(E-195) TRANSCRIPTOMIC INVESTIGATION OF BIOEFFECTS FROM MICROBUBBLE AND FOCUSED ULTRASOUND ASSISTED BLOOD BRAIN BARRIER DISRUPTION

Introduction:: Blood-brain barrier disruption (BBBD) with focused ultrasound and microbubbles (FUS/MB) is a promising drug delivery method due to its noninvasiveness and reversibility of the procedure, however, a deeper understanding of inflammatory processes that occur in response to disruption is still required. Local mechanical forces exerted from an oscillating or cavitating microbubble under ultrasound can unfasten tight junctions between endothelial cells creating pores. However, inflammation can be triggered in response to BBBD known as the sterile immune response (SIR). It is important to understand the extent and duration of the sterile immune response (SIR) following FUS/MB BBBD. Prior research indicates that the release of damage associated molecular proteins and cytokines shortly after BBBD activates inflammatory pathways such as NF-kB and initiates SIR cascades, especially after 6 hours.1 Our group is seeking to better understand the biological effects of ultrasound parameters, unified by the mechanical index (MI), and microbubble volume dose (MVD)2 on SIR following FUS/MB BBBD. We characterized the amount of BBBD and SIR for a range of microbubble doses (0.1 to 40 μL/kg) and MIs (0.2 to 0.6). The objective of our study is to establish the therapeutic window by defining an onset of significant BBBD and SIR at varying experimental parameters (MI and MVD).


Materials and Methods:: DPSC PEG40s microbubbles were size isolated to a mean diameter of 3 ± 0.3 microns. Mice were first imaged in a 9.4T MRI scanner on the T2 weighted channel to identify the striatum. Animals were then transferred to the RK50 focused ultrasound machine where mice were retro-orbitally co-injected with a gadolinium-based contrast agent (4 mmol/kg) and microbubbles (0.1 to 40 μL/kg). After injection, mice were sonicated (peak negative pressures: 0.246, 0.492, 0.738) for 5 minutes at three pressures: 0.246, 0.492 and 0.738 After FUS treatment, animals were re-imaged in the MRI on a T1 weighted channel where BBBD could be visually confirmed through gadolinium contrast enhancement. Six hours after FUS treatment, mouse brains were dissected out, perfused with ice cold 1x PBS and mRNA was extracted and processed for bulk RNA sequencing.

Results, Conclusions, and Discussions:: As expected, the extent of BBBD (quantified by T1w MRI) and SIR (quantified by bulk RNA sequencing) increased with MI and MVD. Out of the analysis of 50 hallmark gene sets, Figure 1A shows the 6 most significantly upregulated pathways associated with inflammation: Allograft rejection, hypoxia, IL6/JAK/STAT3 signaling, inflammatory response, interferon gamma response and TNFa signaling via NFkB. Figure 1B further investigates these pathways which included the total analysis of 395 combined genes and identification of 144 significantly upregulated genes and 47 most significantly upregulated transcripts. Results from our study will provide a deeper biological understanding of the effects of FUS/MB on BBBD. This will provide a more defined therapeutic window in which an immune response can be predicted, giving key insights into the safety of BBBD for future clinical translation.

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1. 
   Kovacs ZI, Kim S, Jikaria N, Qureshi F, Milo B, Lewis BK, Bresler M, Burks SR, Frank JA. Disrupting the blood-brain barrier by focused ultrasound induces sterile inflammation. Proc Natl Acad Sci U S A. 2017 Jan 3;114(1):E75-E84. doi: 10.1073/pnas.1614777114. Epub 2016 Dec 19. PMID: 27994152; PMCID: PMC5224365.
   
   

2. 
   Song KH, Fan AC, Hinkle JJ, Newman J, Borden MA, Harvey BK. Microbubble gas volume: A unifying dose parameter in blood-brain barrier opening by focused ultrasound. Theranostics. 2017 Jan 1;7(1):144-152. doi: 10.7150/thno.15987. PMID: 28042323; PMCID: PMC5196892.