Abstract

Introduction

More recently, nanotechnology has appeared as an up-and-coming and rapidly advancing field of research, potentially revolutionizing various scientific disciplines [1]. Its widespread application in numerous areas of life has made it increasingly prevalent. Nanotechnology primarily focuses on manipulating nanoparticles, which are atomic or molecular clusters portrayed by their minute size, typically less than 100 nm. These nanoparticles are amended versions of fundamental elements, achieved by altering their atomic and molecular properties. One of these essential elements is Zn, which is vital for maintaining the average central nervous system (CNS) performance. Its oxide form, ZnO, is a widely used form of metal oxides as a feed additive as it includes a more significant percentage of zinc and is less toxic than other zinc compounds [1]. Moreover, zinc oxide was previously used to treat epilepsy [2].

Nowadays, metal oxide nanoparticles are manufactured for a diversity of potential uses. One of them is the zinc oxide nanoparticles (ZnONPs), considered one of the best exploited at nano dimensions and have a vast area of utilization such as in electronic, optoelectronics, and as an effective nanocarrier for conventional drugs due to their cost-effectiveness and benefits of being biodegradable and biocompatible [3]. Furthermore, zinc oxide nanoparticles have been reportedly involved in medicinal and agricultural uses [4]. It also has other potential applications, such as therapeutic carriers, biological sensing, gene transfer, nanomedicine discovery, biological labeling, medical implant coatings, electronic sensors, wastewater treatment, and communication [5–7]. Zinc oxide nanoparticles (ZnONP) exhibit excellent biocompatibility, transparency, and high electron mobility [8]. Metal oxide nanoparticles, such as ZnO-NPs, possess a broad spectrum of biological applications and effects [9]. These encompass antioxidant, antibacterial, antitumor, antidiabetic, and anti-inflammatory properties, as well as the ability to enhance wound healing [10]. The wide range of activities can be attributed to zinc’s crucial involvement in enzyme systems, the synthesis of proteins and nucleic acids, hematopoiesis, cell proliferation, tissue regeneration, and neurogenesis [11]. Additionally, zinc plays a role in metabolic and signaling pathways, protecting against ischemia/reperfusion injury in organs like the kidney [12]. Metal oxide nanoparticles were deducted to show better efficacy [13], credited to their ability to pass the blood-brain barrier, alter metabolic profiles, and trace element levels in certain brain regions [14].

Learning and memory are interconnected processes whereby information is stored in the brain and retrieved as needed [15]. Trace elements play crucial roles in various physiological functions of brain biochemistry. These functions encompass the synthesis of neurotransmitters, the maintenance of antioxidant defense mechanisms, the regulation of intracellular redox processes, and the modulation of neural cells [16]. Imbalances, either in the form of deficiency or excess, of these trace elements can lead to disorders in the CNS [17]. For example, zinc deficiency can lead to memory loss, known as Alzheimer’s [18], in humans and animals [19]. Furthermore, zinc can also influence blood-brain-barrier (BBB) permeability [20].

Researchers also indicated that ZnONPs could affect the function of neural cells [21] and biocompatibility [22]. Zinc oxide nanoparticles (ZnONPs) have been found to impact brain monoamine levels (dopamine, norepinephrine, and serotonin) and various ions (Ca2+, Na+, K+, and Zn2+). They have also demonstrated the ability to modulate synaptic transmission in vitro [23]. Furthermore, Yongling et al. [24] have reported that zinc oxide nanoparticles (ZnONPs) can improve depressive-like behavior and cognitive impairment in mice. This effect is believed to be achieved by upregulating neuronal synaptic plasticity and functions. Thus, the precise impact and safety of zinc oxide nanoparticles (ZnONPs) on cognitive function remain unclear. To address this question, the main objective of the current study was to evaluate the impact of zinc oxide nanoparticles (ZnONPs) on brain function and behavior, specifically comparing it to the bulk form of zinc oxide (BZnO).

Materials and Methods

Results

ZnONP Characterization

ZnONP diameter analysis by the transmission electron microscope indicated the polygonal shape of particles with a diameter less than 50 nm, as identified by the manufacturer (Fig. ​(Fig.11).

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Fig. 1

Image by TEM to ZnONP (A) indicating the shape and diameter below 50 nm (scale bar = 200 nm), and bulk zinc oxide (B)

Brain Oxidative Markers

In Fig. ​Fig.2A,2A, B, and C, a notable elevation in the levels of SOD, CAT, and GPx is evident in the ZnONP-5-treated group compared to the other groups. Similarly, the BZnO-5 group significantly increased compared to the BZnO-10 and ZnONP-10-treated groups. Figure ​Figure2D2D and E reveals a significant decrease in MDA and ROS brain levels in the ZnONP-5 group compared to the other treated groups, although the BZnO-10 group exhibited higher levels. The BZnO-5-treated group demonstrated a significant reduction compared to the ZnONP-10 and control groups. Figure ​Figure3A3A and B shows a notable decrease in the levels of IL-6 and TNF-α in the ZnONP-5 group compared to the other treated groups, particularly the ZnONP-10- and BZnO-10-treated groups. Additionally, the BZnO-5 group exhibited a significant reduction compared to the ZnONP-10 and BZnO-10-treated groups.

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Fig. 2

The effects of different doses of ZnONP and BZnO on antioxidant and oxidant levels in brain tissue were evaluated. Specifically, the levels of SOD (superoxide dismutase), CAT (catalase), GPx (glutathione peroxidase), MDA (malondialdehyde), and ROS (reactive oxygen species) were measured. The results are presented as mean ± SEM (standard error of the mean). Statistical analysis revealed that bars labeled with different letters (a, b, c, d) were significantly different from each other at a significance level of P < 0.05

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Fig. 3

The effects of different doses of ZnONP and BZnO on cytokine and oxidant levels in brain tissue were examined. Precisely, IL-6 (interleukin-6) and TNFα (tumor necrosis factor alpha) were measured. The results are presented as mean ± SEM (standard error of the mean). Statistical analysis revealed that bars labeled with different letters (a, b, c) were significantly different from each other at a significance level of P < 0.05

Brain Neurotransmitters

Figure ​Figure4A,4A, C, and D illustrates a noteworthy rise in the levels of acetylcholine (ACh), serotonin, and dopamine in the ZnONP-5- and BZnO-5-treated groups in comparison to the BZnO-10 and ZnONP-10 groups. However, no significant changes were observed when compared to the control group. In Fig. ​Fig.4B,4B, the brain level of acetylcholinesterase (AChE) was markedly decreased in the ZnONP-5- and BZnO-5-treated groups as opposed to the BZnO-10 and ZnONP-10 groups. However, the BZnO-10 and ZnONP-10 groups exhibited considerably higher levels of AChE.

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Fig. 4

The effects of different doses of ZnONP and BZnO on neurotransmitter levels in brain tissue were investigated. Precisely, the levels of brain AChE (acetylcholinesterase), Ach (acetylcholine), serotonin, and dopamine were measured. The results are presented as mean ± SEM (standard error of the mean). Statistical analysis revealed that bars labeled with different letters (a, b, c) were significantly different from each other at a significance level of P < 0.05

Gene Expression

Figure ​Figure55 displays the outcomes of gene expression analysis, revealing a noteworthy increase in BDNF mRNA expression in the ZnONP-5-treated group compared to the other treated groups. Similarly, the BZnO-5 group exhibited a substantial upregulation compared to the BZnO-10- and ZnONP-10-treated groups. Additionally, Fig. ​Fig.55 demonstrates a significant downregulation of iNOS and NF-κB in the ZnONP-5 and BZnO-5 groups compared to the BZnO-10- and ZnONP-10-treated groups. Concerning apoptosis marker genes, there were notable elevations in the mRNA expression of bcl-2 in the ZnONP-5-treated group compared to the other treated groups. Similarly, the BZnO-5 group showed significant upregulation compared to the control, BZnO-10, and ZnONP-10 groups. However, the Bax mRNA expression was significantly downregulated in the ZnONP-5-treated group compared to the other treated groups, with a significant decrease in the BZnO-5 group compared to the control, BZnO-10, and ZnONP-10 groups.

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Fig. 5

The effects of different doses of ZnONP and BZnO on the mRNA expression of BDNF, iNOS, NF-KB, BAX, and BCL2 were investigated. The results are presented as mean ± SEM (standard error of the mean). Statistical analysis revealed that bars labeled with different letters (a, b, c, d) were significantly different from each other at a significance level of P < 0.05

Histopathological Analysis

The meninges, neurons, and cerebral cortex layer of the control group and ZnONP-5-treated rats displayed a normal histological structure, as observed in Fig. ​Fig.6a6a and b. However, in the ZnONP-10-treated rats, the neurons in the cerebral cortex exhibited characteristics such as cell shrinkage, pyknotic nuclei, and hyper-eosinophilic cytoplasm. Additionally, there was evidence of neuropil widening (Fig. ​(Fig.6c),6c), besides satellitosis and neuronophagia of the affected neuron (Fig. ​(Fig.6d),6d), with focal glial cells aggregating in the gray matter (Fig. ​(Fig.6e)6e) and congestion of the vasculature of choroid plexus (Fig. ​(Fig.6f).6f). However, in the BZnO-5-treated rats, the cerebral cortex, hippocampus, and cerebellum showed nearly normal histological structure (Fig. ​(Fig.6g),6g), while in the BZnO-10-treated rats, the cortical neurons showed severe shrunken, pyknotic, and hyper-eosinophilic cytoplasm with the extreme widening of neuropil (Fig. ​(Fig.6h),6h), satellitosis, and neuronophagia of the exaggerated degenerated neurons (Fig. ​(Fig.6i),6i), beside focal gliosis (Fig. ​(Fig.6j),6j), and focal area of polioencephalomalacia with the glial cell reactions also spotted (Fig. ​(Fig.6k)6k) beside congestion of choroid plexus vasculature and destruction of choroid plexus epithelium (Fig. ​(Fig.66l).

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Fig. 6

Photomicrograph of the cerebrum sections of rats stained by H&E (bar=50μm). a Control rats. b ZnONP-5-treated rats. c, d, e, and (f) ZnONP-10-treated rats. g BZnO-5-treated rats. h, i, j, k, and l BZnO-10-treated rats. Meninges (1), molecular layer (2), external granular layer (3), normal cortical neuron (black arrows), shrunken, pyknotic, and hyper-eosinophilic cytoplasm with widening of neuropil (blue arrows), satellitosis (red arrow) neuronophagia (yellow arrows), gliosis (white star), polioencephalomalacia (black star), and congestion of choroid plexus vasculature (short black arrows) destruction of choroid plexus epithelium (arrowheads)

The cerebellar cortex, including the molecular, Purkinje cell, and granular layers, displayed a normal morphology in the control group and ZnONP-5-treated rats, as observed in Fig. ​Fig.7a7a and b. However, in the ZnONP-10-treated rats, the cerebellar cortex showed mild abnormalities. These abnormalities included slight shrinkage of cells, cytoplasmic hypereosinophilia (an increase in the staining intensity of cytoplasm), and loss of dendritic arborization in Purkinje cells (Fig. ​(Fig.7c);7c); the same lesion was noticed in BZnO-5-treated rats, which is still moderate in severity (Fig. ​(Fig.7d).7d). In the BZnO-10-treated rats, most Purkinje cells showed shrinkage, darkly stained, loss of dendritic arborization, and depletion in number (Fig. ​(Fig.7e7e and f); besides, few numbers of granular cell layer were recorded in few rats.

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Fig. 7

Photomicrographs of the cerebellum sections of rats stained by H&E (bar = 50μm). (a) Control rats. (b) ZnONP-5-treated rats. (c) ZnONP-10-treated rats. (d) BZnO-5-treated rats. (e and f) BZnO-10-treated rats. Granular layer (1), Purkinje cell (2), molecular layer (3), normal Purkinje cell (black arrows), shrinkage, and darkly stained Purkinje cell (blue arrows)

Immunohistochemistry of Caspase-3 Protein Expression

The cerebral cortical neurons in the control group and ZnONP-5-treated rats displayed immune-negative brown staining for caspase-3, as shown in Fig. ​Fig.8a8a and b. However, in the ZnONP-10-treated rats, moderate immune-positive solid brown staining for caspase-3 was observed, as depicted in Fig. ​Fig.8c.8c. On the other hand, in the BZnO-5-treated rats, most cerebral cortical neurons exhibited negative brown staining for caspase-3. In contrast, a few neurons showed weak immune-positive brown staining, as seen in Fig. ​Fig.8d.8d. In contrast, the cerebral cortical neurons in the BZnO-10-treated rats exhibited intense immune-positive brown staining for caspase-3 (Fig. ​(Fig.8e).8e). The cerebellum, including Purkinje cells and granular cells, was subjected to immunohistochemical staining. Purkinje and granular cells exhibited immune-negative brown staining in the control rats, as depicted in Fig. ​Fig.99a.

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Fig. 8

Immunohistochemical staining of the cerebrum was performed to evaluate the expression of caspase-3 protein, as shown in figure. The scale bar represents 50μm. In the control group (a) and ZnONP-5-treated rats (b), the staining displayed negative immunostaining for caspase-3 in cortical neurons, indicated by black arrows. In the ZnONP-10-treated rats (c), moderate immunostaining was observed, represented by red arrows. In the BZnO-5-treated rats (d), most cortical neurons exhibited negative staining (black arrows), while a few neurons showed weak immunostaining (yellow arrows). In the BZnO-10-treated rats (e), intense immunostaining for caspase-3 was observed in cortical neurons, denoted by blue arrows. (f) Area % of caspase-3 protein immunostaining positive cell. All the values are expressed as mean ± standard error of mean. Statistical analysis revealed that bars labeled with different letters (a, b, c, d) were significantly different from each other at a significance level of P< 0.0001

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Fig. 9

Immunohistochemical staining of the cerebellum was performed to assess the expression of caspase-3 protein, with a scale bar of 50μm. In the control rats (a), both neurons and granular cells exhibited negative immunostaining for caspase-3, indicated by black arrows and arrowheads. In the ZnONP-5-treated rats (b), the Purkinje cells displayed negative brown staining (black arrow), while the granular cells showed intense immunopositive brown staining (blue arrow). In the ZnONP-10-treated rats (c), both Purkinje cells and granular cells exhibited moderate to weak immunopositive brown staining, represented by red arrows and arrowheads. In the BZnO-5-treated rats (d), the Purkinje cells showed negative brown staining (black arrow), while the granular cells displayed moderate immunopositive brown staining (yellow arrow). Moreover, in the BZnO-10-treated rats (e), both Purkinje cells and granular cells exhibited moderate immunopositive solid brown staining, denoted by red arrows and arrowheads. (f) Area % of caspase-3 protein immunostaining positive cell. All the values were expressed as mean ± standard error of mean. Statistical analysis revealed that bars labeled with different letters (a, b, c, d, e) were significantly different from each other at a significance level of P< 0.0001

Conversely, in ZnONP-5-treated rats, Purkinje cells showed negative brown staining, while granular cells displayed intense immune-positive brown staining, as shown in Fig. ​Fig.9b.9b. In ZnONP-10-treated rats, Purkinje and granular cells exhibited moderate to weak immune-positive brown staining, as seen in Fig. ​Fig.9c.9c. In the BZnO-5-treated rats, Purkinje cells showed negative brown staining, while granular cells displayed moderate immune-positive brown staining, as shown in Fig. ​Fig.9d.9d. Furthermore, in the BZnO-10-treated rats, Purkinje and granular cells exhibited moderate immune-positive solid brown staining, as depicted in Fig. ​Fig.9e.9e. The average percentage of cells expressing caspase-3 protein through immunostaining in the cerebrum and cerebellum showed a notable reduction in ZnONP-5, followed by BZnO-5 rats. Conversely, a significant increase was observed in ZnONP-10 and BZnO-10 rats compared to the control rats.

Discussion

Previous research has documented a range of advantageous effects associated with ZnONPs. However, its adverse effects have been increasingly studied [37], and the exact dose and safety of ZnONPs on cognitive function the precise mechanisms and consequences of ZnONPs have not been fully elucidated. To answer the question, this study evaluated the impact of ZnONPs on brain function and behavior with different doses compared to its bulk form.

The current study revealed that the ZnONP at 5mg/kg showed improvement in the general behavioral patterns, including feeding, drinking, standing, movement, and investigation, whereas lying, licking, and scratching behaviors decreased than the bulk form with a similar dose. Furthermore, it also showed the best locomotor activity and the least fearfulness in the open field test. Also, ZnONP at 5mg/bwt treated rats were the most anxiolytic, indicated by shorter entry time to open arms, multiple entries in open arms, and longer presence time in the elevated plus maze. The learning ability of rats measured by the Morris water maze indicated that ZnONP with 5mg/kg treated rats had the shortest time to locate the hidden platform and a longer time of presence in the target quadrant. On the contrary, ZnONP with 10 mg/kg treated rats showed decreased general behaviors, locomotor activity, and learning ability. These indicated that ZnONPs had a toxic effect at higher doses. However, the worst impact was for the BZnO-10 group on behavioral patterns, locomotor activity, and learning ability.

The anxiolytic effect of ZnONPs at a dosage of 5 mg/kg may be attributed to the involvement of zinc in anxiety-related neurochemical systems. Zinc plays a role in the co-release with glutamate in presynaptic spaces, which exerts an inhibitory neuromodulatory effect on glutamate signaling [19]. Glutamate was found to be an essential element in anxiety and anxiogenic behavior, so blocking of NMDA glutamate receptors by zinc could elicit an anxiolytic effect [38]. Additionally, zinc has been found to promote the release of gamma-aminobutyric acid (GABA) from interneurons in the hippocampus. This enhances GABA’s inhibitory effect, thereby reducing glutamate’s presynaptic release [39].

Consequently, maintaining a balance between the inhibitory effects of GABA receptors and the excitatory effects of glutamate receptors could play a crucial role in regulating the behavioral and physiological responses related to anxiety [40]. Similar to our study, Torabi et al. [41] stated that the better anxiolytic and locomotive effect of ZnONPs 5mg/kg than its conventional form. These could be due to the greater mobility of ZnONPs and their uptake across biological membranes, increasing their interaction with biological tissues [42]. ZnO nanoparticles have a larger surface area than the conventional form, resulting in increased reactivity due to the higher number of exposed reactive groups [43]. These findings suggest that even the lowest dose of ZnONPs is sufficient to release an amount of zinc that can interact with the corresponding receptor structures. However, higher doses only lead to saturation of serum zinc levels and reduced anxiolytic effects. Additionally, the performance improvement observed in both the control group and the rats treated with BZnO at 5 mg/kg may be attributed to their cognitive flexibility [44].

Earlier studies have demonstrated that the administration of nanoparticles to rats or mice, either systemically or intracerebrally, can result in cognitive function alterations and neurotoxicity. However, the extent of these effects depends on factors such as the type of nanoparticles used, the dosage administered, and the duration of exposure [45]. For example, Goma et al. [46] reported neurobehavioral toxicity for CuO-NPs.

The central nervous system (CNS) is sensitive to interactions with ions in physiological and pathological conditions [47]. For example, ZnONPs could leach zinc ions that diffuse to cells and/or organs like CNS [48]. CNS is protected by the blood-brain barrier (BBB), a critical barrier for regulating the transport of substances into the brain. ZnONPs were reported to cause alteration in the integrity and permeability of the BBB due to inflammation [49]. Furthermore, previous reports stated that ZnO dissolution is enhanced in the case of nanoparticles [50], which could have a role in ZnONPs toxicity [51].

Neurodegenerative illnesses have been linked to the accumulation of metals like copper (Cu), aluminum (Al), and zinc (Zn). These conditions are characterized by the loss of function and damage to cells in the affected areas [52]. Moreover, studies have reported that ZnONPs can disrupt zinc homeostasis in synapses. This disruption can result in hyperactive long-term potentiation and inadequate depotentiation, potentially affecting the normal functioning of neuronal synapses [53]. Furthermore, ZnONPs have been observed to induce intracellular calcium accumulation by enhancing sodium influx and increasing neuronal excitability. The concentration of intracellular calcium is crucial for long-term potentiation and long-term depotentiation in the induction and maintenance phases. These findings suggest that the toxicity of ZnONPs depends on the dosage rather than the size of the nanoparticles [54].

Like our study, De Souza et al. [55] indicated anxiogenic behavior in male mice subjected to ZnONPs by 300 mg/kg intraperitoneally for 5 days, which could be due to Zn accumulation in the brain or the impairment of anxiety-related neurological circuit function that can be attributed to the ability of these nanoparticles to interfere with normal neuronal activity. Torabi et al. [41] showed that ZnONP exposure by 20 mg/kg induced anxiety-like behaviors and reduced locomotor activity in rats after an intraperitoneal injection. However, a study demonstrated that administering ZnONPs with a size range of 20–30 nm at a dosage of 25 mg/kg via intraperitoneal injection for ten consecutive days resulted in minor alterations in the exploratory behavior of rats. However, no significant differences were observed in the anxiety index compared to the control group. These findings suggest that the effects of ZnONP exposure depend on factors such as the species being studied, the route and duration of exposure, and the concentrations or doses administered [56]. Another study by Xie et al. [24] showed that exposure to 5.6 mg/kg ZnONPs in mice damaged spatial cognition.

Furthermore, Han et al. [53] reported disturbance in the spatial cognition by ZnONPs within 20–80 nm injected intraperitoneally in rats by 4 mg/kg biweekly for 8 weeks that could be due to alteration of synaptic plasticity, enhanced long-term potentiation, and rigid cognitive flexibility due to repeated exposure. Zhao et al. [23] also reported that ZnONPs could potentially influence neurotransmitter disorders in the central nervous system for their regulatory role in the physiological functions of neurons. However, Chuang et al. [57] studied acute exposure to ZnONPs by 5 and 10 mg/kg but reported no significant changes in spatial cognition, learning ability, or anxiety. In contrast, our study, similar to previous research, found that the administration of 10 mg/kg of ZnONPs resulted in a decrease in the time spent in the open arms, indicating increased anxiety-like behavior.

As a cofactor for many enzymes, zinc is essential for many biological processes, including antioxidant defense mechanisms. This research looked into zinc oxide nanoparticles’ antioxidant and anti-inflammatory properties. Antioxidant enzymes such as glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) were found to have significantly increased levels. Furthermore, compared to the other treatment groups, those given ZnONP-5mg/kg showed substantially lower levels of MDA (a marker of oxidative stress) and the pro-inflammatory cytokines IL-6 and TNF-α. These findings suggest that ZnONPs possess antioxidant and anti-inflammatory properties. These results line up with what has been found before, suggesting the beneficial effects of zinc nanoparticles in modulating antioxidant and anti-inflammatory responses [58, 59]; according to their results, ZnONPs were found to reduce oxidative stress in testicular tissue by decreasing levels of MDA and increasing levels of antioxidants such as SOD, C.A.T., GPx, and GR. As well as, [60] reported that ZnONPs have a protective effect against cognitive impairment and anxiety caused by aging, mitigating associated oxidative damage. Zinc is essential for functioning antioxidant enzymes like SOD and is known to protect sulfhydryl groups by displacing other metals from catalytic sites. Therefore, ZnONPs can help preserve the integrity of cell membranes by shielding them from oxidative injury, increasing antioxidant levels, and reducing free radicals [61].

Compared to other tissues, the brain has a lower level of antioxidant enzymes, higher oxygen consumption, and more lipids, making it more susceptible to oxidative damage. Reactive oxygen species produced during aerobic metabolism mainly contribute to age-related neuronal damage [62]. The presence of ROS and their byproducts can have detrimental effects on DNA, proteins, and lipids. This can result in modifications to enzyme systems and trigger cell death pathways [60]. Our result revealed that The ZnONP-5 mg/kg treated group exhibited a notable reduction in brain levels of ROS related to the other treated groups. However, the BZnO-10 mg/kg group had higher levels of ROS. Conversely, in the BZnO-5 mg/kg treated group, a significant reduction in reactive oxygen species (ROS) levels was observed compared to the ZnONP-10 mg/kg and control groups.

The administration of ZnONPs to aged rats improved their oxidative status, bringing it closer to the levels observed in adult control rats. This indicates that zinc has a benefit in reducing oxidative stress [63]. Previous studies have shown that very low and very high zinc levels are correlated with increased oxidative injury. Nonetheless, moderate levels have been found to have a neuroprotective effect. This finding aligns with the current study, where an average dose of ZnONPs was utilized, providing a plausible explanation for the positive results observed with zinc supplementation [60].

The ZnONP-5 mg/kg group substantially reduced IL-6 and TNF-α levels compared to the other experimental groups, specifically the ZnONP-10 mg/kg and BZnO-10 mg/kg groups. Moreover, the BZnO-5 mg/kg group exhibited substantially lower levels of IL-6 and TNF-α than the ZnONP-10 mg/kg and BZnO-10 mg/kg treated groups. These results align with what other research has shown about the anti-inflammatory effects of zinc nanoparticles at lower doses [64]. For instance, a previous study reported that ZnONPs possess potent antioxidant activity, which can scavenge 45.46% of DPPH at a concentration of 1 mg/ml. Additionally, the study showed that ZnONPs dose-dependently suppressed the mRNA and protein expressions of vital inflammatory markers such as iNOS, COX-2, IL-1β, IL-6, and TNF-α. These findings collectively suggest that ZnONPs possess effective anti-inflammatory properties.

AChE is an essential enzyme involved in cholinergic neurotransmission, responsible for breaking down acetylcholine (ACh) to terminate synaptic transmission. Changes in AChE activity can directly impact ACh levels. Altered AChE activity has been associated with neurological disorders like Alzheimer’s disease [65]. Our study observed a substantial reduction in AChE activity in the brains of rats treated with ZnONPs. This suppressed AChE activity can hinder the degradation of ACh in the synaptic cleft, potentially leading to increased ACh accumulation. This finding supports our results regarding elevated ACh levels in the brain and suggests that the suppression of AChE activity is another potential mechanism contributing to this effect; our impact was also strengthened by [66] in which they found that the reduced activity of AChE in aged rats could be ascribed to the formation of free radicals during the aging process, which disrupts the balance between prooxidants and antioxidants in the brain. This could explain the beneficial effect of ZnONPs, which act as antioxidants, on the activity of AChE in aged rats. This effect brings the AChE activity levels closer to those observed in the control adult group [60].

Dopamine is an ample neurotransmitter in the CNS and is crucial in various cognitive functions [67]. Catecholamines, including dopamine, are essential for learning and memory [68]. Similarly, serotonin levels have been linked to learning and memory consolidation [69]. In this investigation, only animals displayed high BZnO-10 mg/kg doses, and ZnONP-10 mg/kg exhibited reduced neurotransmitter levels (dopamine and serotonin) in the brain tissue. Indeed, no substantial differences were observed in the BZnO-5 mg/kg and ZnONP-5 mg/kg treated groups compared to the control groups. This suggests that higher doses of zinc nanoparticles may be harmful, as the deposit of zinc ions in post-synaptic neurons can result in toxicity to nerve cells [70]

Brain-derived neurotrophic factor (BDNF) is an essential member of the neurotrophin family and plays a significant role in promoting neuron growth, survival, and differentiation. Research conducted on live organisms has demonstrated that BDNF enhances cholinergic neurotransmission in the central nervous system, influences neuronal plasticity, and protects against neurodegenerative disorders. BDNF achieves this protective effect by increasing the activity of endogenous neuroprotective strategies, including antioxidant pathways [71]. The gene expression analysis revealed a significant increase in the mRNA expression of BDNF in the ZnONP-5 mg/kg treated group linked to the other treatment groups. Similarly, the BZnO-5 mg/kg group showed a considerable upregulation of BDNF mRNA expression compared to the BZnO-10 mg/kg and ZnONP-10 mg/kg treated groups. Consistent with prior studies, these results show that zinc nanoparticles have them, particularly at lower doses, to enhance the expression of BDNF, which is associated with neuroprotection and neuronal plasticity [72]. Their study proved that ZnONP treatment leads to increased BDNF gene expression, counteracting the hippocampal neurotoxicity induced by type 2 diabetes in rats.

The administration of monosodium glutamate and ZnO nanoparticles significantly increased brain tissue levels of BDNF in rats [73]. This suggests that zinc has a positive influence on reducing oxidative stress. Previous studies have demonstrated that both zinc deficiency and excessive zinc levels are associated with elevated oxidative stress. However, moderate levels of zinc have been found to have neuroprotective effects. The current findings explain the beneficial effects observed in this study, where an appropriate dose of ZnONPs was administered [74]. Therefore, it can be suggested that the neuroprotection offered by ZnONPs may be directly related to their antioxidant pursuit.

Our findings were consistent with [75] study, in which they showed a noteworthy reduction in the expression levels of iNOS (inducible nitric oxide synthase) and NF-κB (nuclear factor-kappa B) in both the ZnONP-5 mg/kg and BZnO-5 mg/kg groups associated to the BZnO-10 mg/kg and ZnONP-10 mg/kg groups. These consistent results provide additional evidence supporting the potential anti-inflammatory properties of lower doses of zinc nanoparticles. Previous studies, such as the one conducted by Kim [75], have reported that ZnO nanoparticles exhibit anti-inflammatory consequences by stopping the boost in inflammatory cytokines and inhibiting the activation of NF-κB. Activation of the NF-κB pathway fronts to the induction of various inflammatory cytokines and chemokines. Once activated, NF-κB translocates to the nucleus and binds to specific DNA sequences, thereby promoting the production of inflammatory mediators such as cyclooxygenase-2, inducible nitric oxide synthase, tumor necrosis factor-α, interleukin-1β, and interleukin-6. The ability of zinc nanoparticles to inhibit NF-κB activation and suppress the creation of these inflammatory mediators suggests their potential as anti-inflammatory agents [64, 76]

The ZnONP-5 mg/kg group showed a considerable enhancement in the anti-apoptotic gene bcl-2 expression, while a substantial reduction in the expression of the pro-apoptotic gene Bax was observed. The BZnO-5 mg/kg bwt group also exhibited similar gene expression patterns compared to the control, BZnO-10 mg/kg bwt, and ZnONP-10 mg/kg bwt groups; these findings aligned with Tuzcu et al. [77]. They suggested that ZnONPs can mitigate the effects of cisplatin by preventing apoptosis. This is achieved by stabilizing biomembranes with zinc and its indirect antioxidant activity. In the same context, the immunohistochemistry of caspase-3 protein expression strengthens our finding concerning apoptosis, as reported by Barakat et al. [78]. They said that the treatment with ZnONPs demonstrated a protective effect on the kidneys against the toxic effects of cisplatin by its antioxidant, anti-inflammatory, and antiapoptotic properties; zinc nanoparticles exhibit a range of beneficial effects. These properties allow zinc nanoparticles to counteract oxidative stress, reduce inflammation, and prevent cell death.

The histological findings are consistent with the behavioral, molecular, and biochemical data. The histological examination revealed that the control and ZnONP-5 mg/kg treated rats had normal meninges, neurons, and cerebral cortex structures. However, ZnONP-10 mg/kg treated rats accompanied considerable abnormalities such as shrinkage, pyknosis, hyper-eosinophilic cytoplasm, widening of neuropil, satellitosis, neuronophagia, glial cell aggregates, and vascular congestion in the choroid plexus. The rats treated with BZnO-5 mg/kg exhibited cerebral cortex, hippocampus, and cerebellum with histological structures that were almost normal. On the other hand, the rats treated with BZnO-10 mg/kg showed significant shrinkage of cortical neurons, loss of dendritic arborization in Purkinje cells, gliosis (an abnormal increase in glial cells), and areas of tissue damage.

Additionally, there were a few instances of a reduced number of granular cells in the cerebellar cortex. The histopathological analysis revealed no changes in the rats treated with ZnONPs linked to the control groups. These results align with previous studies conducted by Wang et al. [79], Zheng et al. [80], and Hamza et al. [73], which furthermore demonstrated the lack of toxic effects of ZnONPs on brain tissue. In contrast, Win-Shwe et al. reported poisonous results of ZnONPs on the CNS, including cytotoxicity, inflammation, and induction of oxidative stress leading to neurodegeneration [81]. In contrast, Gantedi et al. conducted a study and reported that a dose of 5 mg/kg of ZnONPs did not lead to noticeable alterations in the brain histology of rats after one month of treatment [82]. It is essential for both laboratory and clinical studies to carefully consider the duration, dosage, and route of administration when evaluating the cellular toxicity of ZnONPs in different organs, including nerve tissue. These factors significantly contribute to determining the specific characteristics and potential toxicity associated with the nanoparticles.

Conclusion

This study pioneered investigating the effects of different forms of zinc salt, explicitly comparing the impact of zinc oxide nanoparticles (ZnONPs) and bulk zinc on various aspects of rat neurobehavior, antioxidant levels, gene expression, and brain histopathology. The results indicated that a lower dosage of 5 mg/kg ZnONPs had a beneficial effect on multiple aspects of behavior and brain function, including memory, recognition, learning ability, and neurotransmitter levels. A higher dosage of 10 mg/kg could be detrimental. Furthermore, the study emphasizes the necessity for additional investigations to ascertain the safety profile of ZnONPs on cognitive function and brain health fully.

Author contribution

Conceptualization, Amira A. Goma, Hossam G. Tohamy. Methodology, Amira A. Goma, Hossam G. Tohamy, Rashed R. Rashed, Mustafa Shukry, Alyaa R. Salama, Sara E. El-Kazaz. Software, Amira A. Goma, Mustafa Shukry, Hossam G. Tohamy. Validation, Amira A. Goma, Hossam G. Tohamy, Mustafa Shukry. Formal analysis, Amira A. Goma, Alyaa R. Salama, Hossam G. Tohamy, Mustafa Shukry. Investigation, Amira A. Goma, Hossam G. Tohamy, Mustafa Shukry , Sara E. El-Kazaz, Alyaa R. Salama, Rashed R. Rashed. Resources, Amira A. Goma, Hossam G. Tohamy, Sara E. El-Kazaz, Mustafa Shukry. Data Curation, Amira A. Goma, Hossam G. Tohamy, Mustafa Shukry. Writing - Original Draft, Amira A. Goma, Sara E. El-Kazaz, Hossam G. Tohamy, Mustafa Shukry, Alyaa R. Salama. Writing - Review & Editing, Amira A. Goma, Hossam G. Tohamy, Rashed R. Rashed, Mustafa Shukry, Alyaa R. Salama, Sara E. El-Kazaz. Visualization, Amira A. Goma, Hossam G. Tohamy, Mustafa Shukry. Supervision, Amira A. Goma, Hossam G. Tohamy, Mustafa Shukry. Project administration, Amira A. Goma, Mustafa Shukry, Hossam G. Tohamy.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Footnotes

References