Acute Effects of Focused Ultrasound-Induced Blood-Brain Barrier Opening on Anti-Pyroglu3 Abeta Antibody Delivery and Immune Responses

Abstract

Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid plaques and hyperphosphorylated tau in the brain. Currently, therapeutic agents targeting amyloid appear promising for AD, however, delivery to the CNS is limited due to the blood-brain-barrier (BBB). Focused ultrasound (FUS) is a method to induce a temporary opening of the BBB to enhance the delivery of therapeutic agents to the CNS. In this study, we evaluated the acute effects of FUS and whether the use of FUS-induced BBB opening enhances the delivery of 07/2a mAb, an anti-pyroglutamate-3 Aβ antibody, in aged 24 mo-old APP/PS1dE9 transgenic mice. FUS was performed either unilaterally or bilaterally with mAb infusion and the short-term effect was analyzed 4 h and 72 h post-treatment. Quantitative analysis by ELISA showed a 5-6-fold increase in 07/2a mAb levels in the brain at both time points and an increased brain-to-blood ratio of the antibody. Immunohistochemistry demonstrated an increase in IgG2a mAb detection particularly in the cortex, enhanced immunoreactivity of resident Iba1+ and phagocytic CD68+ microglial cells, and a transient increase in the infiltration of Ly6G+ immune cells. Cerebral microbleeds were not altered in the unilaterally or bilaterally sonicated hemispheres. Overall, this study shows the potential of FUS therapy for the enhanced delivery of CNS therapeutics.

Keywords: focused ultrasound; microglia; pyroglutamate-3 Aβ.

Figure 1

Study overview and the bioavailability of the 07/2a mAb in the brain (A) Experimental design and timeline of the experiment (image generated using BioRender). For unilateral FUS, 24 months old APP PS1dE9 mice were i.v. administered with single dose of 300 µg 07/2a mAb with microbubble i.v. infusion and FUS-BBBD on the brain’s right hemisphere. Mice received 2% Trypan blue dye prior to euthanasia and were taken down at 4 h (n = 5) and 72 h (n = 4) after mAb infusion; saline perfused and brains were collected for coronal sections. Non-sonicated left hemisphere was identified with a small needle mark. For Bilateral FUS cohort, both hemispheres received ultrasound and the same procedure has been followed but without Trypan dye injection. Collected brains were bisected; right hemispheres were used for ELISA and left hemisphere for histology. Representative pictures for trypan blue extravasation (arrows) on the sonicated right hemisphere across Bregma −0.94 mm to −1.64 mm from the unilateral FUS study. (B) Anti pGlu3-42 Aβ mAb levels from the half brain homogenates after 4 h and 72 h post treatment in the bilateral FUS cohort were analyzed by ELISA (C) Brain-to-blood concentration ratio of 07/2a mAb at 4 h and 72 h. Black dots represent data from individual mice. Data are expressed as mean ± SEM. Two-Way ANOVA, with Bonferroni’s multiple comparison test # p = 0.1, *, p < 0.05, **, p < 0.005.

Figure 2

Focused ultrasound and IgG2a immunoreactivity (IR). (A) Representative DAB staining (anti mouse-IgG2a) for the left and right hemispheres in the unilateral FUS cohort. (B) Triple immunofluorescence labelling for Iba1, S97 (general Aβ) and anti-IgG2a indicating presence of antibody with Aβ plaques on the FUS side (right hemisphere). Unilateral FUS on the right hemisphere showed (C) increased anti-IgG2a IR in the cortex at 4 h (p < 0.05) and 72 h (p < 0.05) after 07/2a treatment and (D) no significant changes in the hippocampus; paired-t-test. (E,F) Cortical and hippocampal anti-IgG2a IR in the bilateral FUS cohort at 4 h and 72 h post-treatment. Arrowheads indicating IgG2a IR with some possible staining around plaques. Increased anti-IgG2a IR was found in the (G) cortex and (H) hippocampi after combination treatment at both time points, however, a significant increase was found in the cortex at 4 h. Black dots represent data from individual mice. Two-Way ANOVA, with Bonferroni’s multiple comparison test *, p < 0.05. Scale bar, (A) = 200µm and (B,E) = 20 µm.

Figure 3

Acute immune response after single dose of 07/2a and FUS treatment. (A) Representative images (20×) for Iba1 and CD68 immunoreactivity after 4 h of 07/2a administration and unilateral sonication on the right hemisphere indicating increased microglial area coverage in the cortex compared to the non-FUS contralateral left hemisphere. (B) Quantitative analysis for general microglial marker (Iba1) and phagocytic marker (CD68) coverage in the cortical region from unilateral FUS cohort. After 07/2a mAb administration and sonication on the right hemisphere an increase in glial activity was seen compared to the contralateral left hemisphere (No FUS) control. Paired t-tests (two-tailed, α = 0.05). In the bilateral FUS cohort, 07/2a mAb treated alone or 07/2a + FUS treated mice after 4 h and 72 h post treatment showed a slight increase in glial activity. Data are represented as mean ± SEM. Two-Way ANOVA with Bonferroni’s multiple comparison test. (C) Representative pictures (4×) of Ly6G staining after 4 & 72 h treatment in the sonicated right hemisphere and contralateral non sonicated hemisphere. White arrowheads indicate Ly6G⁺ staining. (D) Area positive for Ly6G measured in the 4 h & 72 h unilateral FUS cohort expressed as percentage cerebral area, Paired t-tests (two-tailed, α = 0.05). Black dots represent data from individual mice. # p = 0.05, * p < 0.05, ** p < 0.005. Scale bar (B) = 50µm, (D) = 100 µm.

Figure 4

Prussian blue-positive cerebral microhemorrhages (CMH). (A) Representative pictures for the Perls’ Prussian blue staining in the cortex after unilateral sonication. Blue colored pigments (arrowheads) indicate hemosiderin deposits and these microhemorrhages are observed around capillaries. (B) Unilateral FUS cohort with sonication on right hemisphere did not show any significant microhemorrhages compared to the contralateral non sonicated left-hemisphere. Paired t-tests (two-tailed, α = 0.05). (C) In the bilateral FUS brain tissue sections no significant CMH were observed both at 4 h and 72 h post treatment. Two-way ANOVA with Bonferroni’s multiple comparison. Data are represented as mean ± SEM. n = 3–5 mice per group. Scale bar (A) = 200 µm.