Advanced physiological aging is associated with impaired cognitive performance and the inability to induce long-term potentiation (LTP), an electrophysiological correlate of memory. Here, we demonstrate in the physiologically aged, senescent mouse brain that scanning ultrasound combined with microbubbles (SUS+MB), by transiently opening the blood–brain barrier, fully restores LTP induction in the dentate gyrus of the hippocampus. Intriguingly, SUS treatment without microbubbles (SUSonly), i.e., without the uptake of blood-borne factors, proved even more effective, not only restoring LTP, but also ameliorating the spatial learning deficits of the aged mice. This functional improvement is accompanied by an altered milieu of the aged hippocampus, including a lower density of perineuronal nets, increased neurogenesis, and synaptic signaling, which collectively results in improved spatial learning. We therefore conclude that therapeutic ultrasound is a non-invasive, pleiotropic modality that may enhance cognition in elderly humans.
Rationale: The blood-brain barrier (BBB) while functioning as a gatekeeper of the brain, impedes cerebral drug delivery. An emerging technology to overcome this limitation is focused ultrasound (FUS). When FUS interacts with intravenously injected microbubbles (FUS +MB ), the BBB opens, transiently allowing the access of therapeutic agents into the brain. However, the ultrasound parameters need to be tightly tuned: when the acoustic pressure is too low there is no opening, and when it is too high, tissue damage can occur. We therefore asked whether barrier permeability can be increased by combining FUS +MB with a second modality such that in a clinical setting lower acoustic pressures could be used. Methods: Given that FUS +MB achieves BBB opening in part by disruption of tight junction (TJ) proteins such as claudin-5 of brain endothelial cells, we generated a stable MDCK (Madin-Darby Canine Kidney) II cell line (eGFP-hCldn5-MDCK II) that expresses fluorescently tagged human claudin-5. Two claudin-5 binders, the peptide mC5C2 and cCPEm (truncated form of an enterotoxin), reported previously to weaken the barrier, were synthesized and assessed for their abilities to enhance the permeability of cellular monolayers. We then performed a comparative analysis of single and combination treatments, measuring transendothelial electrical resistance (TEER) and cargo leakage, combined with confocal image analysis. Results: We successfully generated a novel cell line that formed functional monolayers as validated by an increased TEER reading and a low (< 0.2%) permeability to sodium fluorescein (376 Da). We found that the binders exerted a time- and concentration-dependent effect on barrier opening when incubated over an extended period, whereas FUS +MB caused a rapid opening followed by recovery after 12 hours within the tested pressure range. Importantly, preincubation with cCPEm prior to FUS +MB treatment resulted in greater barrier opening compared to either FUS +MB or cCPEm alone as measured by reduced TEER values and an increased permeability to fluorescently labelled 40 kDa dextran (FD40). Conclusion: The data suggest that pre incubation with clinically suitable binders to TJ proteins may be a general strategy to facilitate safer and more effective ultrasound-mediated BBB opening in cellular and animal systems and potentially also for the treatment of human diseases of the brain.
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