smaller nanoAg particles have been shown to be more active in exerting toxicological responses, inducing organ toxicity and inflammatory responses after repeated oral administration (Park et al, 2010). Current findings indicate that nanoAg may easily cross the BBB and accumulate in the rodent brain after oral or intravenous administration (Kim et al. 2008; Tang et al. 2010). Using an in vitro model, Trickler et al. (2010) showed that by releasing proinflammatory cytokines, nanoAg may induce inflammation in rat brain microvessels, which additionally enhances the breakdown of the BBB. Dysfunction of the BBB facilitates further penetration of nanoAg into brain tissue which causes synaptic (Skalska et al., 2015) and neuronal (Tang et al., 2009) degeneration. When administered intranasally, 3
nanoAg alters spatial reference memory (Davenport et al., 2015) and locomotor activity in neonatal rats (Yin et al., 2015). NanoAg has also been reported to affect concentrations of the neurotransmitters dopamine and 5-hydroxytryptamine (5-HT) in the rat brain (Hadrup et al. 2012) and to impair spatial cognition in rats via their impact on hippocampal synaptic plasticity (Liu al. 2012). Except for a few neurotoxicological reports, the exact influence of nanoAg on CNS processes and underlying mechanism(s) of the action are not well understood. It has been reported that larger nanoAg particles (50-60 nm) in a relatively high dose of 50 mg/kg b.w. may cause myelin damage as reflected by myelin basic protein (MBP) immunostaining (Sharma et al., 2013). However, extensive research on the toxic impacts on myelin in adult organisms as well as a comprehensive assessment of behavioral effects have not yet been conducted. Thus, the aim of the present study was to investigate whether prolonged oral administration of a low dose (0.2 mg/kg b.w.) of nanoAg (10 nm) or ionic silver (Ag+) influences the behavior of rats and affects myelin ultrastructure and expression of myelin-specific proteins. A wide range of behavioral assessments was applied to assess the effects of nanoAg. Measurements of body weight and body temperature and tests of locomotor activity, motor coordination, nociceptive reaction, memory performance and anxiety-like behavior were performed. These measurements provide generally-accepted assessments of behavior in investigations of bioactivity of new compounds. Studies on behavioral impacts were combined with examination of expression of several myelin-specific proteins: myelin/oligodendrocyte glycoprotein (MOG), myelin-associated glycoprotein (MAG) and 2ʹ-3ʹ-cyclic nucleotide 3ʹphosphodiesterase (CNP). Since proteins are integral components of myelin membranes and contribute to their structure and stability, ultrastructural observations were also performed using transmission electron microscopy (TEM). 4
2. Materials and methods 2.1. Chemicals Most of the chemicals used in the experiments, including nanoAg particles, were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). All reagents were of analytical grade. NanoAg particles (10 ± 4 nm in diameter) (CAS No.730785) are defined by the manufacturer as a colloidal solution stabilized in sodium citrate to prevent sedimentation and to maintain the dispersed state. Characterization of the degree of dispersion and size distribution of the nanoAg particles was accomplished by transmission electron microscopy (JEM-1200EX, JEOL) using a digital camera MORADA and iTEM 1233 software (Olympus Soft Imaging Solutions GmbH, Germany) according to a standard method developed for non-biological preparations as described previously (Skalska et al., 2015). 2.2. Animals and experimental design of silver exposure Male Wistar rats weighing 140-160 g obtained from the Farm of Labolatory Animals, Z. Lipiec, Brwinow, Poland were used throughout the study. All animals were caged in groups of six and maintained on a 12 h light-dark cycle (lights on at 6:00 h) at a controlled temperature (21°C). During the experiments, the animals were allowed free access to drinking water and a standard laboratory diet known as the Murigan diet (Agropol, Motycz, Poland). All experiments were carried out according to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and the European Community Directive for the Care and Use of Laboratory Animals, as of 24 November 1986 (86/609/EEC), and were approved by the Local Ethic Committee (35/2014). Rats were randomly divided into three groups, each consisting of 12 animals and representing: (i) a negative control group (treated with saline), (ii) a nanoAg-treated group and 5
(iii) an ionic silver-treated group (silver citrate, Ag+). Solutions of nanoAg stabilized in citrate buffer or silver citrate were administered via a gastric tube in a dose of 0.2 mg/kg b.w. per day for 14 days (0.02 mg nanoAg or Ag+/mL). The rats of the control groups received the same dose of saline. During the experiment, body temperature and body weight were assessed. Body temperature in normothermic rats was measured at the animal’s rectum with a thermistor thermometer twice daily on the 1st, 8th and 14th days of the experiment to check the acute effects of administration (the second measurement was taken 1 h after administration). Body temperature changes (Δt) were calculated according to the formula: Δt = t2 – t1, where t1 and t2, were the presubstance and post-substance body temperature, respectively (Vogel, 2008). Simultaneously, on the 1st, 8th and 14th day of the experiment, each animal was weighed. 2.3. Performance of behavioral tests Behavioral tests were initiated after the final treatment on day 14. Locomotor activity and motor coordination were measured on the 15th day of the experiment. Anxiety and memory tests were carried out on the 16th day. On the last day of the study and before decapitation, the nociception reaction was assessed. 2.3.1. Locomotor activity test Locomotor activity of individual rats was recorded using a photocell device (plexiglass boxes - square cages, 60 cm on each side; Porfex, Bialystok, Poland) in a sound-attenuated experimental room, under moderate illumination (10 lux). Ambulatory activity (distance traveled) was measured by two rows of infrared light-sensitivity photocells, installed along the long axis, 45 and 100 mm above the floor. Total horizontal activity (the distance traveled in meters) was recorded for a 30-minute period (Koltunowska et al. 2013). 2.3.2. Rotarod performance test 6
To evaluate the muscle –relaxant or ataxic effects, rats were tested on a rotating rod (rotarod) apparatus (Multiserv, Lublin, Poland). Two days before the experiment, the animals were trained on a rotarod with a 6 cm diameter and 50 cm length, subdivided into four areas by disks 25 cm in diameter, at a constant rotating speed of 9 rpm. The rats that did not fall off the rotarod within 1 min were assigned the maximum score of 60 s (Kotlinska et al., 2012). 2.3.3. Novel object recognition test The apparatus included a square open box, made of plexiglass (63 cm long x 44.5 cm high x 44 cm wide) and illuminated by a lamp (light intensity – 10 lux), suspended 50 cm above the box. The objects to be discriminated were constructed of either of wood or plastic were in the shape of a block and a ball and were too heavy to be displaced by the animals. The object recognition test was performed as described elsewhere (Bertaina-Anglade et al. 2006). The day before the test, each rat was placed in the empty box for 2 minutes to become acclimatized to the environment. On the day of the experiment, the animals were subjected to two trials, spaced apart by a 1-hour interval. The first trial (acquisition trial, T1) lasted 5 minutes and the second trial (test trail, T2) was 3 minutes long. During T1, the apparatus contained two identical objects (wooden blocks), placed in two opposite corners, 10 cm from the sidewall. The rat was placed in the middle of the box. After T1, the rat was returned to its home cage. Subsequently, after 1 h, T2 was performed. During T2, a new object (N) replaced one of the two objects presented in T1. Therefore, the rats were re-exposed to two objects: a familiar object (F) and a new object (N). In order to avoid the presence of olfactory trails, the apparatus and the objects were cleaned after each test. Exploration behavior was judged to include directing the nose toward the object at a distance of no more than 2 cm and/or touching the object with the nose. Turning around or sitting on the object was not considered as exploratory behavior. The time periods spent by rats in exploring each object during the T1 and T2 tests, were recorded 7
manually with a stopwatch. Recognition memory was evaluated using a recognition index calculated for each animal by the formula: (N-F/N+F) x100 corresponding to the difference between the time exploring the novel and the familiar object, corrected for total time exploring both objects. A higher recognition index is considered to reflect stronger memory retention for familiar objects (Bertaina-Anglade et al., 2006). 2.3.4. Elevated plus-shaped maze apparatus The maze was made of wood and positioned on a height of 50 cm above the floor in a quiet laboratory surrounding. Two opposite arms were open (50 cm × 10 cm) and the other two were enclosed by walls (50 cm × 10 cm × 40 cm). The level of illumination was approximately 100 lx at the floor level of the maze. The maze experiment was initiated by placing the rat in the center of the maze facing an open arm, after which the number of entries and time spent in each of the two arms were recorded for 5 min. An “arms entry” was recorded when the rat entered the arm with all four paws. The maze was carefully cleaned with tap water after each test session. The open arms activity was quantified as (a) the time spent in the open arms as a percent of the total time spent on exploring both the open and closed arms, and (b) the number of entries into the open arms as a percentage of the total number of entries into both open and closed arms (Kotlinska et al., 2012; Lister, 1987). 2.3.5. Tail-immersion test To determine the nociceptive reaction, the tail of the rat was placed in a water bath heated to 52°C, and the latency of response (in seconds; reflexive withdrawal of the distal half of the tail after its immersion in water) was measured. The cut-off time was set to 20 s to avoid strong pain or tissue damage. The antinociceptive effects were expressed as a percent maximum possible effect (MPE%), which was calculated according to the following equation: [(T1-T0)/ (20-T0)] x100. The mean value from the first two measurements (15th and 16th day of the experiment) was 8
assumed as baseline latency response (T0). T1 indicates latency for the tail-immersion response on the 17th day (Kotlinska et al., 2009; Le et al., 2001). 2.4. TEM analysis of brain samples. The animals were anesthetized with Nembutal (80 mg/kg b.w.) and perfused with 0.9% NaCl in 0.01 M sodium-potassium phosphate buffer pH 7.4 and then with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 Tissue was sampled from the forebrain cortex of all rat groups. After fixation in the above ice-cold fixative solution, specimens were post-fixed in 1% OsO4 solution, then dehydrated in the ethanol gradient and embedded in epoxy resin (Epon 812). Then ultra-thin sections (60 nm) were stained with 9% uranyl acetate and lead nitrate. Analysis of blinded brain sections was done by transmission electron microscopy (JEM-1200EX, Jeol, Japan) regarding morphological features of myelin. 2.5. Preparation of myelin fraction The myelin fraction was prepared according to the procedure of Norton and Poduslo (1973). Briefly, after decapitation, brains were removed and homogenized in 0.32 M sucrose. Then samples were centrifuged at 75,000 x g for 30 min in a 0.32 M/0.85 M sucrose gradient. The crude myelin pellet was dispersed in water and centrifuged several times at 12,000 x g for 10 min. To obtained purified myelin, centrifugation in 0.32 M sucrose was performed. The pellet consisting of purified myelin was suspended in water with protease inhibitors, 2 mM EDTA and, 2 mM EGTA, and frozen at -80°C for further experiments. 2.6. Western blot analysis Protein concentration in myelin fractions was determined by the method of Lowry at al., (1951). Equal amounts of protein (50 µg) were subjected to SDS-PAGE electrophoresis and then transferred to nitrocellulose membranes. Primary antibody anti-MAG (1:200), anti-MOG (1:1000) and anti-CNP (1:500) was used followed by a secondary antibody conjugated to HRP 9
(1:200). Polyclonal anti-β actin (1:1000) antibody was used as an internal standard. Bands were visualized on Hyperfilm ECL using chemiluminescence ECL kit (Amersham). The films were scanned and quantified using Image Quant TL v2005 software. 2.7. Determination of the mRNA levels of CNP, MAG and MOG by RT-qPCR Total RNA was extracted from the brain cortex of rats according to the method of Chomczyński and Sacchi (1987) using TRI Reagent (Sigma, St. Louis, MO, USA). 2 µg of total RNA were subjected to reverse transcription using random primers and AMV reverse transcriptase (Applied Biosystems, Forest City, CA, USA). The RT-PCR reaction was conducted under conditions: reverse transcription at 42°C for 45 min and denaturation at 94°C for 30 s. Rat myelin protein-specific primers for MOG Rn 00575354_m1, MAG Rn02586362, CNPase Rn01399463_m1 and the corresponding probes were obtained from Applied Biosystems (Forest City, CA, USA). The pre-validated TaqMan assay reagents (Applied Biosystems, Forest City, CA, USA) were used to determine the mRNA expression levels of myelin proteins and actin. Real-time PCR was conducted on an ABI Prism 7500 system using 5 µL of RT product, TaqMan PCR Master Mix, primers, and a TaqMan probe in a total volume of 20 µL. The PCR cycle conditions were as follows: initial denaturation at 95°C for 10 min, 50 cycles of 95°C for 15 s, and 60°C for 1 min. The relative expression level of the myelin protein mRNA, normalized to actin, was calculated using the standard curve method. 2.8. Statistical analysis The results are expressed as mean ± SEM from 3-8 experiments as indicated in the legends of respective figures. Inter-group comparisons were made using one-way analysis of variance (ANOVA) with post-hoc Dunnett’s test. The results of behavioral tests were analyzed by one-way or two-way ANOVA followed by the Bonferroni’s post hoc test. Each group of animals consisted of 6-12 rats. The level of p<0.05 was considered as statistically significant. 10
Analysis was done using the GraphPad Prism version 5.00 for Windows, GraphPad Software (San Diego, California, USA).
3. Results Commercially-available citrate-stabilized nanoAg particles of a small size (10±4 mm) were applied in a concentration of 0.02 mg/mL. It was demonstrated previously using TEM that nanoAg particles do not agglomerate in citrate buffer and are spherical in shape and of homogenous size (Skalska et al., 2015). About 95% of the particles were approximately 10 nm in diameter, with the remaining 5% being larger or smaller. Using inductively coupled plasma mass spectrometry (ICP-MS), it was shown in the applied model of oral exposure to a low dose of nanoAg, that silver is quickly absorbed from the gastrointestinal tract into the serum compartment, regardless of its form. After 2 weeks of exposure, the level of Ag in serum was found to be in the range of 11-13 µg/L in the experimental group. However, it was found to be below the detection limit of the applied method in brain homogenates of rats (0.241 mg/kg in solid tissue) (Skalska et al., 2015). 3.1. Body parameters of exposed rats One-way ANOVA revealed significant [F (8, 93) =34.05; p<0.001] differences in the body weight of rats on the 1st, 8th and 14th day. Post hoc analysis showed that body weights of the controls and the Ag+-treated rats were similar at selected time points whereas the rats of the group treated with nanoAg had higher body weights compared to those of the control group (p<0.01) on the 14th day of the experiment (Fig. 1). Changes in body temperature were recorded for all groups of rats. Two-way ANOVA revealed statistically significant effects of the exposure [F (2, 54) = 4.39; p=0.0171] and a significant time effect [F (2, 54) = 7.25; p=0.0016]. A significant increase in body temperature was observed in rats after chronic exposure to both 11
nanoAg (p<0.01) and silver citrate (p<.0.05) on the 8th day of the experiment relative to the control group. Moreover, post hoc analysis (Bonferroni’s test) revealed that the rats of the group subjected to chronic exposure of Ag+ had higher body temperatures compared to the control group on the 14th day of the experiment (p<0.05) (Fig. 2). A similar tendency was observed on the 14th day of exposure in the group treated with nanoAg. However, this observation was found to be statistically insignificant. 3.2. Effect of exposure to a low dose of AgNPs/Ag+ on behavior of animals NanoAg exposure did not significantly influence locomotor activity, motor coordination or memory performance of rats. No differences among the groups were observed, regarding the effects of administration of saline (50.81±3.101), Ag+ (54.86±7.41) and nanoAg (54.09±5.806) on the total distance traveled by rats within 30 min (one-way ANOVA). Similarly, the results of the rotarod experiments did not differ among the groups (53.25±4.634, 50.18±4.863, 58.18±1.818 for saline-, Ag+- and nanoAg-treated animals, respectively; one-way ANOVA). Moreover, the recognition index of rats in NOR task was similar in all groups, regarding the effects of administration of saline (74.92±1.961), Ag+ (81.69±6.698) and nanoAg (89.16±3.12) (one-way ANOVA). However, it was found that chronic administration changed the degree of behavioral anxiety in the rats. We observed significant differences among the groups concerning the percentage of time spent by rats in the open arms of the plus-maze [F (2, 29) =4.171; p=0.0256; Table 1A] but no substantial differences in the percentage of open arm entries (Table 1B). Post hoc analysis (Bonferroni’s test) showed that chronic administration of Ag+ but not nanoAg increased the time spent by rats in the open arms (p<0.05) compared to the saline group (Table 1). The nociceptive reaction, as assessed by the tail immersion test, was found to be altered in rats exposed to Ag+ and nanoAg. One-way ANOVA revealed significant differences among 12
groups in the percent maximum possible effect (% MPE) [F (2, 27) =6.231; p=0.006; Table 2]. Post hoc analysis (Bonferroni’s test) showed that the group treated chronically with Ag+ was less sensitive to the noxious stimuli compared to the saline-administered rats (p<0.05) (Table 2). 3.4. Changes in ultrastructure and myelin proteins after administration of silver Since a low dose of applied silver was found to be insufficient to detect in brain homogenates of exposed rats with ICP-MS, the presence of nanoAg was demonstrated in brain tissue indirectly, using a modified TEM method described previously. The nanosized granules were observed to be located i.a. between lamellae of myelin sheaths in specimens from brains of AgNPs- but not in Ag+ -treated and control animals (Skalska et al., 2015). Current TEM analysis of brain specimens of control rats indicated normal appearance of neuropil as well as myelin which exhibited lamellar ultrastructure with proper, well-defined plasma membranes adhering to each other (Fig. 3A). In micrographs of brain tissue obtained from Ag+- and nanoAg-treated groups, pathological changes in myelin were observed (Fig. 3BD). The types and degree of damage were found to be very similar. A vast majority of myelin sheaths were found to have irregularities and to exhibit interruptions of compact multilamellar structure. We determined the levels of selected myelin-specific proteins - CNP, MAG and MOG which play roles in the stabilization of myelin structure. A significant decrease in the level of all examined proteins was observed in rats treated with Ag+ and nanoAg (Fig. 4). The relative level of each of these proteins reached about 60%-70% of the control value (*p<0.01 vs. control), although the changes between both silver-treated groups were found to be statistically insignificant (Figs. 4A-4C). Conversely, with real-time PCR, we observed increased mRNA levels of proteins in myelin fractions isolated from the Ag+- and nanoAg-treated groups relative to the control group (Fig. 5). Enhancement of mRNA of all investigated proteins was noted in the 13
case of rats treated with nanoAg, which differed significantly from control as follows: about 30%, 20% and 15% for CNP, MAG and MOG, respectively. As opposed to nanoAg, Ag+ administration, increased only the expression of CNP mRNA by about 30%. This is significantly different from the control value (*p<0.05).
4. Discussion The vast majority of research indicates that, despite the undeniable benefits of nanoAg particles, they may pose a threat to various organisms (Ulm et al., 2015, Davenport et al., 2015, Gomes et al. 2013, McCarthy et al., 2013; Sharma at al. 2009; Strużyński et al., 2014). It is therefore important to investigate the toxic effects of nanoAg to understand the mechanisms of toxicity, with particular attention to neurotoxicity. Mechanistic studies on nanoAg neurotoxicity have been mostly conducted using in vitro systems with high doses of nanoparticles. Thus, it is essential to characterize these pathomechanisms in animal models because it is not possible to replicate the complexity of the nervous system in cultures. Such efforts are expected to provide meaningful data about the response of a biological system to nanoAg. Persistent accumulation of small (10 nm) citrate-stabilized nanoAg particles has been observed by TEM in brains isolated from orally-exposed rats (Lee et al., 2013; Skalska et al., 2015). Moreover, it is known that the biological half-life of silver in the CNS is longer than in other organs (Hadrup, 2012). Since the neurotoxic effects of in vivo exposure to nanoAg have not been extensively studied, the present study was designed to specifically investigate the behavioral effects of prolonged exposure of adult rats to low doses of small nanoAg particles with regard to ultrastructural and biochemical changes in cerebral myelin. The objective was to test whether nanoAg in such a low concentration will induce pathological processes in myelin sheaths and whether these potential changes may result in behavioral alterations. The effects of prolonged 14
exposure of rats to nanoAg were compared with the effects of Ag+, based on the existing conviction that the toxicity of nanoAg relies (almost partially) on the liberation of free Ag+. Such an approach allows us to evaluate the contribution of ions to the overall toxic effects of nanoAg, since it was suggested that the acidic microenvironment of cellular lysosomes promotes the release of ions from the surfaces of the particles (Loeschner et al., 2011). The results of the current study revealed some differences while comparing nanoAg and Ag+ with respect to body parameters and behavioral performance of rats. Body weight of animals is quite sensitive to repeated oral exposure to nanoAg. Exposed animals have increased body weight on the 14th day of the experiment relative to control group. Conversely, weight gain was not observed in Ag+-exposed animals. These findings are in contrast to the results of an earlier study, indicating loss of body weight after three consecutive days of intravenous administration of 5 nm nanoAg particles (Zhang et., 2013). Loss of weight was also considered an indicator of neurotoxicity of metal nanoparticles other than nanoAg (Cao et al., 2013b; Zhang et al., 2010). However, other reports have not revealed any changes in body weight after chronic (14 or 28 days) inhalation and oral nanoAg exposure (18-20 and 60 nm), respectively (Kim et al., 2008; Sung et al., 2011). The above conflicting results might be explained by differences in experimental design (e.g. route of administration, size of the nanoAg and duration of experiments). Based on available data, it is difficult to define whether the observed increase in body weight is the result of nanoAg-induced CNS alterations. Further studies measurement of food consumption are necessary. In addition, even one-week-administration of both Ag+ and nanoAg was found to cause a substantial increase in the body temperature of normothermic rats. An important role in the central regulation of body temperature has been assigned to 5-HT (Wujec et al., 2014). It was also found that the concentration of 5-HT was changed in the rat brain under conditions of 15
chronic oral administration of nanoAg, but not Ag+ (Hadrup et al., 2012). In the light of the above-cited data, we suspect that the currently observed altered body temperature may be due to the impairment of the serotonergic system. The antinociceptive activity of the examined compounds was investigated in the commonly used tail immersion test (Kotlinska et al., 2009; Le et al., 2001). The tail immersion procedure generates spinal cord reflexes and does not affect visceral or musculoskeletal tissues (Le et al., 2001). The results of the present study showed that the nociceptive reaction generated by this test was not affected by nanoAg whereas Ag+ induces hyperalgesia after a two-week exposure period. Hyperalgesia, an increased sensitivity to painful stimuli, is commonly associated with inflammation. Consistent with this observation, Ag+ has been shown to interact with microvasculature leading to a proinflammatory cascade (Marchi et al., 2004). Many molecules have been identified including lipids, purines, protons, cytokines and growth factors which are produced in the context of this inflammatory process and can contribute to heightened pain states due to their ability to sensitize nociceptors (Woolf and Ma, 2007). No behavioral differences were observed in the EPM procedure, suggesting a lack of nanoAg-induced alterations in the anxiety response. Conversely, the rats from Ag+-exposed group spent more time in the open arms of the maze, indicating an anxiolytic effect. Regulation of anxiolytic activity depends on different neurotransmitters (glutamine, GABA, dopamine or 5HT). Disturbances in any of these systems may influence anxiety-like behavior (Zarrindast and Khakpai, 2015). According to Powers et al. (2011), the characteristics of the nanoparticles are critical determinants of their behavioral effects. This may explain the more substantial behavioral impact of Ag+ relative to nano form of Ag. The weak depressive effect of Ag+ on the CNS observed in these experiments is an interesting result. However, nanoAg and Ag+ did not cause impairment of motor coordination, 16
locomotor activity and memory. These observations are consistent with previous studies, in which locomotor activity of rats did not appear to be sensitive to nanoAg treatment even at doses much higher than used in the current study (5 and 10 mg/kg) (Zhang et al., 2010). Proper transmission of electrical impulses in the CNS is made possible by the myelin sheath surrounding most of the axons. Apart from enhancing the speed of neuronal transmission, the main function of this membrane structure is to protect and insulate axons. Disturbances in myelin integrity may result in inappropriate signal transfer and behavioral abnormalities. The important role of myelin in the human CNS is highlighted by an array of severe neurological diseases such as leukodystrophies, multiple sclerosis (MS) and peripheral neuropathies, in which myelin is impaired (Baumann and Pham-Dinh, 2001). Marked myelin degradation was previously observed based on myelin basic protein (MBP) immunostaining in brains of rats exposed to relatively high dose (50 mg/kg b.w.) of large nanoAg particles (50-60 nm) (Sharma et al., 2013). Myelin disintegration may consequently result in degeneration of myelinated fibers. Our previous study demonstrated considerable nanoAg-induced synaptic degeneration in the applied model of low-dose exposure (Skalska et. al., 2015). Thus, it was interesting to investigate whether myelin is also affected. Examination of the myelin ultrastructure indicates that pathological changes of myelin sheaths are clearly visible in the Ag+- and nanoAg-treated groups (Fig. 3). Focal detachment of the myelin lamellae and increased volumes of cytoplasm within the regions of compacted myelin can be observed. Interestingly, the characteristics and the degree of myelin damage are similar in both of the exposed groups. Moreover, ultrastructural changes are similar to those reported to occur under conditions of exposure to metals exhibiting neurotoxic potential such as mercury, lead or cooper (Cao et al. 2013a, Dabrowska-Bouta et al., 1999, Viquez et al., 2009; Rai et al., 2013).
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A number of morphological observations have been made in context of the biochemical studies. Significant decreases, reaching about 60%-75% of control value, in relative level of all of the examined proteins were noted in rats treated with Ag+- and nanoAg, wherein CNP and MOG appeared to be slightly more sensitive to nanoAg treatment than MAG. Analysis of mRNA expression revealed that each of the tested proteins is significantly over-expressed by about 25% - 35% compared to the controls in nanoAg-treated rats (Fig. 5). In the case of rats treated with Ag+, significantly increased expression of CNP mRNA was observed. The changes observed at the molecular level may suggest that the compensative reaction takes place and the repair processes are launched at the gene expression level, mainly under nanoAg exposure. In the case of Ag+, increased gene expression did not compensate for the levels of all proteins. However, it cannot be excluded that in a longer period, repair mechanisms will be fully effective. Both deficiency and excess of the examined proteins may influence formation and maintenance of proper myelin structure. In transgenic mice, over-expressed CNPase perturbs myelin formation and leads to aberrant oligodendrocyte membrane expansion (Gravel et al., 1996). It should be also highlighted that myelin glycoproteins MOG and MAG are important in the light of their interactions with the immune system. MOG, located on the outermost lamellae of compact myelin sheaths, has received significant attention in studies on autoimmune diseases as a protein inducing auto-antibodies with demyelinating potency (Ben-Nun et al., 1999). MAG is glycoprotein whose extracellular region contains five segments of homology resembling immunoglobulin domains, one of which is strikingly homologous to a domain of the NCAM (Baumann and Pham-Dinh, 2001). Also, the previously shown association of MOG and CNP with different elements of the complement cascade, may be relevant to connections between the immune system and the nervous system and may have pathological implications in demyelinating
18
diseases (Baumann and Pham-Dinh, 2001). Hence, the link between the nervous system and the immune system under conditions of nanoAg exposure should be considered. In summary, the results indicate that nanoAg administered at a low concentration does not cause considerable toxicity, as reflected by behavioral tests, although observed body temperature and body weight alterations suggest that the CNS may be a target of low-level toxicity of nanoAg and warrant further studies. However, similar toxic effects of Ag+ and nanoAg towards myelin are evident at the biochemical level and the ultrastructural level. The results clearly demonstrate morphological abnormalities in myelin sheaths and altered expression of myelin proteins. Therefore, it is reasonable to draw attention to the risk of neurotoxicity caused by low-level exposure to nanoAg.
5. Acknowlegements The study was supported by statutable funds provided by the Polish Ministry of Science and Higher Education for Mossakowski Medical Research Centre, Polish Academy of Sciences and for Medical University of Lublin (DS 21/2015).
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Ulm, L., Krivohlavek, A., Jurašin, D., Ljubojević, M., Šinko, G., Crnković, T., Žuntar, I., Šikić, S., Vinković, Vrček I., 2015. Response of biochemical biomarkers in the aquatic crustacean Daphnia magna exposed to silver nanoparticles. Environ. Sci. Pollut. Res. Int. 22, 19990-9. Viquez, O.M., Lai, B., Ahn, J.H., Does, M.D., Valentine, H.L., Valentine, W.M., 2009. N,Ndiethyldithiocarbamate promotes oxidative stress prior to myelin structural changes and increases myelin copper content. Toxicol. Appl. Pharmacol. 239, 71-79. Woolf, C.J., Ma, Q., 2007. Nociceptors--noxious stimulus detectors. Neuron 55, 353-364. Wujec, M., Kedzierska, E., Kuśmierz, E., Plech, T., Wrobel, A., Paneth, A., Orzelska, J., Fidecka, S., Paneth, P., 2014. Pharmacological and structure-activity relationship evaluation of 4aryl-1-diphenylacetyl(thio)semicarbazides. Molecules 19, 4745-4759. Yang, Z., Liu, Z.W., Allaker, R.P., Reip, P., Oxford, J., Ahmad, Z., Ren, G., 2010. A review of nanoparticle functionality and toxicity on the central nervous system. J. R. Soc. Interface. 7, Suppl 4, S411-22. Yin, N., Zhang, Y., Yun, Z., Liu, Q., Qu, G., Zhou, Q., Hu, L., Jiang, G., 2015. Silver nanoparticle exposure induces rat motor dysfunction through decrease in expression of calcium channel protein in cerebellum. Toxicol. Lett. 237, 112-120. Zarrindast, M.R., Khakpai, F., 2015. The Modulatory Role of Dopamine in Anxiety-like Behavior. Arch. Iran. Med. 18, 591-603. Zhang, X.D., Wu, H.Y., Wu, D., Wang, Y.Y., Chang, J.H., Zhai, Z.B., Meng, A.M., Liu, P.X., Zhang, L.A., Fan, F.Y., 2010. Toxicologic effects of gold nanoparticles in vivo by different administration routes. Int. J. Nanomed. 5, 771-781. Zhang, Y., Ferguson, S.A., Watanabe, F., Jones, Y., Xu, Y., Biris, A.S., Hussain, S., Ali, S.F., 2013. Silver nanoparticles decrease body weight and locomotor activity in adult male rats. Small 9, 1715-1720.
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Figure legends Fig. 1:Effect of chronic administration of Ag+ and nanoAg on the body weight of rats measured on the 1st, 8th and 14th day of exposure. Data are expressed as mean ±SEM values obtained from 12 animals. *P<0.01 vs. control (saline group) on the 14th day of the experiment (Bonferroni’s test).
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Fig. 2:Effects of chronic administration of Ag+ and nanoAg on the body temperature of rats. Data are expressed as mean ±SEM values obtained from 12 animals. *P<0.05 and **p<0.01 vs. control (saline group), 8th day of the experiment; #p<0.05 vs. control (saline group), 14th day of the experiment (Bonferroni’s test).
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Fig.3:Representative electron micrographs of myelin sheaths in forebrain specimens obtained from control (A), Ag+-treated rats (B) and nanoAg-treated rats (C, D). Arrows indicate disintegrated lamellar structure of myelin membranes.
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Fig.4:Representative immunoblots show the expression of myelin-specific proteins: CNP, MAG and MOG in myelin fractions isolated from brains of control, Ag+-treated and nanoAg-treated rats. The graphs provide the results of densitometric analysis of eight independent immunoblots, each performed using a separate myelin sample. The results are normalized to β-actin and expressed as percentage of control. The values represent the means ± SEM, *p< 0.01 (one-way ANOVA test with post-hoc Dunnetts’ test).
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Fig.5:Expression of mRNA of myelin proteins in the forebrains of control, Ag+-treated and nanoAg-treated rats. The mRNA levels of A) CNP, B) MAG and C) MOG were determined by quantitative real time-PCR and normalized against actin. Graphs indicate the results expressed as arbitrary units from four independent experiments, each performed using distinct brain samples. The values represent the means ± SEM, * p < 0.05, significantly different vs. control rats (oneway ANOVA with post-hoc Dunnett’s test).
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Table 1 Effects of chronic exposure to silver on anxiety-like performance of rats estimated in the elevated plus-maze apparatus A
B
percent of the time spent
percent of the open arms
in the open arms
entries
Control (saline)
9.811±3.059
26.76±6.143
Silver citrate (Ag+)
33.48±11.10*
34.03±9.308
nanoAg
10.22±3.068
25.93±7.591
Exposure to:
Data are expressed as mean ±SEM from 12 animals. *P<0.05 vs. control group (Bonferroni’s test).
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Table 2 Effects of chronic exposure to Ag+ and nanoAg on the nociceptive reaction as indicated by the tail immersion test. Exposure to:
MPE%
Saline
0.8823±1.054
silver citrate (Ag+) -4.199±1.055* nanoAg
0.8463±1.435
Data are expressed as mean ±SEM from 12 animals, *p<0.05 vs. control group (Bonferroni’s test); MPE% - a percent maximum possible effect.
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S0300-483X(16)30124-X http://dx.doi.org/doi:10.1016/j.tox.2016.07.007 TOX 51704
To appear in:
Toxicology
Received date: Revised date: Accepted date:
25-5-2016 11-7-2016 13-7-2016
Please cite this article as: D˛abrowska-Bouta, Beata, Zi˛eba, Mateusz, Orzelska-G´orka, Jolanta, Skalska, Joanna, Sulkowski, Grzegorz, Frontczak-Baniewicz, Małgorzata, Talarek, Sylwia, Listos, Joanna, Stru˙zy´nska, Lidia, Influence of a low dose of silver nanoparticles on cerebral myelin and behavior of adult rats.Toxicology http://dx.doi.org/10.1016/j.tox.2016.07.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Influence of a low dose of silver nanoparticles on cerebral myelin and behavior of adult rats.
Beata Dąbrowska-Bouta1a, Mateusz Zięba2b, Jolanta Orzelska-Górka2, Joanna Skalska1, Grzegorz Sulkowski1, Małgorzata Frontczak-Baniewicz3, Sylwia Talarek2, Joanna Listos2, Lidia Strużyńska1*
1
Laboratory of Pathoneurochemistry, Department of Neurochemistry, Mossakowski Medical Research Centre, Polish Academy of Sciences,5 Pawińskiego str., 02-106 Warsaw, Poland 2 Department of Pharmacology and Pharmacodynamics, Medical University of Lublin, 4a Chodźki str., 20-093 Lublin, Poland 3 Electron Microscopy Platform, Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawińskiego str., 02-106 Warsaw, Poland
* Corresponding author: Lidia Strużyńska, PhD, Laboratory of Pathoneurochemistry, Department of Neurochemistry, Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawińskiego str., 02-106 Warsaw, Poland. E-mail: [email protected] a, b - Authors equally contributed to the manuscript
Abstract Nanoscale particles have large surface to volume ratio that significantly enhances their chemical and biological reactivity. Although general toxicity of nano silver (nanoAg) has been intensively studied in both in vitro and in vivo models, its neurotoxic effects are poorly known, especially those of low-dose exposure. In the present study we assess whether oral administration of nanoAg influences behavior of exposed rats and induces changes in cerebral myelin. We examine the effect of prolonged exposure of adult rats to small (10 nm) citrate-stabilized nanoAg particles at a low dose of 0.2 mg/kg b.w. (as opposed to the ionic silver) in a comprehensive behavioral analysis. Myelin ultrastructure and the expression of myelin-specific proteins are also investigated. The present study reveals slight differences with respect to behavioral effects of Ag+- but not nanoAg-treated rats. A weak depressive effect and hyperalgesia were observed after Ag+ exposure whereas administration of nanoAg was found to specifically increase body weight and body temperature of animals. Both nanoAg and Ag+ induce morphological disturbances in myelin sheaths and alter the expression of myelin-specific proteins CNP, MAG and MOG. These results suggest that the CNS may be a target of low-level toxicity of nanoAg.
Keywords: nanosilver; nanotoxicity; neurotoxicity; CNP; MAG; MOG; behavioral tests
2
1. Introduction Nanosilver (nanoAg), one of the most commonly used metal-derived nanomaterials, has broad-spectrum antibacterial and antifungal properties. These qualities have promoted its use in many life domains. NanoAg is currently used in a wide array of commercial goods, biomedical products (such as medical implants, catheters and wound dressings), as well as in food production and bioengineering (Marambio-Jones and Hoek, 2010; Cushen et al., 2014). One of the main aims of nanomedicine is to enhance drug availability within the central nervous system (CNS) by providing a mechanism for delivery of drugs past the blood-brain-barrier (BBB) to increase therapeutic efficacy (Sharma et al. 2013; Chaloupka et al. 2010). NanoAg is regarded as a nanoparticulate drug-carrier system for delivery of drugs to CNS (Leite et al., 2015). The increasing production and use of engineered nanomaterials may inevitably lead to contamination of the environment and thus warrants risk assessment. On the other hand, medical applications also carry potential risks of toxic effects in humans. Hence, there is a need for research on low-level toxicity of nanoAg. A number of studies, both in vitro and in vivo, have indicated toxic properties of nanoAg in a wide range of concentrations and sizes. Smaller nanoAg particles have been shown to be more active in exerting toxicological responses, inducing organ toxicity and inflammatory responses after repeated oral administration (Park et al, 2010). Current findings indicate that nanoAg may easily cross the BBB and accumulate in the rodent brain after oral or intravenous administration (Kim et al. 2008; Tang et al. 2010). Using an in vitro model, Trickler et al. (2010) showed that by releasing proinflammatory cytokines, nanoAg may induce inflammation in rat brain microvessels, which additionally enhances the breakdown of the BBB. Dysfunction of the BBB facilitates further penetration of nanoAg into brain tissue which causes synaptic (Skalska et al., 2015) and neuronal (Tang et al., 2009) degeneration. When administered intranasally, 3
nanoAg alters spatial reference memory (Davenport et al., 2015) and locomotor activity in neonatal rats (Yin et al., 2015). NanoAg has also been reported to affect concentrations of the neurotransmitters dopamine and 5-hydroxytryptamine (5-HT) in the rat brain (Hadrup et al. 2012) and to impair spatial cognition in rats via their impact on hippocampal synaptic plasticity (Liu al. 2012). Except for a few neurotoxicological reports, the exact influence of nanoAg on CNS processes and underlying mechanism(s) of the action are not well understood. It has been reported that larger nanoAg particles (50-60 nm) in a relatively high dose of 50 mg/kg b.w. may cause myelin damage as reflected by myelin basic protein (MBP) immunostaining (Sharma et al., 2013). However, extensive research on the toxic impacts on myelin in adult organisms as well as a comprehensive assessment of behavioral effects have not yet been conducted. Thus, the aim of the present study was to investigate whether prolonged oral administration of a low dose (0.2 mg/kg b.w.) of nanoAg (10 nm) or ionic silver (Ag+) influences the behavior of rats and affects myelin ultrastructure and expression of myelin-specific proteins. A wide range of behavioral assessments was applied to assess the effects of nanoAg. Measurements of body weight and body temperature and tests of locomotor activity, motor coordination, nociceptive reaction, memory performance and anxiety-like behavior were performed. These measurements provide generally-accepted assessments of behavior in investigations of bioactivity of new compounds. Studies on behavioral impacts were combined with examination of expression of several myelin-specific proteins: myelin/oligodendrocyte glycoprotein (MOG), myelin-associated glycoprotein (MAG) and 2ʹ-3ʹ-cyclic nucleotide 3ʹphosphodiesterase (CNP). Since proteins are integral components of myelin membranes and contribute to their structure and stability, ultrastructural observations were also performed using transmission electron microscopy (TEM). 4
2. Materials and methods 2.1. Chemicals Most of the chemicals used in the experiments, including nanoAg particles, were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). All reagents were of analytical grade. NanoAg particles (10 ± 4 nm in diameter) (CAS No.730785) are defined by the manufacturer as a colloidal solution stabilized in sodium citrate to prevent sedimentation and to maintain the dispersed state. Characterization of the degree of dispersion and size distribution of the nanoAg particles was accomplished by transmission electron microscopy (JEM-1200EX, JEOL) using a digital camera MORADA and iTEM 1233 software (Olympus Soft Imaging Solutions GmbH, Germany) according to a standard method developed for non-biological preparations as described previously (Skalska et al., 2015). 2.2. Animals and experimental design of silver exposure Male Wistar rats weighing 140-160 g obtained from the Farm of Labolatory Animals, Z. Lipiec, Brwinow, Poland were used throughout the study. All animals were caged in groups of six and maintained on a 12 h light-dark cycle (lights on at 6:00 h) at a controlled temperature (21°C). During the experiments, the animals were allowed free access to drinking water and a standard laboratory diet known as the Murigan diet (Agropol, Motycz, Poland). All experiments were carried out according to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and the European Community Directive for the Care and Use of Laboratory Animals, as of 24 November 1986 (86/609/EEC), and were approved by the Local Ethic Committee (35/2014). Rats were randomly divided into three groups, each consisting of 12 animals and representing: (i) a negative control group (treated with saline), (ii) a nanoAg-treated group and 5
(iii) an ionic silver-treated group (silver citrate, Ag+). Solutions of nanoAg stabilized in citrate buffer or silver citrate were administered via a gastric tube in a dose of 0.2 mg/kg b.w. per day for 14 days (0.02 mg nanoAg or Ag+/mL). The rats of the control groups received the same dose of saline. During the experiment, body temperature and body weight were assessed. Body temperature in normothermic rats was measured at the animal’s rectum with a thermistor thermometer twice daily on the 1st, 8th and 14th days of the experiment to check the acute effects of administration (the second measurement was taken 1 h after administration). Body temperature changes (Δt) were calculated according to the formula: Δt = t2 – t1, where t1 and t2, were the presubstance and post-substance body temperature, respectively (Vogel, 2008). Simultaneously, on the 1st, 8th and 14th day of the experiment, each animal was weighed. 2.3. Performance of behavioral tests Behavioral tests were initiated after the final treatment on day 14. Locomotor activity and motor coordination were measured on the 15th day of the experiment. Anxiety and memory tests were carried out on the 16th day. On the last day of the study and before decapitation, the nociception reaction was assessed. 2.3.1. Locomotor activity test Locomotor activity of individual rats was recorded using a photocell device (plexiglass boxes - square cages, 60 cm on each side; Porfex, Bialystok, Poland) in a sound-attenuated experimental room, under moderate illumination (10 lux). Ambulatory activity (distance traveled) was measured by two rows of infrared light-sensitivity photocells, installed along the long axis, 45 and 100 mm above the floor. Total horizontal activity (the distance traveled in meters) was recorded for a 30-minute period (Koltunowska et al. 2013). 2.3.2. Rotarod performance test 6
To evaluate the muscle –relaxant or ataxic effects, rats were tested on a rotating rod (rotarod) apparatus (Multiserv, Lublin, Poland). Two days before the experiment, the animals were trained on a rotarod with a 6 cm diameter and 50 cm length, subdivided into four areas by disks 25 cm in diameter, at a constant rotating speed of 9 rpm. The rats that did not fall off the rotarod within 1 min were assigned the maximum score of 60 s (Kotlinska et al., 2012). 2.3.3. Novel object recognition test The apparatus included a square open box, made of plexiglass (63 cm long x 44.5 cm high x 44 cm wide) and illuminated by a lamp (light intensity – 10 lux), suspended 50 cm above the box. The objects to be discriminated were constructed of either of wood or plastic were in the shape of a block and a ball and were too heavy to be displaced by the animals. The object recognition test was performed as described elsewhere (Bertaina-Anglade et al. 2006). The day before the test, each rat was placed in the empty box for 2 minutes to become acclimatized to the environment. On the day of the experiment, the animals were subjected to two trials, spaced apart by a 1-hour interval. The first trial (acquisition trial, T1) lasted 5 minutes and the second trial (test trail, T2) was 3 minutes long. During T1, the apparatus contained two identical objects (wooden blocks), placed in two opposite corners, 10 cm from the sidewall. The rat was placed in the middle of the box. After T1, the rat was returned to its home cage. Subsequently, after 1 h, T2 was performed. During T2, a new object (N) replaced one of the two objects presented in T1. Therefore, the rats were re-exposed to two objects: a familiar object (F) and a new object (N). In order to avoid the presence of olfactory trails, the apparatus and the objects were cleaned after each test. Exploration behavior was judged to include directing the nose toward the object at a distance of no more than 2 cm and/or touching the object with the nose. Turning around or sitting on the object was not considered as exploratory behavior. The time periods spent by rats in exploring each object during the T1 and T2 tests, were recorded 7
manually with a stopwatch. Recognition memory was evaluated using a recognition index calculated for each animal by the formula: (N-F/N+F) x100 corresponding to the difference between the time exploring the novel and the familiar object, corrected for total time exploring both objects. A higher recognition index is considered to reflect stronger memory retention for familiar objects (Bertaina-Anglade et al., 2006). 2.3.4. Elevated plus-shaped maze apparatus The maze was made of wood and positioned on a height of 50 cm above the floor in a quiet laboratory surrounding. Two opposite arms were open (50 cm × 10 cm) and the other two were enclosed by walls (50 cm × 10 cm × 40 cm). The level of illumination was approximately 100 lx at the floor level of the maze. The maze experiment was initiated by placing the rat in the center of the maze facing an open arm, after which the number of entries and time spent in each of the two arms were recorded for 5 min. An “arms entry” was recorded when the rat entered the arm with all four paws. The maze was carefully cleaned with tap water after each test session. The open arms activity was quantified as (a) the time spent in the open arms as a percent of the total time spent on exploring both the open and closed arms, and (b) the number of entries into the open arms as a percentage of the total number of entries into both open and closed arms (Kotlinska et al., 2012; Lister, 1987). 2.3.5. Tail-immersion test To determine the nociceptive reaction, the tail of the rat was placed in a water bath heated to 52°C, and the latency of response (in seconds; reflexive withdrawal of the distal half of the tail after its immersion in water) was measured. The cut-off time was set to 20 s to avoid strong pain or tissue damage. The antinociceptive effects were expressed as a percent maximum possible effect (MPE%), which was calculated according to the following equation: [(T1-T0)/ (20-T0)] x100. The mean value from the first two measurements (15th and 16th day of the experiment) was 8
assumed as baseline latency response (T0). T1 indicates latency for the tail-immersion response on the 17th day (Kotlinska et al., 2009; Le et al., 2001). 2.4. TEM analysis of brain samples. The animals were anesthetized with Nembutal (80 mg/kg b.w.) and perfused with 0.9% NaCl in 0.01 M sodium-potassium phosphate buffer pH 7.4 and then with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 Tissue was sampled from the forebrain cortex of all rat groups. After fixation in the above ice-cold fixative solution, specimens were post-fixed in 1% OsO4 solution, then dehydrated in the ethanol gradient and embedded in epoxy resin (Epon 812). Then ultra-thin sections (60 nm) were stained with 9% uranyl acetate and lead nitrate. Analysis of blinded brain sections was done by transmission electron microscopy (JEM-1200EX, Jeol, Japan) regarding morphological features of myelin. 2.5. Preparation of myelin fraction The myelin fraction was prepared according to the procedure of Norton and Poduslo (1973). Briefly, after decapitation, brains were removed and homogenized in 0.32 M sucrose. Then samples were centrifuged at 75,000 x g for 30 min in a 0.32 M/0.85 M sucrose gradient. The crude myelin pellet was dispersed in water and centrifuged several times at 12,000 x g for 10 min. To obtained purified myelin, centrifugation in 0.32 M sucrose was performed. The pellet consisting of purified myelin was suspended in water with protease inhibitors, 2 mM EDTA and, 2 mM EGTA, and frozen at -80°C for further experiments. 2.6. Western blot analysis Protein concentration in myelin fractions was determined by the method of Lowry at al., (1951). Equal amounts of protein (50 µg) were subjected to SDS-PAGE electrophoresis and then transferred to nitrocellulose membranes. Primary antibody anti-MAG (1:200), anti-MOG (1:1000) and anti-CNP (1:500) was used followed by a secondary antibody conjugated to HRP 9
(1:200). Polyclonal anti-β actin (1:1000) antibody was used as an internal standard. Bands were visualized on Hyperfilm ECL using chemiluminescence ECL kit (Amersham). The films were scanned and quantified using Image Quant TL v2005 software. 2.7. Determination of the mRNA levels of CNP, MAG and MOG by RT-qPCR Total RNA was extracted from the brain cortex of rats according to the method of Chomczyński and Sacchi (1987) using TRI Reagent (Sigma, St. Louis, MO, USA). 2 µg of total RNA were subjected to reverse transcription using random primers and AMV reverse transcriptase (Applied Biosystems, Forest City, CA, USA). The RT-PCR reaction was conducted under conditions: reverse transcription at 42°C for 45 min and denaturation at 94°C for 30 s. Rat myelin protein-specific primers for MOG Rn 00575354_m1, MAG Rn02586362, CNPase Rn01399463_m1 and the corresponding probes were obtained from Applied Biosystems (Forest City, CA, USA). The pre-validated TaqMan assay reagents (Applied Biosystems, Forest City, CA, USA) were used to determine the mRNA expression levels of myelin proteins and actin. Real-time PCR was conducted on an ABI Prism 7500 system using 5 µL of RT product, TaqMan PCR Master Mix, primers, and a TaqMan probe in a total volume of 20 µL. The PCR cycle conditions were as follows: initial denaturation at 95°C for 10 min, 50 cycles of 95°C for 15 s, and 60°C for 1 min. The relative expression level of the myelin protein mRNA, normalized to actin, was calculated using the standard curve method. 2.8. Statistical analysis The results are expressed as mean ± SEM from 3-8 experiments as indicated in the legends of respective figures. Inter-group comparisons were made using one-way analysis of variance (ANOVA) with post-hoc Dunnett’s test. The results of behavioral tests were analyzed by one-way or two-way ANOVA followed by the Bonferroni’s post hoc test. Each group of animals consisted of 6-12 rats. The level of p<0.05 was considered as statistically significant. 10
Analysis was done using the GraphPad Prism version 5.00 for Windows, GraphPad Software (San Diego, California, USA).
3. Results Commercially-available citrate-stabilized nanoAg particles of a small size (10±4 mm) were applied in a concentration of 0.02 mg/mL. It was demonstrated previously using TEM that nanoAg particles do not agglomerate in citrate buffer and are spherical in shape and of homogenous size (Skalska et al., 2015). About 95% of the particles were approximately 10 nm in diameter, with the remaining 5% being larger or smaller. Using inductively coupled plasma mass spectrometry (ICP-MS), it was shown in the applied model of oral exposure to a low dose of nanoAg, that silver is quickly absorbed from the gastrointestinal tract into the serum compartment, regardless of its form. After 2 weeks of exposure, the level of Ag in serum was found to be in the range of 11-13 µg/L in the experimental group. However, it was found to be below the detection limit of the applied method in brain homogenates of rats (0.241 mg/kg in solid tissue) (Skalska et al., 2015). 3.1. Body parameters of exposed rats One-way ANOVA revealed significant [F (8, 93) =34.05; p<0.001] differences in the body weight of rats on the 1st, 8th and 14th day. Post hoc analysis showed that body weights of the controls and the Ag+-treated rats were similar at selected time points whereas the rats of the group treated with nanoAg had higher body weights compared to those of the control group (p<0.01) on the 14th day of the experiment (Fig. 1). Changes in body temperature were recorded for all groups of rats. Two-way ANOVA revealed statistically significant effects of the exposure [F (2, 54) = 4.39; p=0.0171] and a significant time effect [F (2, 54) = 7.25; p=0.0016]. A significant increase in body temperature was observed in rats after chronic exposure to both 11
nanoAg (p<0.01) and silver citrate (p<.0.05) on the 8th day of the experiment relative to the control group. Moreover, post hoc analysis (Bonferroni’s test) revealed that the rats of the group subjected to chronic exposure of Ag+ had higher body temperatures compared to the control group on the 14th day of the experiment (p<0.05) (Fig. 2). A similar tendency was observed on the 14th day of exposure in the group treated with nanoAg. However, this observation was found to be statistically insignificant. 3.2. Effect of exposure to a low dose of AgNPs/Ag+ on behavior of animals NanoAg exposure did not significantly influence locomotor activity, motor coordination or memory performance of rats. No differences among the groups were observed, regarding the effects of administration of saline (50.81±3.101), Ag+ (54.86±7.41) and nanoAg (54.09±5.806) on the total distance traveled by rats within 30 min (one-way ANOVA). Similarly, the results of the rotarod experiments did not differ among the groups (53.25±4.634, 50.18±4.863, 58.18±1.818 for saline-, Ag+- and nanoAg-treated animals, respectively; one-way ANOVA). Moreover, the recognition index of rats in NOR task was similar in all groups, regarding the effects of administration of saline (74.92±1.961), Ag+ (81.69±6.698) and nanoAg (89.16±3.12) (one-way ANOVA). However, it was found that chronic administration changed the degree of behavioral anxiety in the rats. We observed significant differences among the groups concerning the percentage of time spent by rats in the open arms of the plus-maze [F (2, 29) =4.171; p=0.0256; Table 1A] but no substantial differences in the percentage of open arm entries (Table 1B). Post hoc analysis (Bonferroni’s test) showed that chronic administration of Ag+ but not nanoAg increased the time spent by rats in the open arms (p<0.05) compared to the saline group (Table 1). The nociceptive reaction, as assessed by the tail immersion test, was found to be altered in rats exposed to Ag+ and nanoAg. One-way ANOVA revealed significant differences among 12
groups in the percent maximum possible effect (% MPE) [F (2, 27) =6.231; p=0.006; Table 2]. Post hoc analysis (Bonferroni’s test) showed that the group treated chronically with Ag+ was less sensitive to the noxious stimuli compared to the saline-administered rats (p<0.05) (Table 2). 3.4. Changes in ultrastructure and myelin proteins after administration of silver Since a low dose of applied silver was found to be insufficient to detect in brain homogenates of exposed rats with ICP-MS, the presence of nanoAg was demonstrated in brain tissue indirectly, using a modified TEM method described previously. The nanosized granules were observed to be located i.a. between lamellae of myelin sheaths in specimens from brains of AgNPs- but not in Ag+ -treated and control animals (Skalska et al., 2015). Current TEM analysis of brain specimens of control rats indicated normal appearance of neuropil as well as myelin which exhibited lamellar ultrastructure with proper, well-defined plasma membranes adhering to each other (Fig. 3A). In micrographs of brain tissue obtained from Ag+- and nanoAg-treated groups, pathological changes in myelin were observed (Fig. 3BD). The types and degree of damage were found to be very similar. A vast majority of myelin sheaths were found to have irregularities and to exhibit interruptions of compact multilamellar structure. We determined the levels of selected myelin-specific proteins - CNP, MAG and MOG which play roles in the stabilization of myelin structure. A significant decrease in the level of all examined proteins was observed in rats treated with Ag+ and nanoAg (Fig. 4). The relative level of each of these proteins reached about 60%-70% of the control value (*p<0.01 vs. control), although the changes between both silver-treated groups were found to be statistically insignificant (Figs. 4A-4C). Conversely, with real-time PCR, we observed increased mRNA levels of proteins in myelin fractions isolated from the Ag+- and nanoAg-treated groups relative to the control group (Fig. 5). Enhancement of mRNA of all investigated proteins was noted in the 13
case of rats treated with nanoAg, which differed significantly from control as follows: about 30%, 20% and 15% for CNP, MAG and MOG, respectively. As opposed to nanoAg, Ag+ administration, increased only the expression of CNP mRNA by about 30%. This is significantly different from the control value (*p<0.05).
4. Discussion The vast majority of research indicates that, despite the undeniable benefits of nanoAg particles, they may pose a threat to various organisms (Ulm et al., 2015, Davenport et al., 2015, Gomes et al. 2013, McCarthy et al., 2013; Sharma at al. 2009; Strużyński et al., 2014). It is therefore important to investigate the toxic effects of nanoAg to understand the mechanisms of toxicity, with particular attention to neurotoxicity. Mechanistic studies on nanoAg neurotoxicity have been mostly conducted using in vitro systems with high doses of nanoparticles. Thus, it is essential to characterize these pathomechanisms in animal models because it is not possible to replicate the complexity of the nervous system in cultures. Such efforts are expected to provide meaningful data about the response of a biological system to nanoAg. Persistent accumulation of small (10 nm) citrate-stabilized nanoAg particles has been observed by TEM in brains isolated from orally-exposed rats (Lee et al., 2013; Skalska et al., 2015). Moreover, it is known that the biological half-life of silver in the CNS is longer than in other organs (Hadrup, 2012). Since the neurotoxic effects of in vivo exposure to nanoAg have not been extensively studied, the present study was designed to specifically investigate the behavioral effects of prolonged exposure of adult rats to low doses of small nanoAg particles with regard to ultrastructural and biochemical changes in cerebral myelin. The objective was to test whether nanoAg in such a low concentration will induce pathological processes in myelin sheaths and whether these potential changes may result in behavioral alterations. The effects of prolonged 14
exposure of rats to nanoAg were compared with the effects of Ag+, based on the existing conviction that the toxicity of nanoAg relies (almost partially) on the liberation of free Ag+. Such an approach allows us to evaluate the contribution of ions to the overall toxic effects of nanoAg, since it was suggested that the acidic microenvironment of cellular lysosomes promotes the release of ions from the surfaces of the particles (Loeschner et al., 2011). The results of the current study revealed some differences while comparing nanoAg and Ag+ with respect to body parameters and behavioral performance of rats. Body weight of animals is quite sensitive to repeated oral exposure to nanoAg. Exposed animals have increased body weight on the 14th day of the experiment relative to control group. Conversely, weight gain was not observed in Ag+-exposed animals. These findings are in contrast to the results of an earlier study, indicating loss of body weight after three consecutive days of intravenous administration of 5 nm nanoAg particles (Zhang et., 2013). Loss of weight was also considered an indicator of neurotoxicity of metal nanoparticles other than nanoAg (Cao et al., 2013b; Zhang et al., 2010). However, other reports have not revealed any changes in body weight after chronic (14 or 28 days) inhalation and oral nanoAg exposure (18-20 and 60 nm), respectively (Kim et al., 2008; Sung et al., 2011). The above conflicting results might be explained by differences in experimental design (e.g. route of administration, size of the nanoAg and duration of experiments). Based on available data, it is difficult to define whether the observed increase in body weight is the result of nanoAg-induced CNS alterations. Further studies measurement of food consumption are necessary. In addition, even one-week-administration of both Ag+ and nanoAg was found to cause a substantial increase in the body temperature of normothermic rats. An important role in the central regulation of body temperature has been assigned to 5-HT (Wujec et al., 2014). It was also found that the concentration of 5-HT was changed in the rat brain under conditions of 15
chronic oral administration of nanoAg, but not Ag+ (Hadrup et al., 2012). In the light of the above-cited data, we suspect that the currently observed altered body temperature may be due to the impairment of the serotonergic system. The antinociceptive activity of the examined compounds was investigated in the commonly used tail immersion test (Kotlinska et al., 2009; Le et al., 2001). The tail immersion procedure generates spinal cord reflexes and does not affect visceral or musculoskeletal tissues (Le et al., 2001). The results of the present study showed that the nociceptive reaction generated by this test was not affected by nanoAg whereas Ag+ induces hyperalgesia after a two-week exposure period. Hyperalgesia, an increased sensitivity to painful stimuli, is commonly associated with inflammation. Consistent with this observation, Ag+ has been shown to interact with microvasculature leading to a proinflammatory cascade (Marchi et al., 2004). Many molecules have been identified including lipids, purines, protons, cytokines and growth factors which are produced in the context of this inflammatory process and can contribute to heightened pain states due to their ability to sensitize nociceptors (Woolf and Ma, 2007). No behavioral differences were observed in the EPM procedure, suggesting a lack of nanoAg-induced alterations in the anxiety response. Conversely, the rats from Ag+-exposed group spent more time in the open arms of the maze, indicating an anxiolytic effect. Regulation of anxiolytic activity depends on different neurotransmitters (glutamine, GABA, dopamine or 5HT). Disturbances in any of these systems may influence anxiety-like behavior (Zarrindast and Khakpai, 2015). According to Powers et al. (2011), the characteristics of the nanoparticles are critical determinants of their behavioral effects. This may explain the more substantial behavioral impact of Ag+ relative to nano form of Ag. The weak depressive effect of Ag+ on the CNS observed in these experiments is an interesting result. However, nanoAg and Ag+ did not cause impairment of motor coordination, 16
locomotor activity and memory. These observations are consistent with previous studies, in which locomotor activity of rats did not appear to be sensitive to nanoAg treatment even at doses much higher than used in the current study (5 and 10 mg/kg) (Zhang et al., 2010). Proper transmission of electrical impulses in the CNS is made possible by the myelin sheath surrounding most of the axons. Apart from enhancing the speed of neuronal transmission, the main function of this membrane structure is to protect and insulate axons. Disturbances in myelin integrity may result in inappropriate signal transfer and behavioral abnormalities. The important role of myelin in the human CNS is highlighted by an array of severe neurological diseases such as leukodystrophies, multiple sclerosis (MS) and peripheral neuropathies, in which myelin is impaired (Baumann and Pham-Dinh, 2001). Marked myelin degradation was previously observed based on myelin basic protein (MBP) immunostaining in brains of rats exposed to relatively high dose (50 mg/kg b.w.) of large nanoAg particles (50-60 nm) (Sharma et al., 2013). Myelin disintegration may consequently result in degeneration of myelinated fibers. Our previous study demonstrated considerable nanoAg-induced synaptic degeneration in the applied model of low-dose exposure (Skalska et. al., 2015). Thus, it was interesting to investigate whether myelin is also affected. Examination of the myelin ultrastructure indicates that pathological changes of myelin sheaths are clearly visible in the Ag+- and nanoAg-treated groups (Fig. 3). Focal detachment of the myelin lamellae and increased volumes of cytoplasm within the regions of compacted myelin can be observed. Interestingly, the characteristics and the degree of myelin damage are similar in both of the exposed groups. Moreover, ultrastructural changes are similar to those reported to occur under conditions of exposure to metals exhibiting neurotoxic potential such as mercury, lead or cooper (Cao et al. 2013a, Dabrowska-Bouta et al., 1999, Viquez et al., 2009; Rai et al., 2013).
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A number of morphological observations have been made in context of the biochemical studies. Significant decreases, reaching about 60%-75% of control value, in relative level of all of the examined proteins were noted in rats treated with Ag+- and nanoAg, wherein CNP and MOG appeared to be slightly more sensitive to nanoAg treatment than MAG. Analysis of mRNA expression revealed that each of the tested proteins is significantly over-expressed by about 25% - 35% compared to the controls in nanoAg-treated rats (Fig. 5). In the case of rats treated with Ag+, significantly increased expression of CNP mRNA was observed. The changes observed at the molecular level may suggest that the compensative reaction takes place and the repair processes are launched at the gene expression level, mainly under nanoAg exposure. In the case of Ag+, increased gene expression did not compensate for the levels of all proteins. However, it cannot be excluded that in a longer period, repair mechanisms will be fully effective. Both deficiency and excess of the examined proteins may influence formation and maintenance of proper myelin structure. In transgenic mice, over-expressed CNPase perturbs myelin formation and leads to aberrant oligodendrocyte membrane expansion (Gravel et al., 1996). It should be also highlighted that myelin glycoproteins MOG and MAG are important in the light of their interactions with the immune system. MOG, located on the outermost lamellae of compact myelin sheaths, has received significant attention in studies on autoimmune diseases as a protein inducing auto-antibodies with demyelinating potency (Ben-Nun et al., 1999). MAG is glycoprotein whose extracellular region contains five segments of homology resembling immunoglobulin domains, one of which is strikingly homologous to a domain of the NCAM (Baumann and Pham-Dinh, 2001). Also, the previously shown association of MOG and CNP with different elements of the complement cascade, may be relevant to connections between the immune system and the nervous system and may have pathological implications in demyelinating
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diseases (Baumann and Pham-Dinh, 2001). Hence, the link between the nervous system and the immune system under conditions of nanoAg exposure should be considered. In summary, the results indicate that nanoAg administered at a low concentration does not cause considerable toxicity, as reflected by behavioral tests, although observed body temperature and body weight alterations suggest that the CNS may be a target of low-level toxicity of nanoAg and warrant further studies. However, similar toxic effects of Ag+ and nanoAg towards myelin are evident at the biochemical level and the ultrastructural level. The results clearly demonstrate morphological abnormalities in myelin sheaths and altered expression of myelin proteins. Therefore, it is reasonable to draw attention to the risk of neurotoxicity caused by low-level exposure to nanoAg.
5. Acknowlegements The study was supported by statutable funds provided by the Polish Ministry of Science and Higher Education for Mossakowski Medical Research Centre, Polish Academy of Sciences and for Medical University of Lublin (DS 21/2015).
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Figure legends Fig. 1:Effect of chronic administration of Ag+ and nanoAg on the body weight of rats measured on the 1st, 8th and 14th day of exposure. Data are expressed as mean ±SEM values obtained from 12 animals. *P<0.01 vs. control (saline group) on the 14th day of the experiment (Bonferroni’s test).
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Fig. 2:Effects of chronic administration of Ag+ and nanoAg on the body temperature of rats. Data are expressed as mean ±SEM values obtained from 12 animals. *P<0.05 and **p<0.01 vs. control (saline group), 8th day of the experiment; #p<0.05 vs. control (saline group), 14th day of the experiment (Bonferroni’s test).
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Fig.3:Representative electron micrographs of myelin sheaths in forebrain specimens obtained from control (A), Ag+-treated rats (B) and nanoAg-treated rats (C, D). Arrows indicate disintegrated lamellar structure of myelin membranes.
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Fig.4:Representative immunoblots show the expression of myelin-specific proteins: CNP, MAG and MOG in myelin fractions isolated from brains of control, Ag+-treated and nanoAg-treated rats. The graphs provide the results of densitometric analysis of eight independent immunoblots, each performed using a separate myelin sample. The results are normalized to β-actin and expressed as percentage of control. The values represent the means ± SEM, *p< 0.01 (one-way ANOVA test with post-hoc Dunnetts’ test).
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Fig.5:Expression of mRNA of myelin proteins in the forebrains of control, Ag+-treated and nanoAg-treated rats. The mRNA levels of A) CNP, B) MAG and C) MOG were determined by quantitative real time-PCR and normalized against actin. Graphs indicate the results expressed as arbitrary units from four independent experiments, each performed using distinct brain samples. The values represent the means ± SEM, * p < 0.05, significantly different vs. control rats (oneway ANOVA with post-hoc Dunnett’s test).
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Table 1 Effects of chronic exposure to silver on anxiety-like performance of rats estimated in the elevated plus-maze apparatus A
B
percent of the time spent
percent of the open arms
in the open arms
entries
Control (saline)
9.811±3.059
26.76±6.143
Silver citrate (Ag+)
33.48±11.10*
34.03±9.308
nanoAg
10.22±3.068
25.93±7.591
Exposure to:
Data are expressed as mean ±SEM from 12 animals. *P<0.05 vs. control group (Bonferroni’s test).
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Table 2 Effects of chronic exposure to Ag+ and nanoAg on the nociceptive reaction as indicated by the tail immersion test. Exposure to:
MPE%
Saline
0.8823±1.054
silver citrate (Ag+) -4.199±1.055* nanoAg
0.8463±1.435
Data are expressed as mean ±SEM from 12 animals, *p<0.05 vs. control group (Bonferroni’s test); MPE% - a percent maximum possible effect.
30