Alzheimer's Disease (AD) is a progressive and fatal neurodegenerative disorder (NDD), and the leading cause of dementia globally, with females being more susceptible than males. Existing animal models for AD are primarily pharmacologically induced or transgenic, yet many fail to recapitulate the full spectrum of human AD pathology and thereby elucidating its sex-based differences. This underscores the need for a cost-effective and robust experimental model that reliably mimics the multifactorial nature of AD taking into account the differences that arise due to sex. In recent years, the zebrafish (Danio rerio) has emerged as a promising model organism for studying central nervous system (CNS) disorders, including AD, owing to its high genetic and physiological homology to humans, transparent embryonic development, and amenability to high-throughput screening. This study aims to establish a novel chronic neurotoxicity induced ZF model, using AlClas an inducing neurotoxic agent. The hypothesis centers on AlCl₃-induced oxidative stress, cholinergic pathway dysfunction, and gut pathophysiological changes as drivers of AD-like pathology. Adult zebrafish, of both sexes were exposed to chronic AlCl₃ treatment over a 28-day period. Post-treatment assessments included histopathological, biochemical, and behavioural analyses to evaluate changes in brain and gut tissues, oxidative stress biomarkers, and cognitive performance. Zebrafish exposed to AlCl₃ exhibited distinct pathological changes in both brain and gut tissues compared to controls. In the brain, hallmarks such as pyknotic neurons, neuronal vacuolisation, and neural tissue necrosis was observed. Gut tissue displayed significant abnormalities, including reduced villi number, epithelial cell loss, and fused or shortened villi. Biochemical analyses revealed elevated oxidative stress, evidenced by altered levels of catalase (CAT), glutathione (GSH), and lipid peroxidation (LPO). Additionally, disruption of the cholinergic system was evident. Behavioural analyses using locomotor tracking revealed marked cognitive deficits, including reduced average speed, decreased distance travelled, and increased immobility. Lastly, our sex specific differences revealed that females were more affected by the biochemical, histological and neurobehavioural parameters as compared to males, thereby indicating that females pose a greater susceptibility towards developing AD. The AlCl₃ -induced zebrafish model successfully replicates key features of human neurotoxicity, which may lead to AD like features including oxidative stress, cholinergic dysfunction, neurodegeneration, and gut-brain axis alterations. This novel and cost-effective model provides a comprehensive platform for exploring sex-mediated neurotoxicity experimental animal model and offers potential utility for screening therapeutic interventions and understanding disease-modifying mechanisms. Keywords: Alzheimer's Disease, Chronic Neurotoxicity, Gut-brain axis, Zebrafish, Sex differences, Alumnium chloride. 3
