Introduction

The Industrial Revolution, coupled with rapid technological progress, has greatly intensified the release of hazardous substances in recent decades. This escalation has created a complex interplay between environmental degradation and public health, exacerbating the challenges faced in managing both issues (Andra et al. 2017; Andjelkovic et al. 2019).

To effectively evaluate risks, it is crucial to deeply understand the biochemical interactions of pesticides and their environmental impacts, which forms the foundation for precise risk assessment (Sakthiselvi et al. 2020; Zhao et al. 2020). As awareness grows regarding the potential impacts of pesticide use on ecosystems and human health, it is evident that their application comes with significant environmental and health risks, including acute and chronic poisoning (Fang et al. 2019; Bishop et al. 2020; Utami et al. 2020).

Neonicotinoids, a prominent class of pesticides, are particularly concerning due to their widespread use and potential for inducing oxidative stress in both humans and animals (Asghari et al. 2017; Wang et al. 2017). These pesticides, which include Imidacloprid, are used extensively in agriculture and on domestic pets, as well as for soil treatment and crop protection (Douglas and Tooker 2015; Cimino et al. 2017). Since their introduction in the late 1980s, neonicotinoids have been recognized for their efficacy but also for their potential to disrupt nervous system development and function (Simon-Delso et al. 2015; Julvez et al. 2016).

Neurotoxic effects from neonicotinoids can manifest through various behavioral, biochemical, physiological, and pathological changes in both the central and peripheral nervous systems. Imidacloprid, a widely used neonicotinoid, has been a key focus of studies due to its significant role in modern pest management and its potential to disrupt environmental and human health (Jeschke et al. 2011; Karahan et al. 2015). However, excessive use of these pesticides can lead to severe environmental imbalances and health hazards (Wang et al. 2018).

Mitochondria and lysosomes are crucial cellular organelles with significant roles in maintaining cell function and integrity. Disruptions in their functionality have been linked to various neurological disorders (Fraldi et al. 2016; Fivenson et al. 2017). Mitochondria, essential for energy production and calcium signaling, are particularly sensitive to damage, which can lead to a cascade of cellular dysfunctions and diseases (Pryde and Hirst 2011; Sun et al. 2016). Recent research suggests that mitochondrial impairment may be a primary target of neonicotinoid toxicity, with alterations in mitochondrial calcium homeostasis and respiration contributing to oxidative stress and cell death (Miao et al. 2022; Xu et al. 2022; Cestonaro et al. 2023).

Lysosomes, involved in the enzymatic degradation of cellular substances, are also affected by environmental stressors. The stability of lysosomal membranes can be assessed using neutral red, a cationic dye that accumulates in lysosomes and indicates membrane integrity (Duve 1963; Cesen et al. 2012). Changes in neutral red retention can reveal lysosomal damage and dysfunction in response to various chemicals and pollutants. (Lowe et al. 1992).

This study aims to investigate the neurotoxic effects of Imidacloprid exposure in Wistar rats by examining its impact on oxidative stress, mitochondrial function, and lysosomal stability. It explores the relationship between pesticide exposure and neurotoxicity through both biochemical and microscopic analyses. The research offers novel insights into the mechanisms of neurotoxicity induced by Imidacloprid, focusing on its effects on mitochondrial and lysosomal functions. By combining biochemical assessments with detailed microscopic observations, it enhances our understanding of how neonicotinoids disrupt cellular processes and affect neurological health. For comprehensive risk assessment, it is crucial to consider the standard permissible limits of neonicotinoids in environmental matrices, as established by organizations such as the EPA, WHO, and FAO, which are essential for evaluating the safety and regulatory compliance of pesticide use (Table 1).

Table 1 The standard permissible limits of neonicotinoids in various matrices
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Materials and methods

Experiment design

In order to carry out our research study, we employed a particular experimental animal model, specifically adult male "Wistar albino rats." These rats were procured from the Algeria Pasteur Institute (API), which is renowned for its contributions to scientific research. The rats selected for the study had an average weight ranging between 240 and 300 g and were of similar size within the study parameters. Subsequently, we divided the animals into two distinct groups: a control group and a treatment group. were housed in spacious cages. Thirty rats were used in the study, and they were divided into three groups of ten rats each. upon arrival and maintained under controlled conditions: a temperature of 24 ± 4 °C, relative humidity of 60 ± 10%, and a 12-h light/dark cycle. They had unrestricted access to food and water and were kept under standard hygiene conditions. After a four-week adaptation period, the rats were divided according to the studies by Duzguner and Erdogan (2010) and Kara et al. (2015).

The treatment involved daily gastric gavage with prepared solutions for 90 days. The groups were:

  1. 1.

    Control Group (CT): Rats in this group received only distilled water (1ml) via gavage for the entire 90-day period.

  2. 2.

    Group II (IMI D1 group): Rats in this group received a daily aqueous solution of Imidacloprid at a dose of 5 mg/kg/day.

  3. 3.

    Group III (IMI D2 group): Rats in this group received a daily dose of 50 mg/kg/day of Imidacloprid.

The study was conducted at the Toxicology Laboratory, Faculty of Applied Biology, Tebessa University, Algeria, adhering to Institutional Animal Care and Use Committee (IACUC) policies (Accreditation number: D01N01UN130120150006/2019).

Chemical product

Imidacloprid, a systemic neonicotinoid insecticide from the chloronicotinyl nitroguanidine family, was used in this study (CAS No. 138261–41-3, molecular formula C9H10ClN5O2). The chemical was sourced from Sigma Aldrich, Germany (Tomlin 2006).

Dose selection

Two doses of Imidacloprid (5 mg/kg and 50 mg/kg body weight) were administered to the animals for 90 days, dissolved in distilled water. Dose selection was based on previous studies, including those by Kara et al. (2015) and Duzguner and Erdogan (2010), and were within the non-toxic range, calculated relative to the LD50 values (424–475 mg/kg) to ensure safe daily exposure.

Mitochondria isolation from tissue

Mitochondria were isolated at 4°C using the following procedure: Resuspend the cell pellet from 1–2 mL of washed cells in ice-cold RSB hypo buffer (10 mM NaCl, 1.5 mM MgCl2, 10 mM Tris–HCl, pH 7.5). Transfer the suspension to a Dounce or Potter–Elvehjem homogenizer and allow the cells to swell. After swelling, disrupt the cells with a tight-fitting pestle and verify lysis. Add 2.5 × MS homogenization buffer (210 mM mannitol, 70 mM sucrose, 5 mM Tris–HCl, pH 7.5, 1 mM EDTA) to the homogenate and transfer to a centrifuge tube for differential centrifugation. Centrifuge at 1,300 g for 5 min to remove nuclei, unbroken cells, and large debris. Collect the supernatant and repeat the centrifugation if necessary. Finally, centrifuge the supernatant at 7,000–17,000 g for 15 min to pellet the mitochondria, then wash and resuspend them in an appropriate buffer. For long-term storage, keep the mitochondria at -80°C for up to one year. Refer to Clayton and Shadel (2014) for detailed buffer recipes and protocol information.

Assessment of redox status markers

After a 3-month exposure period, rats were euthanized by decapitation, and the brain was collected and rinsed with cold phosphate-buffered saline (PBS). The redox status of mitochondrial matrix fractions was assessed by measuring protein content using the method of Bradford (1976), with bovine serum albumin as the reference standard.

Glutathione S-transferase (GST) activity was measured using the method of Habig et al. (1974), with absorbance recorded at 340 nm every 30 s for 3 min. Glutathione peroxidase (GPx) activity was determined by the method of Flohe (1984), with absorbance read at 420 nm. Catalase (CAT) activity was assessed following Aebi (1984), by monitoring the decomposition rate of H2O2 with absorbance at 240 nm. Superoxide dismutase (SOD) activity was evaluated using the method of Beauchamp and Fridovich (1971), where 50 μL of the matrix fraction was mixed with 2 ml of a reactive medium containing sodium cyanide (10–2 M), NBT solution (1.76 × 10–4 M), EDTA (66 mmol), methionine (10–2 M), riboflavin (2 μmol), and adjusted to pH 7.8 and exposed to light for 30 min to induce riboflavin's photoreaction. The resulting blue formazan was measured at 560 nm. Reduced glutathione (GSH) concentration was determined according to Weckbercker and Cory (1988), with absorbance at 412 nm, and malondialdehyde (MDA) levels were measured by the method of Warso and Lands (1983), detecting a red-colored complex with peak absorbance at 530 nm. Enzymatic activities were quantified in international units per milligram of protein.

Assessment of swelling, permeability and mitochondrial respiration

The impact of imidacloprid (IMI) on mitochondrial permeability transition pore (MPTP) was assessed by evaluating mitochondrial membrane permeability in the brain. Mitochondria were isolated from fresh brain tissue at 4°C and analyzed for swelling using the method described by Li et al. (2014). Equal volumes of isolated mitochondria were placed in quartz cells, and absorbance was monitored at 540 nm. A decrease in absorbance indicated increased mitochondrial swelling and loss of MPTP integrity. Mitochondrial respiration levels were measured using an Oxygraph (Hansatech) following the methods of Rouabhi et al. (2015) and Henine et al. (2016).

Isolation of lysosomes from rat brain

Based on De Duve and Wattiaux's 1963 technique, lysosomes were isolated from rat brain tissue as follows: The brain was collected, rinsed with cold phosphate-buffered saline (PBS), and free of meninges and blood. The tissue was cut into small fragments and homogenized in a pre-chilled glass or Potter–Elvehjem homogenizer with 0.25 M sucrose and 10 mM HEPES (pH 7.4). The homogenate was centrifuged at 1,000 g for 10 min at 4°C to remove large debris and nuclei. The supernatant was transferred to a new tube and centrifuged at 10,000 g for 20 min at 4°C to pellet the lysosomes. The lysosome pellet was resuspended in an appropriate buffer for further use. Throughout the procedure, maintaining a low temperature (4°C or on ice) was critical to prevent enzymatic degradation and preserve lysosome integrity.

Assessment of brain lysosomal stability by determination of neutral red retention time (NRRT)

Neutral red dye retention assay

Neutral red (CI 50040; Toluylene red chloride) was sourced from Sigma for the assay. This method is widely used to assess cell viability and cytotoxicity, particularly in biomedical and environmental studies (Borenfreund and Puerner 1984; Repetto and Sanz 1993). The assay evaluates lysosomal membrane integrity, as neutral red selectively accumulates in lysosomes; damage reduces dye uptake. Following exposure to toxins, cells were fixed with formaldehyde, and neutral red was extracted using an acetic acid/ethanol mixture. Absorption was quantified using spectrophotometry at 540 nm, based on the protocol of Lowe et al. (1995) and PNEU/RAMOGE (1999).

Neutral red solution preparation

Dissolve 20 mg of Neutral Red powder in 1 ml of dimethyl sulfoxide (DMSO) to prepare the stock solution. Store in a dark container at room temperature. For the working solution, dilute 5 µL of the stock solution in 995 µL of phosphate-buffered saline (PBS). Prepare PBS by dissolving 4.77 g HEPES, 25.48 g NaCl, 13.06 g MgSO4, 0.75 g KCl, and 1.4 g CaCl2 in 1 L of distilled water, adjust pH to 7.36 with 1M NaOH, and store in the refrigerator. Use the working solution at room temperature and store any unused solution in the dark at 4°C.

Toxicity determined In Vitro by morphological alterations and neutral red absorption

Microscopic study

The microscopic study of toxicity using morphological alterations and neutral red absorption involves observing cells under a light microscope with phase-contrast optics at × 400 magnification to identify structural changes indicative of cellular damage. Key alterations include cell shrinkage, membrane blebbing, and loss of adherence, vacuolization, and lysosomal damage, such as swelling or rupture. The neutral red dye accumulates in healthy lysosomes, so diminished uptake reflects lysosomal membrane disruption and overall cellular toxicity. These morphological observations provide critical insights into the extent and mechanisms of toxicity in vitro.

Histological tests

The histological tests were conducted following the method described by Houlot (1984), which involves several essential steps. First, the tissue samples are fixed in formalin and then placed in special perforated cassettes that allow the circulation of the liquids necessary for preparation. Next, the samples undergo progressive dehydration through a series of ethanol baths of increasing concentrations (70%, 95%, and 100%) and are then cleared in xylene to render the tissues transparent.

Once dehydrated, the tissues are embedded in liquid paraffin at 60°C. This step enables the paraffin to penetrate and impregnate the tissues. The following step is embedding, where the impregnated tissues are enclosed in paraffin blocks that, once solidified, facilitate cutting. These blocks are then sectioned into thin slices of 4 to 7 microns using a microtome. The resulting sections are placed onto glass slides for staining.

The standard staining method involves hematoxylin to stain the nuclei blue/violet, followed by eosin, which stains the cytoplasm and extracellular structures pink. After staining, the sections are mounted with cover slips and examined under a microscope for detailed tissue structure analysis. The sections are also photographed using a camera connected to a binocular magnifier. Histopathological anomalies, such as lesions, inflammation, or necrosis, can thus be accurately visualized.

Qualitative assessment of lysosomal ph variations

Brain organ samples are prepared by isolating and slicing the organ into 100–200 μm thick sections, which are then placed in a PBS buffer solution (1 g PBS powder per 100 mL distilled water) to preserve viability. pH-indicating probes are introduced by adding a working solution to the brain sections and incubating for 30 min to 1 h. Following incubation, the samples are washed and homogenized to remove excess probe. The homogenized sample is optionally centrifuged at 1,000 to 5,000 × g, and the supernatant is transferred to a micro-plate or cuvette for spectrophotometric pH measurement. pH variations are then assessed to determine lysosomal pH levels, following methods detailed by Fujiwara et al. (2009) for qualitative and quantitative analysis.

Statistical analysis

Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using Minitab® software version 18.1 and Microsoft Excel version 19.0. Significant differences between treatment effects were evaluated using one-way ANOVA, followed by Tukey's post-hoc test for multiple comparisons. A p-value < 0.05 was considered statistically significant. Additionally, Student's t-test was used to compare group differences, with significance thresholds defined as: *0.05 ≥ p ≥ 0.01 (significant), **0.01 ≥ p ≥ 0.001 (highly significant), and ***p < 0.001 (very highly significant).

Results

Clinical signs of intoxication

The clinical signs of intoxication observed in rats exposed to chronic Imidacloprid are summarized in Table 2. Rats in the control group showed no significant clinical signs, maintaining normal activity and behavior. In the low-dose group (IMI D1, 5 mg/kg), mild symptoms were observed, including occasional lethargy, slight reductions in activity, occasional alterations in grooming behavior, minimal weight loss, and mild fur ruffing. Tremors, gait abnormalities, and respiratory distress were not commonly observed in this group. In contrast, the high-dose group (IMI D2, 50 mg/kg) exhibited pronounced neurotoxic effects, such as marked lethargy, frequent tremors, significant weight loss, impaired motor coordination, and altered exploratory behavior. Additional symptoms included frequent gait abnormalities, pronounced fur ruffing, and occasional seizures. Despite these pronounced symptoms, no mortalities were recorded in any of the groups. The severity and nature of clinical signs in the high-dose group underscore the dose-dependent neurotoxic impact of chronic Imidacloprid exposure, aligning with the observed biochemical and functional impairments in the study.

Table 2 Clinical signs and mortality in rats exposed to chronic imidacloprid
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Assessment of redox status markers in brain mitochondria

The results of assessing various markers of mitochondrial redox status in the brains of rats after chronic exposed to imidacloprid at doses of 5 mg/kg and 50 mg/kg body weight are presented in Table 3. Glutathione (mitGSH), a crucial antioxidant tripeptide in brain mitochondria, showed a very highly significant (p ≤ 0.0001) reduction in IMI-treated group (5/50 mg/kg/day) compared to the normal group. Additionally, the Table 3 indicates a significant increase (P ≤ 0.01) in the enzymatic activity of the matrix glutathione S -transferase (mitGST) at 5 mg/kg/day and a very highly significant increase (p ≤ 0.0001) at 50 mg/kg/day following a chronic exposition of the rats to imidacloprid in comparison with the control group. Furthermore, the enzymatic activity of glutathione peroxidase (mitGPx), a key antioxidant enzyme regulating ROS levels in the mitochondrial matrix, has been decreased with very highly significant manner (p ≤ 0.0001), (Table 3) in IMI-treated rats at the higher dose compared to control group. A very highly significant decrease (p ≤ 0.0001) was also observed in brain mitochondrial catalase activity in the IMI-treated group at 50 mg/kg/day compared to the control. Moreover, the activity of superoxide dismutase enzyme (mitSOD) was highly significantly (p ≤ 0.01) decreased at 5mg / kg / day and showed a very highly significant decrease (p ≤ 0.0001) at 50 mg/kg/day after 90 days of imidacloprid exposure compared to the control group. Levels of Malondialdehyde (mitMDA) in brain mitochondria, indicating oxidative stress impact on lipid compounds, were very highly (p ≤ 0.0001) significantly increased in brain mitochondria at 50 mg/kg/day and highly significantly increased (p ≤ 0.01) at 5mg / kg / day in the IMI-treated rats compared to the control group (Table 3). These findings demonstrate that the severity of these changes is dose-dependent.

Table 3 Variation of mitochondrial oxidative stress parameters in rats’ brain in control and treated rats after three months of treatment under the effect of imidacloprid chronic exposure at the dose of 5 and 50 mg/kg body weight
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Study of mitochondrial apoptosis parameters

The toxicological effects of imidacloprid on mitochondrial integrity and function parameters (Including swelling, permeability, respiration) in the brains of Wistar rats is presented in Table 4. The study results indicate a very highly significant increase (p ≤ 0.0001***) in mitochondrial swelling at a dose of 50 mg/kg/day of IMI (Table 4; Fig. 1), which is directly proportional to the absorbance values. Additionally, there was a significant increase (p ≤ 0.05*) in mitochondrial swelling when rats were exposed to 5 mg/kg/day of IMI for an extended period compared to the control group (Fig. 2). On the other hand, the assessment of mitochondrial permeability measured as the change of mitochondrial size over time, following the addition of calcium to mitochondrial suspension (Table 4; Fig. 3). In addition, the results of this essay are represented by a kinetic curve (Table 4) which demonstrated a significant difference between IMI-treated group and normal group with significance degree of p. Furthermore, the evaluation of respiratory function of brain mitochondria revealed a highlighted a significant decrease in oxygen consumption rate in IMI-treated rats compared to non-IMI-exposed animals (Table 4; Fig. 2).

Table 4 Brain swelling, respiration, and mitochondrial permeability in control and treated rats following three months of treatment
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Fig. 1
figure 1

Effect of IMI on mitochondrial swelling at OD540 nm after 90 days. Each value is expressed as mean ± standard deviation, we use Student test. Batch Compare treated with Imidacloprid (IMI) and control group. (P ≤ 0.05): significant (*), (p ≤ 0.0001): very highly significant (***), P > 0.05: not significant (ns)

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Fig. 2
figure 2

Effect of oral administration of IMI at different doses on mitochondrial respiration (nmol/ml) after 90 days. n = 6 rats/group. Results in each group represent mean ± SEM; (p ≤ 0.01): (**), (p ≤ 0.0001): (***)

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Fig. 3
figure 3

Effect of IMI on mitochondrial permeability after 90 days. Results in each group represent mean ± SEM., (p ≤ 0.0001): (***), (n = 6 rats/group)

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Assessment of brain lysosomal stability by determination of neutral red retention time (NRRT)

The effect of imidacloprid exposure on the rate of incorporation of the RN by the viable cells after 24, 48 h indicates significant differences (P ≤ 0.05) at the specified concentrations compared to the control at both time points. Experiments (n = 6) were performed in triplicate, and the data are presented as mean ± standard error (SE) in Table 5; Fig. 4.

  • Control group: In this group, absorbance at 540 nm is measured for brain cells without treatment. The initial absorbance gives a reference to compare with the other groups.

  • Group treated with 5 mg/kg of IMI (Imidacloprid): After 24 h, we observed an increase in absorbance compared to the control group. This could indicate that brain cells have absorbed the neutral red or undergone changes in lysosomes. After 48 h, absorbance could decrease as lysosomes destabilize, releasing neutral red into the cytosol and resulting in cell death.

  • Group treated with 50 mg/kg of IMI: After 24 h, we see a more pronounced increase in absorbance compared to the control group due to the higher dose of IMI. After 48 h, a more significant decrease in absorbance occurs, indicating faster lysosome destabilization and more pronounced cell death. In summary, after 24 h, absorbance increase in the treated groups due to the absorption of neutral red by lysosomes. After 48 h, absorbance decrease due to lysosome destabilization and the release of neutral red into the cytosol, leading to cell death.

Table 5 Assessment of imidacloprid's effects on absorbance at 540 nm: Comparative analysis across variable groups and doses over time
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Fig. 4
figure 4

Effect of Imidacloprid in different treatment groups, on the brain cells of retention of neutral red. indicate significant differences (p < 0.05; Tukey's test) between means ± SE. absorbance values (540 nm) measured with the neutral red assay to evaluate the lysosome destabilization of brain cells incubated for A 24 h and B 48 h (n = 6). Error bars present standard deviation

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Morphological alterations [microscopic observations (× 400)]

The results of Microscopic examination under a light microscope equipped with phase-contrast optics at a magnification of × 400 following exposure to imidacloprid after a neutral red assay, were presented in Fig. 5, 6, 7 and 8. Showed the morphological alterations following the designated exposure periods, cells are carefully observed with a specific focus on identifying indications of disruption in the structural integrity of the lysosomal membrane.

Fig. 5
figure 5

Indications of a disturbance in the structural integrity of the lysosomal membrane

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Fig. 6
figure 6

Temporal metamorphosis: the trilogy of transformative events in sample evolution

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Fig. 7
figure 7

Lysosomal dynamics: the retention and release of neutral red

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Fig. 8
figure 8

The correlation between the time of incubation with neutral red and the subsequent absorbance at 540 nm following the neutral red assay

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An NRR assay on Wistar rat brain cells exposed to Imidacloprid yielded intriguing findings: [1] Exposed cells exhibited "rounding-up," indicating a change in shape towards a more spherical form. [2] The rounding-up appeared linked to the leakage of neutral red dye into cell cytosol, suggesting a disruption in cellular membrane integrity due to Imidacloprid exposure. [3] The presence of granulocytes with small lysosomes was noted, hinting at potential impacts on these white blood cells, including alterations in lysosomal structures and functions as a consequence of imidacloprid exposure.

Over time, this sample underwent a dynamic temporal transformation characterized by a trilogy of events: [1] firstly, an expansion and augmentation of the lysosomal compartment; [2] secondly, the gradual seepage of NR dye into the cytoplasm; and ultimately, culminating in a cell-rounding phenomenon as the final evolutionary progression [3].

Undoubtedly, the lysomotropic dye, neutral red, exhibits a proclivity for lysosomal retention, a phenomenon characterized by a progressive escalation in lysosomal volume during the incubation period. This trend persists until it reaches a critical point where the lysosomal membrane experiences destabilization, subsequently leading to the efflux of neutral red into the cytosolic milieu.

The study examines the relationship between incubation time and absorbance at 540 nm after a neutral red assay, following exposure to Imidacloprid, at various time intervals (0h, 2h, 4h, 6h, and 8h).

The initial time point (0h), establishes a baseline measurement reflects the immediate impact of Imidacloprid exposure. At 2 h (2h), early cellular responses and adaptations are observed, potentially indicating changes in lysosomal volume and neutral red leakage. The 4-h (4h) mark represents a pivotal stage in the incubation period, reflecting significant cellular alterations. By 6 h (6h), late-stage responses or stabilization of observed effects becomes apparent. The final time point, 8 h (8h), offers insights into cumulative and late-stage responses and the saturation of cellular changes.

Histopathological examination

Figure 9 illustrates histopathological changes in the brains of Wistar rats after 90 days of Imidacloprid exposure. The control group shows normal neuronal architecture with no signs of degeneration or inflammation. In the low-dose group (5 mg/kg/day), there is marked disorganization of nerve fibers, vacuolization, neuronal loss, and mild glial cell infiltration, indicating neurotoxicity and an inflammatory response. The high-dose group (50 mg/kg/day) displays severe neurotoxic effects, including early cerebral necrosis, tissue degradation, and signs of mild tissue edema, suggesting progressive brain damage and disruptions in the blood–brain barrier.

Fig. 9
figure 9

Histological Sections (× 10/40) of the brain after oral administration of imidacloprid in control and treated rats following three months of treatment. A Control, B Treated with IMI 5 mg/kg/day, C Treated with IMI 50 mg/kg/day, (1): Glial Cells; (2): Nerve Fibers. (3): Regularly spaced portal areas. (4): Tissue degradation

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Pathological and morphological changes observed in the brain and other tissues

The Table 6 highlights dose-dependent pathological changes in rats exposed to Imidacloprid. The control group showed no significant changes, while the low-dose group (5 mg/kg) exhibited mild neuronal degeneration and slight vacuolization. In contrast, the high-dose group (50 mg/kg) showed pronounced neuronal degeneration, severe vacuolization, lysosomal destabilization, and disrupted cellular architecture, indicating severe neurotoxic effects. leading to the highest total pathological score. The scores reflect the escalating severity of neurotoxic damage with increasing doses of Imidacloprid.

Table 6 Pathological and morphological changes observed in the brain and other tissues, along with the pathological scores for each dose group
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Qualitative assessment of lysosomal ph variations

Measuring the ph levels inside lysosomes

The results of assessments of lysosomal pH variations during chronic exposure on brain cells in control and IMI-treated rats were presented in Figs. 10 and 11 demonstrated a significant increase in lysosomal pH in brain cells among the IMI-exposed groups, with the magnitude of the increase varying with the IMI dose.

Fig. 10
figure 10

The effect of Imidacloprid on lysosomal pH during chronic exposure on brain cells in control and treated rats. Results are expressed as mean ± SE.t-test was used for multiple comparisons, **P < 0.01, *P < 0.05 statistical significant as compared to control. IMI: Imidacloprid, (n = 5)

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Fig. 11
figure 11

Empirical CDF (cumulative distribution function) of pH represents the cumulative probability of pH values ​​in different groups in the whole brain of rats treated for 3 months with IMI. (n = 15). IMI: Imidacloprid

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Discusion

The current study aimed to evaluate the neurotoxic impact of exposure to Imidacloprid (IMI) on Wistar rat brains (5 and 50mg/kg body weight; orally) for three months. Various parameters, including redox status markers (GST, GPX, GSH, MDA, SOD, CAT) Results were shown in Table 2, Clinical Signs and Mortality. Table 3, mitochondrial stress parameters (Table 4; Figs. 1, 2 and 3), lysosomal stability (Table 5, Fig. 4), cellular morphology (Figs. 5, 6, 7 and 8), histopathological changes (Fig. 9) and pH variations (Figs. 10 and 11), were analyzed, to understand the underlying neurotoxic mechanisms.

Clinical signs and mortality rates observed in our study (Table 3) provided additional insights into the neurotoxic potential of IMI. High-dose exposure (50 mg/kg) was associated with pronounced clinical symptoms, including ataxia, tremors, and lethargy, which reflect central nervous system impairment were consistent with previous reports of neurobehavioral alterations and toxicity associated with IMI exposure. Bomann (1989) and Pauluhn (1988) also documented clinical symptoms such as tremors and ataxia in pesticide-treated animals, similar to the symptoms observed in the current study. In our study, no mortality was observed in any of the treatment groups throughout the experimental period, indicating that the administered doses of Imidacloprid were not lethal to the rats. Conversely, in laboratory studies involving rats, the median lethal dose (LD50) for imidacloprid was found to be approximately 424 mg/kg for male rats and between 450–475 mg/kg for female rats. Mortality was first observed at doses of 400 mg/kg, with a rapid increase to 100% mortality at 500 mg/kg. (Sheets 2001).

Alterations in oxidative balance, such as increased levels of reactive species or reduced efficiency of radical scavenging systems, can compromise cellular integrity. Cellular antioxidant defenses consist of both non-enzymatic antioxidants like GSH and enzymatic antioxidants such as SOD, CAT, and GST, working together to alleviate oxidative stress (Mehta and Gowder 2015).

Our findings reveal a statistically significant increase in Malondialdehyde (MDA) levels in rats treated with Imidacloprid, showing a highly significant elevation (p ≤ 0.0001) at a dose of 50 mg/kg/day and a significant increase (p ≤ 0.01) at 5 mg/kg/day compared to the control group (Table 3). This indicates enhanced oxidative damage and lipid peroxidation. MDA serves as a biomarker for lipid oxidative damage, particularly from the peroxidation of polyunsaturated fatty acids (Zhang et al. 2004). The proposed mechanism suggests that free radicals generated by Imidacloprid exposure inhibit antioxidant enzymes, contributing to increased oxidative stress. These findings enhance our understanding of the potential adverse effects of Imidacloprid on oxidative stress in the brain.

Similarly, previous studies have reported altered oxidative stress and lipid peroxidation markers in rats exposed to Imidacloprid (Mohany et al. 2011; Balani et al. 2011). Additionally, Birsen (2011) demonstrated that exposure to thiacloprid, another neonicotinoid insecticide, at doses of 112.5 and 22.5 mg/kg/day for 30 days significantly elevated MDA levels in lymphoid organs. Ince et al. (2013) also reported that oral administration of Imidacloprid at 15 mg/kg/day for 28 days resulted in a significant increase in MDA levels, along with decreased SOD and catalase activities in mice.

According to our biochemical analysis, the enzymatic activity of glutathione S-transferase (GST) very highly significantly increased (p ≤ 0.0001) in the Imidacloprid-treated group at the higher dose compared to the control group (Table 3). GST utilizes glutathione as a cofactor, and the observed reduction in GST levels in our study may be attributed to the consumption of glutathione as it acts to protect against Imidacloprid-induced toxicity (Danyelle and Kenneth 2003). These findings align with those of Lonare et al. (2014), who reported the activation of detoxification mechanisms in rats following oral administration of Imidacloprid at doses of 45 and 90 mg/kg, noting increased activity of detoxification enzymes, including glutathione (GSH) and GST, compared to controls.

Our results also reveal a significant decrease in the activities of glutathione peroxidase (GPx) and catalase (CAT) in treated rats compared to the control group (Table 3). This decline is likely due to the depletion of cellular antioxidant defenses against reactive oxygen species (ROS) generated by Imidacloprid exposure. Reduced GPx and CAT activity can lead to the accumulation of hydrogen peroxide (H₂O₂), which subsequently generates hydroxyl radicals (OH°) through the Fenton reaction (Cory-Slechta et al. 2005). Multiple independent studies support these observations (Mikolić and Karačonji  2018), consistent with findings from a meta-analysis by Coremen et al. (2022), which highlighted a strong link between pesticide exposure and changes in various biochemical markers in rats.

In this study, a significant decrease in mitochondrial GSH levels was observed in the treated rats compared to the control group (Table 3). GSH plays a crucial role in protecting the brain from oxidative stress, with its regulation and production limited by the transport of thiol amino acids across the blood–brain barrier (Valdovinos-Flores and Gosenbatt 2012). GSH exists in reduced (GSH) and oxidized (GSSG) forms, both of which are essential for neutralizing reactive oxygen species (ROS) (Daubié, 2011).

Our findings are consistent with previous research showing a reduction in mitochondrial GSH levels following 21 days of treatment with various doses of pyrethroids, attributed to excessive ROS generation (Ashar Waheed and Muthu Mohammed 2012; Dar et al. 2013; Mani et al. 2014).

Our findings indicate that rats treated with Imidacloprid (IMI) experienced a very highly significant decrease (p ≤ 0.0001) in superoxide dismutase (SOD) levels at a dosage of 50 mg/kg/day, and highly significant reduction (p ≤ 0.01) at 5 mg/kg/day (see Table 3). This reduction in SOD activity suggests that Imidacloprid plays a role in the conversion of superoxide (O−2) to hydrogen peroxide (H2O2).

Our findings align with Kapoor et al. (2010) that conducted a study examining the effects of sub-chronic dermal exposure to Imidacloprid in female rats, which yielded similar findings related to SOD and catalase (CAT) activities and lipid peroxidation levels. In contrast, EL-Gendy et al. (2010) reported that administering 15 mg/kg of Imidacloprid resulted in significant increases in various antioxidant enzyme activities, including CAT, SOD, glutathione peroxidase, and GST in male mice. Furthermore, Ince et al. (2013) found that mice given oral doses of 15 mg/kg/day of Imidacloprid for 28 days exhibited a significant increase in malondialdehyde (MDA) levels, along with decreased SOD and CAT activities.

By isolating the mitochondria, we were able to illustrate a disturbance in both their structural integrity and energy-related functions, such as oxygen consumption, membrane permeability and swelling. Our findings reveal significant alterations in mitochondrial function (Table 4; Figs. 1, 2 and 3), indicative of neurotoxicity induced by IMI exposure.

Mitochondria, which are vital for energy production and cellular homeostasis, are particularly susceptible to Imidacloprid (IMI) exposure, resulting in significant disruptions to their structural and functional integrity. Our findings demonstrate that IMI exposure leads to impaired mitochondrial functions, including reduced oxygen consumption, increased swelling, and altered membrane permeability, which collectively contribute to mitochondrial dysfunction (Cestonaro et al. 2023). These observations suggest that mitochondria are primary targets of IMI-induced cytotoxicity.

A key concern is the observed increase in mitochondrial swelling, a precursor to cell death, as excessive or prolonged swelling can cause the mitochondrial outer membrane to rupture, releasing pro-apoptotic proteins and initiating apoptosis. This mechanism links IMI exposure to potential neuronal apoptosis or cell death within the central nervous system (CNS) of mammals, paralleling findings in insect CNS studies (Sun et al. 2016; Chapa-Dubocq et al. 2017).

The underlying mechanism of mitochondrial swelling involves calcium overload, which promotes nitric oxide formation and activates enzymes that generate reactive oxygen species (ROS). This cascade disrupts ion homeostasis, releases pro-apoptotic factors, and amplifies oxidative stress, resulting in cellular damage (Sun et al. 2016). The resulting oxidative stress severely impairs mitochondrial structural integrity and function, as evidenced by decreased oxygen consumption rates, consistent with previous research as Gasmi et al. (2017).

Our study also found a highly significant increase (p ≤ 0.001) in cerebral tissue swelling, suggesting potential inflammation and edema in the forebrain region (Figs. 1 and 3). This finding aligns with evidence that IMI exposure disrupts mitochondrial function in the CNS, potentially leading to apoptosis or programmed cell death (Table 4). These results underscore the neurotoxic potential of IMI and provide critical insights into how its exposure might contribute to neuronal damage (Lin and Beal 2006).

The stability of lysosomal membranes can be influenced by different environmental stressors in a dose-dependent manner, and an assay using neutral red can help identify the nature and degree of stress. Neutral red, a cationic probe and lipophilic compound employed as a dye, can penetrate cell membranes (Lowe et al. 1992).

In this work, Lysosomal destabilization was observed through the Neutral Red Retention Time assay, revealing significant changes in absorbance after 24 and 48 h in treated groups (Table 5; Fig. 4), aligning with findings by Sargent et al. 2021, who highlighted lysosomal dysfunction as a consequence of imidacloprid exposure. These results are consistent with previous studies by Martinez-Gomez et al. (2008) and Zhao et al. (2011), examined the effects of Pyrethroid on lysosomal membrane fragility by evaluating the retention time of the neutral red probe within the lysosomal compartment of both control and treated hemocytes.

Moreover, a qualitative assessment of lysosomal pH variations indicated a significant increase in pH in IMI-exposed groupsy (Figs. 10 and 11), emphasizing the impact of chronic pesticide exposure on lysosomal function. When the cell dies or the pH gradient is reduced, the dye cannot be retained (Filman et al 1975).

Microscopic observations are presented in Figs. 5, 6, 7 and 8 further demonstrated morphological alterations in lysosomal structures, suggesting potential membrane disruption. This is consistent with the results reported by Borenfreund and Puerner (1984), who demonstrated Maintaining a lower pH than that of the cytoplasm allows for dye retention within lysosomes, facilitating the differentiation between viable, damaged, or dead cells based on dye uptake and release dynamics.

The NRR assay on brain cells from Wistar rats exposed to 50 mg/kg of Imidacloprid revealed a notable "rounding-up" effect, where cells became more spherical, suggesting compromised cell membrane integrity (Fig. 5). This alteration likely facilitated the leakage of neutral red dye into the cytosol, indicating membrane disruption. Additionally, the presence of granulocytes with small lysosomes suggests that Imidacloprid exposure may also affect lysosomal structure and function in these cells.

The histopathological examination revealed marked neuronal degeneration, gliosis, and nuclear condensation (Fig. 9), confirming the cytotoxic impact of IMI on brain tissues. Our findings align with those of Bhardwaj et al., who observed significant changes in brain sections of rats treated with 80 mg/kg body weight, including pronounced congestion in the cerebellum, degeneration of Purkinje cells with dendritic loss, vacuolation around neurons, and shrunken neurons by the 14th day of the experiment (Soujanya et al. 2013).

Overall, the study provides comprehensive insights into the multi-faceted neurotoxic impacts on the central nervous system induced by Imidacloprid, shedding light on the intricate interplay between oxidative stress, lysosomal integrity, and mitochondrial function in response to pesticide exposure.

Conclusion

This study highlights the harmful effects of Imidacloprid (IMI) on the brains of Wistar rats, revealing significant changes in redox markers, mitochondrial stress, and lysosomal stability. The findings indicate oxidative stress, mitochondrial dysfunction, and lysosomal destabilization, supported by histological alterations such as neuronal disorganization and evidence of neuroinflammation. The observed progression of lysosomal changes, from expansion to dye leakage and cell rounding, emphasizes the dynamic neurotoxic responses to IMI. Overall, these results underscore the complex interactions between oxidative stress and cellular integrity in the central nervous system, reinforcing the need for further research to understand the long-term impacts of neonicotinoid pesticides and their ecological implications.