Amyloid-β plaque reduction, endogenous antibody delivery and glial activation by brain-targeted, transcranial focused ultrasound

Abstract

Noninvasive, targeted drug delivery to the brain can be achieved using transcranial focused ultrasound (FUS), which transiently increases the permeability of the blood-brain barrier (BBB) for localized delivery of therapeutics from the blood to the brain. Previously, we have demonstrated that FUS can deliver intravenously-administered antibodies to the brain of a mouse model of Alzheimer's disease (AD) and rapidly reduce plaques composed of amyloid-β peptides (Aβ). Here, we investigated two potential effects of transcranial FUS itself that could contribute to a reduction of plaque pathology, namely the delivery of endogenous antibodies to the brain and the activation of glial cells. We demonstrate that transcranial FUS application leads to a significant reduction in plaque burden four days after a single treatment in the TgCRND8 mouse model of AD and that endogenous antibodies are found bound to Aβ plaques. Immunohistochemical and western blot analyses showed an increase in endogenous immunoglobulins within the FUS-targeted cortex. Subsequently, microglia and astrocytes in FUS-treated cortical regions show signs of activation through increases in protein expression and changes in glial size, without changes in glial cell numbers. Enhanced activation of glia correlated with increased internalization of Aβ in microglia and astrocytes. Together these data demonstrate that FUS improved the bioavailability of endogenous antibodies and led to a temporal activation of glial cells, providing evidence towards antibody- and glia-dependent mechanisms of FUS-mediated plaque reduction.

Keywords: Alzheimer's disease; Amyloid-beta peptide; Astrocytes; Autoantibodies; Focused ultrasound; Immunoglobulin; Microglia; Transgenic mice.

Copyright © 2013 Elsevier Inc. All rights reserved.

Figures

Fig. 1

MRIgFUS setup and confirmation of BBB disruption. A, Mice were placed in a supine position over a transducer, generating FUS waves upwards to target locations in the brain. B, Target locations along the right hemisphere were chosen using MRI images taken before MRIgFUS treatment (left). Effectiveness of BBB opening along the right hemisphere was confirmed from MRI contrast-enhanced images and the entry of gadolinium agent after MRIgFUS treatment (right hemisphere, black arrows). The image chosen here for representation of gadolinium entry in the brain was taken from a mouse displaying high enhancement levels. MRI intensities were detected and compared between the untreated (C, left hemisphere) and MRIgFUS-treated region with influx of gadolinium (D, right hemisphere). E, The mean intensity in the MRIgFUS-treated cortex was significantly higher than in the untreated contralateral cortex. F, The amount of gadolinium agent entering the MRIgFUS-treated region over time was calculated by subtracting the increase in enhancement seen in the MRIgFUS-treated region to the background intensity in the untreated region. Scale bar: B = 0.5 cm. Data is presented as paired values between left, untreated and right, MRIgFUS-treated, ***p<0.0001.

Fig. 2

MRIgFUS treatment causes a reduction in Aβ plaque pathology. Plaque load was assessed 4 days following MRIgFUS treatment using immunohistochemistry and stereology. A, Plaques were quantified in MRIgFUS-treated (right) and an equivalent untreated (left) region of the cortex. Significant reductions were observed in mean plaque size (B) and total Aβ surface area (C) in the MRIgFUS-treated compared to untreated cortex. D, Plaque counts within the MRIgFUS-targeted region demonstrated a trend of reduction compared to the untreated region. Within the MRIgFUS-treated cortex, endogenous IgG (F) and IgM (G), were found to be co-localized with plaques (E). At the site of plaques in the untreated cortex (H), a small amount of IgG (I) but no IgM (J) was found. White arrows indicate the location of Aβ plaques (E–J). Scale bar: A=0.5 mm, E–J = 50 μm. Data is presented as paired values between left, untreated and right, MRIgFUS-treated, *p<0.05, **p<0.01.

Fig. 3

MRIgFUS facilitates the entry of IgG and IgM into the brain. Using immunohistochemistry, high levels of IgG were detected in MRIgFUS-treated compared to untreated cortex of TgCRND8 (A) and non-Tg (B) mice. Similarly, IgM observed in MRIgFUS-treated cortical regions was greater than that detected in contralateral cortex of TgCRND8 (C) and non-Tg mice (D). Quantitative western blot analysis revealed that compared to untreated cortex, the cortex treated with MRIgFUS had greater levels of IgG in TgCRND8 (E) and non-Tg (F) mice; and of IgM in TgCRND8 (G) and non-Tg (H) mice. Scale bar: A–D= 500 μm. Data are presented as the mean ± SEM, *p<0.05.

Fig. 4

Increases in IgM are proportional to the enhancement in MRIgFUS-treated cortical regions. A, Using enhancement data gathered from MRI post-treatment scans, the estimated maximum enhancement (red) and rate of enhancement (blue) can be determined for all MRIgFUS-treated mice (representative enhancement graph for one mouse). B, The maximum enhancement, representative of the maximum amount of gadolinium able to enter the brain after BBB opening, was positively correlated to the increase in IgM levels measured in the MRIgFUS-treated cortex of the same mice (r2=0.4055, p=0.026, n=12). C, Similarly, the increase of endogenous IgM detected within the MRIgFUS-treated cortex was correlated to the rate of enhancement (r2=0.4310 p=0.020, n=12), which is indicative of the speed at which gadolinium enters the brain. Dashed lines indicate a 95% confidence intervals (B–C).

Fig. 5

A time-dependent increase in Iba1 and GFAP staining in TgCRND8 mice treated with MRIgFUS. At 4 hours post-MRIgFUS treatment, Iba1 staining appears to be slightly increased in the MRIgFUS-treated cortex (A′) compared to the untreated cortex (A). Iba1-immunostaining intensity appeared to be elevated at 4 days (B, B′) and dampened by 15 days (C, C′). D, D′, GFAP expression did not seem affected by the MRIgFUS treatment at 4 hours. In contrast, at 4 (E, E′) and 15 (F, F′) days post-treatment, GFAP-positive staining was found to be increased on the right, MRIgFUS-targeted side compared to the left, untreated side. Scale bar: A–F′ = 200 μm.

Fig. 6

Levels of Iba1 and GFAP protein expression in response to MRIgFUS in TgCRND8 and non-transgenic mice. Iba1 protein levels were significantly increased at 4 hours post-treatment in the MRIgFUS-treated cortex (FUS, grey column) relative to the left, untreated cortex (UT, white column) in TgCRND8 (A) and non-Tg (B) mice. At 4 days, Iba1 levels remained elevated in the MRIgFUS-treated cortex of transgenic (C) and non-Tg mice (D). E–F, At 15 days, no statistical difference was found between treated and untreated cortical levels of Iba1. GFAP protein levels were not different between MRIgFUS-treated and untreated cortex at 4 hours (G–H), but they were significantly increased in the MRIgFUS-targeted cortex at 4 days in TgCRND8 (I) and non-Tg (J) mice. At 15 days post-MRIgFUS treatment, TgCRND8 mice had a significant increase in GFAP levels in the MRIgFUS-treated cortex (K), whereas GFAP levels in the MRIgFUS-treated cortex of non-Tg mice were not statistically different from the untreated cortex (L). Mean ± SEM shown, *p<0.05, **p<0.01.

Fig. 7

Iba1- and GFAP-positive cells located distally to plaques increase in size following MRIgFUS treatment. A, Iba1-positive (green) and GFAP-positive (blue) cell volume surrounding plaques (red) were detected by immunohistochemistry. B, Optical sections were obtained using confocal microscopy and used to create three-dimensional reconstructions. C, Aβ plaque volumes were determined using the surface function and then regions of interest (ROIs) were created, one in contact with the plaque (proximal ROI, dark grey) and second, further from the plaque (distal ROI, light grey). The volume of Iba1-positive cells (D) and GFAP-positive cells (E) were significantly increased in the distal ROI of the MRIgFUS-targeted cortex, compared to the untreated cortex. Similarly, the surface area of microglia (G) and astrocytes (H) were also increased in the distal ROI following MRIgFUS treatment. Scale bar: C = 30μm. Mean ± SEM shown, *p<0.05, **p<0.01.

Fig. 8

MRIgFUS treatment does not change the number of glial cells but increases their volume per cell. Representative images of microglia (A) and astrocytes (B) found in the proximal (dark grey) and distal (light grey) ROIs surrounding a single plaque in the MRIgFUS-treated cortex of a TgCRND8 mouse are shown. The number of Iba1- and GFAP-positive counts were not different between MRIgFUS-treated and untreated cortex (C, D respectively). E, The volume per Iba1-positive cell count, however, was increased significantly in the distal ROI of plaques in the MRIgFUS-targeted cortex compared to the untreated cortex. F, GFAP-positive volume per cell was significantly increased both in proximal and distal ROIs within the cortex treated with MRIgFUS. Mean ± SEM shown, *p<0.05, **p<0.01, ****p<0.0001.

Fig. 9

Aβ internalization by Iba1- and GFAP-positive cells is increased in MRIgFUS-treated cortex. Brain sections were stained for Aβ (red), Iba1 (green) and GFAP (blue). Orthogonal views of confocal imaging show Aβ within Iba1-positive (A, representative Iba1-Aβ co-localization encircled) and GFAP-positive cells (B, representative GFAP-Aβ co-localization encircled) indicating internalization of Aβ at 4 days following MRIgFUS treatment. Imaris software was used to create three-dimensional surfaces of glial cells and detect Aβ within the glia found proximal (Aβ in red) and distal (Aβ in purple) to plaques (C, Aβ within a single cell shown in D). E, Aβ counts within Iba1-positive cells was greater within MRIgFUS-treated cortex in both proximal and distal regions surrounding plaques. F, Similarly, GFAP-positive cells within the MRIgFUS-treated cortex also contained a greater number of Aβ counts than those in the untreated, contralateral cortex. In addition, the total volume of internalized Aβ was greater in Iba1-positive (G) and GFAP-positive (H) cells within MRIgFUS-treated regions, proximal and distal to plaques. Scale bar: A–B = 25 μm. Mean ± SEM shown, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.