Quercetin Abrogates Oxidative Neurotoxicity Induced by Silver Nanoparticles in Wistar Rats

This study aimed to investigate the oxidative neurotoxicity induced by silver nanoparticles (AgNPs) and assess the neuroprotective effects of quercetin against this toxicity. Forty adult male rats were divided into four equal groups: control, AgNPs (50 mg/kg intraperitoneally), quercetin (50 mg/kg orally), and quercetin + AgNPs. After 30 days, blood and brain tissue samples were collected for further studies. AgNP exposure increased lipid peroxidation and decreased glutathione peroxidase, catalase, and superoxide dismutase activities in brain tissue. AgNPs decreased serum acetylcholine esterase activity and γ-aminobutyric acid concentrations. AgNPs upregulated tumor necrosis factor-α, interleukin-1β, and Bax transcript levels. AgNPs reduced the transcripts of claudin-5, brain-derived neurotrophic factor, paraoxonase, nuclear factor-erythroid factor 2 (Nrf2), and Bcl-2. Histopathologically, AgNPs caused various degenerative changes and neuronal necrosis associated with glial cell reactions. AgNPs increased the immunohistochemical staining of glial fibrillary acidic protein (GFAP) in the cerebrum and cerebellum. Oral treatment with quercetin efficiently counteracted the opposing effects of AgNPs on brain tissue via modulation of tight junction proteins, Nrf2, and paraoxonase, and its positive mechanism in modulating pro-inflammatory cytokines and the downregulation of GFAP expression, and the apoptotic pathway. AgNPs also altered the severity of histopathological lesions and modulated GFAP immunostaining in the examined tissue.


Introduction
Nanotechnology has developed many nanoparticles that are very significant in medicine, industry, and agriculture. Silver nanoparticles (AgNPs) are broadly utilized in different industrial applications because of their small size and unique physicochemical features [1]. AgNPs have been employed in clothing, water disinfectants, and cosmetics because of their antibacterial and antifungal properties, and appear in air freshener sprays, wound dressings, sunscreens, hygiene products, and food containers because of their appropriate antibacterial/antifungal characteristics [2].
AgNPs can enter the body by various routes, such as the inhalation of industrial manufacturing dust or shoe sprays, injection, ingestion, and dermal contact. As a result,

Experimental Design
Wistar Albino rats (160-190 g) were bought from the Animal Breeding Unit, Medical Research Institute, Alexandria University. Rats were housed in metal cages with optimal temperature and humidity, a dark/light cycle, and free feed and drinking water access. The worldwide ethical norms for the care and use of laboratory animals were followed to manage the animals. The Experimental Animal Use and Ethics Committee of Alexandria University's Faculty of Veterinary Medicine authorized the research techniques. Four groups of rats were randomly assigned (10 rats each): control; AgNP group (AgNPs 50 mg/kg BW intraperitoneally (i.p.) administered thrice weekly; warranted by a previous study) [19]; quercetin group (50 mg/kg BW orally) [20]; and quercetin + AgNP group (infused with AgNPs and given quercetin orally) as shown in Figure 1. All treatments were given for 30 days. Rats were anesthetized with ketamine/xylazine (7.5-10 mg/kg, 1 mg/kg Life 2022, 12, 578 3 of 18 i.p.) 24 h after the last treatment. The inner canthus was used to collect blood. Separation of the sera for the estimation of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) pro-inflammatory cytokine levels were measured using specific rat IL-6 DuoSet ELISA Kit (DY506; R&D System, Minneapolis, MN, USA) and TNF-α Quantikine ELISA Kit (RTA00; R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions.
groups of rats were randomly assigned (10 rats each): control; AgNP group (AgNPs 50 mg/kg BW intraperitoneally (i.p.) administered thrice weekly; warranted by a previous study) [19]; quercetin group (50 mg/kg BW orally) [20]; and quercetin + AgNP group (infused with AgNPs and given quercetin orally) as shown in Figure 1. All treatments were given for 30 days. Rats were anesthetized with ketamine/xylazine (7.5-10 mg/kg, 1 mg/kg i.p.) 24 h after the last treatment. The inner canthus was used to collect blood. Separation of the sera for the estimation of interleukin (IL)-6 and tumor necrosis factor-α (Tnf-α) proinflammatory cytokine levels were measured using specific rat IL-6 DuoSet ELISA Kit (DY506; R&D System, Minneapolis, MN, USA) and Tnf-α Quantikine ELISA Kit (RTA00; R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions. Rats were subsequently euthanized, and their brains were dissected directly, drenched in normal cold saline (0.9%), and divided into three equal portions. The first section was used for histological and immunohistochemical studies, whereas the second section assessed oxidative indices. The third section was used for gene expression analyses. Quimica Clinica Aplicada S.A. provided the acetylcholine esterase (AChE) assay kit, and γ-aminobutyric acid (GABA) was provided by Abnova.

Antioxidant and Lipid Peroxidation Analysis
A Teflon and a pestle homogenizer were used to homogenize 500 mg of the entire brain tissue in an ice-cold solution (0.1 M phosphate-buffered saline (PBS), pH 7.4). The supernatant was separated after centrifuging the crude homogenate at 14,000 rpm for 10 min at 4 °C ; the supernatant was separated. After the reaction with thiobarbituric acid, lipid peroxide was measured and expressed as nmol malondialdehyde (MDA) per tissue weight [21]. The activity of glutathione (GSH) peroxidase (GPx) was assessed [22], based on the hydrogen peroxide (H2O2) interaction with NADPH, GSH, and GSH reductase. At a wavelength of 340 nm, the absorbance was measured, and the result was reported as IU per tissue weight. Superoxide dismutase (SOD) activity was determined based on a 50% suppression of nitroblue tetrazolium reduction by superoxide anion generated by xanthine and xanthine oxidase; the result was reported as IU per tissue weight [23]. The oxidation rate was used to determine catalase (CAT) activity according to Chiang et al. [24] and reported as IU per tissue weight. The GSH level was assessed according to Ellman Rats were subsequently euthanized, and their brains were dissected directly, drenched in normal cold saline (0.9%), and divided into three equal portions. The first section was used for histological and immunohistochemical studies, whereas the second section assessed oxidative indices. The third section was used for gene expression analyses. Quimica Clinica Aplicada S.A. provided the acetylcholine esterase (AChE) assay kit, and γ-aminobutyric acid (GABA) was provided by Abnova.

Antioxidant and Lipid Peroxidation Analysis
A Teflon and a pestle homogenizer were used to homogenize 500 mg of the entire brain tissue in an ice-cold solution (0.1 M phosphate-buffered saline (PBS), pH 7.4). The supernatant was separated after centrifuging the crude homogenate at 14,000 rpm for 10 min at 4 • C; the supernatant was separated. After the reaction with thiobarbituric acid, lipid peroxide was measured and expressed as nmol malondialdehyde (MDA) per tissue weight [21]. The activity of glutathione (GSH) peroxidase (GPx) was assessed [22], based on the hydrogen peroxide (H 2 O 2 ) interaction with NADPH, GSH, and GSH reductase. At a wavelength of 340 nm, the absorbance was measured, and the result was reported as IU per tissue weight. Superoxide dismutase (SOD) activity was determined based on a 50% suppression of nitroblue tetrazolium reduction by superoxide anion generated by xanthine and xanthine oxidase; the result was reported as IU per tissue weight [23]. The oxidation rate was used to determine catalase (CAT) activity according to Chiang et al. [24] and reported as IU per tissue weight. The GSH level was assessed according to Ellman [25]. GSH interacted with 5.5-dithiobis 2-nitrobenzoic acid to produce a yellow-green color and was colorimetrically quantified at 410 nm and reported as nmol per tissue weight.

Histopathological Examination and Scoring System
Brain tissue specimens (cerebrum and cerebellum) were taken from separate rat groups (n = 5) following necropsy, washed in saline solution, and then put in 10% buffered formalin for at least 24 h (pH 7.4). Fixed tissue specimens were embedded in paraffin, sectioned at 4.5 µm, and stained with Mayer's hematoxylin and eosin (HE). A light microscope was used to examine the stained sections, and a digital camera was used to picture them (Nikon Corporation Co., Ltd., Tokyo, Japan). Five slides of five different rats per group were analyzed to evaluate and grade the various pathological changes found in the brain tissues (cerebrum and cerebellum). A four-point grading system was used to represent the severity of the identified histopathological changes, as follows: (-) Normal regular histology; (x) a mild injury; (xx) Moderate injury; (xxx) Severe injury [10].
The number of the affected slides and the number of regions impacted within each slide determined the extent of the pathological lesions in various groups. Each animal's score was evaluated, and the mean score was calculated for the various pathological alterations per group [10].

Immunohistochemical Studies
For glial fibrillary acidic protein (GFAP) detection as an indicator for astrogliosis, 5-mm-thick paraffin tissue slices of the cerebrum and cerebellum were placed on positively charged slides, deparaffinized in xylene, and rehydrated in ethanol in descending concentrations. Citrate buffer (10 mM; pH 6.0) was boiled at 105 • C for 10-20 min for Life 2022, 12, 578 5 of 18 microwave-assisted antigen retrieval and cooled for 20 min at room temperature. Endogenous peroxidase was deactivated by 3% H 2 O 2 in absolute methanol for 5 min at room temperature after washing with PBS. The nonspecific reaction was inhibited by incubation for 60 min with 10% normal goat serum. Rabbit polyclonal anti-GFAP antibody (1:500 dilution) was incubated overnight at 4 • C on tissue slices. For 60 min, the sections were incubated with the Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC kit. GFAP expression was tagged with horseradish peroxidase HRP and colored with diaminobenzidine (DAB) substrate for 6-10 min to identify the antigen-antibody complex. Finally, a counterstain of Mayer's hematoxylin was applied, followed by dehydration in absolute alcohol, cleaning, and mounting. Using a light microscope, immunoreactivity was observed as dark brown GFAP staining.

Morphometric Investigation
Images of 10 non-overlapped high-power microscopic fields (×400) per section (one section/rat, 5 rats/group) taken with a digital camera (EC3, Leica, Munich, Germany) coupled to a Leica microscope were used to quantify GFAP expression in the cerebrum and cerebellum of control and treated groups (DM500).
The area percent of GFAP positive brown immune-stained cells was calculated from the digital pictures using Image J analysis software (Image J 1.47v, National Institute of Health, Bethesda, MD, USA).

Statistical Analysis
Using SPSS version 22.0 for Windows as a statistical package (IBM, Armonk, NY, USA), data were analyzed using a one-way analysis of variance, followed by Tukey's post hoc multiple comparisons test. The obtained values are assessed as the mean ± standard error. The degree of significance was set at p < 0.05.

Serum Pro-inflammatory Cytokines
The pro-inflammatory cytokine concentrations were assessed to examine the potential neurotoxic effects of AgNPs. AgNPs induced a significant (p < 0.05) increment in TNF-α and IL-6 concentration compared with the control. Cotreatment of quercetin significantly (p < 0.05) decreased the TNF-α and IL-6 concentrations compared with that of the AgNP group, verifying that quercetin has anti-inflammatory activity, as shown in Figure 2.  Figure 3 shows that, compared with control, AgNPs induced a significant (p < 0.05) increase in the MDA quantity, and a reduction in GSH levels and antioxidant enzyme activity (GPx, CAT, and SOD) in the AgNP-treated group, confirming brain tissue injury  Figure 3 shows that, compared with control, AgNPs induced a significant (p < 0.05) increase in the MDA quantity, and a reduction in GSH levels and antioxidant enzyme activity (GPx, CAT, and SOD) in the AgNP-treated group, confirming brain tissue injury due to oxidative damage and reduction of antioxidants in this group. Compared with AgNP-treated rats, cotreatment with quercetin improved the brain oxidative/antioxidative indices, as demonstrated by a significant (p < 0.05) reduction in MDA and increases in different antioxidant enzymes, indicating the antioxidant effects of quercetin.

Brain Neurotransmission
GABA amino acid neurotransmitter mediates fast excitatory and inhibitory neurotransmission in the brain, and ACE is a very fast enzyme that terminates neurotransmission at cholinergic synapses. The brain enzymatic activity of AChE and GABA concentrations is shown in Figure 3. Compared with control, AgNPs treatment induced a significant decrease in (p < 0.05) AChE activity, and GABA concentrations confirmed the disturbance in neuroendocrine control that led to the destructive neuronal consequences of AgNPs. Quercetin coadministration reduced the biochemical assays compared with the AgNP group, as demonstrated by a significant (p < 0.05) increase, as shown in Figure 4.

Brain Neurotransmission
GABA amino acid neurotransmitter mediates fast excitatory and inhibitory neurotransmission in the brain, and ACE is a very fast enzyme that terminates neurotransmission at cholinergic synapses. The brain enzymatic activity of AChE and GABA concentrations is shown in Figure 3. Compared with control, AgNPs treatment induced a significant Life 2022, 12, 578 7 of 18 decrease in (p < 0.05) AChE activity, and GABA concentrations confirmed the disturbance in neuroendocrine control that led to the destructive neuronal consequences of AgNPs. Quercetin coadministration reduced the biochemical assays compared with the AgNP group, as demonstrated by a significant (p < 0.05) increase, as shown in Figure 4.

Lesion Grading and Histopathological Findings
The average scores of pathological lesions in the cerebrum and cerebellum of different rat groups are shown in Table 2. The control and quercetin-treated groups showed regular histological limits. AgNP-treated rat tissue exposed histological changes that are quite severe, and had a high score in all examined characteristics. Nevertheless, cotreatment of quercetin with AgNPs improved most of these changes and revealed mild to moderate pathological abnormalities. Table 2. Cerebral and cerebellar lesion grading of control (CTR), quercetin and/or silver nanoparticles-treated rats.

Organ
Lesions

Lesion Grading and Histopathological Findings
The average scores of pathological lesions in the cerebrum and cerebellum of different rat groups are shown in Table 2. The control and quercetin-treated groups showed regular histological limits. AgNP-treated rat tissue exposed histological changes that are quite severe, and had a high score in all examined characteristics. Nevertheless, cotreatment of quercetin with AgNPs improved most of these changes and revealed mild to moderate pathological abnormalities.
The control and quercetin-treated rats revealed nearly normal histological structures of the cerebrum cortices (Figure 5a,b) and cerebellum (Figure 6a,b). In AgNP-treated rats, cerebral tissue showed meningitis, accompanied by congestion of blood vessels, edema, hemorrhage, and mononuclear inflammatory cell infiltrations (Figure 5c). Many cortical neurons displayed neuronal swelling ( Figure 5d); others were shrunken and intensely stained, associated with increased pericellular spaces. Necrotic neurons had pyknotic nuclei and hypereosinophilic cytoplasm with or without satellitosis and neuronophagia (Figure 5e). The neuropil also displayed variable-size vacuoles and focal areas of gliosis (Figure 5f).
Meninges displayed vascular congestion, edema, hemorrhage (Figure 6c), and infiltration of mononuclear inflammatory cells in the cerebellum. The Purkinje layer was separated from the rest of the brain's layers. Many Purkinje cells with pyknotic and hyperchromatic nuclei were observed (Figure 6d). Others were necrotic and associated with satellitosis and neuronophagia. A selective neuronal loss was also observed. Cellular depletion was evident in the granular cell layer. Also, areas of gliosis, congestion, and hemorrhages ( Figure 6e) were observed. In comparison, AgNPs + quercetin-treated rats' cerebrum cortices (Figure 5f) and cerebellum tissues (Figure 6f) displayed a significant improvement in the previously observed lesions in AgNPs-treated rats as they were less in severity and distribution ( Table 2). cerebral tissue showed meningitis, accompanied by congestion of blood vessels, edema, hemorrhage, and mononuclear inflammatory cell infiltrations (Figure 5c). Many cortical neurons displayed neuronal swelling ( Figure 5d); others were shrunken and intensely stained, associated with increased pericellular spaces. Necrotic neurons had pyknotic nuclei and hypereosinophilic cytoplasm with or without satellitosis and neuronophagia (Figure 5e). The neuropil also displayed variable-size vacuoles and focal areas of gliosis (Figure 5f).   (orange arrow); swollen neurons (red arrow); shrunken darkly stained neurons (white arrow); the area with gliosis (star); necrotic neurons with satellitosis and neuronophagia (yellow arrow); perivascular cuffing (green arrow). Additionally, perivascular cuffing (Figure 5g), congestion, and areas of hemorrhages have been observed.
Meninges displayed vascular congestion, edema, hemorrhage (Figure 6c), and infiltration of mononuclear inflammatory cells in the cerebellum. The Purkinje layer was separated from the rest of the brain's layers. Many Purkinje cells with pyknotic and hyperchromatic nuclei were observed (Figure 6d). Others were necrotic and associated with

Immunohistochemical Analysis and Quantitative Assessment
As demonstrated in Figures 7 and 8, the immunohistochemical staining of the cerebral cortices and the cerebellum of the control (Figures 7a and 8a) and quercetin-treated (Figures 7b and 8b) groups showed brown-yellow small astrocyte cell bodies with thin processes. AgNPs-treated rats' tissues displayed relatively larger cells with prominent processes (Figures 7c and 8c). severity and distribution ( Table 2).

Immunohistochemical Analysis and Quantitative Assessment
As demonstrated in Figures 7 and 8, the immunohistochemical staining of the cerebral cortices and the cerebellum of the control (Figures 7a and 8a) and quercetin-treated (Figures 7b and 8b) groups showed brown-yellow small astrocyte cell bodies with thin processes. AgNPs-treated rats' tissues displayed relatively larger cells with prominent processes (Figures 7c and 8c).  ; Silver nanoparticles-treated (c) silver nanoparticles + quercetintreated groups (d). Arrow represents the astrocytes; (e) Quantification of GFAP expression, the immunohistochemical staining of GFAP was assessed as area (%) across 10 fields/section, n = 5 rat/group. Mean values were significantly different from control (# p < 0.05), Quercetin (* p < 0.05), silver nanoparticles ($ p < 0.05) group.   In contrast, AgNP + quercetin-treated rats significantly reduced astrocytic reactions (Figures 7d and 8d). In line with these remarks, quantitative assessment of the GFAP-immunostained area percentage in the cerebrum and cerebellum of quercetin-treated rats presented no significant (p < 0.05) changes compared with those of control rats (Figures 7e and 8e,  respectively). Meanwhile, AgNPs-treated rats' tissues displayed a substantial (p < 0.05) increment as contrasted to the control group values (Figures 7e and 8e). Conversely, AgNP + quercetin-treated rats displayed a significant (p < 0.05) decrease in the GFAP area percentage. In contrast, AgNP + quercetin-treated rats significantly reduced astrocytic reactions (Figures 7d and 8d). In line with these remarks, quantitative assessment of the GFAPimmunostained area percentage in the cerebrum and cerebellum of quercetin-treated rats presented no significant (p < 0.05) changes compared with those of control rats (Figures 7e and 8e, respectively). Meanwhile, AgNPs-treated rats' tissues displayed a substantial (p < 0.05) increment as contrasted to the control group values (Figures 7e and 8e). Conversely, AgNP + quercetin-treated rats displayed a significant (p < 0.05) decrease in the GFAP area percentage.

Effects of Quercetin and/or AgNP on Gene Expression
In Figure 9, AgNPs significantly downregulated the claudin-5 and brain-derived neurotrophic factor (BDNF) mRNA gene expression and the nuclear factor-erythroid factor 2 (NrF2) and paraoxonase mRNA gene expression. Quercetin cotreated with AgNPs showed a significant upregulation of claudin-5. The AgNP group showed significant normalization of NrF2 and paraoxonase mRNA expression. In Figure 10, AgNPs significantly upregulated the TNF-α, IL-1β, and Bax mRNA expression, with significant downregulation of the Bcl-2 expression that significantly normalized with quercetin treatment.

Effects of Quercetin and/or AgNP on Gene Expression
In Figure 9, AgNPs significantly downregulated the claudin-5 and brain-deriv neurotrophic factor (BDNF) mRNA gene expression and the nuclear factor-erythroid fa tor 2 (NrF2) and paraoxonase mRNA gene expression. Quercetin cotreated with AgN showed a significant upregulation of claudin-5. The AgNP group showed significant no malization of NrF2 and paraoxonase mRNA expression. In Figure 10, AgNPs significan upregulated the Tnf-α, IL-1β, and Bax mRNA expression, with significant downregulati of the Bcl-2 expression that significantly normalized with quercetin treatment.

Discussion
The brain is a susceptible organ; it is vulnerable to the effects of ROS, owing to the high demand for oxygen, the highly peroxidizable unsaturated fatty acids, and the abundance of highly peroxidizable substrates [27]. Moreover, the brain's antioxidant defenses are not particularly generous because of their restricted ability to repair nerve cells that are more vulnerable to toxins [28]. AgNPs have been regularly utilized for many years in consumer products. Cell death can be caused by various methods, such as exposure to high ROS quantities generated by AgNPs [29]. This study aimed to investigate the neuroprotective effects of quercetin on male adult rats exposed to AgNPs.
Oxidative stress leads to BBB disturbance parallel to the changes in cytoskeletal tight junction proteins (TJPs), such as claudin-5 and occludin [30]. Smaller AgNPs can easily penetrate the BBB, a detriment to the barrier's integrity by modifying the cell membrane's endothelial permeability [9]. The deposition of silver over a long time leads to significant brain dysfunction [31]. The BBB plays an essential role in the pathogenic progression [32]. Results revealed that AgNPs significantly downregulated claudin-5 and BDNF mRNA gene expression, inconsistent with Martirosyan et al. [33], who reported that occludin in AgNP-treated cells was disturbed. This study was consistent with Park et al. [34], who reported that AgNPs substantially inhibited BDNF-induced cell survival by increased lactate dehydrogenase secretion and oxidative ROS creation. This study showed that quer-

Discussion
The brain is a susceptible organ; it is vulnerable to the effects of ROS, owing to the high demand for oxygen, the highly peroxidizable unsaturated fatty acids, and the abundance of highly peroxidizable substrates [27]. Moreover, the brain's antioxidant defenses are not particularly generous because of their restricted ability to repair nerve cells that are more vulnerable to toxins [28]. AgNPs have been regularly utilized for many years in consumer products. Cell death can be caused by various methods, such as exposure to high ROS quantities generated by AgNPs [29]. This study aimed to investigate the neuroprotective effects of quercetin on male adult rats exposed to AgNPs.
Oxidative stress leads to BBB disturbance parallel to the changes in cytoskeletal tight junction proteins (TJPs), such as claudin-5 and occludin [30]. Smaller AgNPs can easily penetrate the BBB, a detriment to the barrier's integrity by modifying the cell membrane's endothelial permeability [9]. The deposition of silver over a long time leads to significant brain dysfunction [31]. The BBB plays an essential role in the pathogenic progression [32]. Results revealed that AgNPs significantly downregulated claudin-5 and BDNF mRNA gene expression, inconsistent with Martirosyan et al. [33], who reported that occludin in AgNP-treated cells was disturbed. This study was consistent with Park et al. [34], who reported that AgNPs substantially inhibited BDNF-induced cell survival by increased lactate dehydrogenase secretion and oxidative ROS creation. This study showed that quercetin cotreated with AgNPs normalized claudin-5 and BDNF mRNA expression. This result was consistent with [30,35], who revealed that quercetin preserves and avoids hippocampus neuronal injury through upregulation of the TJPs of the BBB and BDNF. Quercetin significantly normalized NrF2 and paraoxonase mRNA expression. Nrf2 attaches to the antioxidant/electrophilic response factor and governs cytoprotective protein expression, mainly antioxidant GSH producing enzymes [36]. MDA levels were lower, and antioxidant enzyme activity was higher in AgNP-intoxicated rats given quercetin. This study was supported by Xiao et al. [37], who revealed that quercetin decreases brain cell apoptosis and neuronal injury by improving pathways of DJ-1/Nrf2 and enhancing the possibility of antioxidation. In addition, Pereira et al. [38] showed that paraoxonase is related to several oxidative stress diseases. Paraoxonase 2 has anti-inflammatory and neuroprotective properties [39,40]. Costa et al. [41] showed the neuroprotection effects of quercetin through modulation of paraoxonase 2 expressions. AgNPs significantly upregulated TNF-α, IL-1β, and Bax mRNA expression, with significant downregulation of Bcl-2 expression that significantly normalized with quercetin treatment.
This study was consistent with Murphy et al. [42], who proved that, in primary blood monocytes, AgNPs could massively increase the mRNA expression of the proinflammatory cytokines IL-1, IL-6, and TNF-α, which provoked the inflammatory and oxidative effect of AgNPs. Others [43] also showed that quercetin and MSG normalized inducible nitric oxide synthase, IL-1β, and TNF-α. AgNPs decreased Bcl-2 and increased Bax gene expression in hippocampal rat cells [43], supporting the findings on apoptosis. Additionally, [43] found that quercetin has a defensive role by partly controlling Bcl-2/Bax protein expression. Moreover, AChE plays a crucial role in neuromuscular impulse transmission and is considered a potential biomarker of xenobiotic toxicity [10]. In this study, AChE activity was inhibited by AgNPs, consistent with Myrzakhanova et al. [44], who reported that toxicity with AgNPs leads to acetylcholine decrease.
Increased expression of acetylcholine leads to increased ROS and inflammation. GABA is an inhibitory neurotransmitter. When GABA attaches to a GABA receptor, it produces a calming effect [45]. This study reported that AgNPs induced a significant reduction in GABA concentrations, confirming the disturbance in neuroendocrine control that led to the neuronal damaging effects of AgNPs.
This study was supported by Jakaria et al. [46], who revealed that quercetin has a significant anxiolytic activity mediated by the GABAergic nervous system. Therefore, from the aforementioned results, it can be concluded that the protective pathway of quercetin against AgNP toxicity is through the modulation of TJPs, Nrf2, and paraoxonase, and its positive mechanism in modulating the pro-inflammatory cytokines and the apoptotic pathway.
Histological and immunohistochemical studies confirmed the biochemical findings. Various histological abnormalities (i.e., glial cell activation) in the AgNP-treated rat cerebrum and cerebellum were related to neuronal degeneration and necrosis, consistent with previous findings [47,48]. The capacity of AgNPs to cause oxidative stress, ROS production, mitochondrial dysfunction, and cytoskeleton alterations may be due to these lesions and impaired DNA and protein synthesis, resulting in inflammation, cell necrosis, or apoptosis [49,50].
ROS interacts with proteins, DNA, and RNA, among other biological elements. Vital cellular functions are disrupted. Furthermore, free radicals target the cell membrane polyunsaturated fatty acids and trigger the cell membrane's lack of permeability and disintegration [10]. ROS can potentially affect brain vascular functions, resulting in cell death [51]. AgNPs also decrease the interference with the synaptic machinery [9,52,53] and disruption of the BBB [52,53].
GFAP is a type III intermediate filament protein that maintains the astrocyte shape and mechanical strength [54]. Modifying the cell's filament network plays a role in several key activities in the CNS, including cell communication, support for nearby neurons, BBB functions, and mitosis [55]. GFAP serves as a specific marker of astrocytic activity and, during astrogliosis, is significantly expressed in CNS astrocytes [47]. Reactive astrogliosis may impair neuronal survival [56]. Glial cells create neurotoxic chemicals that cause inflammation and neuronal death, for example, reactive oxygen radicals and excitatory amino acids [57]. GFAP immunohistochemical staining in the cerebrum and cerebellum showed that AgNP-treated rats had a high level of GFAP positivity. The cerebrum and cerebellum showed an upsurge in the area percentage of GFAP-positive cells in the quantitative analysis of the AgNP-treated group, consistent with Yin et al. [58]. Reactive astrogliosis may impair neuronal survival [56] by the release of many neurotoxic chemicals (including reactive oxygen radicals and excitatory amino acids) and the consequent induction of inflammation and neuronal death by glial cells [57]. AgNPs may promote reactive gliosis and inflammation by excessive induction of lipid peroxidation, inducing oxidative stress and depletion of antioxidants that enhance the incidence of inflammation [59].
Concurrent administration of quercetin with AgNPs partially ameliorated these histopathological changes, as demonstrated by histopathological scoring. Correspondingly, quercetin downregulated GFAP expression in brain tissue. These data were in line with those of previous studies that reported the ability of quercetin to mitigate histological changes in the brain tissue of rats after traumatic brain injury [60] and various neurotoxicants [61][62][63]. The neuroprotective effects of quercetin might be ascribed to its aptitude to ameliorate oxidative damage, avoid lipid peroxidation, mitigate ER stress, modulate microglial activation [64][65][66], protect against AgNP-induced free radical assault, and boost the overall antioxidant capacity.

Conclusions
Quercetin administration effectively counteracted the adverse effects of AgNPs via modulation of TJPs, Nrf2, and paraoxonase and the expression of pro-inflammatory cytokines and the GFAP apoptotic pathway.  Institutional Review Board Statement: All of the experimental procedures of this study were carried out under the National Institutes of Health Guidelines and regulations for the care and use of laboratory animals. All the steps were followed to decrease and/or minimize the suffering of the experimental animals for project number (AU0122062931).

Informed Consent Statement: Not applicable.
Data Availability Statement: All data sets obtained and analyzed during the current study are available in the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.