AimsIschemic stroke remains a challenge in medical research because of the limited treatment options. Recombinant human tissue plasminogen activator (rtPA) is the primary treatment for recanalization. However, nearly 50% of the patients experience complications that result in ineffective reperfusion. The precise factors contributing to ineffective reperfusion remain unclear; however, recent studies have suggested that immune cells, notably neutrophils, may influence the outcome of rtPA thrombolysis via mechanisms such as the formation of neutrophil extracellular traps. This study aimed to explore the nonthrombolytic effects of rtPA on neutrophils and highlight their contribution to ineffective reperfusion.MethodsWe evaluated the effects of rtPA treatment on middle cerebral artery occlusion in rats. We also assessed neutrophil infiltration and activation after rtPA treatment in vitro and in vivo in a small cohort of patients with massive cerebral ischemia (MCI).ResultsrtPA increased neutrophil infiltration into the brain microvessels and worsened blood-brain barrier damage during ischemia. It also increased the neutrophil counts of the patients with MCI.ConclusionNeutrophils play a crucial role in promoting ischemic injury and blood-brain barrier disruption, making them potential therapeutic targets. Recombinant human tissue plasminogen activator (rtPA) activates neutrophils, enhancing their migration and adhesion, which participate in the side effect of rtPA.image