Ecto-5'-nucleotidase (CD73) is an ecto-enzyme that hydrolyzes adenosine monophosphate to adenosine, which acts through one of four subtypes of adenosine receptors. Increased CD73 activity and expression in chronic neurodegenerative diseases leads to a higher concentration of adenosine in the extracellular space. This results in excessive activation of adenosine A2AR, which triggers proinflammatory signaling pathways that further promote neurodegeneration. Therefore, dual CD73/A2AR inhibition is a potential pharmacologic target in chronic neurodegeneration. In the present study, we used a novel in vitro model of neuroinflammation induced by activation of primary astrocytes with the proinflammatory cytokines TNF, IL-1α and C1q, which together are necessary and sufficient to induce a neurotoxic astrocyte phenotype (TIC-induced reactive astrocytes). To investigate the efficacy of dual CD73/A2AR blockade, the TIC-induced reactive astrocytes were then treated with CD73 inhibitor- adenosine 5'-(α,β-methylene) diphosphate (APCP) and the A2AR antagonist- istradefylline. The TIC model was first used to study the temporal pattern of CD73 and A2AR expression and their membrane localizations to provide a suitable model for testing the efficacy of dual CD73/A2AR blockade. Dual CD73/A2AR inhibition with APCP and istradefylline in a TIC-induced reactive astrocyte model reduces neuroinflammation at the gene level by inducing a decreasing trend in the expression of Vcam1, the marker of TIC-induced reactive astrocytes, and proinflammatory genes such as C3, and also reduces oxidative stress parameters by increasing the activity of antioxidant enzymes. Acknowledgments- This study was supported by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Grant No. 451-03-66/2024-03/ 200178).