SALICYLALDEHYDE BENZOYL HYDRAZONE ALLEVIATES MICROGLIAL DYSFUNCTION AND RESTORES BEHAVIOURAL INTEGRITY <EM>IN VITRO</EM> AND <EM>IN VIVO</EM>​

Neuroinflammation is central to many neuropathologies and is driven by uncontrolled microglial activation, leading to neuronal dysfunction and disease progression. In the degenerating brain, dysfunctional microglia adopt a glycolytic, iron-retentive phenotype that promotes chronic proinflammatory activation. Salicylaldehyde benzoyl hydrazone (SBH) is a Schiff base ligand, with antioxidant and iron-chelating properties. Having previously reported the ability of SBH to mitigate iron-mediated neurotoxicity, here we evaluated its capacity to alleviate inflammation and behavioural dysfunction in vitro and in vivo. BV2 microglia were stimulated with the neurotoxin 6-hydroxydopamine (6-OHDA; 50µM), in the presence and absence of SBH. Inflammatory responses were assessed by nitric oxide production and the release of proinflammatory cytokines (TNF, IL-6). Microglial activation was characterised by gene expression analysis, while intracellular iron and copper accumulation was quantified using metal assays. In vivo, zebrafish larvae (Danio rerio; 4 days post-fertilisation) were challenged with lipopolysaccharide (LPS; 20µg/ml) following intravenous administration of SBH. Pathological outcomes included survival, morphology, touch-startle sensorimotor behaviour, and neutrophil migration as a marker of inflammation. Statistical comparisons were made using two-way ANOVA and Chi-squared analyses. 6-OHDA induced robust inflammatory mediator production in microglia, accompanied by a shift toward an M1-like, glycolytic phenotype, intracellular iron and copper accumulation, and dysregulation of genes involved in ferroptosis (Gpx4, Ascl4) and cuproptosis (Slc31a1, Fdx1). SBH attenuated microglial activation, likely through restoration of iron homeostasis. In zebrafish larvae, SBH reduced LPS-induced inflammation, decreased mortality and non-lethal malformations, and restored sensorimotor behaviour. These findings identify SBH as a promising therapeutic strategy for targeting neuroinflammatory disease.