Extracellular vesicles derived from astrocytes pretreated with melatonin promoted neuro-angiogenesis in mice with ischemic medial prefrontal cortex

Ischemic stroke (IS) is one of the leading causes of death and long-term disability worldwide. In addition to physical deficits, stroke survivors also exhibit cognitive impairment. Reparative properties of mouse astrocyte extracellular vesicles (AS-EVs) have been proven in ischemic conditions. Here, the neuro-angiogenesis properties of melatonin (MT)-astrocyte EVs (MT-AS-EVs) were studied in IS mice. A total volume of 5 µl (~ 1 × 1011 particles) of MT-AS-EVs and AS-EVs was administered into the penumbra of the ischemic medial prefrontal cortex induced by photothrombosis. After 21 days, brain samples were taken, and protein levels of neuroangiogenesis (NeuN and vWF) and synaptogenesis (Homer, Synaptophysin, and Synapsin) were assessed using immunofluorescence (IF) staining and western blotting. The proinflammatory response was monitored by measuring TNF-α and TGF-β in brain samples. Behavioral tests were also conducted to evaluate cognitive performance. From TTC staining confirmed the efficiency of the present protocol for inducing IS in the target region. The injection of AS-EVs, especially MT-AS-EVs, reduced the infarcted area compared to IS mice. IF staining revealed induction of vWF + and NeuN + cells at the ischemic site in mice treated with AS-EVs and MT-AS-EVs, with a significant effect (p < 0.05). The protein levels of Homer, Synaptophysin, and Synapsin increased in the presence of MT-AS-EVs compared to the other experimental group. Additionally, MT-AS-EVs had the potential to attenuate the pro-inflammatory response by reducing TNF-α and TGF-β levels compared with IS mice (p < 0.05). It was noted that the improvement in behavioral parameters in the IS mice after injection of EVs, especially MT-AS-EVs (p < 0.05). AS-EVs are valid biological agents for inducing brain healing under IS conditions. Preconditioning with melatonin can enhance the reparative properties of AS-EVs, particularly in neuro-angiogenesis.