Noninvasive ultrasound surgery can be achieved using focused ultrasound to locally affect the targeted site without damaging intervening tissues. Mechanical ablation and histotripsy use short and intense acoustic pulses to destroy the tissue via a purely mechanical effect. Here, we show that coupled with low-frequency excitation, targeted microbubbles can serve as mechanical therapeutic warheads that trigger potent mechanical effects in tumors using focused ultrasound. Upon low frequency excitation (250 kHz and below), high amplitude microbubble oscillations occur at substantially lower pressures as compared to higher MHz ultrasonic frequencies. For example, inertial cavitation was initiated at a pressure of 75 kPa for a center frequency of 80 kHz. Low frequency insonation of targeted microbubbles was then used to achieve low energy tumor cell fractionation at pressures below a mechanical index of 1.9, and in accordance with the Food and Drug Administration guidelines. We demonstrate these capabilities in vitro and in vivo. In cell cultures, cell viability was reduced to 16% at a peak negative pressure of 800 kPa at the 250 kHz frequency (mechanical index of 1.6) and to 10% at a peak negative pressure of 250 kPa at a frequency of 80 kHz (mechanical index of 0.9). Following an intratumoral injection of targeted microbubbles into tumor-bearing mice, and coupled with low frequency ultrasound application, significant tumor debulking and cancer cell death was observed. Our findings suggest that reducing the center frequency enhances microbubble-mediated mechanical ablation; thus, this technology provides a unique theranostic platform for safe low energy tumor fractionation, while reducing off-target effects.

Inflammatory demyelinating diseases of peripheral nerves are associated with altered nerve conduction and with activation of the coagulation pathway. Thrombin mediates many of its effects through protease-activated receptor 1 (PAR-1). We examined the possibility that thrombin may mediate conduction abnormalities through PAR-1 on rat sciatic nerve. PAR-1 was found to be present by both RT-PCR and Western blot analysis of the sciatic nerve. Activation of PAR-1 by a specific peptide agonist caused a 3-fold increase in phosphorylated extracellular signal-regulated kinase (ERK) in the sciatic nerve indicating the existence of functional receptors in the nerve. By confocal immunofluoresence microscopy of the sciatic nerve using anti-PAR-1 antibody and double staining for the paranodal marker contactin-associated protein 1 (Caspr1) or the nodal markers gliomedin and ezrin, the receptor was localized predominantly to myelin microvilli at the node of Ranvier. Thrombin and the PAR-1-specific agonist were applied to exposed rat sciatic nerve and their effects on nerve conduction were measured. Thrombin at concentrations of 100 and 200 U/ml and PAR-1 agonists 150 and 300 muM produced a conduction block within 30 min of application. This effect was maintained for at least 1 h and was reversible by washing. The function of the nodal non-compacted myelin is not well known. The current results implicate this structure and PAR-1 activation in the pathogenesis of conduction block in inflammatory and thrombotic nerve diseases.

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory diseases of the central nervous system (CNS). Activated coagulation factors are associated with inflammation and are elevated in the plasma of animals with EAE. Thrombin is a key coagulation factor and its major endogenous inhibitors are antithrombin III (ATIII) in the plasma and protease nexin 1 (PN-1) in the brain. We measured the capacity of brain homogenates to inhibit exogenous thrombin and the CNS levels of ATIII and PN-1 during the course of EAE. Acute EAE was induced in SJL/J mice by immunization with mouse spinal cord homogenates. On Days 8, 13, and 22 post-immunization, inhibition of exogenous thrombin activity was measured by a recently developed fluorimetric assay. PN-1 and ATIII were assayed both by immunohistochemistry and by immunoblots in the brain and spinal cord. Total brain thrombin inhibitory activity increased (32%) in EAE mice at the peak of clinical disease (Day 13, P=0.04 compared to controls). Brain ATIII also increased at the peak of disease (2.5-fold higher than controls, P=0.0001), and correlated significantly with clinical scores at all stages of disease (r=0.72, P=0.0068). In contrast, PN-1 elevations were more pronounced at the preclinical stage on Day 8 (3-fold higher than controls, P=0.01) than on Day 13 (1.4-fold higher, P=0.005). Increased brain thrombin inhibition at the clinical peak of EAE probably reflects increased influx of plasma thrombin inhibitors. Early PN-1 changes represent a potential target for thrombin modulating drugs in EAE and MS.

Taking microbubbles contrast agents into the nanoscale holds the promise for noninvasive cancer therapy. However, the nanobubbles small size limits the obtained bioeffects as a result of ultrasound cavitation, when...

Objectives Recent evidence has associated mood disorders with blood‐brain barrier (BBB)/ neurovascular unit (NVU) dysfunction, and reduction in blood vessels coverage by the water channel aquaporin‐4 (AQP4) immunoreactive astrocytes. Lithium is an established treatment for mood disorders, yet, its mechanism of action is partially understood. We investigated the effects of lithium on BBB integrity and NVU‐related protein expression in chronic mild stress (CMS) rat model of depressive‐like behavior. Methods Male Wistar rats were exposed for 5 weeks to unpredictable mild stressors with daily co‐administration of lithium chloride to half of the stressed and unstressed groups. Sucrose preference and open field tests were conducted to validate the depressive‐like phenotype, and dynamic contrast‐enhanced MRI analysis was utilized to assess BBB integrity in brain regions relevant to the pathophysiology of depression. Hippocampal AQP4 and claudin‐5 expression were studied using immunofluorescence, western blot, and enzyme‐linked immunosorbent assays. Results Lithium administration to the stressed rats prevented the reductions in sucrose preference and distance traveled in the open field, and normalized the stress‐induced hippocampal BBB hyperpermeability, whereas lithium administration to the unstressed rats increased hippocampal BBB permeability. Additionally, lithium treatment attenuated the decrease in hippocampal AQP4 to glial fibrillary acidic protein immunoreactivity ratio in the stressed rats and upregulated hippocampal claudin‐5 and BDNF proteins expression. Conclusions Our findings suggest that lithium administration in a rat CMS model of depressive‐like behavior is associated with attenuation of stressed‐induced hippocampal BBB/NVU disruption. These protective effects may be relevant to the mode of action of lithium in depression.