A comparative study of the effects of Aducanumab and scanning ultrasound on amyloid plaques and behavior in the APP23 mouse model of Alzheimer disease.

BACKGROUND: Aducanumab is an anti-amyloid-ß (Aß) antibody that achieved reduced amyloid pathology in Alzheimer's disease (AD) trials; however, it is controversial whether it also improved cognition, which has been suggested would require a sufficiently high cumulative dose of the antibody in the brain. Therapeutic ultrasound, in contrast, has only begun to be investigated in human AD clinical trials. We have previously shown that scanning ultrasound in combination with intravenously injected microbubbles (SUS), which temporarily and safely opens the blood-brain barrier (BBB), removes amyloid and restores cognition in APP23 mice. However, there has been no direct testing of how the effects of SUS compare to immunotherapy or whether a combination therapy is more effective. METHODS: In a study comprising four treatment arms, we tested the efficacy of an Aducanumab analog, Adu, both in comparison to SUS, and as a combination therapy, in APP23 mice (aged 13-22 months), using sham as a control. The active place avoidance (APA) test was used to test spatial memory, and histology and ELISA were used to measure amyloid. Brain antibody levels were also determined. RESULTS: We found that both Adu and SUS reduced the total plaque area in the hippocampus with no additive effect observed with the combination treatment (SUS + Adu). Whereas in the cortex where there was a trend towards reducing the total plaque area from either Adu or SUS, only the combination treatment yielded a statistically significant decrease in total plaque area compared to sham. Only the SUS and SUS + Adu groups included animals that had their plaque load reduced to below 1% from above 10%. There was a robust improvement in spatial memory for the SUS + Adu group only, and in this group the level of Adu, when measured 3 days post-treatment, was 5-fold higher compared to those mice that received Adu on its own. Together, these findings suggest that SUS should be considered as a treatment option for AD. Alternatively, a combination trial using Aducanumab together with ultrasound to increase brain levels of the antibody may be warranted.

Role for caveolin-mediated transcytosis in facilitating transport of large cargoes into the brain via ultrasound.

The blood-brain barrier (BBB) is a dynamic diffusional barrier regulating the molecular and chemical flux between the blood and brain, thereby preserving cerebral homeostasis. Endothelial cells form the core anatomical component of the BBB based on properties such as specialized junctional complexes between cells, which restricts paracellular transport, and extremely low levels of vesicular transport, restricting transcytosis. In performing its protective function, the BBB also constrains the entry of therapeutics into the brain, hampering the treatment of various neurological disorders. Focused ultrasound is a novel therapeutic modality that has shown efficacy in transiently and non-invasively opening the BBB for the targeted delivery of therapeutics to the brain. Although the ability of ultrasound to disrupt the junctional assembly of endothelial cells has been partially investigated, its effect on the transcellular mode of transport has been largely neglected. In this study, we found that ultrasound induces a pronounced increase in the levels of the vesicle-forming protein caveolin-1. In order to investigate the role of vesicle-mediated transcytoplasmic transport, we compared the leakage of various cargo sizes between a mouse model that lacks caveolin-1 and wild-type mice following sonication of the hippocampus. The absence of caveolin-1 did not lead to overt abnormalities in the cerebral vasculature in the mice. We found that caveolin-1 has a critical role specifically in the transport of large (500 kDa), but not smaller (3 and 70 kDa) cargoes. Our findings indicate differential effects of therapeutic ultrasound on cellular transport mechanisms, with implications for therapeutic interventions.

Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound.

Only a small fraction of therapeutic antibodies targeting brain diseases are taken up by the brain. Focused ultrasound offers a possibility to increase uptake of antibodies and engagement through transient opening of the blood-brain barrier (BBB). In our laboratory, we are developing therapeutic approaches for neurodegenerative diseases in which an antibody in various formats is delivered across the BBB using microbubbles, concomitant with focused ultrasound application through the skull targeting multiple spots, an approach we refer to as scanning ultrasound (SUS). The mechanical effects of microbubbles and ultrasound on blood vessels increases paracellular transport across the BBB by transiently separating tight junctions and enhances vesicle- mediated transcytosis, allowing antibodies and therapeutic agents to effectively cross. Moreover, ultrasound also facilitates the uptake of antibodies from the interstitial brain into brain cells such as neurons where the antibody distributes throughout the cell body and even into neuritic processes. In our studies, fluorescently labeled antibodies are prepared, mixed with in-house prepared lipid-based microbubbles and injected into mice immediately before SUS is applied to the brain. The increased antibody concentration in the brain is then quantified. To account for alterations in normal brain homeostasis, microglial phagocytosis can be used as a cellular marker. The generated data suggest that ultrasound delivery of antibodies is an attractive approach to treat neurodegenerative diseases.

Scanning ultrasound in the absence of blood-brain barrier opening is not sufficient to clear ß-amyloid plaques in the APP23 mouse model of Alzheimer's disease.

A major challenge in treating brain diseases is presented by the blood-brain barrier (BBB) that constitutes an efficient barrier not only for toxins but also a wide range of therapeutic agents. In overcoming this impediment, ultrasound in combination with intravenously injected microbubbles has emerged as a powerful technology that allows for the selective brain uptake of blood-borne factors and therapeutic agents by transient opening of the blood-brain barrier. We have previously shown that ultrasound in combination with microbubbles, but in the absence of a therapeutic agent, can effectively clear protein aggregates such as the hallmark lesions of Alzheimer's disease, amyloid-ß (Aß) plaques and Tau-containing neurofibrillary tangles. We have also demonstrated that the associated memory and motor impairments can be ameliorated or even restored. These studies included a negative sham control that received microbubbles in the absence of ultrasound. However, considering that ultrasound on its own is a pressure wave which has bioeffects, the possibility remained that ultrasound, without microbubbles, would also clear amyloid. We addressed this by performing repeated ultrasound only treatments of one brain hemisphere of Aß-depositing APP23 mice, using the contralateral hemisphere as the unsonicated control. This was followed by an extensive histological analysis of fibrillar and non-fibrillar amyloid. We found that ultrasound on its own was not sufficient to clear amyloid. This implies that although ultrasound on its own has neuromodulatory effects, exogenously supplied microbubbles are required for the clearance of Aß deposits.

Measurement of Poisson's ratio of dental composite restorative materials.

The aim of this study was to determine the Poisson ratio of resin-based dental composites using a static tensile test method. Materials used in this investigation were from the same manufacturer (3M ESPE) and included microfill (A110), minifill (Z100 and Filtek Z250), polyacid-modified (F2000), and flowable (Filtek Flowable [FF]) composites. The Poisson ratio of the materials were determined after 1 week conditioning in water at 37 degrees C. The tensile test was performed with using a uniaxial testing system at crosshead speed of 0.5 mm/min. Data was analysed using one-way ANOVA/post-hoc Scheffe's test and Pearson's correlation test at significance level of 0.05. Mean Poisson's ratio (n=8) ranged from 0.302 to 0.393. The Poisson ratio of FF was significantly higher than all other composites evaluated, and the Poisson ratio of A110 was higher than Z100, Z250 and F2000. The Poisson ratio is higher for materials with lower filler volume fraction.