Callosal glia, GABAergic interneurons, and apoptotic profiles in the brains of cypermethrin-exposed male and female rats

Abstract

Objective

Cypermethrin’s effects on the corpus callosum, the brain’s largest white matter tract essential for interhemispheric communication, remain unexplored, particularly regarding sex‑dependent vulnerability. We examined the effects of sub‑chronic cypermethrin exposure (6.25 and 12.5 mg/kg) on corpus callosum integrity in male and female Wistar rats, focusing on glial populations, GABAergic interneurons, and apoptosis, using biochemical (brain‑derived neurotrophic factor [BDNF] and total protein) and immunohistochemical markers (GFAP, IBA‑1, parvalbumin, cleaved caspase‑3).

Results

Cypermethrin caused sex‑dependent behavioral changes: males showed increased rearing frequency while females showed decreased rearing. BDNF concentrations significantly decreased in low‑dose males (p = 0.0079) and females (p = 0.0039), with partial recovery in high‑dose females. GFAP‑positive astrocyte density decreased in exposed males but increased in low‑dose females (p = 0.024). IBA‑1‑positive microglia decreased significantly in both sexes, particularly in low‑dose groups (males: p = 0.0001; females: p = 0.0075). Parvalbumin‑positive GABAergic interneuron density decreased in low‑dose males (p = 0.029) and both female groups. Cleaved caspase‑3 expression increased in all treated groups, with high‑dose males most affected (p < 0.0001). H&E staining revealed dose‑dependent cellular density reductions, greatest in high‑dose males (p = 0.0001). Collectively, cypermethrin induced sex‑dependent white matter neurotoxicity, with males showing greater susceptibility than females.

Introduction

Cypermethrin, a widely used type II pyrethroid insecticide, kills pests by prolonging the opening of voltage‑gated sodium channels, causing hyperexcitability and excitotoxic injury [1, 2]. It also blocks GABAₐ receptors and chloride channels, further upsetting brain inhibition [1]. These actions, together with the oxidative stress generated during its breakdown, trigger neuroinflammation and programmed cell death in the brain.

The corpus callosum is the largest bundle of white matter in mammals, containing about 200–250 million axons that connect the two cerebral hemispheres. Its integrity is essential for attention, processing speed, and executive function, and damage to this tract is linked to schizophrenia, autism, and dementia [3]. Environmental toxicants, including pyrethroids, can harm white matter by causing demyelination, axonal injury, and glial dysfunction.

Within white matter, astrocytes (GFAP⁺) and microglia (IBA‑1⁺) maintain healthy function by clearing neurotransmitters, buffering ions, and performing immune surveillance [4, 5]. Parvalbumin‑expressing GABAergic interneurons send axons into the corpus callosum and fine‑tune communication between the hemispheres, and they are highly sensitive to oxidative stress because of their intense energy demands [6]. The loss of these fast‑spiking cells is a consistent finding in schizophrenia and age‑related cognitive decline.

It is now clear that sex strongly influences how the brain responds to toxicants. Females often benefit from higher antioxidant reserves and oestrogen‑mediated protection [7]. However, sex‑dependent vulnerability varies with the chemical and dose, making sex‑stratified studies essential. We previously showed that λ‑cyhalothrin and cypermethrin produce sex‑specific inflammation and interneuron damage in the rat hippocampus [8, 9]. In a separate work, we demonstrated that thymoquinone or vitamin E combined with valproate could protect white matter and reduce oxidative damage after cypermethrin‑triggered seizures [10–12].

To date, no study has examined whether cypermethrin affects the corpus callosum differently in males and females by simultaneously analysing glial cells, GABAergic interneurons, and apoptosis. We therefore gave male and female rats a 28‑day oral exposure to cypermethrin and assessed behaviour, BDNF levels, astrocyte and microglial populations, parvalbumin‑positive interneurons, and cell death, using the hole‑board test, biochemical assays, immunohistochemistry, and histology.

Materials and methods

Ethical approval

All procedures conformed to the NIH Guide (8th Edition) and were approved by the Ethical Review Committee, University of Ilorin (UIL/ANA/ERC/2023/042).

Chemicals and antibodies

Cypermethrin (>95% purity; Sigma‑Aldrich) was dissolved in corn oil. Primary antibodies: rabbit anti‑GFAP (1:500; Abcam ab7260), rabbit anti‑IBA‑1 (1:400; Wako 019‑19741), mouse anti‑parvalbumin (1:1000; Sigma P3088), rabbit anti‑cleaved caspase‑3 (Asp175) (1:300; Cell Signaling 9661).

Animals and experimental design

Sixty adult Wistar rats (30 males, 30 females; 10–12 weeks old) were obtained from the Animal Holding Facility, Faculty of Basic Medical Sciences, College of Health Sciences, University of Ilorin, Nigeria. The breeding colony was originally established using animals from the Faculty of Veterinary Medicine, University of Ilorin. They were then housed under standard conditions with free access to food and water. After 10 days of handling and habituation to oral gavage, rats were randomly assigned to six groups (n = 10): male and female controls (corn oil, 2 mL/kg/day), low‑dose cypermethrin (6.25 mg/kg), and high‑dose cypermethrin (12.5 mg/kg). Doses were chosen because they produce sub‑chronic neurotoxicity without overt systemic illness. Cypermethrin (> 95% purity; Sigma‑Aldrich) was dissolved in corn oil and given by gavage every morning for 28 consecutive days. Animals were weighed weekly; no treatment‑related weight differences were seen.

Behavioral assessment

Twenty‑four hours after the last dose, each rat was placed individually in a hole‑board box (60 × 60 cm, 16 holes) for 5 min. Two blinded observers scored rearing, head‑dipping, stretch‑attend posture, and grooming from video recordings.

Tissue collection

Rats were euthanized 24 h after the final treatment. For the biochemical cohort (n = 5/group), deep anaesthesia was induced with ketamine (20 mg/kg, intraperitoneal) and the brain was rapidly removed; the corpus callosum was dissected out on ice, and stored frozen. For the histological cohort (n = 5/group), rats were deeply anaesthetised with ketamine (20 mg/kg) and transcardially perfused with cold phosphate‑buffered saline followed by 10% buffered formalin. Brains were removed, post‑fixed overnight, embedded in paraffin, and cut into 8‑μm coronal sections through the full length of the corpus callosum.

Biochemical measurements

Frozen callosal tissue was homogenised in RIPA buffer with protease inhibitors. Total protein was measured with a BCA assay. BDNF was quantified using a rat‑specific ELISA kit (RayBiotech) and normalised to total protein.

Immunohistochemistry

Sections were dewaxed, subjected to antigen retrieval in citrate buffer (95 °C, 10 min), and treated with 0.3% H₂O₂. After blocking, they were incubated overnight at 4 °C with primary antibodies: rabbit anti‑GFAP (1:500; Abcam), rabbit anti‑IBA‑1 (1:400; Wako), mouse anti‑parvalbumin (1:1000; Sigma), or rabbit anti‑cleaved caspase‑3 (1:300; Cell Signaling). Detection used biotinylated secondary antibodies, avidin‑biotin‑peroxidase complex, and DAB, with haematoxylin counterstaining. Additional sections were stained with haematoxylin and eosin for overall morphology.

Image analysis

For each marker, three sections per animal were analysed; three non‑overlapping fields within the body of the corpus callosum were photographed at 400× magnification by a blinded observer. Positively stained cells were counted using ImageJ, and density was expressed as cells/mm².

Statistics

Data are presented as mean ± SEM. Two‑way ANOVA (sex × treatment) with Tukey’s post‑hoc test was performed using GraphPad Prism; p < 0.05 was considered significant.

Results

Behavioral consequences of subchronic cypermethrin exposure in male and female rats

Cypermethrin caused opposite changes in rearing: dose‑related increases in males, decreases in females (sex × treatment interaction, p = 0.025). Head‑dipping fell significantly in high‑dose groups of both sexes (p < 0.05 vs. controls). Stretch‑attend posture and grooming duration increased markedly in high‑dose males and, to a lesser degree, in high‑dose females, indicating heightened anxiety (Fig. 1A–D).

Fig. 1.

Behaviour in the hole‑board test after 28‑day cypermethrin exposure. A Rearing, B head‑dipping, C stretch‑attend posture, D grooming. Symbols: circles = control, squares = low‑dose, triangles = high‑dose. Mean ± SEM; n = 5/group. Two-way ANOVA

Effects of cypermethrin on tropic factor and protein in the brain of exposed male and female rats

Callosal BDNF levels fell in low‑dose males (p = 0.0079) and females (p = 0.0039). High‑dose males remained low, whereas high‑dose females showed partial recovery (sex × treatment interaction, p = 0.0034; Fig. 2A). Total protein concentration showed modest, non‑significant reductions (Fig. 2B).

Fig. 2.

A BDNF concentration (pg/mg protein) and B total protein (mg/mL) in corpus callosum homogenates. Symbols: circles = control, squares = low‑dose, triangles = high‑dose. Mean ± SEM; n = 5/group. Two-way ANOVA

Cleaved caspase-3 expression in the corpus callosum

CC3⁺ cell density increased in all treated groups except low‑dose females. High‑dose males exhibited the greatest elevation (232% increase; p < 0.0001), significantly exceeding high‑dose females (p = 0.012; Fig. 3).

Fig. 3.

Cleaved caspase‑3 immunostaining. A Representative images; scale bar = 50 µm. B Quantitative cell density (cells/mm²). Symbols: circles = control, squares = low‑dose, triangles = high‑dose. Mean ± SEM; n = 5, 9 sections/animal. Two-way ANOVA

Glial fibrillary acidic protein expression in the corpus callosum

Astrocyte density dropped dose‑dependently in males (36.8% loss at high dose; p = 0.023). In striking contrast, low‑dose females showed reactive astrogliosis (37% increase; p = 0.024), which normalised at the high dose (Fig. 4).

Fig. 4.

GFAP immunostaining. A Representative images. B Astrocyte density (cells/mm²); scale bar = 50 µm. B Quantitative cell density (cells/mm²). Symbols: circles = control, squares = low‑dose, triangles = high‑dose. Mean ± SEM; n = 5, 9 sections/animal. Two-way ANOVA

Ionized calcium-binding adapter molecule-1 expression in the corpus callosum

Microglial density plummeted in low‑dose males (49.7% decrease; p = 0.0001) and females (38.8% decrease; p = 0.0075). Both sexes partly recovered at the high dose, but low‑dose males had the lowest count (sex × treatment interaction, p = 0.006). Remaining microglia often assumed an amoeboid shape (Fig. 5).

Fig. 5.

IBA‑1 immunostaining. A Representative images. B Microglial density (cells/mm²). Symbols: circles = control, squares = low‑dose, triangles = high‑dose. Mean ± SEM; n = 5, 9 sections/animal. Two-way ANOVA

Parvalbumin expression in the corpus callosum

PV⁺ cell density decreased dose‑dependently, with greater loss in males. High‑dose males lost 53.8% of PV⁺ cells (p < 0.001) and had significantly lower density than high‑dose females (p = 0.038; Fig. 6).

Fig. 6.

Parvalbumin immunostaining. A Representative images. B PV⁺ interneuron density (cells/mm²). Symbols: circles = control, squares = low‑dose, triangles = high‑dose. Mean ± SEM; n = 5, 9 sections/animal. Two-way ANOVA

Hematoxylin and eosin histological analysis

Total cellular density in the corpus callosum declined dose‑dependently. High‑dose males showed a 40.8% reduction (p < 0.0001) and differed significantly from high‑dose females (p = 0.018). Pathological features included pyknotic nuclei and vacuolation (Fig. 7).

Fig. 7.

H&E staining of the corpus callosum. A Representative images; scale bar = 50 µm. B Total cellular density (cells/mm²). Symbols: circles = control, squares = low‑dose, triangles = high‑dose. Mean ± SEM; n = 5, 9 sections/animal. Two-way ANOVA

Discussion

We show for the first time that sub‑chronic cypermethrin exposure causes pronounced, sex‑dependent damage to the corpus callosum. Male rats consistently suffered greater harm than females across behavioural, neurotrophic, glial, and apoptotic endpoints.

The opposite rearing response, hyperactivity in males, hypoactivity in females, reflects the excitatory and disinhibitory nature of cypermethrin [1, 2]. Elevated anxiety, shown by more stretch‑attend postures and grooming, mirrors the clinical complaints of workers and communities exposed to pyrethroids and aligns with the loss of inhibitory interneurons we observed.

BDNF is essential for the survival of neurons and the formation of myelin by oligodendrocytes [13]. In males, BDNF fell in a dose‑related fashion, matching the progressive cell loss. Females, however, showed a U‑shaped curve: a steep drop at the low dose but recovery at the high dose. This hormetic response is increasingly recognised in toxicology and likely stems from the activation of oestrogen‑triggered protective pathways, including the Nrf2 antioxidant system and a specific oestrogen‑responsive element on the BDNF gene [10, 14, 15]. The BDNF recovery in high‑dose females paralleled normalisation of astrocyte numbers, suggesting co‑ordinated neuroprotective adaptation.

Apoptosis, flagged by cleaved caspase‑3, was highest in high‑dose males. The non‑significant decrease in low‑dose females may indicate a transient activation of pro‑survival signals or faster removal of dying cells by microglia. The marked apoptosis in males fits with cypermethrin’s known ability to trigger mitochondrial dysfunction and caspase cascades.

The astrocytic response was sexually dimorphic: progressive loss in males versus initial reactive proliferation in females. Male astrocyte depletion likely worsens excitotoxicity, because astrocytes are the chief removers of extracellular glutamate [4]. In females, the early reactive increase, a classic protective reaction, was lost at the higher dose, presumably because the defence was overwhelmed or astrocytes themselves began to die.

Microglial density fell drastically at the lower dose, especially in males. This surprising depletion could reflect direct toxicity, impaired self‑renewal, or down‑regulation of IBA‑1 below the detection limit. The partial return of microglia at the high dose suggests either a proliferative burst of surviving cells or invasion of blood‑borne monocytes through a leaky blood‑brain barrier [16]. Despite the lower numbers, the amoeboid shape of remaining cells points to an activated, pro‑inflammatory state that may contribute to bystander injury, as we have previously described in cypermethrin‑exacerbated seizures [11, 17].

Parvalbumin‑positive interneurons were lost in a dose‑dependent manner, and males were more affected. These fast‑spiking cells are crucial for generating gamma‑frequency oscillations that orchestrate information transfer across the hemispheres. Their high energy demand, reliance on NMDA receptors, and weak antioxidant defences make them exquisitely sensitive to cypermethrin’s oxidative and excitotoxic stress⁶. The observation that male loss reached 53.8% is clinically relevant because similar degrees of PV‑interneuron depletion are seen in schizophrenia and bipolar disorder [18].

The consistent male vulnerability can be explained by multiple factors: females express higher levels of certain detoxifying cytochrome P450 enzymes, maintain stronger basal antioxidant defences, and benefit from oestrogen signalling that protects mitochondria and dampens microglial inflammation [7, 19]. Nevertheless, protection was not absolute, high‑dose females still sustained significant callosal damage, indicating that heavy exposure can overcome these safeguards.

Conclusions

A 28‑day oral exposure to cypermethrin produces clear, sex‑dependent neurotoxicity in the rat corpus callosum, with males consistently more affected. The pathology includes altered anxiety‑like behaviour, reduced BDNF support, divergent glial responses, loss of parvalbumin‑positive interneurons, and caspase‑3‑mediated apoptosis. Female resilience appears to rely on oestrogen‑related neuroprotection and more efficient detoxification, but this protection is saturable. Because the corpus callosum is vital for cognitive and emotional processing, our findings raise concerns that current safety limits, which ignore sex, may not adequately protect the most vulnerable.

Limitations

The Sample size per subgroup may limit detection of subtle interactions but allowed robust statistical evaluation, and only a single exposure duration and two doses were examined, preventing time‑course or dose‑response nonlinearity assessments. Immunohistochemical quantification, due to access relied on manual cell counting and could be strengthened by unbiased stereology. Also, the behavioral battery was restricted to one behavioral test; although sufficient, complementary assays of motor coordination and cognition would enhance phenotyping.

Author contributions

O.P. Investigation, Methodology, Formal analysis, Writing—original draft. A.A. Investigation, Methodology. E.F.A. Investigation, Data curation. O.M.O. Investigation. O.R. Investigation. O.P. Investigation. A.A. Methodology, Validation. D.O.A. Formal analysis, Visualization. O.J.O. Investigation. J‑S.R. Investigation. M.S.A. Supervision, Resources. I.A. Conceptualization, Methodology, Formal analysis, Writing, review & editing, Supervision, Project administration.

Funding

This research did not receive any specific grant.

Availability of data and materials

This study generated no datasets that require mandatory deposition. All other data supporting the findings are included in the article, and raw data are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All experimental procedures were approved by the Ethical Review Committee, University of Ilorin, Nigeria (Approval reference: UIL/ANA/ERC/2023/042). This study did not involve human participants; therefore, consent to participate is not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.