Neurotoxicity investigation of Neonicotinoid on Wistar rats and protective role of antioxidant plant extracts

Abstract

The widespread use of Imidacloprid (IMI), a neonicotinoid pesticide, raises concerns about its neurotoxic effects. Chronic exposure induces oxidative stress, mitochondrial dysfunction, and behavioral impairments. Melissa officinalis L. (MOE), a medicinal plant known for its antioxidant properties, may offer neuroprotective effects. The present study investigates the dose-dependent neurotoxic effects of IMI and the protective role of MOE in Wistar rats, focusing on regional brain differences. In the first phase, the aqueous extract of MOE was analyzed for its active constituents. Phytochemical screening revealed high levels of polyphenols, flavonoids, and condensed tannins—compounds known for their potent antioxidant activity. In vitro assays, including DPPH, ABTS, CUPRAC, and FRAP, confirmed significant free radical scavenging activities (85% and 80% scavenging in DPPH and ABTS assays, respectively). Structural analysis through X-ray diffraction (XRD), infrared spectroscopy (IR), and scanning electron microscopy (SEM) validated the presence of bioactive compounds. The second phase assessed the neurotoxic effects of chronic IMI exposure (5 mg/kg/day and 50 mg/kg/day) in Wistar rats for 90 days. IMI induced neurotoxicity in a dose-dependent manner, with more severe damage in the striatum and hippocampus. IMI exposure led to metabolic disruptions, including a significant reduction in body weight and brain mass at the higher dose (50 mg/kg/day, p ≤ 0.001), with MOE administration partially counteracting these effects. Additionally, IMI exposure caused significant depletion of oxidative stress markers (SOD, CAT, GPx, GSH, GST, MDA) and mitochondrial dysfunction, evidenced by increasing mitochondrial swelling and reduced oxygen consumption (p ≤ 0.001) and altering membrane permeability. Neurobehavioral assessments included tests for anxiety (open field and labyrinth tests), cognition (novel object and olfactory recognition tests), social behavior (Vsoc-Maze), and anhedonia (sucrose preference test). showed substantial cognitive and motor dysfunction in IMI-exposed rats. With a 40% decrease in memory retention and a 50% decrease in motor coordination at the higher dose (p ≤ 0.001). Lysosomal stability was compromised by IMI exposure, as evidenced by a 45% reduction in Neutral Red Retention Time (NRRT) (p ≤ 0.001) with MOE treatment restoring lysosomal stability. Lysosomal pH variations were also measured to assess functional changes, with lysosomal destabilization further confirmed by morphological alterations observed under light microscopy. These changes included lysosomal expansion, leakage of neutral red dye, and cell rounding, all of which indicated progressive lysosomal damage. The degree of damage progressed over time, from minor structural disruptions to more severe alterations, which were clearly visible at the microscopic level. Histopathological examination revealed severe neuronal damage, particularly at the higher IMI dose, whereas MOE co-treatment preserved neuronal integrity. AChE activity was significantly inhibited, especially in the hippocampus and striatum, and was partially restored with MOE administration.