Characterization of focused ultrasound blood-brain barrier disruption effect on inflammation as a function of treatment parameters

Abstract

The technology of focused ultrasound-mediated disruption of the blood-brain barrier (FUS-BBB opening) has now been used in over 20 Phase 1 clinical trials to validate the safety and feasibility of BBB opening for drug delivery in patients with brain tumors and neurodegenerative diseases. The primary treatment parameters, FUS intensity and microbubble dose, are chosen to balance sufficient BBB disruption to achieve drug delivery against potential acute vessel damage leading to microhemorrhage. However, other safety considerations due to second order effects caused by BBB disruption, such as inflammation and alteration of neurovascular function, are only beginning to be understood. This study builds on previous work that has investigated the inflammatory response following FUS-BBB opening. In this study, we characterize the effect of FUS intensity, microbubble dose and single vs multiple treatments on the extent of BBB disruption, observed level of microhemorrhage, and degree of inflammatory response at acute post-treatment time points in the wild-type mouse brain. Results show that upregulation of pro-inflammatory markers is primarily driven by microbubble dose, with peak effects seen at 24 hours post-treatment. We additionally saw significantly elevated levels of cytokine and chemokine markers in female vs male mice, despite no sex differences in level of BBB disruption or microglia activation. Multiple treatments did not result in increased levels of pro-inflammatory markers compared to single treatment baseline. However, we did see an interesting elevation of the anti-inflammatory molecule eNOS after multiple treatments, indicating active mechanisms were at work to restore homeostasis in the brain environment.

Keywords: Blood-brain barrier; Focused ultrasound; Inflammation.

Fig. 1.

Literature reports of BBB opening and inflammation as a function of FUS intensity and microbubble dose treatment parameters. Colored circles represent experimental groups at a particular FUS intensity and microbubble dose combination for each given publication. Background shaded regions indicate whether the publication reported no BBB opening, BBB opening only, or BBB opening plus evidence of an inflammatory response for the treatment parameters used. Colored circles on a boundary indicate results were equivocal for this parameter combination. Dashed black circles represent the treatment parameters considered in this study. The vertical dashed blue line indicates the currently approved clinical dose level of 10 μL/kg of Definity.

Fig. 2.

Trypan Blue fluorescence imaging for quantification of BBB opening area. A) Example images of trypan blue fluorescence imaging following FUS-BBB opening for each FUS intensity and time point are shown on top. Blue outlines delineate ROIs drawn to quantify area of BBB opening. Area of BBB opening as a percentage of total hemisphere area are shown in the bar graphs. Significant differences between groups are based on Two-way ANOVA analysis with Tukey’s multiple comparisons test (* = p < 0.05, ** = p < 0.01, *** = p < 0.001). B) Similar example images and bar graphs quantifying BBB opening for the microbubble dose groups. Male mice are plotted as squares, female mice as circles. Note that the example images shown for the 0.36 MPa / 20 μL/kg cases are the same in A) and B) as these mice were used in both sets of analyses.

Fig. 3.

Giemsa staining for quantification of microhemorrhage. A) Representative images of giemsa-stained brain sections showing the FUS-targeted right striatum for mice treated at 0.32, 0.36 and 0.40 MPa FUS intensity. Insets show areas of hemorrhage acquired at higher resolution. B) log10 transform of microhemorrhage area, collapsed over time points. C – D) Similar representative images and bar graphs quantifying microhemorrhage area for the microbubble dose groups. (* = p < 0.05, **** = p < 0.0001).

Fig. 4.

A) Heat map depicting mRNA levels of all pro-inflammatory markers measured by qPCR for the FUS intensity groups. No FUS control is marked as time zero. Color scale shows fold change of control. Based on two-way ANOVA analysis, * denotes a significant effect of the main factor of time, † denotes a significant effect of the main factor of FUS intensity, and • denotes a group significantly different than control following Tukey’s multiple comparison test, all at p < 0.05. B) Similar heat map depicting mRNA levels of all pro-inflammatory markers measured by qPCR for the microbubble dose groups.

Fig. 5.

Bar plots show comparisons of mRNA levels in male vs female mice from the 40 μL/kg microbubble dose groups. A) Measures of BBB opening area and microhemorrhage area do not show any differences between the sexes. B) The primary markers for astrocytosis (GFAP) and microgliosis (Iba1) do not show any differences between the sexes. NLRP3 does show significantly greater elevation in female mice compared to male mice, while IKBa is an outlier showing greater elevation in male mice. C) Of the nine cytokine, chemokine and adhesion molecule markers measure, eight showed significantly greater elevation in female mice compared to male mice for at least one time point. Seven of the markers had significance of p < 0.0001.

Fig. 6.

Western blot analysis of GFAP (A), ICAM (B) levels in male vs female mice from the 40 μL/kg microbubble dose groups. Bars represent densitometric quantification expressed as fold change relative to control (n = 3 per time point per sex).

Fig. 7.

A) Heat map depicting mRNA levels of all anti-inflammatory markers measured by qPCR for the FUS intensity groups. No FUS control is marked as cntrl. Color scale shows fold change of control. As in Fig. 4, * denotes a significant effect of the main factor of time, † denotes a significant effect of the main factor of FUS intensity, and • denotes a group significantly different than control following Tukey’s multiple comparison test, all at p < 0.05. B) Similar heat map depicting mRNA levels of all anti-inflammatory markers measured by qPCR for the microbubble dose groups.

Fig. 8.

5x vs 1x FUS treatments. A) Select examples comparing mRNA levels of pro-inflammatory markers in male mice after 1x or 5x FUS treatments, with sacrifice at 24 hours after final FUS treatment. Of the 13 pro-inflammatory markers measure, none showed a significant difference between the 1x and 5x groups based on a non-paired two-tailed t-test. B) Similar comparisons for mRNA levels of anti-inflammatory markers. Only eNOS was significantly higher in the 5x treated group compared to the 1x treated group. * * p < 0.01.

Fig. 9.

Differential effects of FUS intensity and microbubble dose on BBB opening, microhemorrhage and mRNA levels at 24 hours post treatment. Area of BBB opening (A), area of microhemorrhage (B) and upregulation of pro-inflammatory makers (C) are compared for 24-hour groups treated at the lowest and highest levels of FUS intensity and microbubble dose. The graph in (C) shows each pro-inflammatory marker plotted as fold change for the low/high FUS intensity group on the x-axis and fold change for the low/high microbubble groups on the y-axis.