silver nanoparticles ( ag - nps ) have been widely used in health industries such as water purification , tooth paste , nursing bottles and other environmental applications ( 1 ) . these nanoparticles are also used for the treatment of wounds and burns , and as a contraceptive ( 2 ) . despite the increasing use of ag - nps , there is little information about their possible toxic effects on the nervous system . because of their small size , nanomaterials can easily pass into the cells and affect cellular function ( 3 , 4 ) . moreover , there have been some reports indicating that nanoparticles move into the brain by traversing the blood - brain barrier ( bbb ) ( 1 , 4 ) . so , nanoparticles have potential toxic effects on human health since they can pass through biological membranes ( 5 ) . a recent study reported that 28 days of oral exposure to ag - nps produced changes in blood chemistry and caused hepatotoxicity ( 6 ) . rahman et al suggested that ag - nps have neurotoxic effects by generating oxidative stress ( 1 ) . these studies also showed that after exposure of ag - nps , these nanoparticles accumulate in various organs including liver , kidney , testis and brain ( 6 ) . it is known that the biological half - life of silver in central nervous system ( cns ) is longer than that in other organs ( 5 ) . moreover , it has been shown that ag - nps can induce the production of reactive oxygen species ( ros ) in the hippocampus ( 1 ) . on the other hand , the brain has a high metabolic activity , and therefore it is highly vulnerable to oxidative stress ( 2 ) . many investigators have shown that ros can induce apoptosis in many organs ( 7 ) . the hippocampus is one of the most important parts of the brain that plays a role in learning and memory ( 4 , 8 , 9 ) . it was shown that hippocampal lesions disrupt memory and learning ( 6 , 10 ) . moreover , it is reported that the rats memory was obstructed after exposure to ag - nps ( 9 ) . thus , ag - nps exposure could possibly affect this important region of the brain and induce cytological changes such as dark neuron and apoptosis . apoptosis is a form of cell death that is responsible for maintaining balance in the number of cells in a tissue . this type of cell death that occurs in normal tissue can also occur due to pathological problems . apoptosis , which is characterized by morphological characteristics including cell shrinkage , cytoplasmic condensation and chromatin segregation , can be easily detected using tunel staining ( 11 ) . dark neuron ( dn ) is a type of cell degeneration ( 12 , 13 ) that in histological studies is recognized by hyperbasophilia and hyper - electron density properties . we hypothesized that hippocampal ros accumulation and memory impairment caused by ag - nps would be associated with hippocampal apoptosis and dns formation . in this regard , in this study , the neurotoxic effect of ag - nps exposure on the rat hippocampus was investigated by toluidine blue ( for dns ) and tunel staining ( for apoptosis ) . male wistar rats aged 3 to 4 months and weighting 25020 g were purchased from the animal center of mashhad university of medical sciences . the rats were housed under controlled temperature ( 202 c ) and lighting ( 12 hr light : 12 hr dark photoperiod ) conditions and permitted free access to food and water . ag - nps used in this study were purchased from sigma - aldrich , usa ( cat . it is a kind of nano powder with particle size of < 100 nm , purity 99% and density 10.49 g / cm . ag - nps suspended in distilled water and were administered by gavage to the rats . 12 male wistar rats were randomly divided into two groups ( 6 rats in each group ) . animals in the experimental group received ag - nps ( 30 mg / kg ) orally ( gavage ) for 28 consecutive days ( 6 ) . experimental procedures were carried out according to mashhad university of medical sciences , ethical committee acts . the collected brains from each group were fixed in 10% neutral buffered formalin for 5 days . after fixation , the specimens were dehydrated with an ascending ethanol series , cleared with xylene and embedded in paraffin . the brain tissue blocks were cut into 5 m coronal serial sections ( 6 sections in each animal at 150 m intervals ) and stained with toluidine blue ( 16 ) . coronal serial sections ( 5 m thick ) were cut at the level of the dorsal hippocampus ( 34 mm posterior from bregma ) using microtome , and 6 sections from each animal at 150 m intervals were collected . then the sections were deparaffinized with xylene , rehydrated through descending concentrations of ethanol and rinsed in phosphate buffered saline ( pbs ) twice for 10 min at room temperature . then the tunel reaction was carried out by the in situ cell death detection kit ( roche diagnostics , mannheim , germany ) . the photography was performed from different regions of the hippocampus using the microscope ( olympus bx51 , japan ) . then on the computer monitor , the number of dns and apoptotic cells were counted under 40  objectives and by using rectangular grids placed randomly on the investigated areas . morphometrical methods were used to count dns and apoptotic cells per unit area in ca1 , ca2 , ca3 and dentate gyrus ( dg ) of the hippocampus . finally , the mean of dn and apoptotic positive cells number per unit area ( na ) was calculated in different regions of hippocampus using the following formula ( 19 ) . in this formula , q  is the sum of counted particles appeared in sections ,  a / f  is the area associated with each frame , and p  is the sum of frame associate points hitting the reference ( 18 ) . the results obtained from the chemically - treated group was compared to the control group . the values were compared using the independent t - test . the p - value less than 0.05 was considered to be statistically significant . male wistar rats aged 3 to 4 months and weighting 25020 g were purchased from the animal center of mashhad university of medical sciences . the rats were housed under controlled temperature ( 202 c ) and lighting ( 12 hr light : 12 hr dark photoperiod ) conditions and permitted free access to food and water . ag - nps used in this study were purchased from sigma - aldrich , usa ( cat . 484059 ) . it is a kind of nano powder with particle size of < 100 nm , purity 99% and density 10.49 g / cm . ag - nps suspended in distilled water and were administered by gavage to the rats . 12 male wistar rats were randomly divided into two groups ( 6 rats in each group ) . animals in the experimental group received ag - nps ( 30 mg / kg ) orally ( gavage ) for 28 consecutive days ( 6 ) . experimental procedures were carried out according to mashhad university of medical sciences , ethical committee acts . the collected brains from each group were fixed in 10% neutral buffered formalin for 5 days . after fixation , the specimens were dehydrated with an ascending ethanol series , cleared with xylene and embedded in paraffin . the brain tissue blocks were cut into 5 m coronal serial sections ( 6 sections in each animal at 150 m intervals ) and stained with toluidine blue ( 16 ) . coronal serial sections ( 5 m thick ) were cut at the level of the dorsal hippocampus ( 34 mm posterior from bregma ) using microtome , and 6 sections from each animal at 150 m intervals were collected . then the sections were deparaffinized with xylene , rehydrated through descending concentrations of ethanol and rinsed in phosphate buffered saline ( pbs ) twice for 10 min at room temperature . the sections were rinsed in pbs subsequently . then the tunel reaction was carried out by the in situ cell death detection kit ( roche diagnostics , mannheim , germany ) . the photography was performed from different regions of the hippocampus using the microscope ( olympus bx51 , japan ) . then on the computer monitor , the number of dns and apoptotic cells were counted under 40  objectives and by using rectangular grids placed randomly on the investigated areas . morphometrical methods were used to count dns and apoptotic cells per unit area in ca1 , ca2 , ca3 and dentate gyrus ( dg ) of the hippocampus . finally , the mean of dn and apoptotic positive cells number per unit area ( na ) was calculated in different regions of hippocampus using the following formula ( 19 ) . in this formula , q  is the sum of counted particles appeared in sections ,  a / f  is the area associated with each frame , and p  is the sum of frame associate points hitting the reference ( 18 ) . the results obtained from the chemically - treated group was compared to the control group . the values were compared using the independent t - test . the p - value less than 0.05 was considered to be statistically significant . the numbers of dns per unit area ( na ) of the ca1 , ca2 , ca3 and dg subdivisions of the hippocampus were counted . then the mean number of dns in the hippocampus of experimental group compared with the control group . a few dns were found in different regions of hippocampus in control group . in comparison to control group , the mean of dns number per unit area in ca1 , ca2 , ca3 and dg of hippocampus was significantly increased in ag - nps group ( p<0.05 for ca1 and p<0.01 for other areas ) ( figure 1 , 2 ) . photomicrographs of the adult rat hippocampus , exposure to ag - nps increased formation of dns in the hippocampus . ( a ) ca1 control group , ( b ) ca1 ag - nps group , ( c ) ca2 control group , ( d ) ca2 ag - nps group , ( e ) ca3 control group , ( f ) ca3 ag - nps group , ( g ) dg control group , ( h ) dg ag - nps group . scale bars , 150 m ag - nps exposure for 28 days induces dns production in the adult rat hippocampus . bars marked with an asterisk or double asterisks mean that it is significantly different from the control at the 5% or 1% confidence level , respectively . the data are presented as the mean  sem the numbers of tunel positive cells per unit area ( na ) of the ca1 , ca2 , ca3 and dg subdivisions of the hippocampus were counted . in comparison to control group , the mean of tunel positive cells number per unit area in ca1 , ca2 , ca3 and dg of hippocampus was increased significantly in ag - nps group ( p<0.01 ) , ( figures 3 and 4 ) . photomicrographs of the adult rat hippocampus , exposure to ag - nps increased apoptotic cells in the hippocampus . ( a ) ca1 control group , ( b ) ca1 ag - nps group , ( c ) ca2 control group , ( d ) ca2 ag - nps group , ( e ) ca3 control group , ( f ) ca3 ag - nps group , ( g ) dg control group , ( h ) dg ag - nps group , arrow heads point to representative tunel positive cells with dark brown nucleus . scale bars , 150 m the influence of ag - nps exposure on the adult rat hippocampal cells ; these nanoparticles induce apoptosis in the hippocampus . bars marked with double asterisks mean that it is significantly different from the control at the 1% confidence level . the numbers of dns per unit area ( na ) of the ca1 , ca2 , ca3 and dg subdivisions of the hippocampus were counted . then the mean number of dns in the hippocampus of experimental group compared with the control group . a few dns were found in different regions of hippocampus in control group . in comparison to control group , the mean of dns number per unit area in ca1 , ca2 , ca3 and dg of hippocampus was significantly increased in ag - nps group ( p<0.05 for ca1 and p<0.01 for other areas ) ( figure 1 , 2 ) . photomicrographs of the adult rat hippocampus , exposure to ag - nps increased formation of dns in the hippocampus . ( a ) ca1 control group , ( b ) ca1 ag - nps group , ( c ) ca2 control group , ( d ) ca2 ag - nps group , ( e ) ca3 control group , ( f ) ca3 ag - nps group , ( g ) dg control group , ( h ) dg ag - nps group . scale bars , 150 m ag - nps exposure for 28 days induces dns production in the adult rat hippocampus . bars marked with an asterisk or double asterisks mean that it is significantly different from the control at the 5% or 1% confidence level , respectively . the numbers of tunel positive cells per unit area ( na ) of the ca1 , ca2 , ca3 and dg subdivisions of the hippocampus were counted . in comparison to control group , the mean of tunel positive cells number per unit area in ca1 , ca2 , ca3 and dg of hippocampus was increased significantly in ag - nps group ( p<0.01 ) , ( figures 3 and 4 ) . photomicrographs of the adult rat hippocampus , exposure to ag - nps increased apoptotic cells in the hippocampus . ( a ) ca1 control group , ( b ) ca1 ag - nps group , ( c ) ca2 control group , ( d ) ca2 ag - nps group , ( e ) ca3 control group , ( f ) ca3 ag - nps group , ( g ) dg control group , ( h ) dg ag - nps group , arrow heads point to representative tunel positive cells with dark brown nucleus . scale bars , 150 m the influence of ag - nps exposure on the adult rat hippocampal cells ; these nanoparticles induce apoptosis in the hippocampus . bars marked with double asterisks mean that it is significantly different from the control at the 1% confidence level . the aim of this study was to assess the effect of ag - nps on dns formation and apoptosis induction in the rat hippocampus . our data indicate that administration of ag - nps to adult rats can induce apoptosis and dns formation in the hippocampus . our body can receive ag - nps via three routes including inhalation , dermal contact , and ingestion . among the above mentioned ways , oral ingestion may be important in using consumer products such as toothpaste , nursing nipples and toys ( 1 , 2 ) . because of this , we have used orally administration for ag - nps in the present study . previous studies have shown that a daily intake of silver nanoparticles could lead to the accumulation of these particles in different organs ( 6 , 18 ) . nanomaterials are able to enter in to cells in different ways including passive diffusion , receptor - mediated endocytosis and clarthrin mediated endocytosis ( 17 ) . tang et al examined the distribution and toxicity of ag - nps in rats following subcutaneous injection . they found that these nanoparticles were translocated to the circulation and finally distributed to the brain and some other target organs including kidney , liver and lung . because ag - nps can induce blood on the other hand , hippocampal cells are highly sensitive to oxidative stress following exposure to nanoparticles ( 20 ) . it can cause damage to cellular components such as membranes and proteins ( 21 ) . in fact , one possible mechanism for ag - nps cytotoxicity is free radical formation , particularly ros generation ( 22 , 23 ) . moreover , inflammatory responses were significantly induced by oral administration of ag - nps for 28 days . it was shown that production of cytokines including il-1 , il-6 , il-4 , il-10 , il-12 , and tgf- are increased after silver nanoparticles exposure ( 24 ) . so , another mechanism of damage by these nanoparticles is the induction of inflammatory reactions . dn formation is a type of cell death that is neither apoptosis nor necrosis ( 25 ) . dns formations have been reported in ischemia , epilepsy and hypoglycemia ( 26 ) . in all of these pathological conditions , the excitatory effects of ag - nps on hippocampal neurons are similar with glutamate , which plays a key role in the pathological process of brain disorders ( 27 ) . thus in the present study , apoptosis induction and dns formation in the hippocampus after ag - nps treatment may occur due to oxidative stress , inflammation and glutamate increasing . results of a recent study indicated that the rats spatial memory was obstructed after ag - nps exposure ( 9 ) it has been proved that hippocampal lesions are associated with impairments in learning and memory ( 4 , 6 ) , and in our study , it is shown that ag - nps increased dns and apoptotic cells in the hippocampus . based on the results of this study , it is suggested that ag - nps have neurotoxic effect and induce apoptosis and dns formation in hippocampus .
objective(s):silver nanoparticles ( ag - nps ) are used widely in bedding , water purification , tooth paste and toys . 
 these nanoparticles can enter into the body and move into the hippocampus . 
 the aim of this study was to investigate the neurotoxicity of silver nanoparticles in the adult rat hippocampus.materials and methods:12 male wistar rats were randomly divided into two experimental and control groups ( 6 rats in each group ) . 
 animals in the experimental group received ag - nps ( 30 mg / kg ) orally ( gavage ) for 28 consecutive days . 
 control group in the same period was treated with distilled water via gavage . at the end of experiment , 
 animals were deeply anesthetized , sacrificed , and their brains were collected from each group . finally the brain sections were stained using toluidine blue and tunel . then to compare the groups , dark neurons ( dns ) and apoptotic neurons were counted by morphometric method.results:results showed that the numbers of dns and apoptotic cells in the ca1 , ca2 , ca3 , and dentate gyrus ( dg ) of hippocampus significantly increased in the ag - nps group in comparison to the control group ( p<0.05).conclusion : exposure to ag - nps can induce dark neuron and apoptotic cells in the hippocampus .