In Vitro Evaluation of a Hydroxytyrosol‐Rich Olive Mill Wastewater Extract for Neuroprotection Against Hyperglycemia‐Induced Cortical Damage in Tunisian <i>Psammomys obesus</i>

Cerebral ischemia represents a significant cause of morbidity and mortality worldwide, particularly in diabetic patients, where hyperglycemia exacerbates ischemic brain injury. This study examined whether hydroxytyrosolricholive mill wastewater (HT-OMWW) could protect against glucose-induced neuronal damage in primary cortical cultures from Psammomys obesus, a natural model of Type 2 diabetes. Cortical cells were exposed to hyperglycemic conditions of 25 and 40 mM glucose for 5 days, followed by HT-OMWW treatment (1-100 µg/mL) for 48 h. Cell viability was assessed using trypan blue exclusion and 4'6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, whereas immunofluorescence was used to analyze glial activation and cytoskeletal organization. Results demonstrated dose-dependent neuroprotection with optimal effects at 5-20 µg/mL concentrations. HT-OMWW significantly enhanced neuronal survival, reduced glial activation, and preserved cytoskeletal integrity under hyperglycemic stress. These findings suggest that olive mill wastewater represents a promising renewable source of neuroprotective compounds for managing ischemic brain injury in diabetic contexts through antioxidant, anti-inflammatory and cytoprotective mechanisms.