Table 2.

Summary of neurotoxic injuries of AgNPs in CNS

Types of AgNPs	Animal models	Route, dosage and exposure time of administration	Results	Ref.
PVP-coated AgNPs	Male rats: 140–150 g	Oral: 30 mg/kg b.w./day, lasted for 8 weeks	-Significantly changed the level of neurotransmitters and amino acids in brain. ­ Oxidative stress occurs in brain. - The transcriptional levels of NMDA receptors, MAO-A/B and MT-III significant increased. - Astrogliosis and demyelination of neurons accompanied by neuronal degeneration and vacuolation have been observed. - Oral administration of rutin overcomes neurotoxic effects through improving the antioxidant status in brain, correcting the imbalance of neurotransmitters.	[30]
AgNPs: 45–120 nm	Adult male rats: 7–9 weeks old	ig.: 10 and 30 mg/kg b.w./day, lasted for 28 days	- The enhancement of Txnip and FMO2 gene expression changed the redox homeostasis in rat brain, Conduciving to activate the inflammatory responses. - Ddit4 and Txnip mRNA levels significantly increased at all exposed dose levels, FMO2 mRNA level highly significant increases in the high-dose group, Ddit4 and JNK protein expression were up-regulated to enhanced apoptosis in the cerebellar cortex. - Presence of considerable amounts of sliver in the cerebellar cortex increased in a dose-dependent manner. -The architecture of the cerebellar cortex were more severely disrupted in the high-dose group than the low-dose group.	[59]
AgNPs: 3–30 nm	Female SD rats: 6 weeks old	ig.: 1 and 10 mg/kg b.w./day, lasted for 2 weeks	- Silver content significantly increased to 7.3- and 7.7-fold in the blood and 60- and 312-fold in the brain in the low- and high-dose groups compared to the control group. - Neuronal degeneration, astrocyte swelling, and IL-4 significantly increased in brain even in a low-dose group.	[60]
CT-coated AgNPs: 7 nm	Female rats: 16 weeks old, 280 - 310 g	i.v.: 5 mg/kg b.w. AgNPs, 0.0003 mg/kg b.w. Ag+ 5% sucrose solution in control group, Rats were sacrificed 24 h after injection	- The permeability of BBB of Rats exposed to AgNPs were not changed, but the expression of Claudin-4 significantly decreased. - Observed astrocyte foot swelling, neuron shrinkage, and AgNPs-like particles in the hippocampus of AgNPs-exposed group. - Ag+ was only detected in the hippocampus of rats exposed to AgNPs, while not detected in the control group and the Ag+ group, meanwhile, Ag was not detected in the blood after 24 h injection in all groups. - Ca2+ signaling pathway and neuroactive ligand-receptors (Grin2a, Drd2, and Adra1d) affected by AgNPs damaged the cognitive and neural development of the brain.	[73]
CT-coated AgNPs: 26.9 nm	Adult male mice: 3 months old, 28–32 g	Oral: 100 and 1000 mg/kg b.w./day, 28 days	- Total Ag+ contents in whole brain tissue significantly increased in a dose-dependent manner. - The levels of monoamines (Dopamine and 5-HT) in the brain and the value of enzyme activity of AChE in the cerebral cortex of mice exposed to AgNPs significantly decreased in a dose-dependent manner. - The cerebral cortex of mice exposed to AgNPs showed that neuron pyknosis and increase of neuroglia cells.	[87]
CT-coated AgNPs: 25 nm	Adult male mice	i.p.: 0, 100, 500 and 1,000 mg/kg b.w., Mice were sacrificed 24 h after injection	- Changed the expression of genes related to oxidative stress in the caudate nucleus, frontal cortex and hippocampus of mice.	[103]
PVP-coated AgNPs: 8.7 ± 0.4 nm	mice: 2 months and 5 months old	Oral: 50 µg/day, per animal lasted for 60 days	- The behavior of younger and elder mice both exposed to AgNPs showed a decrease in locomotor activity with age. - Elder mice exposed to AgNPs exhibited certain improvements in behavioral functions compared to those of the exposed younger ones. - The silver accumulation levels in the brains of younger and elder mice are equal. - The adaptive homeostasis in CNS exposed to AgNPs non-linearly changes with age increase.	[105]
AgNPs: 10 ± 4 nm,	Adult male rats: 140–160 g	ig.: 0.2 mg/kg b.w. /day, once a day, lasted for 14 days	- The level of sliver in serum in the range of 11–13 µg/L, while it was below the detection limit of the applied method in brain homogenates of rats (0.241 mg/kg in solid tissue) after 2 weeks. - The locomotor activity, motor coordination, and memory performance of rats exposed to AgNPs lasting for 14 days didn’t have significant influence. - Brain of rats exposed to AgNPs showed morphological disturbances in myelin sheaths and changed the expression of myelin-specific proteins (CNP, MAG, and MOG), those proteins significantly decreased, while the mRNA level increased.	[109]
PVP-coated AgNPs: 34 ± 2 nm	Male mice: 8 weeks old, 19–27 g	Oral: Suspended AgNPs in distilled water (50 µg/day); lasted for 30 days, 60 days, 120 days and 180 days,	- Mice experienced two adaptation periods to toxic AgNPs, that is anxiety and the development of research instincts, but ultimately failed because AgNPs eventually led to the degradation of long-term memory. - The relatively short (30–60 days) and long administration time (more than 120 days) after exposure to AgNPs is the most dangerous period of brain damage.	[112]
PVP-coated AgNPs: 34 ± 5 nm	Male mice: 8 weeks old	Oral: Suspended AgNPs in distilled water (50 µg/day); lasted for 30 days, 60 days, 120 days and 180 days	- The concentration of sliver in the hippocampus, cerebellum, and cortex at 120 days, and the remaining brain tissue at 180 days increases in a step-like manner. - The irregular and rarefied appearance stratum in the pyramidale and the dispersed arrangement of neurons in the CA2 region in brain were observed. - The long-term contextual fear memory tested 24 h after training of mice in the group after AgNPs-exposed for 180 days were impaired. - Memory and behavioral changes are caused by the accumulation of silver in the brain and neuronal damage to the CA2 subregion of the hippocampus.	[113]
CT-coated AgNPs: 10 nm	Adult male rats: 6 weeks old, 180–210 g	ig.: 0.2 mg/kg b.w./day, once a day for 14 days	- The level of ROS, MDA and GPx activity in brain tissue of the group exposed to AgNPs were significantly higher than control group receiving saline. - AgNPs and Ag+ were both causing a significant decrease in the reduced-to-oxidized glutathione ratio in brain. -Rats exposed to a very low dose of AgNPs produce mild oxidative stress in their brain but not in the liver, indicating the oxidative stress induced by AgNPs in brain may cause neurotoxicity.	[130]
PVP-coated AgNS, PVP-coated AgNC	Male rats: 7 weeks old	Oral: 3.6 mg/kg b.w./day, lasted for 14 days	Anxiety-like and possibly stereotypical behaviors of male rats exposed to short-term and low-dose AgNPs increased, and those changes of exposure to AgNS were more pronounced.	[169]
BSA-coated AgNS: 20 ± 5 nm	Male rats: 10 weeks old	ig.: 1 and 30 mg/kg b.w./day, lasted for 28 days	- The memory and cognitive coordination processes of rats exposed to low dose AgNPs have been affected. - The presence of Ag+ in different brain regions, especially in the hippocampus, plays a crucial role in AgNPs induced impairment of advanced brain function.	[134]
CT-coated AgNPs: 10 ± 4 nm	Wistar rat pups: 2 weeks old	Oral gavage with a gastric probe: once daily at a dose of 0.2 mg/kg b.w./day for 21 consecutive days	- Silver concentrations in brain were 0.15 ± 0.01 mg/kg w.w. in AgNPs-treated group and 0.23 ± 0.03 mg/kg in Ag citrate-treated group measured by ICP-MS. - TEM analysis of the rat brains exposed to AgNPs revealed ultrastructural features indicative of the presence of ER stress. - The unfolded protein response (UPR) pathway mediates protective mechanisms in immature rat brains exposed to low doses AgNPs.	[170]
CT-coated AgNPs: 10 nm	Male mice: 4–5 weeks old	ig.: 0.25 and 1 mg/kg b.w./day AgNPs, silver acetate (AgAc) at a dose of 1.55 mg/kg b.w./day, once a day for 5 days per week, lasted for 4 weeks	- Abnormal behaviors and significant difference in body/organ weight were not observed. - The content of sliver accumulation in the brain tissue in AgAc- and AgNPs-exposed groups were dose-dependent at the end of treatment (EoT), ant, it was a significant difference in mice treated with AgAc and AgNPs compared with the control group treated with sterile water. After the recovery period, the total sliver in brain of mice treated with 0.25 mg/kg b.w. AgNPs, 1 mg/kg b.w. AgNPs and 1.55 mg/kg b.w. AgAc were reduced by 31%, 38%, and 50%, respectively. - The immunoreactive GFAP+ expression of astrocytes was significantly increased in the hippocampus of mice treated with AgNPs at the EoT, while there were no differences that could be observed after recovery. - The immunoreactive Iba1+ expression of microglial cells was significantly increased in the cortex of mice treated with 1 mg/kg b.w./day AgNPs at the EoT, and it significant decreased in all treated groups after recovery. - The split of the basement membrane of capillaries and the swelling of astrocytic perivascular endfeet were observed in the hippocampus of mice treated with 1 mg/kg b.w. AgNPs- and AgAc at the EoT, while no changes were found in the cortex. - The impact on glial cells and ultrastructural changes of the BBB caused by AgNPs affected the accumulation and slow clearance of silver in the brain.	[171]
AgNPs: 20–25 nm	Neonatal SD rats	intranasal instillation: 0.1, 0.2, 0.5, and 1 mg/kg b.w./day, once a day for 14 consecutive weeks	- Significantly decreased body weight. - The GFAP protein expression of cerebellar tissue in rats treated with AgNPs were dramatically increased, while it is significantly reduced in rats treated with AgNPs-Vitamin E compared with rats treated with AgNPs alone - The levels of activated caspase-3 in cerebella of rats treated with AgNPs significantly increased. - AgNPs exposure cause neurotoxic injury to the rat cerebellum by activating neuroglial cells and destroying the cerebellum granular layer, Vitamin E supplementation attenuates AgNPs-induced those neurotoxic injuries.	[27]
CT-coated AgNPs: 10 ± 4 nm	Rats: 2 weeks old, 30–40 g	ig.: 0.2 mg/kg b.w./ day, lasted for 21 days from postnatal day 14 (PND14)	- Silver content in brain were 0.15 ± 0.01 mg/kg w.w. in PND35 group and 0.17 ± 0.03 mg/kg w.w. in PND64 group measure by ICP-MS. - Behaviors of AgNPs-exposed rats were changed, AgNPs-exposed rats of PND14 group in a short time have pro-depressive and anti-anxiety reactions, but not in PND64 group.	[81]
AgNPs:	Adults male Wistar rats, average weight: 152 ± 20 g	Oral: 0.5, 5 and 10 mg/kg b.w./day, once a day for 14 consecutive weeks	- Minimal oxidative stress in the cortex and hippocampus in rat brains. - The reduction of AchE activity and the level of 5-HT and NE in rat brains were inhibited by AgNPs in a dose-dependent manner. - Brain tissues of rats treated with a dose of 0.5 mg/kg b.w. showed slight structural changes in the myelinated axons and their cytoplasm, and 5 mg/kg b.w. showed higher concentrations of AgNPs in the myelin lamella and their increased structural disturbance.	[99]
Uncoated AgNPs and PVP-coated AgNPs	Pregnant rats: 3 months old, Male offspring: 5 weeks after birth, the 1st day after copulation as the Gestation day 0 (GD0)	i.p.: 20 mg AgNPs/ml, 1 ml each time, once time every two days from GD10 to GD18	- The silver content in hippocampus of offsprings of pregnant rats exposed to uncoated AgNPs group (17.51 μg/g) were significantly higher than exposed to PVP-coated AgNPs (4.13 μg/g) and silver nitrate (5.31 μg/g). - The levels of GAP-43 protein expression in the hippocampus of offsprings brain of pregnant rats exposed to uncoated AgNPs were significantly lower than those exposed to PVP-coated AgNPs and silver nitrate, but there was no significant difference in the later two groups. - Pregnant rats exposed to uncoated AgNPs impaired spatial learning and memory ability in their offspring, while exposed to PVP-coated AgNPs protected from the toxic injury of their offspring.	[106]
AgNPs: 20 nm	Neonatal SD rats	Intranasal instillation: 0.1, 0.2, 0.5 and 1 mg/kg b.w./day, once a day for 14 consecutive weeks	- The protein and the mRNA levels of CACNA1A in CGCs decrease in a dose-dependent manner. - Cerebellar ataxia like symptoms (Dysfunction of motor coordination and impairment of locomotor activity) occurred in rats associated with CACNA1A expression decrease for the AgNPs-induced neurotoxicity. - The level of silver in cerebellum tissue increased from 12.2 ± 3.8 to 32.8 ± 5.2 μg/g by dose-related accumulation.	[120]
CT-coated AgNPs: 32 ± 6.6 nm	Female mice: 6–7 weeks old, Adult offsprings	s.c.: 0, 0.2 and 2 mg/kg b.w./day, female mice repeated administration once every 3 days from the 3rd day of gestation until parturition	- Cognitive behaviors in the Morris water maze of adult offspring significantly impaired; - Pregnant mice exposed to AgNPs show the number of defecations and leanings in the open field assay and number of passages in the light-dark box were greater in their offspring, Most of neurotoxic impairments were more apparent in their offspring which had been prenatally exposed to high-dose of AgNPs, especially female ones. - Pregnant animals exposed to AgNPs may cause neurobehavioral disorders in their offspring.	[172]
CT-coated AgNPs 9–21 nm, SN-coated AgNPs: 11–32 nm	Pregnant rats, Male offsprings: 6 weeks old	Oral: administration from the 10th day to the 21st day of gestation	- Pregnant rats exposed to AgNPs affected behaviors, and increased anxiety and higher levels of nitro-oxidative stress in their male offsprings. - The levels of nitro-oxidative stress and apoptosis in the hippocampus of CT-coated AgNPs group were higher than SN-coated AgNPs group, indicating functionalizing AgNPs with natural antioxidants can improve its stability and biocompatibility, and reduce toxic injury to CNS.	[173]
CT-coated AgNPs: 10 nm	Wistar rat pups: 2 weeks old	ig.: 0.2 mg/kg b.w./ day, Starting administration to pups at postnatal day 14 (PND14) and lasted for 21 consecutive days	- The concentrations of silver accumulated in brain tissue in AgNPs-exposed group were significantly higher than CT-coated AgNPs and saline exposure group at PND35 and PND90, and it below the detection limit value (0.011 mg/kg w.w.) in control group exposed to saline. - AgNPs and Ag+ accumulated in brains of immature rats affected the ultrastructure of the synapse. - Down-regulated the expression level of NMDA receptor complex-related proteins (GluN1 and GluN2B subunits, PSD95 and SynGAP). - The down-regulation of the GluN2B-PSD95-nNOS-cGMP signaling pathway, which maintains the process of LTP/LTD during development as a result of learning and memory formation, was the mechanism of the changes in NMDA receptors mediated by AgNPs. - The downregulation and density reduction of NMDA receptors and the disturbed molecular pattern of synaptic plasticity caused by disintegration of glutamatergic synapses were related to neurotoxicity in brain of immature rats exposed to Ag+ or AgNPs.	[174]
AgNPs: 18 ± 1.8 nm	Female mice: 4–5 weeks old, Offspring	Oral: 3 mg/kg b.w./day, Administration from 1 week before the mating of female and male mice to the end of parturition	- Increased likelihood of engaging in certain repetitive behaviors. - Decreased resident microglial cells in brain of offspring. - Increased body fat in the offspring.	[175]

AgNS Sphere-shaped AgNPs, AgNC Cube-shaped AgNPs, BSA Bovine serum albumin, i.v. intravenous, i.p. intraperitoneal, s.c. subcutaneous, b.w. body weight