Microglia change at the micro- and nano-scopic scales in response to therapeutic focused ultrasound blood-brain barrier modulation




Gonçalves de Andrade, Elisa

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Transcranial focused ultrasound sonication in combination with intravenously injected microbubbles (FUS+MB) has the unique ability to modulate blood-brain barrier (BBB) permeability with high spatial precision and in a minimally invasive manner. This process induces endothelial mechanical stress and the transient infiltration of blood-derived molecules, collectively triggering an acute inflammation in the targeted region. As the resident immune cells of the central nervous system (CNS), microglia are at the forefront of the acute inflammatory response triggered by FUS+MB BBB modulation. Notably, in inflammatory and non-inflammatory contexts, microglia are highly dynamic and continuously survey the brain parenchyma by extending their processes and interacting with surrounding elements, such as synapses and blood vessels. In response to stressors, microglia rapidly alter their cellular and molecular profile to help facilitate the return to homeostasis. While the underlying mechanisms by which FUS+MB alters microglial function remain largely unknown, several studies in adult mouse models of Alzheimer’s disease pathology have reported changes in the gene expression of microglia, e.g. ionized calcium binding adaptor molecule 1 (Iba1), and in their phagocytic activity, e.g. increased uptake of the toxic protein amyloid beta, in several CNS areas, including the hippocampus where emotional, cognitive and memory processing takes place. Characterizing the baseline responses of microglia to FUS+MB in healthy models is, however, still required to understand possible outcomes of this technology in physiological processes such as microglial parenchymal surveillance, maintenance of the BBB and synaptic plasticity. To address this need, I provide the first cellular (i.e., density, distribution, and morphology) and subcellular (i.e., ultrastructure) description of microglial changes at 1 hour and 24 hours after magnetic resonance imaging-guided focused ultrasound sonication with microbubbles (MRIgFUS) targeting the ipsilateral (ipsi-) ventral hippocampus of adult male mice. Using brightfield imaging of a double immunoperoxidase staining for immunoglobulin G and Iba1, I show that MRIgFUS causes the entering of IgG into the ipsi- Cornu ammonis 1 (CA1) parenchyma at 1 hour and 24 hours, where it correlates with the proximity between Iba1 positive (+) cells. Moreover, I found that specific states within the ipsi- microglial CA1 stratum lacunosum moleculare (LMol) population are more responsive to MRIgFUS compared to the contra- LMol, adjusting their soma, cell body and arborization parameters to a rod-like shape, while collectively, most LMol microglia adopt an elongated soma shape after MRIgFUS. Notably, I observed that the LMol nanoscale structure of microglia changes at 1 hour and 24 hours after MRIgFUS, increasing their interactions with the BBB in vessels with bigger areas and presenting swollen astrocytic endfeet. By contrast, MRIgFUS induces less frequent interactions between microglia, pre-synaptic elements and extracellular space, associated with trogocytosis or nibbling of synaptic elements. Collectively, my research suggests that, at 1 hour and 24 hours after MRIgFUS, ventral hippocampal modifications are mostly restricted to a microglia subset, warranting further mechanistic investigations but simultaneously supporting the overall application of this technology.



Blood-brain barrier, Microglia, Focused ultrasound with microbubbles, Scanning electron microscopy, Brightfield microscopy