Histological and immunohistochemical alterations in the brain tissues induced by the subchronic toxicity of gold nanoparticles: In vivo study

Abstract

Background

Gold nanoparticles (Au NPs) have emerged as major contributors for innovative technologies and have been used extensively in various biomedical and industrial fields with little, if any, known about their neurotoxicity.

Objective

The current study aims to explore the nanotoxicity of Au NPs on the brain tissues.

Materials and methods

Two experimental groups, a control one and an Au NPs-treated group, each comprising 10 adult male Wistar albino rats, were used. Nanoparticle-treated rats received 28 intraperitoneal injections of 10 nm Au NPs at a daily dosage of 2 mg/kg.

Results

Brain tissue specimen for each rat under study was subjected to histological, immunohistochemical, and morphometric examination for alterations that might be induced by Au NPs exposure. Compared with control animals, brain tissue of rats treated with Au NPs exhibited neuronal shrinkage and pyknosis, perineuronal spacing, glial cell proliferation, vascular congestion, and neurons with lipofuscin pigmentation. Moreover, the hippocampus exhibited shrunken neurons, vascular congestion, and perivascular edema. Furthermore, the cerebellum showed degenerated Purkinje cells, cerebellar congestion, and perivascular spacing. In addition, the neuronal tissue demonstrated decreased autophagy, astrogliosis, and apoptosis presented by substantially decreased immunohistochemical protein expression of Beclin 1, increased expression of GFAP and caspase 3, respectively.

Conclusion

The findings suggest that exposure to Au NPs has the potential to cause histological, immunohistochemical, and morphometric changes in brain tissue, which could impact the function of this vital organ. Further endeavors are necessary for more understanding of the potential risks of Au NPs to human health.

Introduction

Gold nanoparticles (Au NPs) are a class of metallic nanomaterials that have gained widespread application in medical diagnostics, therapeutics, and industrial processes due to their unique physicochemical properties, including small size, large surface area-to-volume ratio, and tunable surface chemistry.^1,2 They are incorporated into photodynamic therapy, drug delivery systems, photothermal therapy, biosensors, and X-ray imaging modalities. ³ In oncology, Au NPs have been investigated for targeted gene delivery and cancer therapy, particularly in conditions such as benign prostatic hyperplasia and renal carcinoma.^4,5

One of the notable advantages of Au NPs is their capacity to cross the blood-brain barrier (BBB), a property that has expanded their potential in diagnosing and treating brain disorders, including gliomas and neurodegenerative diseases.^6,7 Their high atomic number and associated Auger effect enhance their efficacy in imaging and radiation-based therapies. ⁸ Au NPs have also been applied in retinal molecular imaging and cell labeling. ⁹

Despite these promising applications, concerns regarding the safety profile of Au NPs are growing. Evidence indicates that these nanoparticles exhibit biological reactivity distinct from their bulk counterparts and can accumulate in critical organs such as the brain.^10–12 Their small size and increased surface reactivity raise the possibility of crossing biological barriers and interacting directly with cellular components.^13,14 Several studies have demonstrated their capacity to infiltrate neural tissues and localize in neurons, astrocytes, and microglia.^15,16 Moreover, they may trigger neurotoxic mechanisms including oxidative stress, mitochondrial dysfunction, inflammatory responses, and interference with neural signaling pathways.^17,18

Importantly, Au NP-induced oxidative stress has been associated with reactive astrogliosis, impaired axonal regeneration, and apoptotic neurodegeneration. These effects are thought to involve dysregulation of key cellular processes such as apoptosis and autophagy, which are critical in maintaining neuronal homeostasis. ¹⁹ The neurobehavioral consequences of these disruptions include altered memory, cognition, and attention. Additionally, systemic toxicity involving hepatic, renal, and cardiac tissues has also been observed.^20–22

Some reports on the mechanisms through which Au NPs may induce neurotoxicity suggested oxidative stress, apoptosis, inflammation, and DNA damage.^10,12 However, mechanisms involved in Au NPs neurotoxic effects, particularly following subchronic exposure, remain insufficiently characterized with little data, if any, on their histopathological and immunohistochemical impact on brain tissues are still limited.

Neurotoxicity can be indicated by several biomarkers including Beclin-1, GFAP, and Caspase-3. Beclin-1 plays a crucial role in autophagy initiation by contributing to the formation of the isolation membrane, a double-membrane structure that engulfs cytoplasmic components to form the autophagosome. ²³ Glial fibrillary acidic protein (GFAP) is a structural protein expressed by astrocytes widely used as a marker of astrogliosis and neuroinflammation triggered by toxic insult, neural injury, and oxidative stress. ²⁴ Caspase-3 plays a key role in the apoptotic pathway of cell death and is often increased in response to mitochondrial damage or accumulation of ROS. ²⁵

Based on previous evidences, we hypothesize that subchronic exposure to Au NPs induces neurotoxic effects by disrupting autophagy, triggering apoptosis, and promoting astrogliosis. These alterations are expected to manifest as distinct histopathological and immunohistochemical changes in brain tissues. Accordingly, this study was designed to test this hypothesis and elucidate the underlying mechanisms.

Materials and methods

Gold nanoparticles

Spherical colloidal monodisperse Au NPs (10 nm, the most usable size in nanomedicine) suspended in 0.1 mM PBS were purchased from Sigma-Aldrich (St Louis Missouri, Millipore, USA). According to the manufacturer’s certificate, Au NP dispersion met the following specifications: 5.98 × 10^12 nanoparticles/ml; 1.01 × 10^8 M Ext M-1 Cm-1 concentration; 10 nm size; 3.16 × 10-12 (cm²) surface area for each particle; reactant-free; and absorption at about 520 nm.

Experimental Animals

In the current study, 20 healthy male Wistar albino rats (Rattus norvegicus) weighing 175–195 g and between 11 and 12 weeks of age were used. All animals were kept in polypropylene cages in a well-ventilated room under controlled circumstances (50%–60% humidity; 24 ± 2°C temperature; 12 h/12 h light/dark cycle) with 1-week acclimation prior to the experimental study. Animals had free access to standard rodent laboratory diet and water ad libitum.

Experimental protocol and tissue sampling

The rats were divided into two experimental groups, each with 10 rats: a control group and a treated group. Group size was chosen to provide a balance between statistical power, biological variability, ethical considerations, and to provide meaningful results without excessive animal use. Animals were randomized into control and AuNP-treated groups using a random number generator, with body weight stratification (≤10% variation) to minimize variability. Group allocation was concealed, and all investigators were blinded to treatment assignments.

The treated rats received intraperitoneal injections of 2 mg/kg bw of Au NPs once daily, 5 days per week (Saturday to Wednesday), for a total of 28 injections administered over 38 days. Prior to injection, gold nanoparticles were warmed to 37°C. Using the same protocol, 0.1 ml of 0.1 mM PBS was given to rats of the control group as a vehicle at 37°C. The dosage was selected based on earlier studies on the nanotoxicity of Au NPs. ²⁶ AuNPs were selected due to their increasing biomedical applications, particularly in diagnostics and drug delivery, as well as their ability to cross the blood-brain barrier. However, concerns have been raised regarding their potential neurotoxic effects. Our study aims to evaluate these possible toxicological outcomes.

Twenty-four hours after the last experimental dose, the rats were anesthetized using Xylazine/Ketamine at a dose of 80/8 mg/kg dissolved in normal saline via the intraperitoneal route. Once deep anesthesia was confirmed, the animal heads were perfused with paraformaldehyde (2%) prepared in 0.01 M PBS. Euthanasia was performed by exsanguination via severing the carotid arteries and jugular veins in the neck following the American Veterinary Medical Association (AVMA) Guidelines on euthanasia of rats and in accordance with CCAC and ARRIVE guidelines. Confirming an animal’s death through the cessation of heartbeat and respiration was assessed by observing the chest for lack of movement and palpating the thorax to ensure the cessation cardiac activity prior to brain tissue collection. Confirming an animal’s death is a standard practice to ensure humane euthanasia. Then, the skull vaults were opened, and the entire brains were quickly extracted and fixed in neutral buffered formalin (10%) for 36 h.

All animal procedures have been reviewed and approved by the local Ethics Committee of Jerash University for the Use of Experimental Animals (Approval number 1/10/2023/2024 issued by the Jerash University Animal Ethics Committee) in compliance with the Canadian Council on Animal Care’s (CCAC, 2019) requirements, and in accordance with the ARRIVE guidelines. Moreover, the study design followed the 3 Rs (Replacement, Reduction, and Refinement) principles. The number of animals used was minimized while ensuring statistical power, and all procedures were refined to reduce pain and discomfort.

Histological processing

Formalin-fixed midsagittal sectioned brain tissue specimens were subjected to dehydration using ascending grades of ethanol, clearance in chloroform, impregnation and embedding in paraffin wax with a melting point of 58°C.^27,28 Paraffin sections, 4 µm thick, were prepared from all samples and stained with hematoxylin and eosin (H&E), examined under a light microscope and photographed for potential alterations in the frontal cortex, hippocampus, and cerebellar cortex.

Immunohistochemistry and morphometric analyses

Immunohistochemical stains were applied via the streptavidin-biotin-peroxidase technique according to the procedures described by Torlakovic et al.. ²⁹ Formalin-fixed brain specimens were processed and analyzed to assess the expression of protein markers: Beclin-1, GFAP, and Caspase 3. For this analysis, the following primary antibodies were used: Beclin-1 was detected using a rabbit polyclonal antibody (Cat #3738, Cell Signaling Technology), diluted 1:100 and incubated at 4°C overnight. GFAP was identified using a mouse monoclonal antibody (Cat #SC-58,766, Santa Cruz Biotechnology), with a dilution of 1:50 and incubation at 4°C overnight. Caspase-3 expression was assessed using a rabbit polyclonal antibody (Cat #PA1-26,426, Fisher Scientific, Lab Vision Corporation Laboratories), diluted 1:100 and incubated overnight at 4°C. To validate staining specificity, negative control sections were prepared by following all identical steps and reagents, except for the omission of the specific primary antibodies. All histological and immunohistochemical sections were coded to conceal group identity. Assessments were performed independently by two blinded observers to minimize bias.

For morphometric analysis, Beclin-1, GFAP, and Caspase 3 protein expression was quantified by measuring immunoreactive area in five non-overlapping high-power fields at 400× magnification per brain region, per animal. The area % of Beclin1, GFAP, and Caspase 3 protein expressions were estimated using FIJI/ImageJ software (Version 1.51; NIH, USA) in accordance with Alsemeh et al.. ³⁰

Statistical analysis

Version 28 of the Statistical Package for the Social Sciences (SPSS) from IBM Corp. (Armonk, NY) was used to analyze the collected data. The morphometry data were summarized as mean ± standard deviation (SD). To perform the comparisons between control and Au NPs-treated groups, the independent t-test was employed. Statistical significance was defined as p < 0.05. (*) indicates a statistically significant difference between the control and Au-NPs treated groups when p < 0.05 while (***) denotes a highly statistically significant difference when p < 0.0001.

Results

No mortality or signs of illness were noted in all rats under study. In comparison to the control rats, herein the histological as well as the immunohistochemical alterations in brain tissues caused by long-term exposure to 10 nm Au NPs.

Histological alterations

Exposure to 28 injections of 10 nm Au NPs (2 mg/kg) induced the following histological changes in the brain tissue.

Frontal cortex histological changes

Frontal cortex of the control rats demonstrated normal neurons appeared as small spherical neurons in the outer cortical layers but large pyramidal in shape in the deep cortex. In addition, the neurons were arranged in a regular pattern with large rounded and vesicular open face nuclei. The cerebral neurons toward the white matter appeared more developed (Figure 1(a), 1(c), 1(e)). On the other hand, Au NPs-treated rats’ cerebral cortex exhibited a heterogeneous pattern of neurons. Shrunken darkly stained neurons with lipofuscin pigmentation and pyknotic nuclei occasionally surrounded by pericellular hallos among normal neurons with normal rounded nuclei were observed. Furthermore, glial cells proliferation, dilated congested capillaries, wide perivascular spacing, and vacuolated neuropil were also noted (Figure 1(b), (d) and (f)).

Figure 1.

(a–f). Photomicrographs of frontal cortex sections displaying (a–b) the external cortical layers, (c–d) the internal cortical layers, and (e–f) the deepest cortex at the junction with white matter. (a, c, e) The control sections display normal neurons (blue arrow), glial cells (blue arrowhead), capillaries (blue elbow arrow), and neuropil (blue*). (b, d, f) Sections of AuNP-treated rats showing neurons with nuclear pyknosis, occasional lipofuscin deposits, and perineuronal halos (white arrow); glial proliferation (white arrowhead); capillary congestion with wide perivascular spacing (white elbow arrow); and neuropil vacuolation (white*). H&E ×400; Scale bar = 50 μm.

Hippocampus histological changes

The first (CA1) and third (CA3) partitions of Cornu Ammonis (CA) of the hippocampus in the control rats displayed well-defined three layers; polymorphic, pyramidal cell (main layer), and molecular layers (Figure 2(a) and (c)). The pyramidal cell layer demonstrated small rounded neurons in CA1 but larger triangular shaped neurons in CA3. The neurons exhibit large rounded vesicular nuclei. The polymorphic and molecular layers demonstrate glial cells and blood capillaries. Similarly, the dentate gyrus in the control hippocampus (Figure 2(e)) presented three layers: polymorphic; granular (the principal cell layer being formed of small rounded neurons), and molecular cell layers.

Figure 2.

(a–f). Photomicrographs of hippocampal sections displaying (a–b) the CA1 region and (c–d) the CA3 region both show well-defined 3 layers, including the polymorph (PO), pyramidal (P), and molecular (M) layers. The dentate gyrus (e–f) shows well-defined 3 layers, including the polymorph (PO), granular (G), and molecular (M) layers. (a, c, e) Control sections show well-defined layers with normal pyramidal and granular neurons (blue arrow) and glial cells (blue arrowhead). (b, d, f) Sections of AuNP-treated rats show neurons with nuclear pyknosis and irregular dark staining (white arrow); glial proliferation with deep staining (white arrowhead); vascular congestion with perivascular spacing (white elbow arrow); and disrupted hippocampal architecture. H&E ×400; Scale bar = 50 μm.

Comparable to the control rats, the hippocampus of Au NPs-treated rats showed shrunken irregular deeply stained neurons with pyknotic nuclei. In addition, the polymorphic and molecular layers exhibited numerous deeply stained glial cells, vascular congestion, and perivascular edema (Figure 2(b), (d) and (f)).

Cerebellar histological changes

Sections of the cerebellar cortex obtained from the control rats exhibited three layers; granular, Purkinje, and molecular cell layers. The Purkinje cells were arranged in a single row of large neurons characterized by large rounded pale prominent nuclei (Figure 3(a)).

Figure 3.

(a–d). Photomicrographs of cerebellar cortex sections. (a) Control section showing distinct molecular (M), Purkinje (P), and granular (G) layers, with normal Purkinje cells (blue arrow) and capillaries (blue elbow arrow). (b–d): Sections of Au NPs-treated rat. (b) Reveals reduced numbers of Purkinje cells, pyknotic neurons (white arrow), and vacuolation within the cerebellar cortex. Notably, one Purkinje cell (short arrow) is entirely displaced within the granular layer. (c) Shows Purkinje cells with wrinkled cell boundaries and indistinct nuclear profiles (white arrow), arranged in more than one layer. (d) Exhibits vascular congestion and a wide perivascular space (white elbow arrow). H&E ×400; Scale bar = 50 μm.

In comparison with the control rats, the cerebellar cortex of the Au NPs-treated ones demonstrated changes that included reduced numbers of Purkinje cells, pyknotic neurons, and vacuolation within the cerebellar cortex. Occasional instances of complete dislodgement within the granular layer (Figure 3(b)). In addition, some Purkinje cells presented corrugated cell boundaries with indistinct nuclear profile appeared to be arranged in more than one layer (Figure 3(c)). The cells of the granular layer exhibited darkly stained nuclei with vascular congestion and wide perivascular spacing were noted (Figure 3(d)).

Immunohistochemical alterations

Immunohistochemistry and morphometry measurement of expression area (%) in the selected brain regions of all rats under study were investigated to examine the expression of particular genes involved in various biological processes. Compared with the control rats. Au NPs-treated rats demonstrated the following immunohistochemical changes.

Autophagy reduction

Beclin 1 protein immunostaining showed an average immune expression in the selected brain regions, including the frontal cortex, hippocampus (CA1, CA3, and dentate gyrus), and cerebellar cortex of the control rats, as shown in Figure 4(a), (c), (e), (g), and (i), respectively. However, exposure to Au NPs was associated with a highly significant decrease (P < 0.0001) in Beclin 1 protein immunoexpression in the corresponding brain regions relative to those of the control animals, as shown in Figure 4(b), (d), (f), (h), (j), represented in Bar Charts 4 (I, II, III, IV, V), and summarized in Table 1 (A), respectively.

Figure 4.

(a–j). Photomicrographs of Beclin1-immunostained sections: Subfigure (a, c, e, g, i) of the frontal cortex, CA1, CA3, dentate gyrus, and cerebellar cortex of the control rats, respectively, exhibiting an average immune expression (black arrow). Subfigure (b, d, f, h, j) of the frontal cortex, CA1, CA3, dentate gyrus, and cerebellar cortex of Au NPs-treated rats, respectively, demonstrating diminished expression relative to the corresponding control ones (blue arrow). Beclin1 immunostaining ×400, Scale bar = 50 μm. Bar charts (I–V): showing statistical assessment of Beclin1 immune expression in the selected brain regions of the two studied groups. Values are expressed as mean ± standard deviation (SD). (***) denotes a high statistically significant difference when p < 0.0001.

Table 1.

Morphometric assessment of area % (Mean ± SD) relevant to Beclin1, GFAP, and Caspase 3 immunohistochemical protein expression in the selected brain regions.

Marker	Brain region	Control (Mean ± SD)	AU NPs (Mean ± SD)	Independent T-test (p value)
A- Beclin 1 (mean area % ± SD)	Frontal cortex	4.886 ± 0.758	1.1875 ± 0.293***	<0.0001
	CA1	6.612 ± 1.174	1.458 ± 0.224***	<0.0001
	CA3	4.885 ± 0.592	2.399 ± 0.331***	<0.0001
	Dentate	11.449 ± 1.514	3.269 ± 0.719***	<0.0001
	Cerebellum	4.953 ± 0.687	1.825 ± 0.391***	<0.0001
B- GFAP (ZMean area % ± SD)	Frontal cortex	1.846 ± 0.558	8.643 ± 1.155***	<0.0001
	CA1	3.549 ± 0.912	16.927 ± 2.227***	<0.0001
	CA3	4.471 ± 0.922	11.304 ± 1.664***	<0.0001
	Dentate	4.111 ± 1.549	14.392 ± 2.462***	<0.0001
	Cerebellum	1.954 ± 0.413	10.314 ± 1.817***	<0.0001
C- caspase 3 (mean area % ± SD)	Frontal cortex	7.032 ± 0.798	20.477 ± 2.007***	<0.0001
	CA1	2.218 ± 0.640	11.997 ± 1.056***	<0.0001
	CA3	5.015 ± 0.549	16.304 ± 1.664***	<0.0001
	Dentate	3.111 ± 0.469	13.028 ± 2.806***	<0.0001
	Cerebellum	1.979 ± 0.517	14.695 ± 1.946***	<0.0001

Values are expressed as Mean area % (percentage of immunopositive area). Independent t-test was used for comparisons between control and AuNP-treated groups (n = 10 each). Significance: “*p < 0.05; ***p < 0.0001 vs. control. Highly significant differences are indicated by p < 0.0001.

Astrogliosis induction

Compared to the immuneexpression of GFAP protein in the control rats, exposure to Au NPs was highly significant (P < 0.0001) associated with increased immuneexpression of this protein in the corresponding brain regions. This was seen in the frontal cortex, hippocampus (CA1, CA3, dentate gyrus), and cerebellar cortex, as observed in Figure 5(a), (c), (e), (g), (i) and 5(b), (d), (f), (h), (j), represented in Bar Charts 5(I, II, III, IV, V), and summarized in Table 1(B).

Figure 5.

(a–j). Photomicrographs of GFAP-immunostained sections: Subfigure (a, c, e, g, i) of the frontal cortex, CA1, CA3, dentate gyrus, and cerebellar cortex of the control rats, respectively, exhibiting a few minor-sized astrocytes with small, thin processes (black arrow). Subfigure (b, d, f, h, j) of the frontal cortex, CA1, CA3, dentate gyrus, and cerebellar cortex of Au NPs-treated rats, respectively, revealing numerous prominent astrocytes with dense processes (blue arrows). GFAP immunostaining ×400, Scale bar = 50 μm. Bar charts (I–V): showing statistical valuation of GFAP immunoexpression in the different brain regions of both control and AU NPs-treated groups. Values are expressed as mean ± standard deviation (SD). (***) indicates a high statistically significant difference when p < 0.0001.

Apoptosis induction

In comparison with the control rats, exposure to Au NPs was highly significantly associated with increased Caspase 3 protein immunoexpression (p < 0.0001). This increase was observed in various brain regions, including the frontal cortex, CA1, CA3, dentate gyrus, and cerebellar cortex. These findings are shown in Figure 6(a), (c), (e), (g), (i) and (b), (d), (f), (h), (j). They are also illustrated in Bar Charts 6 (I, II, III, IV, V). A summary of the data is provided in Table 1(C).

Figure 6.

(a–j). Photomicrographs of caspase 3 immunostained sections of rat brain. Subfigure (a, c, e, g, i) of the frontal cortex, CA1, CA3, dentate gyrus, and cerebellar cortex of the control rats, respectively, exhibiting minimal expression (black arrow). Subfigure (b, d, f, h, j) of the frontal cortex, CA1, CA3, dentate gyrus, and cerebellar cortex of Au NPs-treated rats, respectively, revealing strong expression (blue arrow). Caspase 3 immunostaining ×400, Scale bar = 50 μm. Bar charts (I–V): showing statistical valuation of Caspase 3 immunoexpression in the selected brain regions of both study groups. Values are represented as mean ± standard deviation (SD). (***) specifies a high statistically significant difference when p < 0.0001.

Discussion

There is a growing use of Au NPs in the diagnosis and treatment of brain cancer and neurodegenerative diseases due to their capability to pass through the blood-brain barrier and facilitate targeted drug delivery.^31–33 In addition, Au NPs can traverse the respiratory epithelium and translocate into the brain tissue, ³⁴ raising concerns about potential neurotoxicity. Moreover, previous studies stated that Au NPs could induce injury to the vital body organs, including the liver and kidney.^20–22 The authors suggested that the affected tissues were unable to manage the accumulated residues might be resulted from metabolic and structural disturbances caused by these nanoparticles, primarily through oxidative stress and disruption of antioxidant defenses. The neurotoxic effects of Au NPs observed in the current study are consistent with previous studies,^11,35,36 who attributed Au NP neurotoxic effects to neural structure changes and/or alterations in the activity of the nervous system that can occur through oxidative stress mechanisms. Similar neuronal cell loss and disintegration were observed in a study involving silica nanoparticles, ¹⁹ where similar toxicity mechanisms involving oxidative stress, glutathione depletion, and lipid peroxidation were reported.

The findings of the current study suggest that Au NPs exposure could lead to significant changes in both the structure and function of the brain. Some studies suggest that organs rich in blood flow, including the brain, are particularly vulnerable to Au NPs toxicity owing to their prolonged circulation in the bloodstream.^31,37

The susceptibility of the brain may stem from the reliance of the neural tissue on external energy sources, high level of unsaturated fatty acids, coupled with its high metabolic demand, accounting for approximately 15% of cardiac output and due to the relatively low levels of antioxidant enzymes. ³⁸

The findings of the present study showed that the neurons of Au NPs-treated rats exhibited shrinkage and pyknotic nuclei, mainly seen in the frontal cerebral cortex, and in the hippocampus. This alteration could slow neurotransmitter systems that may affect memory, attention, cognition, learning, and behavioral disruption leading to anxiety, depression, and mood swings. ³⁹ The detection of lipofuscin pigment within neurons of the frontal cortex may indicate cellular aging or metabolic stress. Lipofuscin is a product of lipid peroxidation and is often considered a marker of cellular damage and oxidative stress. ⁴⁰

The present findings revealed that subjection to Au NPs could induce Purkinje cells shrinkage, degeneration with corrugated cell boundaries as well as pyknosis of granular cell nuclei. Such changes were associated with Caspase 3 positive immune reaction in the Purkinje and granular cells indicating apoptosis induction associated with Au NPs exposure. Purkinje cells’ disarray may have been caused by cell shrinkage, which was followed by the withdrawal of their protoplasmic processes due to the cytoskeletal elements’ disintegration. ⁴¹ Previous research revealed that granular cell disruption could be secondary to Purkinje neuronal degeneration which in turn led to gradual loss of synchronization between both neurons. ⁴²

The injury of neuroglia is indicative of various neurological disorders most likely affects coordinated motor movement, posture, motor skills, balance and may manifest muscle weakness and ataxia. ⁴³ In addition, the findings of the current work showed that Au NPs could induce neuroinflammation, hippocampal edema and vascular congestion. Previous studies confirmed that neuroinflammation is associated with the release of cytokines could affect the blood brain barrier function, which can lead to severe neurological disorders.^35,44 Moreover, hippocampal edema may specify that Au NPs exposure could induce fluid accumulation in the neural tissues that may lead to memory problems, concentration difficulties, and confusion. ⁴⁵

Gliosis is considered as a response to CNS injury especially astrocytes and microglia as a protective response to injury by increased glial fibrillary proteins. ⁴⁶ The findings of the present study revealed that exposure to gold nanomaterials may induce glial cell proliferation (gliosis). This finding agrees with that of Shi, ⁴⁷ where inhalation of carbon nanoparticles led to inflammatory response by activating microglia cells. It has been reported that brain insult due to Au NPs exposure triggers the microglia to release pro-inflammatory cytokines, promoting neuronal degeneration. ⁴⁸ Moreover, gliosis activates cytokines and chemokines release to exacerbate inflammation and could lead to cognitive deficits, sensory disturbances, and motor impairments.^49,50 Furthermore, the notable dilated congested capillaries and perivascular spacing in Au NPs treated brain tissue were largely associated with the inflammatory response elicited by astrocytes. Some previous studies reported that Au NPs could trigger and elicit reactive astrogliosis in mice’s brain with evident mutual correlation between the glial cell proliferation and the redox state in the brain tissue.^51,52

The present study’s findings showed that exposure to Au NPs induced reactive astrocytosis by the apparent increase of glial cells as a defensive reaction. ⁵³ In addition, Au NPs-treated rats, demonstrated neuronal degeneration confirmed by activation and proliferation of astrocytes represented as the concomitant upregulation of GFAP immune-expression. This gene is included in astrocytes cytoskeleton giving these cells structural support. Reactive astrocytes limit damage by erecting barriers to stop inflammation spread to support post lesion regeneration. ⁵⁴ As a response to an insult affecting astrocytes-nearby neurons, astrocytes start reactive astrocytosis detrimentally by releasing pro-inflammatory cytokines, ROS, and neurotoxins. ⁵⁵ A previous study reported that exposure to Au NPs has been associated with neuroinflammatory responses and disruptions in the BBB function, which could potentially lead to vascular leakage and perivascular edema in the nervous tissues. ⁵⁶ There is an evident mutual correlation between astrocyte proliferation and the redox state in the brain tissue. Reactive astrogliosis was defined in numerous oxidative stress conditions in which astrocytes actively liberate pro-inflammatory cytokines and ROS that result in neuronal damage. ⁵⁰ On the other side, ROS that were released during the oxidative stress state could induce the release of inflammatory factors signaling pathways in the astrocytes. ⁵⁷

Autophagy is an intracellular process by which cells break down and unwanted components are recycled using lysosomes. This process is essential for preserving cellular homeostasis by removing damaged proteins, organelles, and other waste. ⁵⁸ When autophagy is disrupted, protein buildup can occur, which is a common feature in neurodegenerative diseases. ⁵⁹ The current work’s findings indicate that Au NPs exposure could reduce the capability of Beclin-1 in autophagy regulation and membrane endocytic trafficking in brain tissue suggesting neurotoxicity via reducing autophagy. This might be resulted from lysosomal malfunction as autophagosome formation is subsequently inhibited. ⁶⁰ Furthermore, stress induced by exposure to Au NPs may impair the cellular adaptive systems including autophagy. ⁶¹ However, autophagy may be inhibited by both oxidative and ER stresses leading to cellular stress accumulation and promotes neuronal vulnerability to degeneration.^62–64

The findings of the present work could indicate that Au NPs might increase the immunoexpression of Caspase 3. This suggests a potential link to neuronal apoptosis.

Such alterations were also mirrored by neuronal shrinkage and nuclear pyknosis. ⁶⁵

These effects were reinforced by a significantly raised mean area (%) of Caspase 3 protein expression. This result is consistent with recent studies that exposure to Au NPs is associated with apoptosis induction, excessive production of ROS, and oxidative stress.^66,67 Apoptosis is probable a downstream effect of continuous glial activation and autophagy suppression, which together tip the cellular balance toward apoptosis. ⁶⁸

Reactive astrogliosis (GFAP ↑) reflects a heightened glial response that promotes inflammatory cytokine and ROS release, hence intensifying neuronal stress. ⁶⁹ In parallel, reduced Beclin-1 suppresses autophagic clearance, leading to the accumulation of damaged organelles and proteins, which in turn shifts the balance toward apoptotic signaling. ⁷⁰ Activated Caspase-3 not only executes apoptosis but also further impairs autophagy through degradation of autophagy proteins such as Beclin-1. ⁷¹ Collectively, these pathways establish a feed-forward loop in which neuroinflammation, autophagy suppression, and apoptosis reinforce one another, driving progressive neuronal injury. The Au NP-induced alterations observed in this study (GFAP upregulation, Beclin-1 reduction, and Caspase-3 activation) thereby reflect a mechanistic cascade consistent with known neurotoxic responses to environmental stressors and may underlie Au NP-induced neurodegeneration.

Conclusion

The present study demonstrates that exposure to gold nanoparticles induces marked neurotoxic effects, reflected by significant histological and immunohistochemical alterations in brain tissue. These effects appear to be mediated through impaired autophagy, astrogliosis, neuroinflammation, and apoptosis. Further studies are needed to clarify the health risks of gold nanoparticles across different biological levels, from organs and tissues to cells and molecular pathways.

Limitations

This study has several limitations that should be acknowledged. First, the lack of behavioral assessments limits our ability to directly correlate the observed histopathological and immunohistochemical alterations with functional neurological outcomes. Second, the absence of a broader molecular profile, including markers of oxidative stress (e.g., SOD, MDA), proinflammatory cytokines (e.g., TNF-α, IL-6), and autophagy-related genes (e.g., LC3, p62), restricts a more detailed mechanistic interpretation of the findings. Third, ultrastructural analyses, such as transmission electron microscopy, were not conducted, which would have provided direct evidence of nanoparticle localization within neural tissues. Fourth, dose–response studies were not performed, limiting our understanding of the relationship between nanoparticle concentration and neurotoxic effects.