Effect of Focused Ultrasound Applied With an Ultrasound Contrast Agent on the Tight Junctional Integrity of the Brain Microvascular Endothelium

Abstract

Previous studies have investigated a potential method for targeted drug delivery in the central nervous system that uses focused ultrasound bursts combined with an ultrasound contrast agent to temporarily disrupt the blood-brain barrier (BBB). The purpose of this work was to investigate the integrity of the tight junctions (TJs) in rat brain microvessels after this BBB disruption. 1.5-MHz ultrasound bursts in combination with a gas contrast agent (Optison) was applied at two locations in the brain in 25 rats to induce BBB disruption. Using immunoelectron microscopy, the distributions of the TJspecific transmembrane proteins occludin, claudin-1, claudin-5, and of submembranous ZO-1 were examined at 1, 2, 4, 6 and 24 h after sonication. A quantitative evaluation of the protein expression was made by counting the number of immunosignals per micrometer in the junctional clefts. BBB disruption at the sonicated locations was confirmed by the leakage of intravenously administered horseradish peroxidase (HRP, m.w. 40,000 Da) and lanthanum chloride (La 3+ , m.w. ~ 139 Da). Leakage of these agents was observed at 1 and 2 h and in a few vessels at 4 h after ultrasound application. These changes were paralleled by the apparent disintegration of the TJ complexes, as evidenced by the redistribution and loss of the immunosignals for occludin, claudin-5 and ZO-1. Claudin-1 seemed less involved. At 6 and 24 h after sonication, no HRP or lanthanum leakage was observed, and the barrier function of the TJs, as indicated by the localization and density of immunosignals, appeared to be completely restored. This study provides the first direct evidence that ultrasound bursts combined with a gas contrast agent cause disassembling of the TJ molecular structure, leading to loss of the junctional barrier functions in brain microvessels. The BBB disruption appears to last up to 4 h after sonication and permits the paracellular passage of agents with molecular weights up to at least 40 kDa. These promising features can be exploited in the future development of this method that could enable the delivery of drugs, antibodies or genes to targeted locations in the brain.

Reversible and irreversible vascular bioeffects induced by ultrasound and microbubbles in chorioallantoic membrane model

“…This could be due to bioeffects such as red blood cell lysis (13) or thrombus formation (14). Similar transient vascular damage has previously been reported for blood brain barrier vasculature resulting in erythrocyte extravasation (15)(16)(17). Acoustic research is currently in progress to use USMB for thrombolysis (18,19) however the results from this study indicate that USMB may also cause temporary thrombus formation.…”

Disrupting the blood–brain barrier by focused ultrasound induces sterile inflammation

“…Alterations in metabolism and in the structure of cerebral vascular endothelium led to the impairment of the BBB function (i.e., disrupted homeostasis) and to the leakage of blood products into the parenchyma microenvironment (14,15,17,33 (41,42). Another explanation for the BBBD resulting from pFUS+MB exposure is that pFUS+MB increases membrane permeabilization or deformation (30) at the adluminal surfaces of endothelial cells, causing leakage or transcytosis of Gd chelates into the parenchyma and making it possible to visualize the changes noninvasively by MRI (11).…”

“…The rapid increase in TNFα and other proinflammatory factors indicates an acute involvement of NVU elements in response to hypoxia or injury (14,48). Previous studies have suggested that the mechanism of action by which pFUS+MB exposure results in BBBD was stable acoustic cavitation forces acting at the level of TJP (i.e., vessel centric) stretching apart endothelial cells (6,20,28,30,41). However, this proposed mechanism does not address how the cavitation forces interact directly with other cells in the NVU.…”

Reorganization of Gap Junctions after Focused Ultrasound Blood–Brain Barrier Opening in the Rat Brain

“…Blockade of the Connexin 43 binding site leads to a significant increase in plaque size (Hunter et al, 2005). Sheikov et al (2008) found evidence for a reversible disintegration of the tight-junctional molecular complexes with a transient loss of immunosignals for ZO-1 after ultrasound-induced opening of the BBB. Consistent with our findings, changes in tight-junctional molecule expression appeared a few hours after insonation, with full restoration at 24 hours (Sheikov et al, 2008).…”