Theodorou, A. | bioRxiv Science

bioRxiv2025

Characterization of Microbubble Cavitation in Theranostic Ultrasound-mediated Blood-Brain Barrier Opening and Gene Delivery

RationaleThe characterization of microbubble activity has proven critical in assessing the safety and efficacy of ultrasound-mediated blood-brain barrier (BBB) opening and drug and gene delivery. In this study, we build upon our previous work on theranostic ultrasound (ThUS)-mediated BBB opening (ThUS-BBBO) and conduct for the first time a comprehensive characterization of the role of microbubble cavitation in ThUS-BBBO, as well as its impact on gene delivery with adeno-associated viruses (AAV). MethodsA repurposed imaging phased array was used throughout the study to generate focused transmits and record microbubble activity through high-resolution power cavitation imaging (PCI). The cavitation of microbubbles under ThUS pulses was first characterized in flow phantom using pulse lengths ranging from 1.5 to 20 cycles and under varying microbubble flow rates using a separate single-element transducer a passive cavitation detector (PCD). A comprehensive in vivo study in mice was then conducted to characterize the in vivo microbubble activity under ThUS and correlate the resulting cavitation with AAV-mediated transgene delivery and expression. The transcranial microbubble activity was first detected in two mice using a PCD, to confirm the findings of the flow phantom study. Next, three mouse studies were conducted to evaluate the relationship between cavitation and AAV delivery; one with three different microbubble size distributions using polydisperse and size-isolated microbubbles, one with variable burst length and burst repetition frequency, and one with different AAV serotypes and injection doses. Electronic beam steering was used to induce bilateral BBB opening with 1.5 cycle on the left and 10 cycles on the right hemisphere. Cavitation dose was correlated with BBB opening volume, AAV transgene expression was evaluated with immunofluorescence staining and histological safety was assessed with T2* imaging and Hematoxylin and Eosin staining. ResultsFrequency domain analysis in the phantoms revealed a broadband-cavitation dominance at the shorter pulse lengths, while harmonic cavitation components are significantly increased for longer pulses. The PCD was better at detecting higher frequency harmonics, while the signal received by the theranostic array was more broadband dominated. Analysis of signals in the time domain showed that the longer pulses induce higher microbubble collapse compared to short pulses. In the transcranial in vivo experiments, the PCD was able to detect increased harmonic cavitation for 10-cycle pulses. The microbubble study showed that 3-5 m microbubbles resulted in the largest cavitation doses, BBBO volumes and AAV transgene expression compared to the smaller microbubble sizes. The burst sequence study revealed that the sequences with shorter bursts and faster burst repetition frequencies induce larger BBBO volumes and AAV transduction due to faster microbubble replenishment in the focal volume. Increased erythrocyte extravasation was observed on the hemisphere sonicated with 10-cycle pulses. Transgene expression was also increased with injection dose, without notable side effects during the three-week survival period. Finally, AAV9 was shown to be the serotype with the highest transduction efficiency compared to AAV2 and AAV5 at the same injected dose. ConclusionsThis is the first comprehensive study into the microbubble cavitation under theranostic ultrasound. The phantom and in vivo studies show that the mechanism of ThUS-BBBO is mainly transient cavitation dominant, as microbubble collapse increases with pulse length despite the increased harmonic frequency response. Increased cavitation dose resulted in larger BBBO volumes and transgene expression in vivo. While ThUS induced microhemorrhage for most of the studied conditions, it did not have an impact on the survival and behavior of the mice.

bioengineering↗

bioRxiv2023

Better together: Objects in familiar constellations evoke high-level representations of real-world size

High-level vision is frequently studied at the level of either individual objects or whole scenes. An intermediate level of visual organisation that has received less attention is the "object constellation" - a familiar configuration of contextually-associated objects (e.g., plate + spoon). Recent behavioural studies have shown that information from multiple objects can be integrated to support observers high-level understanding of a "scene" and its constituent objects. Here we used EEG in human participants (both sexes) to test when the visual system integrates information across objects to support recognition, using representations of objects real-world size as a proxy for recognition. We briefly presented masked object constellations consisting of object silhouettes of either large (e.g., chair + table) or small (e.g., plate + spoon) real-world size, while independently varying retinal size. As a control, observers also viewed each silhouette in isolation. If object context facilitates object recognition, real-world size should be inferred more effectively when the objects appear in their contextually-associated pairs than in isolation, leading to the emergence of real-world size information in multivariate EEG patterns. Representational similarity analysis revealed that neural activity patterns captured information about the real-world size of object constellations from [~]200 ms after stimulus onset. This representation was stronger for, and specific to, object pairs as compared to single objects, and remained significant after regressing out visual similarity models derived from computational models. These results provide evidence for inter-object facilitation of visual processing, leading to a qualitatively different high-level representation of object pairs than single objects. Significance StatementThis study used electroencephalography decoding to reveal the neural timecourse of inter-object facilitation present for contextually-associated groups of objects (e.g., chair + table). Although ubiquitous in daily life, the object constellation level of representation has rarely been examined compared to isolated objects or entire scenes. By shedding new light on facilitatory interactions between objects, arising before 200ms of visual processing, our results provide insight into the continuum along which objects and scenes exist. At the same time, this work advances the current understanding of the neural basis of real-world size, using strict visual controls to show that inferred information about objects spatial scale in the real world emerges around 200 ms after stimulus onset.

neuroscience↗