Study design
APP23 mice express human APP751 with the Swedish double mutation (KM670/671NL) under the control of the neuron-specific mThy1.2 promoter. As they age, these mice exhibit memory deficits [25], amyloid plaque formation which is initiated in the cortex, and cerebral amyloid angiopathy (CAA) [26]. In this study, APP23 mice, aged 13 months, were assigned to four treatment groups: sham (N = 10), SUS (N = 11), Adu (5 mg/kg delivered retroorbitally, N = 11), or SUS + Adu (5 mg/kg retroorbitally, N = 10). Assignment to treatment groups was based on matching performance of spatial memory (number of shocks) on day 5 of the active place avoidance (APA) test. We have previously shown that this approach reduces variability because mice yield similar results when repeatedly tested (as revealed by a main effect of subject) [17], and repeated APA testing of the same mouse can detect the effect of hippocampal injury and exercise, demonstrating the intra-animal validity of this approach [27]. A group of wild-type mice (N = 12) was also included. APP23 mice were ranked from those receiving the fewest shocks to those receiving the most shocks on day 5 and were assigned to the four treatment groups (sham, SUS, Adu, SUS + Adu) in rank order. Each group received a total of nine treatments (an APA retest was performed after the fourth treatment), with the final treatment in the Adu and SUS + Adu groups using fluorescently labeled antibody (2.5 mg/kg Alexa Fluor 647-labeled Adu and 2.5 mg/kg unlabeled Adu) (Fig. 1a). Three days after the final treatment, the mice were administered an overdose of sodium pentobarbitone and perfused with phosphate-buffered saline (PBS). The right hemisphere of the brain was fixed in 4% paraformaldehyde for histology, while the cortex and hippocampus of the left hemisphere were dissected and frozen in liquid nitrogen for subsequent analysis. Due to the increased mortality of this strain [28], the numbers of mice surviving to 22 months for histological and biochemical analysis were N = 10 sham, N = 9 Adu, N = 8 SUS, and N = 9 SUS + Adu. Assessment of outcomes was performed with the researcher blinded to the treatment group. All animal experimentation was approved by the Animal Ethics Committee of the University of Queensland (approval number QBI/554/17). Sample sizes for the experiment were selected based on our earlier studies [17]. Due to availability, mostly male mice were used (males/females: sham = 9/1, Adu = 8/1, SUS 7/1, SUS + Adu 7/2, Wild-type = 9/3 in the mice that survived to 22 months). We were unable to perform a third APA test as 22-month old APP23 mice are unable to physically perform the task. Data was collected for all mice that survived until the end of the experiment and all data was included.
SUS equipment
An integrated focused ultrasound system (Therapy Imaging Probe System, TIPS, Philips Research) was used. This system consisted of an annular array transducer with a natural focus of 80 mm, a radius of curvature of 80 mm, a spherical shell of 80 mm with a central opening of 31 mm diameter, a 3D positioning system, and a programmable motorized system to move the ultrasound focus in the x and y planes to cover the entire brain area [17]. A coupler mounted to the transducer was filled with degassed water and placed on the head of the mouse with ultrasound gel for coupling, to ensure unobstructed propagation of the ultrasound to the brain.
Production of microbubbles
Microbubbles comprising a phospholipid shell and octafluoropropane gas core were prepared in-house. 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DSPE-PEG2000) (Avanti Polar Lipids) were mixed in a 9:1 M ratio and dissolved in chloroform (Sigma), after which the chloroform solvent was evaporated under vacuum. The dried phospholipid cake was then dissolved in PBS with 10% glycerol to a concentration of 1 mg lipid/ml and heated to 55 °C in a sonicating water bath. The solution was placed in a 1.5 ml glass HPLC vial with the air in the vial replaced with octafluoropropane gas (Arcadophta). The microbubbles were activated on the day of the experiment by agitation of the vial in a dental amalgamator at 4000 rpm for 45 s. Activated microbubbles were measured with a Multisizer 4e coulter counter which reported a mean diameter of 1.885 μm and a concentration of 9.12 × 108 microbubbles/ml. These microbubbles were also observed to be polydisperse under a microscope (Supplementary Figure 1).
SUS application
Mice were anesthetized with ketamine (90 mg/kg) and xylazine (6 mg/kg), and the hair on their head was shaved and depilated. They were then injected retro-orbitally with 1 μl/g body weight of microbubble solution and placed under the ultrasound transducer with the head immobilized. A heating pad was used to maintain normal body temperature. Parameters for the ultrasound delivery were 1 MHz center frequency, 0.7 MPa peak rarefactional pressure, 10 Hz pulse repetition frequency, 10% duty cycle, and a 6-s sonication time per spot. The focus of the transducer was 1.5 mm × 12 mm in the transverse and axial planes, respectively. The motorized positioning system moved the focus of the transducer array in a grid with 1.5 mm spacing between individual sites of sonication so that ultrasound was delivered sequentially to the entire brain as described previously [17, 18]. Mice typically received a total of 24 spots of sonication in a 6 × 4 raster grid pattern. For the sham treatment, mice received all injections and were placed under the ultrasound transducer, but no ultrasound was emitted. When the animals were treated with Adu antibody, the solution was mixed briefly with the microbubble solution and injected into the retro-orbital sinus before the mouse was placed under the ultrasound transducer. The time between injecting microbubbles and commencing ultrasound delivery was 60 ± 10 s and the duration of sonication was approximately 3 min (total time from microbubble injection approximately 4 min). We assumed that 100% of the antibody reached the circulation where it circulated with a half-life of 2.5 days [5] and that there was no interference from mixing with the microbubbles which have a half-life of 2 min.
Production of the Aducanumab analog
VH and VL sequences were identified in Biogen Idec’s patent submission for BIIB-037 WO2014089500 A1 and were cloned into mouse IgG2a and kappa pcDNA3.1 vectors (GenScript). Murine chimeric Aducanumab (Adu) was produced using the Expi293 expression system, purified using protein A chromatography and verified to be endotoxin-free by LAL assay (Thermo Fisher).
Antibody affinity ELISA
The EC50 of Adu was determined by direct-binding ELISA. Aβ1-42 fibrils were generated by incubating 0.1 mM Aβ1-42 peptide (JPT Peptide Technologies) in 10 mM HCl for 3d at 37 °C. A MaxiSorp ELISA plate was coated with 2 μ/ml Aβ1-42 fibrils in 0.1 M sodium bicarbonate buffer and then blocked with 1% bovine serum albumin. The EC50 was determined by incubating the wells with serial dilutions of Adu, followed by washing and detection of bound Adu with a rabbit anti-mouse horseradish-peroxidase-conjugated antibody (Dako) and 3,3′,5,5′-tetramethylbenzidine substrate. The 6E10 antibody [29] was used as a positive control for Aβ binding and its EC50 was determined for comparison with Adu using the same methods (Supplementary Figure 2).
Antibody labeling
Adu was covalently conjugated with Alexa Fluor 647 dye (Thermo Fisher Scientific) in PBS with 0.1 M sodium bicarbonate as previously described [22]. The protein concentration and degree of labeling were determined by measuring absorbance at 280 nm and 650 nm, respectively.
Tissue processing
Mice were deeply anesthetized with pentobarbitone before being perfused with 30 ml of PBS, after which their brains were dissected. One hemisphere of the brain was fixed overnight in a solution of 4% wt/vol paraformaldehyde, and then cryoprotected in 30% sucrose and sectioned coronally at 40 μm thickness on a freezing-sliding microtome (SM2000R, Leica). A one-in-eight series of sections was stored in PBS containing 0.01% sodium azide at 4 °C for subsequent staining.
Assessment of amyloid plaques
For the assessment of amyloid plaque load, an entire one-in-eight series of coronal brain sections taken from the start of the anterior commissure to the ventral hippocampus of one hemisphere at 40 μm thickness was stained using the Campbell-Switzer silver stain protocol that discriminates fibrillar from less aggregated amyloid as previously described [17]. Stained sections were mounted onto microscope slides and imaged with a × 10 objective on a Metafer bright-field VSlide scanner (MetaSystems) using Zeiss Axio Imager Z2. Analysis of amyloid plaque load was performed on all stained sections using ImageJ. Separate regions of interest were drawn around the cortex and dorsal hippocampus. As both black and amber plaques are present in the sections representing different types of amyloid compactness, they were analyzed separately using a color deconvolution method and automated thresholding to distinguish the two types of amyloid plaques. For the analysis of black plaques, a color deconvolution vector was used followed by the MaxEntropy auto thresholding function in ImageJ. As black plaques consist mainly of diffuse fibrils, no size filter was applied. To measure amber plaques, a second color deconvolution vector was used, followed by invert function and automated thresholding using the triangle method in ImageJ, fill-holes function, and a 60-μm2 size filter was applied. Using this method, plaque number, total plaque area, average plaque size, and % area covered by plaque were obtained for both the black and amber plaques and summed to give total plaque area for the cortex and hippocampus. We were unable to analyze the hippocampus of one mouse in the Adu-treated group because of folds in the tissue.
Assessment of cerebral amyloid angiopathy
To assess CAA, a one-in-eight series of Campbell-Switzer silver-stained sections was examined. Regions of interest were drawn manually around areas of CAA in the cortex, which were distinguished from plaques by having a rod-like structure indicative of blood vessels and a diameter greater than 15 μm. Meningeal CAA which has a ring-shaped structure and occurred close to the edge of the section was also measured. The number of CAA deposits per section, the average size, and the % area of the brain sections positive for CAA staining were determined.
Assessment of cerebral microbleeds
Prussian blue staining was performed using freshly prepared 5% potassium ferrocyanide and 5% hydrochloric acid (Sigma) for 30 min. Cerebral microbleeds were identified at a × 20 magnification as focal clusters of blue hemosiderin deposits which were smaller than 50 μm wide and appeared to have a perivascular location. A randomly selected subset of 5 mice per treatment group were stained and 4 sections were analyzed from each mouse.
Immunofluorescence labeling
Coronal 40 μm sections were co-stained with the 4G8 antibody against Aβ (1:1000, Covance) and against Iba1 (1:1000 Wako), followed by goat anti-mouse and goat anti-rabbit Alexa Fluor-conjugated secondary antibodies (1:2000, Thermo Fisher). Alexa Fluor 647-conjugated Adu was detected in situ without additional amplification. Sections were cover-slipped and imaged with a fluorescence slide scanner (Metafer).
Enzyme-linked immunosorbent assay for Aβ
Frozen cortices were homogenized in 10 volumes of a solution containing 50 mM NaCl, 0.2% diethylamine (DEA) with complete protease inhibitors, and Dounce homogenized by passing through 19 and 27 gauge needles. The samples were then centrifuged at 21,000×g for 90 min at 4 °C. The supernatant was retained as the DEA-extracted soluble Aβ fraction. The remaining pellets were resuspended in 10 volumes of 5 M guanidine HCl, sonicated, and centrifuged at 21,000×g for 30 min at 4 °C. The resultant supernatant was retained as the guanidine-extracted insoluble Aβ fraction. The concentrations of Aβ40 and Aβ42 were determined in brain lysates using ELISA kits according to the manufacturer’s instructions (human Aβ40 and Aβ42 brain ELISA, Merck).
Active place avoidance test
The active place avoidance (APA) task is a test of hippocampus-dependent spatial learning. We used a repeated APA paradigm, where mice were tested in the APA one time and the performance of each mouse was used to assign that mouse to one of four treatment groups. This was done by ranking all the mice based on their performance and assigning them to the four groups in order so that the APA performance of each treatment group was the same. Following this, mice received either sham, SUS, Adu, or SUS + Adu treatment and 3 days after the last treatment mice were retested in the APA to assess whether there was an improvement in APA performance due to the treatment the mouse had received. For each APA test, APP23 mice and non-transgenic littermate controls were tested over 6 days in a rotating elevated arena (Bio-Signal group) that had a grid floor and a 32-cm-high clear plastic circular fence enclosing a total diameter of 77 cm. High-contrast visual cues were present on the walls of the testing room. The arena and floor were rotated at a speed of 0.75 rpm, with a mild shock (500 ms, 60 Hz, 0.5 mA) being delivered through the grid floor each time the animal entered a 60-degree shock zone, and then every 1500 ms until the animal left the shock zone. The shock zone was maintained at a constant position in relation to the room. Recorded tracks were analyzed with Track Analysis software (Bio-Signal group). A habituation session was performed 24 h before the first training session during which the animals were allowed to explore the rotating arena for 5 min without receiving any shocks. A total of five training sessions were held on consecutive days, one per day with a duration of 10 min. After day 5 of the first APA (test), APP23 mice were divided into four groups with mice matched so that the performance (number of shocks) of the four groups of mice on day 5 of the task was the same, for the retest. Following four once-a-week SUS or Adu treatments, the mice underwent the APA test again (reversal learning). The retest was held in the same room as the initial test. However, the shock zone was switched to the opposite side of the arena, the visual cues were replaced with different ones, and the platform was rotated clockwise rather than counterclockwise. The number of shocks, numbers of entries to the shock zone, time to first entry, time to second entry, and proportion of time spent in the opposite quadrant of the shock zone for sham, SUS, Adu, and SUS + Adu-treated groups were compared over the days of testing.
Statistical analysis
Statistical analyses were conducted with Prism 8 software (GraphPad). Values were always reported as mean ± SEM. One-way ANOVA followed by the Holm-Sidak multiple comparisons test, or t test was used for all comparisons except APA analyses where two-way ANOVA with day as a repeated measures factor and group as a between subjects factor was performed, followed by the Holm-Sidak multiple comparisons test for simple effects to compare group performance on different days. The model assumption of equal variances was tested by Brown-Forsyth or Bartlett tests, and the assumption of normality was tested by Kolmogorov-Smirnov tests and by inspecting residuals with QQ plots. All observations were independent, with allocation to groups based on active place avoidance where mice were ranked on performance and assigned to one of the four groups (sham, SUS, Adu, SUS + Adu) in order of number of shocks on day 5 listed from most to least shocks.