Exposure to silver nanoparticles does not affect cognitive outcome or hippocampal neurogenesis in adult mice

Ecotoxicology and Environmental Safety 87 (2013) 124–130

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Exposure to silver nanoparticles does not affect cognitive outcome or hippocampal neurogenesis in adult mice Peidang Liu a,b, Zhihai Huang c, Ning Gu a,c,n a

Department of Toxicology, School of Public Health, Southeast University, Nanjing 210009, PR China Institute of Neurobiology, School of Medicine, Southeast University, Nanjing 210009, PR China c School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 May 2012 Received in revised form 12 October 2012 Accepted 19 October 2012 Available online 10 November 2012

Due to the unique antimicrobial and many other broad spectrum biotechnological advantages, silver nanoparticles (Ag-NPs) are widely used in biomedical and general applications. However, the current knowledge about the impact of Ag-NPs on the central nervous system is extremely limited. To assess whether Ag-NPs influence spatial cognition and adult hippocampal neurogenesis, male ICR mice received intraperitoneal administration of Ag-NPs (10, 25, and 50 mg/kg body weight) or vehicle every day for 7 days. At the end of this time period, Morris water maze test was performed for the spatial learning and memory. Subsequently, mice were injected with bromodeoxyuridine and sacrificed 1 day or 28 days after the last injection in order to evaluate cell proliferation, survival and differentiation in the hippocampus. Results showed that compared with the control group, both reference memory and working memory were not impaired in Ag-NPs exposed groups. In addition, no differences were observed in hippocampal progenitor proliferation, new born cell survival or differentiation. These data reveal that exposure to Ag-NPs does not affect spatial cognition or hippocampal neurogenesis in mice. & 2012 Elsevier Inc. All rights reserved.

Keywords: Silver nanoparticles Spatial learning Adult neurogenesis In vivo Mice Neurotoxicity

1. Introduction Nanomaterials are defined to be either nano-objects or nano¨ structured materials (Loestam et al., 2010). Of various nanomaterials, silver nanoparticles (Ag-NPs) are emerging as one of the most commonly used nanomaterials (Chen and Schluesener, 2008). Ag-NPs are now in daily use including bedding, washers, toothpaste, shampoo and fabrics (Wijnhoven et al., 2009). Moreover, the use of Ag-NPs in medical applications is rapidly expanding due to the beneficial physiochemical features they offer (Chaloupka et al., 2010; Wong and Liu, 2010). However, the adverse affects of commercialized Ag-NPs have not been fully examined, especially on the central nervous system (CNS). It has been reported that Ag-NPs (50–100 nm) may alter the action potential of hippocampal CA1 neurons by depressing voltage-gated sodium current (Liu et al., 2009). In the CNS,

Abbreviations: Ag-NPs, silver nanoparticles; BrdU, bromodeoxyuridine; BW, body weight; CNS, central nervous system; DA, dopamine; DLS, dynamic light scattering; GFAP, glial fibrillary acidic protein; ICP-MS, inductively coupled plasma-mass spectrometry; MWM, Morris water maze; NeuN, neuronal nuclear protein; PBS, phosphate-buffered saline; ROS, reactive oxygen species; TEM, transmission electron microscopy n Corresponding author at: School of Biological Science and Medical Engineering, Southeast University, 4 Sipailou, Nanjing 210096, PR China. Fax: þ86 25 83272476. E-mail address: [email protected] (N. Gu). 0147-6513/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2012.10.014

voltage-gated sodium current is responsible for modifying the excitability of neuronal cells, neuronal activity and function. Thereby, potential modulation of the current by Ag-NPs would be expected to alter neuronal functions. Furthermore, in vitro studies focused on PC-12 cells, a neuroendocrine cell line with the capability to produce the neurotransmitter dopamine (DA) and contain functional DA metabolism pathways, have shown that AgNPs (15 nm) significantly reduce DA and its metabolite (dihydroxyphenylacetic acid) concentrations, and the expression level of genes associated with the dopaminergic system (Hussain et al., 2006; Wang et al., 2009). Many in vivo studies have demonstrated that Ag-NPs administered systematically may cross the blood brain barrier and accumulate in CNS (Yang et al., 2010; Win-Shwe and Fujimaki, 2011). The long retention of silver in brain has been observed in spite of low concentrations of silver detected in brain compared to other tissues (Lankveld et al., 2010; van der Zande et al., 2012). Further, intraperitoneal, intravenous, intracarotid or subcutaneous administration of Ag-NPs (50–100 nm) induces edema formation, neuronal degeneration, astrocyte swelling as well as myelin damage (Sharma, 2007; Sharma and Sharma, 2007; Sharma et al., 2009a, 2010; Tang et al., 2009). The leakage of Evans blue albumin can be found in several brain regions including hippocampus after Ag-NPs (50 nm) treatment (Sharma, 2007). Importantly, Rahman et al. (2009) have revealed significant alterations in gene expression associated with the toxicity caused

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by Ag-NPs (25 nm) in the caudate, frontal cortex, and hippocampus of mice. It is believed that reactive oxygen species (ROS) and oxidative stress results from an increased generation of ROS or from poor antioxidant defense systems may be responsible for the neurotoxicity of Ag-NPs (Hussain et al., 2006; Rahman et al., 2009; Wang et al., 2009). Oxidative damage has been implicated in age-related decline in cognitive processes and in the pathogenesis of many neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease (Halliwell, 1992; Cadet and Brannock, 1998; Varadarajan et al., 1999; Ahlemeyer and Krieglstein, 2000). Thus, it can be speculated that Ag-NPs might have deleterious effect on the CNS functions including cognition. However, the effect of Ag-NPs on spatial cognition is a matter of debate. Some studies found that exposure to Ag-NPs induced impairment of reference or working memory (Hritcu et al., 2011; Liu et al., 2012), while other studies showed Ag-NPs had no effects on cognition (Sharma and Sharma, 2007; Sharma et al., 2009b). Although discrepancies exist between these studies using rats, due to the difference of animal species, this impact needs to be further evaluated in mice. On the other hand, in the adult mammalian hippocampus, cumulative evidence has shown that neurogenesis persists throughout life (Altman and Das, 1965; Gould et al., 1997). Dynamic changes in hippocampal neural precursor cells may contribute to underlying substrates of learning and memory (Gould et al., 1999; Shors et al., 2001; Deng et al., 2010). However, neurogenesis can be influenced by a number of different factors such as environmental enrichment, physical activity, reduced caloric intake, psychosocial stress, aging, and hormones (Gould et al., 1999; Kempermann et al., 2002; Shors et al., 2007; Nagata et al., 2009). To date, little information is available about the impact of exposure to nanomaterials on adult neurogenesis. The aim of the present study was to assess whether Ag-NPs exposure could result in spatial learning and memory deficits, as well as to assess the effects on cell proliferation, survival and differentiation in the dentate gyrus in mice. These findings will provide an important theoretical basis for evaluating the neurotoxicity underlying impacts of nanomaterials on animals and human. 2. Materials and methods 2.1. Silver nanoparticles Noncoated Ag-NPs with a diameter of 25 nm were purchased from Amresco, USA. The suspension of the Ag-NPs was prepared in deionized water, and dispersed by ultrasonic vibration (KQ2200E, Kunshan, China) for 30 min in icy water, followed by stirring on a vortex agitator before every use. The morphologies of Ag-NPs were studied by transmission electron microscopy (TEM, JEM2000EX, JEOL, Japan), and the size statistical distributions were determined by counting more than one hundred Ag-NPs in TEM photographs. Dynamic light scattering (DLS) and zeta potential measurements were carried out at room temperature using a Zetaplus Analyzer (Zetaplus, Brookhaven, USA). 2.2. Animals Adult male ICR mice, weighing 32–35 g at the beginning of the experiments, were purchased from National Rodent Laboratory Animal Resources (Shanghai Branch, China). All animals were housed in separate cages, with access to standard laboratory food and water ad libitum, and kept in a regulated environment (22– 22 1C) under a 12-h:12-h light/dark cycle starting at 7:00 AM. The use of animals in this study was approved by the Institutional Animal Care and Use Committee of the University. All efforts were made to minimize animal suffering and to reduce the number of animals used. 2.3. Experimental design Mice were randomly divided into five groups including control group (n¼ 10) which received vehicle (0.9 percent normal saline), and three experimental groups (n¼ 15 in each group): 10, 25, and 50 mg/kg body weight (BW) Ag-NPs. The fifth

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group (n¼10) received scopolamine (3 mg/kg BW) as a positive control for the behavioral studies. Ag-NPs suspension or the same volume vehicle was administered intraperitoneally once daily in the morning for 7 consecutive days. One day after the last administration, five mice from each experimental group were sacrificed, and the hippocampi were taken out for silver content analysis. At the same time point, Morris water maze (MWM) test was started. 24 h after the behavioral test, mice (n¼ 5 in each group/time point) from the control and experimental groups were injected with bromodeoxyuridine (BrdU) and sacrificed 1 day or 28 days after the last BrdU injection. 2.4. Determination of tissue silver content Hippocampi were rapidly separated from brains, weighed, and digested in nitric acid overnight. After adding 1 ml H2O2, the mixed solutions were heated at about 180 1C until the samples were completely digested. Then, the solutions were heated at 120 1C to remove the remaining nitric acid until the solutions were colorless and clear. At last, the remaining solutions were diluted to 5 ml with 2 percent nitric acid. Inductively coupled plasma-mass spectrometry (ICP-MS, Elan 9000, PE, USA) was used to analyze the silver concentration in the samples. The concentration of silver in the tissue was expressed as mg/g wet weight. 2.5. Behavioral testing The spatial learning and memory ability was assessed using an MWM according to the protocol by Vorhees and Williams (2006). Briefly, before mouse testing, a circular pool (120 cm diameter) was filled to a depth of 50 cm with water at 23 7 2 1C. A target platform (7 cm diameter) was hidden 1 cm below the water surface in the southeast (fourth) quadrant. In the training task, mice were given 90 s to reach the hidden platform. Four starting positions were used randomly and each mouse was trained with four trials per day. After reaching the platform, the mouse was allowed to remain on it for 20 s. If the mouse failed to find the hidden platform within 90 s, the trial was terminated and the animal was put on the platform for 20 s. The training task was performed at 8:00 AM for consecutive 5 days. Swimming paths were recorded using a computer system with a video camera (AXIS-90, Target/2, Neuroscience). Latency to reach the platform and average swimming velocity were scored on all trials. On the day following the last hidden platform trial, a probe test was done in a single 90 s trial in which the submerged platform was removed from the pool, and the animal released from the quadrant opposite the fourth quadrant. The percentage of time spent in the target quadrant and the number of platform crossing were recorded for each mouse. Subsequently, a working memory test was performed for 3 consecutive days, which was consisted of five trials per day. Except that the platform location was changed daily, the working memory test was similar, procedurally, to the standard training of the water maze test. The hidden platform remained in the same position across trials on a given test day. The first trial of each session was an informative sample trial in which the mouse was allowed to swim to the platform in its new location and to remain there for 15 s. The latency and distance swum, required to locate the hidden platform, were recorded as above. Spatial working memory was assessed as the mean performance in the second trial of 3 consecutive days. 2.6. Bromodeoxyuridine administration and sample collection After MWM test, mice from the control and experimental groups were intraperitoneally injected with BrdU (Sigma-Aldrich, St. Louis, MO), which can be incorporated into the DNA of dividing cells during S phase, at 100 mg/kg/day for 2 consecutive days. 1 day and 28 days after the last BrdU injection, the animals were anesthetized by a peritoneal injection with 5 ml/g of 7.5 percent chloral hydrate and perfused transcardially with 4 percent paraformaldehyde in phosphate-buffered saline (PBS). Brains were removed from the skulls, and postfixed overnight at 4 1C in 4 percent paraformaldehyde. Next day, the brains were transferred to a 30 percent sucrose in PBS solution for 48 h at 4 1C. Consecutive coronal sections with a thickness of 40 mm were cut using a cryostat microtome (CM1900, Leica, Germany). 2.7. Immunohistochemistry Biotinylated-BrdU immunostaining was carried out as described previously (Karishma and Herbert, 2002). Briefly, free-floating sections were rinsed repeatedly in PBS (pH 7.4) between steps. The sections were prepared with 50 percent formamide-2  SSC, at 65 1C for 2 h, denatured by incubating in 2 mol/l hydrochloric acid for 30 min at 37 1C, and then washed for 30 s with boric acid (pH 8.5). Nonspecific binding sites were blocked in blocking solution (PBS in 3 percent bovine serum albumin) containing 1 percent Triton X-100 for 1 h. Mouse monoclonal antibody against BrdU (Chemicon International) was diluted 1:200 in blocking solution containing 0.5 percent Triton X-100 and incubated at 4 1C for

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24 h. After being washed in PBS, the sections were incubated with a biotinylated secondary antibody for 4 h. They were washed and further incubated with a streptavidin–biotin–peroxidase complex (Vector Laboratories). Brown pigmentation to demarcate regions of immunostaining was produced using 3,3-diaminobenzidine. Negative control sections received identical preparations for immunohistochemical staining, except that primary antibody was omitted. Double immunofluorescence staining for BrdU and neuronal nuclear protein (NeuN), or BrdU and glial fibrillary acidic protein (GFAP) was performed to identify the survival and phenotypes of proliferated cells as described previously (Yagita et al., 2001). After pre-treatment as described above, sections were incubated with an anti-BrdU antibody and antibodies for each cell marker at 4 1C for 24 h. After several washes with PBS, the sections were incubated for 2 h in a mixture of FITCor rhodamine-labeled secondary antibodies. The following primary antibodies were used for immunofluorescence: rat monoclonal anti-BrdU antibody (1:200; Harlan Sera-Labo), mouse monoclonal anti-NeuN antibody (1:200; Chemicon), rabbit poly-clonal anti-GFAP antibody (1:200; Sigma Chemical Co).

50 nm

40 2.8. Counting labeled cells

2.9. Statistical analysis All data were expressed as means7 SEM. Data obtained over training days from the hidden platform trial and repeated acquisition test were analyzed by repeated measures ANOVA. Data from probe trial and neurogenesis were analyzed by one-way ANOVA followed by a Tukey–Kramer post hoc test. Values of p o0.05 were considered statistically significant.

3. Results 3.1. Characterization of nanoparticles TEM imaging of the Ag-NPs was performed to confirm the primary particle size, observe the general morphology, and obtain a size distribution of the particles. The Ag-NPs were generally spherical in morphology (Fig. 1A) with an average size of 36.371.2 nm (Fig. 1B). The DLS size distribution by number was 51.4 70.5 nm, which was consistent with the TEM results. The value of zeta potential was  19.670.7 mV. 3.2. Silver concentration in hippocampus To determine whether the Ag-NPs can accumulate in hippocampus, we measured the concentration of silver by ICP-MS. The results showed that silver accumulated in the hippocampus in a dose-dependent manner (F(2,12) ¼5.848; p o0.05) (Fig. 2), indicating that the peritoneally absorbed silver from nanoparticles is able to enter the blood circulation and be distributed to hippocampus. 3.3. Effect of Ag-NPs on spatial cognition To assess the effect of Ag-NPs on spatial memory ability, we examined the MWM tasks in the Ag-NPs exposed mice and control mice. Spatial reference memory was evaluated using the Morris water navigation test which included place and probe trials. Our results showed significant main effects of group (F(4,45) ¼166.144; po0.001) and day (F(4,180) ¼48.027; po0.001) in the place navigation test, but mice exposed to Ag-NPs at doses of 10, 25, and 50 mg/kg BW did not

Frequency(%)

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Fig. 1. TEM characterization of silver nanoparticles. Particles were spotted onto carbon-coated Cu grids and dried under air prior to TEM imaging. The particle size distributions were determined by measuring the nanoparticles from micrographs using Image J with n4100. (A) TEM image of the Ag-NPs. (B) Size distribution of the nanoparticles.

Concentration of Ag (μg/g wet weight)

The total number of BrdU positive cells throughout the entire hippocampus was estimated by using an unbiased stereological procedure in a blinded fashion. BrdU positive cells in the subgranular zone, granular cell layer, and hilus of the bilateral dentate gyrus were exhaustively counted in one out of six serial coronal sections using a conventional light microscope (PD70) with a 100  objective. When labeled cells were found in clusters, the focal plane was changed to maximize the ability to distinguish individual cells. The number of BrdU positive cells was calculated by multiplying the total number counted by 6 (because every sixth section was used). Fluorescent-stained preparations underwent essentially the same procedure. Every labeled cell within the outlined dentate gyrus was counted. Data are presented as the percentage of BrdU-NeuN or BrdU-GFAP out of the total number of BrdU positive cells. Each group data contained five mice.

0.6 *

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Fig. 2. Silver concentration in hippocampus. Values are the means 7SEM of determinations in five animals of each group. npo 0.05 compared with the 10 mg/kg BW Ag-NPs group.

show a significantly longer escape latency to locate the platform compared to the control mice (Fig. 3A, p40.05). Swimming velocity was also not affected by Ag-NPs throughout the testing (data not shown). Consistent with latency measures, probe trials revealed that scopolamine decreased the number of platform crossing and percentage of time spent searching in the target quadrant (F(4,45) ¼4.315 and F(4,45) ¼ 3.525; po0.01 and po0.05), with no difference between control and experimental groups (Fig. 3B and C, p40.05). Spatial working memory was assessed as the mean performance in the second trial over days. There were significant main effects of group (F(4,45) ¼67.534; po0.001) and trial (F(4,180) ¼78.048; po0.001), however, no significant differences between control and Ag-NPs exposed groups were found (Fig. 3D, p40.05). These data suggested that Ag-NPs exposure did not induce dysfunction in spatial or memory performance.

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Fig. 3. Effects of Ag-NPs on performance in the behavioral tasks. (A) The mean escape latency in the place navigation test. (B) The number of platform crossing in the probe trials. (C) The percentage of time in the target quadrant in the probe trials. (D) The mean performance in the second trial of working memory test. Values are the means7 SEM of determinations in 10 animals of each group. np o 0.05, nnp o 0.01, nnnp o 0.001 compared with the control group. Scop.: scopolamine.

To determine whether exposure to Ag-NPs impairs hippocampal progenitor proliferation, the expression level of BrdU positive cells were investigated by immunohistochemical staining 1 day after the last BrdU injection. BrdU, a thymidine analog, is considered the most popular marker currently used to detect cell proliferation. Results showed that a number of brownly stained BrdU positive cells were evident in the dentate gyrus in all four groups of animals (Fig. 4A and B). However, the number of BrdU positive cells in Ag-NPs treated mice was not altered significantly compared with that in control mice (F(3,16) ¼0.593; p40.05) (Fig. 4C). To further assess the effect of Ag-NPs on survival and differentiation of the newly generated cells, the total number of BrdU, BrdU-NeuN, and BrdU-GFAP positive cells was estimated using immunofluorescence staining 28 days after the last administration of BrdU. NeuN and GFAP are proteins selectively expressed in neuronal and glial cells, respectively. The results revealed that most of the BrdU positive cells had been incorporated into the granule cell layer. No overall differences on survival and differentiation were noted between groups (F(3,16) ¼1.902, F(3,16) ¼0.326, and F(3,16) ¼0.566; p40.05) (Fig. 5A–C), despite variability in the data likely precludes detecting a difference in the number of BrdU-GFAP positive cells. Taken together, these data indicate that exposure to Ag-NPs did not modify hippocampal neurogenesis.

4. Discussion It is well known that Ag-NPs have superior optical, electrical, and thermal properties relative to other nanostructured metal particles (Ganeev et al., 2004; Majeed Khan et al., 2011). The adverse effects of Ag-NPs on living cells have been investigated widely (Stensberg et al., 2011; You et al., 2012). However, the current knowledge about the

Total number of BrdU positive cells

3.4. Effect of Ag-NPs on hippocampal neurogenesis

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Fig. 4. Proliferation in the hippocampus 1 day after the last Bromodeoxyuridine injection. (A) Representative image of BrdU positive cells from the control group. (B) Representative image of BrdU positive cells from one Ag-NPs treatment group. (C) Total number of BrdU positive cells in the hippocampus. Values are the means7 SEM of determinations in 5 animals of each group. Scale bar: 200 mm.

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Total number of BrdU positive cells

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Fig. 5. Survival and differentiation of new born cells in the hippocampus 28 days after the last administration of BrdU. (A) Total number of BrdU positive cells. (B) Percentage of BrdU-NeuN double labeled cells from total BrdU positive cells. (C) Percentage of BrdU-GFAP double labeled cells from total BrdU positive cells. In the hippocampus, some of the survived neural cells differentiated to the mature neurons, while a few of the cells stopped dividing and differentiated to astrocytes. Values are the means7 SEM of determinations in five animals of each group.

impact of Ag-NPs on the CNS in vivo is extremely limited. In addition, Ag-NPs remain one of the most controversial research areas as regards their toxicity to biological systems, due to some issues under debate such as the effects of Ag-NPs on lung and air pathways (Sung et al., 2008, 2009; Hussain and Schlager, 2009; Kim et al., 2010; Foldbjerg et al., 2011), and the mechanisms by which Ag-NPs exert toxicity (Powers et al., 2011; Wang et al., 2012; Yang et al., 2012). Therefore, in the present study, we examined the potential influence of commercialized Ag-NPs on cognition as well as neurogenesis. Sex differences in performance of the MWM have been observed in rodent models with male animals showing an advantage in spatial learning and memory (Berger-Sweeney et al., 1995; Perrot-Sinal et al., 1996). On the other hand, although

a gender-related difference in the accumulation of silver was noted in the kidneys, with a twofold increase in the female kidneys when compared with the male kidneys, there were no overall differences in accumulation of silver content in other organs, blood parameters, and tissue distribution between male and female animals after subacute exposure to Ag-NPs (Kim et al., 2008, 2009). Thereby, in the current study, male adult mice were chosen to expose to Ag-NPs to test the hypothesis that Ag-NPs induce spatial learning and memory impairments, and that these may be contributed by reduced hippocampal neurogenisis. The MWM is one of the most frequently used tasks to measure spatial learning and memory of rodents (Morris et al., 1982; Morris, 1984). The MWM results of the present study revealed that exposure to Ag-NPs did not alter either reference memory or working memory in mice. Our data are consistent with the findings reported by Sharma and Sharma (2007) and Sharma et al. (2009b) where cognitive functions were not influenced significantly in rats assessed by Rota-Rod treadmill, following intraperitoneal administration of Ag-NPs (50 nm, 30 or 50 mg/kg) once daily for 1 week. However, the present results are not in agreement with two recent reports. One study showed that, after once every 2 days exposure to Ag-NPs (50–100 nm, 3 or 30 mg/kg) for 2 weeks through the nasal administration, reference memory was impaired in rats evaluated by MWM test (Liu et al., 2012). The other one found that laboratory rats exhibited a selective impairment of working memory-based spatial learning, while sparing reference memorybased learning, following intraperitoneal injection of silverpoly (amidehydroxyurethane) coated Ag-NPs (23 or 29 nm, 5 or 10 mg/kg) for 7 days, assessed by radial arm-maze (Hritcu et al., 2011). These controversial results may be due to differences in the coating of Ag-NPs, the way Ag-NPs are administered, the learning protocol used, or even animal species. It has been established that Ag-NPs cause toxicity in size and dose dependent manners, thus, it is worthy to note that relatively small sized and high dosed AgNPs were used in the present study. In addition, the toxicity of coating and/or its metabolites in living body, the shape and aggregation potential of Ag-NPs should not be discarded. Adult neurogenesis has been extensively studied in mammalian brains in relation to learning and memory processes (Kempermann, 2008; Zhao et al., 2008), which can be positively or negatively regulated by a number of factors and conditions, such as hippocampal-dependent learning, exercise, chronic stress, and aging (Balu and Lucki, 2009). Indeed, impairment of hippocampal neurogenesis by oxidative stress has been found in rodents (Herrera et al., 2003; Limoli et al., 2004). It is believed that Ag-NPs mediate its deleterious effects primarily through the production of oxidative stress (Hussain et al., 2006; Rahman et al., 2009; Wang et al., 2009). However, in the present study, no effects of Ag-NPs were observed in cell proliferation, survival and differentiation, indicating that the presence of Ag-NPs in the brain did not interfere with the incorporation of BrdU into the nucleus of dividing cells, and that oxidative stress induced by Ag-NPs was not sufficient to modify neurogenesis given low silver concentrations in the hippocampus. Unchanged neurogenesis observed in the dentate gyrus is coincident with unaltered cognitive outcome. In summary, results from the present study suggest that, in mice, exposure to Ag-NPs does not affect neurocognitive outcome, or hippocampal neurogenesis.

Acknowledgments This work was supported by the Natural Science Foundation of Jiangsu Province (Grant no. BK2009013), and the National Important Science Research Programs of China (Grant nos. 2006CB705602, 2011CB933500).

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References Ahlemeyer, B., Krieglstein, J., 2000. Inhibition of glutathione depletion by retinoic acid and tocopherol protects cultured neurons from staurosporine-induced oxidative stress and apoptosis. Neurochem. Int. 36, 1–5. Altman, J., Das, G.D., 1965. Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J. Comp. Neurol. 124, 319–335. Balu, D.T., Lucki, I., 2009. Adult hippocampal neurogenesis: regulation, functional implications, and contribution to disease pathology. Neurosci. Biobehav. Rev. 33, 232–252. Berger-Sweeney, J., Arnold, A., Gabeau, D., Mills, J., 1995. Sex differences in learning and memory in mice: effects of sequence of testing and cholinergic blockade. Behav. Neurosci. 109, 859–873. Cadet, J.L., Brannock, C., 1998. Free radicals and the pathobiology of brain dopamine systems. Neurochem. Int. 32, 117–131. Chaloupka, K., Malam, Y., Seifalian, A.M., 2010. Nanosilver as a new generation of nanoproduct in biomedical applications. Trends Biotechnol. 28, 580–588. Chen, X., Schluesener, H.J., 2008. Nanosilver: a nanoproduct in medical application. Toxicol. Lett. 176, 1–12. Deng, W., Aimone, J.B., Gage, F.H., 2010. New neurons and new memories: how does adult hippocampal neurogenesis affect learning and memory? Nat. Rev. Neurosci. 11, 339–350. Foldbjerg, R., Dang, D.A., Autrup, H., 2011. Cytotoxicity and genotoxicity of silver nanoparticles in the human lung cancer cell line, A549. Arch. Toxicol. 85, 743–750. Ganeev, R.A., Baba, M., Ryasnyansky, A.I., Suzuki, M., Kuroda, H., 2004. Characterization of optical and nonlinear optical properties of silver nanoparticles prepared by laser ablation in various liquids. Opt. Commun. 240, 437–448. Gould, E., McEwen, B.S., Tanapat, P., Galea, L.A., Fuchs, E., 1997. Neurogenesis in the dentate gyrus of the adult tree shrew is regulated by psychosocial stress and NMDA receptor activation. J. Neurosci. 17, 2492–2498. Gould, E., Beylin, A., Tanapat, P., Reeves, A., Shors, T.J., 1999. Learning enhances adult neurogenesis in the hippocampal formation. Nat. Neurosci. 2, 260–265. Halliwell, B., 1992. Reactive oxygen species and the central nervous system. J. Neurochem. 59, 1609–1623. Herrera, D.G., Yague, A.G., Johnsen-Soriano, S., Bosch-Morell, F., Collado-Morente, L., Muriach, M., Romero, F.J., Garcia-Verdugo, J.M., 2003. Selective impairment of hippocampal neurogenesis by chronic alcoholism: protective effects of an antioxidant. Proc. Natl. Acad. Sci. USA 100, 7919–7924. Hritcu, L., Stefan, M., Ursu, L., Neagu, A., Mihasan, M., Tartau, L., Melnig, V., 2011. Exposure to silver nanoparticles induces oxidative stress and memory deficits in laboratory rats. Cent. Eur. J. Biol. 6, 497–509. Hussain, S.M., Schlager, J.J., 2009. Safety evaluation of silver nanoparticles: inhalation model for chronic exposure. Toxicol. Sci. 108, 223–224. Hussain, S.M., Javorina, A.K., Schrand, A.M., Duhart, H.M., Ali, S.F., Schlager, J.J., 2006. The interaction of manganese nanoparticles with PC-12 cells induces dopamine depletion. Toxicol. Sci. 92, 456–463. Karishma, K.K., Herbert, J., 2002. Dehydroepiandrosterone (DHEA) stimulates neurogenesis in the hippocampus of the rat, promotes survival of newly formed neurons and prevents corticosterone-induced suppression. Eur. J. Neurosci. 16, 445–453. Kempermann, G., 2008. The neurogenic reserve hypothesis: what is adult hippocampal neurogenesis good for? Trends Neurosci. 31, 163–169. Kempermann, G., Gast, D., Gage, F.H., 2002. Neuroplasticity in old age: sustained fivefold induction of hippocampal neurogenesis by long-term environmental enrichment. Ann. Neurol. 52, 135–143. Kim, W.Y., Kim, J., Park, J.D., Ryu, H.Y., Yu, I.J., 2009. Histological study of gender differences in accumulation of silver nanoparticles in kidneys of Fischer 344 rats. J. Toxicol. Environ. Health A 72, 1279–1284. Kim, Y.S., Song, M.Y., Park, J.D., Song, K.S., Ryu, H.R., Chung, Y.H., Chang, H.K., Lee, J.H., Oh, K.H., Kelman, B.J., Hwang, I.K., Yu, I.J., 2010. Subchronic oral toxicity of silver nanoparticles. Part. Fibre Toxicol. 7, 20. Kim, Y.S., Kim, J.S., Cho, H.S., Rha, D.S., Kim, J.M., Park, J.D., Choi, B.S., Lim, R., Chang, H.K., Chung, Y.H., Kwon, I.H., Jeong, J., Han, B.S., Yu, I.J., 2008. Twenty-eight-day oral toxicity, genotoxicity, and gender-related tissue distribution of silver nanoparticles in Sprague–Dawley rats. Inhal. Toxicol. 20, 575–583. Lankveld, D.P., Oomen, A.G., Krystek, P., Neigh, A., Troost-de Jong, A., Noorlander, C.W., Van Eijkeren, J.C., Geertsma, R.E., De Jong, W.H., 2010. The kinetics of the tissue distribution of silver nanoparticles of different sizes. Biomaterials 31, 8350–8361. Limoli, C.L., Giedzinski, E., Rola, R., Otsuka, S., Palmer, T.D., Fike, J.R., 2004. Radiation response of neural precursor cells: linking cellular sensitivity to cell cycle checkpoints, apoptosis and oxidative stress. Radiat. Res. 161, 17–27. Liu, Y., Guan, W., Ren, G., Yang, Z., 2012. The possible mechanism of silver nanoparticle impact on hippocampal synaptic plasticity and spatial cognition in rats. Toxicol. Lett. 209, 227–231. Liu, Z., Ren, G., Zhang, T., Yang, Z., 2009. Action potential changes associated with the inhibitory effects on voltage-gated sodium current of hippocampal CA1 neurons by silver nanoparticles. Toxicology 264, 179–184. ¨ ¨ Loestam, G., Rauscher, H., Roebben, G., Kluttgen, B.S., Gibson, N., Putaud, J., Stamm, H., 2010. Considerations on a Definition of Nanomaterial for Regulatory Purposes. Joint Research Centre. European Commission, Luxembourg. Majeed Khan, M.A., Kumar, S., Ahamed, M., Alrokayan, S.A., Alsalhi, M.S., 2011. Structural and thermal studies of silver nanoparticles and electrical transport study of their thin films. Nanoscale Res. Lett. 6, 434.

129

Morris, R., 1984. Developments of a water-maze procedure for studying spatial learning in the rat. J. Neurosci. Methods 11, 47–60. Morris, R.G., Garrud, P., Rawlins, J.N., O’Keefe, J., 1982. Place navigation impaired in rats with hippocampal lesions. Nature 297, 681–683. Nagata, K., Nakashima-Kamimura, N., Mikami, T., Ohsawa, I., Ohta, S., 2009. Consumption of molecular hydrogen prevents the stress-induced impairments in hippocampus-dependent learning tasks during chronic physical restraint in mice. Neuropsychopharmacology 34, 501–508. Perrot-Sinal, T.S., Kostenuik, M.A., Ossenkopp, K.P., Kavaliers, M., 1996. Sex differences in performance in the Morris water maze and the effects of initial nonstationary hidden platform training. Behav. Neurosci. 110, 1309–1320. Powers, C.M., Badireddy, A.R., Ryde, I.T., Seidler, F.J., Slotkin, T.A., 2011. Silver nanoparticles compromise neurodevelopment in PC12 cells: critical contributions of silver ion, particle size, coating, and composition. Environ. Health Perspect. 119, 37–44. Rahman, M.F., Wang, J., Patterson, T.A., Saini, U.T., Robinson, B.L., Newport, G.D., Murdock, R.C., Schlager, J.J., Hussain, S.M., Ali, S.F., 2009. Expression of genes related to oxidative stress in the mouse brain after exposure to silver-25 nanoparticles. Toxicol. Lett. 187, 15–21. Sharma, H.S., 2007. Nanoneuroscience: emerging concepts on nanoneurotoxicity and nanoneuroprotection. Nanomedicine (Lond) 2, 753–758. Sharma, H.S., Sharma, A., 2007. Nanoparticles aggravate heat stress induced cognitive deficits, blood–brain barrier disruption, edema formation and brain pathology. Prog. Brain Res. 162, 245–273. Sharma, H.S., Ali, S.F., Hussain, S.M., Schlager, J.J., Sharma, A., 2009a. Influence of engineered nanoparticles from metals on the blood–brain barrier permeability, cerebral blood flow, brain edema and neurotoxicity. An experimental study in the rat and mice using biochemical and morphological approaches. J. Nanosci. Nanotechnol. 9, 5055–5072. Sharma, H.S., Hussain, S., Schlager, J., Ali, S.F., Sharma, A., 2010. Influence of nanoparticles on blood–brain barrier permeability and brain edema formation in rats. Acta Neurochir. Suppl. 106, 359–364. Sharma, H.S., Ali, S.F., Tian, Z.R., Hussain, S.M., Schlager, J.J., Sjoquist, P.O., Sharma, A., Muresanu, D.F., 2009b. Chronic treatment with nanoparticles exacerbate hyperthermia induced blood–brain barrier breakdown, cognitive dysfunction and brain pathology in the rat. Neuroprotective effects of nanowiredantioxidant compound H-290/51. J. Nanosci. Nanotechnol. 9, 5073–5090. Shors, T.J., Miesegaes, G., Beylin, A., Zhao, M., Rydel, T., Gould, E., 2001. Neurogenesis in the adult is involved in the formation of trace memories. Nature 410, 372–376. Shors, T.J., Mathew, J., Sisti, H.M., Edgecomb, C., Beckoff, S., Dalla, C., 2007. Neurogenesis and helplessness are mediated by controllability in males but not in females. Biol. Psychiatry 62, 487–495. Stensberg, M.C., Wei, Q., McLamore, E.S., Porterfield, D.M., Wei, A., Sepulveda, M.S., 2011. Toxicological studies on silver nanoparticles: challenges and opportunities in assessment, monitoring and imaging. Nanomedicine (Lond) 6, 879–898. Sung, J.H., Ji, J.H., Yoon, J.U., Kim, D.S., Song, M.Y., Jeong, J., Han, B.S., Han, J.H., Chung, Y.H., Kim, J., Kim, T.S., Chang, H.K., Lee, E.J., Lee, J.H., Yu, I.J., 2008. Lung function changes in Sprague–Dawley rats after prolonged inhalation exposure to silver nanoparticles. Inhal. Toxicol. 20, 567–574. Sung, J.H., Ji, J.H., Park, J.D., Yoon, J.U., Kim, D.S., Jeon, K.S., Song, M.Y., Jeong, J., Han, B.S., Han, J.H., Chung, Y.H., Chang, H.K., Lee, J.H., Cho, M.H., Kelman, B.J., Yu, I.J., 2009. Subchronic inhalation toxicity of silver nanoparticles. Toxicol. Sci. 108, 452–461. Tang, J., Xiong, L., Wang, S., Wang, J., Liu, L., Li, J., Yuan, F., Xi, T., 2009. Distribution, translocation and accumulation of silver nanoparticles in rats. J. Nanosci. Nanotechnol. 9, 4924–4932. van der Zande, M., Vandebriel, R.J., Van Doren, E., Kramer, E., Herrera Rivera, Z., Serrano-Rojero, C.S., Gremmer, E.R., Mast, J., Peters, R.J., Hollman, P.C., Hendriksen, P.J., Marvin, H.J., Peijnenburg, A.A., Bouwmeester, H., 2012. Distribution, elimination, and toxicity of silver nanoparticles and silver ions in rats after 28-day oral exposure. ACS Nano 6, 7427–7442. Varadarajan, S., Yatin, S., Kanski, J., Jahanshahi, F., Butterfield, D.A., 1999. Methionine residue 35 is important in amyloid beta-peptide-associated free radical oxidative stress. Brain Res. Bull. 50, 133–141. Vorhees, C.V., Williams, M.T., 2006. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat. Protoc. 1, 848–858. Wang, J., Rahman, M.F., Duhart, H.M., Newport, G.D., Patterson, T.A., Murdock, R.C., Hussain, S.M., Schlager, J.J., Ali, S.F., 2009. Expression changes of dopaminergic system-related genes in PC12 cells induced by manganese, silver, or copper nanoparticles. Neurotoxicology 30, 926–933. Wang, Z., Chen, J., Li, X., Shao, J., Peijnenburg, W.J., 2012. Aquatic toxicity of nanosilver colloids to different trophic organisms: contributions of particles and free silver ion. Environ. Toxicol. Chem. 31, 2408–2413. Wijnhoven, S.W.P., Peijnenburg, W.J.G.M., Herberts, C.A., Hagens, W.I., Oomen, A.G., Heugens, E.H.W., Roszek, B., Bisschops, J., Gosens, I., Meent, D.V.D., Dekkers, S., De Jong, W.H., Zijverden, M.V., Sips, A.J.A.M., Geertsma, R.E., 2009. Nano-silver—a review of available data and knowledge gaps in human and environmental risk assessment. Nanotoxicology 3, 109–138. Win-Shwe, T.T., Fujimaki, H., 2011. Nanoparticles and neurotoxicity. Int. J. Mol. Sci. 12, 6267–6280. Wong, K.K.Y., Liu, X., 2010. Silver nanoparticles—the real ‘‘silver bullet’’ in clinical medicine? Med. Chem. Commun. 1, 125–131. Yagita, Y., Kitagawa, K., Ohtsuki, T., Takasawa, K., Miyata, T., Okano, H., Hori, M., Matsumoto, M., 2001. Neurogenesis by progenitor cells in the ischemic adult rat hippocampus. Stroke 32, 1890–1896.

130

P. Liu et al. / Ecotoxicology and Environmental Safety 87 (2013) 124–130

Yang, X., Gondikas, A.P., Marinakos, S.M., Auffan, M., Liu, J., Hsu-Kim, H., Meyer, J.N., 2012. Mechanism of silver nanoparticle toxicity is dependent on dissolved silver and surface coating in Caenorhabditis elegans. Environ. Sci. Technol. 46, 1119–1127. 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. Roy. Soc. Interface 7, S411–422.

You, C., Han, C., Wang, X., Zheng, Y., Li, Q., Hu, X., Sun, H., 2012. The progress of silver nanoparticles in the antibacterial mechanism, clinical application and cytotoxicity. Mol. Biol. Rep. 39, 9193–9201. Zhao, C., Deng, W., Gage, F.H., 2008. Mechanisms and functional implications of adult neurogenesis. Cell 132, 645–660.