The chronic loss of brain cortex was reduced by the hEPO+MBs/FUS treatment. These results indicated that MBs/FUS enhanced the hEPO entry even 5 h after I/R, which resulted in neuron protection in both acute and chronic phases. Although stroke itself might alter hEPO delivery, the amount of hEPO entering the infarction area did not produce significant therapeutic effect. As hEPO combined with MBs/FUS, it can result in a significant neuroprotection on both acute and chronic phases. It has been demonstrated that intracerebraventricular administration of hEPO inhibits the I/R-induced brain injury. However, direct injection of hEPO into the brain is not a practical approach to have an appropriate hEPO distribution in the entire infarcted region. In the meanwhile, this kind of interstitial method can result in severe hemorrhages and brain trauma. On the contrary, systemic delivery of hEPO can have a much more uniform distribution of hEPO in the infarcted SB431542 volume but may be limited by the therapeutic time window. In this study, transcranial, noninvasive FUS technology was demonstrated to be a useful modality to transiently open the localized BBB for the targeted delivery of neuroprotectant to treat the ischemic stroke-induced brain injury beyond the conventional therapeutic time window. Brines et al. reported that animals receiving hEPO,3 h after occlusion showed significant reduction of necrosis volume compared with controls. Animals receiving hEPO 6 h after occlusion exhibited a significant decrease in injury volume, but the effect was substantially smaller compared with animals receiving hEPO earlier. Gan et al. reported that EPO exerted significantly neuroprotective effects when administered up to 4 h after I/R in MCAO model, but the effects were significantly diminished and lost when administered 6 h after I/R. In our study, we employed 3VO for 50 min and injected EPO at 5 h after reperfusion and the result showed that there was no significant neuroprotection. These might be due to different stroke models with various occlusion and ischemic duration would produce different levels of impact on the brain. EPO-TAT administered at the onset of post-stroke reperfusion showed the ability across the BBB for neuroprotection. Derivatives of EPO such as CEPO had the neuroprotection ability only within 4 h after occlusion, which is equal to 3 h after reperfusion, in a rat model of focal ischemia. BBB is more permeable for these EPO derivatives within 3 h after reperfusion due to leaky vascularity. Mutant EPO exerted neuroprotective effects up to 4 h after reperfusion but gradually lose its efficacy as time went by. However, the neuroprotective effects were diminished and lost when the mutant EPO was administered 6 h after reperfusion. Ischemia is an acute pathological process and cells die rapidly within first several hours after ischemia. Therefore, neuroprotective drugs must be delivered within their therapeutic window. In this study, we demonstrated that MBs/FUS had the ability to enhance EPO into the brain at 5 h after reperfusion.