Reorganization of gap junctions following focused ultrasound blood-brain barrier opening in the rat brain

Abstract

Background: MR-guided focused ultrasound (US) is a new technology for drug delivery across the BBB to stimulate neurogenesis and angiogenesis after stroke. Mechanisms of action and related bioeffects, however, are largely unknown. In this study we examined the effect of US-induced BBB opening on plaque size distribution of gap junctions in cortical neurons (Connexin36) and astrocytes (Connexin43), which are important for information transfer between adjacent cells and also for tissue homeostasis. Methods: 16 male Wistar rats were treated with 1Mhz US (intracranial pressure 1,27 MPa, pulse length 10 ms, repetition frequency 1Hz, duration 1 min) together with IV microbubbles (Optison, 0.1ml/kg). The cortex of one hemisphere was insonated, the contralateral hemisphere was used as control. Immunofluorescence and immunohistochemistry for detection of Connexin36 and Connexin43 were performed. Results: Quantification of immunofluorescence signals revealed that compared to non-insonicated hemispheres, small size Connexin43 and Connexin36 gap junctional plaques were markedly reduced in areas with BBB breakdown after three to six hours (34.02±6.04% vs. 66.49±2.16%, p=0.02 for Connexin43; 33,80±1.24 vs. 36.77±3.43%, p=0.07 for Connexin36). Complementary, we found significant increases in large size gap junctional plaques (5.76±0.96% vs 1.02±0.84%, p=0.05 for Connexin43; 5.62±0.22% vs. 4.65±0.80%, p=0.02 for Connexin36). This effect was reversible 24 hours after US exposure. Western blot analyses did not show any change in the total Connexin amount. Conclusion: These results indicate that ultrasound-induced BBB opening leads to a transient reorganization of gap junctional plaques in both neurons and astrocytes. Plaque-size increase may be a cellular response to imbalances in extracellular homeostasis following BBB leakage.