The Glial Response to Focused Ultrasound-mediated Permeabilization of the Blood-brain Barrier: Evaluating the Potential Contribution of Microglia and Astrocytes to Pathology and Regeneration

Focused ultrasound in the presence of intravenous microbubbles (FUS) facilitates the non-invasive temporary permeabilization of the blood-brain barrier (BBB) for delivering therapeutics to the brain. FUS-mediated BBB modulation is known to activate microglia and astrocytes, which can exacerbate pathology or promote neuroprotection. I hypothesized that FUS induced a transient activation of microglia and astrocyte that is consistent with pro-regenerative functions. Firstly, I developed a method, MORPHological assessment of Outlier clusters (MORPHIOUS), to quantify microglial and astrocytic activation. MORPHIOUS is a machine learning tool that detects clusters of activated glia in histological sections by referencing control sample cell morphologies. As validation, identified clusters of activated microglia and astrocytes exhibited hallmark morphological and marker changes. Indeed, activated microglia were deramified and upregulated transforming growth factor beta 1 (Tgfβ1), and activated astrocytes were hypertrophic and expressed nestin. MORPHIOUS can therefore sensitively detect activated glia. Secondly, I used MORPHIOUS to characterize microglial and astrocytic activation from 1 to 30 days (D) post-FUS. Microglial activation peaked at 1D and was progressively attenuated between 4D and 30D post-FUS. Activated microglia predominantly colocalized with Tgfβ1, indicative of anti-inflammatory and neuroprotective functions; but not with the inflammation-associated cluster of differentiation 68. Astrocytic activation peaked at 4D and was significantly attenuated by 30D post-FUS. Activated astrocytes did not significantly proliferate or upregulate glial scar components. Thus, post-FUS glial activation was transient, and not indicative of pathology. Thirdly, to evaluate the regenerative potential of the brain environment post-FUS, I characterized neural progenitor cell (NPC) proliferation and oligodendrogenesis. NPC proliferation increased between 4D and 7D post-FUS and coincided with the activated astrocytic upregulation of insulin-like growth factor 2. Moreover, an increased proliferation of oligodendrocyte progenitors increased between 1D and 4D, promoted the number of newly developed oligodendrocytes at 30D. Therefore, post-FUS glial activation is permissive of broader brain regenerative programs. Collectively, this data supports my hypothesis by demonstrating that the FUS-mediated activation of microglia and astrocytes is transient, upregulates regenerative factors, and occurs alongside enhanced neurogenesis and oligodendrogenesis. This work positions FUS-mediated BBB permeabilization as a promising component of multimodal therapies, with potential to aid in the treatment of neurodegenerative disorders.

Department dc:contributor.department

Laboratory Medicine and Pathobiology

Year dc:date.issued

2021

Author dc:creator

• Silburt, Joseph Leib

Advisor dc:contributor.advisor

• Aubert, Isabelle

• Astrocytes
• Blood-brain barrier
• Brain Regeneration
• Focused Ultrasound
• Machine Learning
• Microglia

Handle dc:identifier.uri

http://hdl.handle.net/1807/130756

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/130756