Permethrin exposure affects neurobehavior and cellular characterization in rats' brain

Abstract

This study investigated the neurotoxic effects of permethrin on the cerebellum, hippocampus and prefrontal cortex of Wistar rats and its effects on some behavioral patterns. Fifteen adult male Wistar rats were grouped into three categories: Group A received 0.1 mL normal saline (control), and Groups B and C received mixed feed with 500 mg/kg and 1,000 mg/kg of 0.6% permethrin, respectively, for 14 days. The animals were assessed for memory, anxiety and exploratory locomotion and thereafter anesthetized and transcardially perfused with normal saline and 4% paraformaldehyde (PFA). Cerebellum, hippocampus and prefrontal cortex were excised from the whole brain and processed for tissue histology, histochemistry and immunohistochemistry. Oxidative status and lipid peroxidation were also assessed using catalase, glutathione peroxidase, superoxide dismutase and malondialdehyde as biomarkers. Results revealed dosedependent decrease in body weights but increase in cerebellar and prefrontal weights, depletion of endogenous antioxidant markers, cognitive deficits, reduced locomotor activities, degenerative changes in the microarchitecture at high doses and presence of chromatolytic cells at both low and high doses of permethrin. Astrocytes were activated while synaptophysin expression was downregulated. Permethrin causes dose-dependent neurotoxicity on the morphology, neurochemistry and oxidative status of different brain regions, and these could affect behavioral performance and other neurologic functions.

Keywords: behavior; brain morphology; neurotoxicity; oxidative stress; permethrin.

Figure 1

Outcome of behavioral analyses of animals after being exposed. Behavioral paradigm of memory (A1: escape latency period, and A2: % correct alternation), spatial exploration (B1: centered square entry, and B2: rearing frequency), anxiety (C1: time spent in the dark chamber, and C2: freezing duration) and locomotion (D1: latency of turning, and D2: number of lines crossed). High dose of permethrin (1000 mg/kg) significantly increased the escape latency period and reduced the % correct alternation relative to the control group in the Morris water maze and Y maze, respectively. * p<0.05.

Figure 2

Activities of superoxide dismutase (SOD; A1–A3), catalase (CAT; B1–B3), glutathione peroxidase (GPx; C1–C3) and lipid peroxidation using malon-dialdehyde (MDA; D1–D3) in the prefrontal cortex, hippo-campus and cerebellum. Permethrin showed dose-dependent affectation of the activities of these enzymes in almost all parts of the brain. Permethrin significantly depleted the levels of CAT in all three brain regions when compared to the control while increasing MDA in the prefrontal cortex. *p<0.05, **p <0.01.

Figure 3

Representative photomicrographs of the cerebellum, prefrontal cortex and hippocampus of animals that received standard diet (control), low dose permethrin diet (500 mg/kg) and high dose permethrin diet (1000 mg/kg). The control presented with the typical histomorphology of the three brain regions. The cerebellar cortex showed densely packed internal granular layer separated from the external molecular layer by a single-celled layer of Purkinje cells (black arrow) in the control section, while the low dose group revealed a reduced staining intensity of Purkinje cells. The Purkinje layer was markedly destroyed in the high dose group, with possible absence of Purkinje cells (red arrows), and the cellular density in the molecular layer was reduced. The prefrontal cortex showed external granular layer made up of fine granule cells (black arrow), with increased perinuclear spaces in the low dose group and reduction in cell population in the high dose group. The dentate gyrus of the hippocampus revealed densely and finely packed granule cells (control; black arrow), however, cellular density reduced in permethrin-treated rats with increased intercellular spaces, and cellular arrangement was disorganized especially in the high dose group (Hematoxylin and Eosin; Scale bar: 25 μm).

Figure 4

Representative photomicrographs of Nissl profile of cerebellum, prefrontal cortex and dentate gyrus of experimental animals treated with standard diet (control), low dose permethrin (500 mg/kg) and high dose permethrin (1000 mg/kg). The control animals presented with typical Nissl staining intensity across the three brain regions. The neurons were deeply stained (black arrows) and well situated in their respective neuropils. The low dose and high dose treated groups presented with chromatolytic cells (red arrows). The dentate gyrus of the high dose group particularly appeared poorly stained (Cresyl fast violet; Scale bar is 25 μm).

Figure 5

Representative photomicrographs of the cerebellum, prefrontal cortex and hippocampus of experimental animals treated with standard diet (control), low dose permethrin (500 mg/kg) and high dose permethrin (1000 mg/kg) showing anti-GFAP immunopositivity (red arrow) and nuclei of inherent neurons as counterstained by hematoxylin (black arrow) (scale bar: 25 μm).

Figure 6

Cell counting using Image-J software. A, B and C represented Nissl profile, anti-GFAP immunopositivity and anti-synaptophysin immune-positivity, respectively. *p<0.05.

Figure 7

Representative photomicrographs of the cerebellum, prefrontal cortex and hippocampus of experimental animals treated with standard diet (control), low dose permethrin (500 mg/kg) and high dose permethrin (1000 mg/kg) showing anti-synaptophysin immunopositivity (red arrow) and nuclei of other neuronal cells as counterstained by hematoxylin (black arrow). (Scale bar: 25 μm)