Animals
Male and female hTau mice 12–14 weeks old (weight 19–25 g) were sourced from Jackson Laboratories (Bar Harbor, ME). The animals were housed under standard laboratory conditions (14-h light/10-h dark cycle, 23 ± 1C, 50 ± 5% humidity) with free access to food and water. All procedures were carried out under Institutional Animal Care and Use Committee (IACUC) approval and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Experimental groups and study design
A total of 72 mice were randomly assigned to 1 of 6 groups (n = 12 per group): repetitive sham/vehicle, repetitive sham/nilvadipine, repetitive sham/ARC031, repetitive injury/vehicle, repetitive injury/nilvadipine and repetitive injury/ARC031. The r-mTBI (total of 5 hits with an inter-concussion interval of 48 h) was administrated to mice as previously described [18, 19, 21, 33, 34]. Sham injured animals (5 anesthesia, 48 h apart) underwent the same procedures and were exposed to anesthesia for the same length of time as the mTBI animals. Either nilvadipine, ARC031 or vehicle (PBS:PEG/1:1) were injected intraperitoneally (100 μL, i.p.) daily for 21 days, with the first injection administered immediately following the last injury. The behavior analysis began 24 h after the last mTBI/anesthesia for each group, as shown in Fig. 1. Euthanasia was performed 22 days after the last mTBI/sham procedure. Researchers were blind to animal group assignments during both neurobehavioral experiments and immunohistochemistry.
Injury protocol
All animals were anesthetized with 1.5 L/min of oxygen and 3% isoflurane prior to r-mTBI or sham injury. The heads were shaved, and mice were placed on a heating pad to maintain body temperature at 37C to prevent hypothermia. The head of each animal was fixed in a stereotaxic frame, and the blunt impactor tip (3 mm diameter) was positioned midway to the sagittal suture. The injury was triggered at 5 m/s velocity and 1.0 mm depth, with a dwell time of 200 ms, using a myNeuroLab controller device (Impact \({\hbox {One}}^{\mathrm{TM}}\) Stereotaxic Impactor, Richmond, IL). All mice experienced short-term apnea (\(<20\) s) and showed no skull fractures. All animals were allowed to recover from anesthesia on a heating pad and then returned to their cages with water and soft food access. Sham animals received anesthesia alone for the same duration of time as the r-mTBI mice, to control for the effects of repeated anesthesia. Mice were monitored daily for any abnormalities in behavior.
Treatment
For convenience, racemic nilvadipine will be referred to as “nilvadipine”, while (−)-nilvadipine enantiomer as “ARC031” . All mice received either nilvadipine, ARC031 or vehicle via i.p. injections for 21 days, starting immediately after the last injury/sham (the first injection was administered while animals were still under anesthesia). The treated groups received 2 mg/kg nilvadipine (equivalent to a human oral dose of 8 mg conferring antihypertensive activity) or 2 mg/kg ARC031 dissolved in a 1:1 solution of PBS and PEG vehicle solution [23]. The injection volume (100 μL) was calculated based on the average animal’s weight (0.028 kg). Untreated animals underwent the same procedure but received vehicle solution only (PBS:PEG/1:1). All solutions were freshly prepared every day before the injections.
Motor and cognitive function assessment
Motor function was assessed using the Rotarod apparatus, and the latency to fall from an accelerating rotating rod was measured. Baseline performance was recorded 1 day prior to the first injury/sham. Rotarod assessment started on the day after the last injury/sham procedure and was carried out on days 1, 3, 5 and 7 post-last r-mTBI/sham. An acclimation period involved 3 trials with a duration of 3 min each and a 3 min rest interval in the animal’s home cage between the trials (velocity = 5 rpm, no acceleration). The mice were placed back on the bar during the acclimation period if they fell. All experimental trials, including the baseline trials, lasted for 5 min and were conducted with acceleration from 5 to 50 rpm over the 5 min period. Each animal underwent 3 trials per day, with a 3 min rest interval between each trial. The fall time of each mouse was recorded in seconds. To ensure that fall time would correlate with motor coordination, rather than purely grip strength, if a mouse clung to the bar for more than 5 consecutive rotations on the accelerating rod without walking or making forward progress against the rotation of the bar, the time of the \(5{\mathrm{th}}\) rotation was considered to be the fall time and was recorded as such.
Barnes Maze (BM) was initiated on day 8 post-last mTBI/sham and lasted for 7 consecutive days to assess cognitive function. For 6 days, animals were trained to find the target hole which had a black escape box underneath. The walls in the room were equipped with visual cues and the brightness of the room was consistent throughout testing (7 days). The BM table was 1.2 m in diameter and has 18 equally spaced holes around the perimeter. Every mouse had 4 acquisition trials per day, with a duration of 1.5 min each. The starting position for each trial during acquisition rotated, beginning at one of 4 cardinal directions of the maze and rotating, first clockwise (until reaching the initial position again) and then counterclockwise. If an animal did not find the target hole or did not go inside the box within the time limit, the mouse was guided to the target hole by hand. Regardless of their success, mice then spent 30 s in the box before returning to their cage. On the last day, 24 h following the final acquisition trial, a probe trial was conducted during which the animals were placed in the middle of the maze and had 60 s to find the target hole, from which the escape box had been removed. The cumulative distance from the target hole, total distance travelled, time to find the target hole, and velocity were calculated using Noldus Ethovision XT software and analyzed to assess spatial memory and learning. Cumulative distance was measured as the sum of the distance between the center point of the mouse and the center of the target hole for every video frame from each trial at 30 frames per second. This distance stopped accumulating when the trial ended, either when the total time had elapsed or when the mouse entered the target box. Data were presented as the raw values of the cumulative distance.
Tissue collection and processing
Animals were euthanized on the day after the last injection (22 days post-last injury/sham). Mice were anesthetized with 3\(\%\) isoflurane and perfused transcardially with phosphate-buffered saline (PBS), pH-7.4. After perfusion, the brains were post-fixed in a solution of 4% paraformaldehyde (PFA) at 4C for 48 h and paraffin-embedded for immunohistochemistry. Brain samples fixed in PFA were processed in paraffin using the Tissue-Tek VIP (Sakura, USA). Sagittal sections were cut at 8 μm using a Leica RM2235 microtome and mounted on positively charged glass slides. Prior to staining, sections were deparaffinized in xylene and rehydrated in ethanol solutions of decreasing concentrations.
Immunohistochemistry
Non-fluorescent staining for GFAP
Following rehydration, slides were processed with hydrogen peroxide for 15 min and heated in citric acid buffer (pH-6) for antigen retrieval. Slides were then blocked with normal goat serum, washed with PBS, and incubated in a primary antibody for GFAP overnight at 4C (GFAP7857983, Aves Labs, Inc., 1:10,000). On the next day, slides were processed using the anti-chicken VectaSTAIN ABC Kit and developed with 3,3′-Diaminobenzidine (DAB) before mounting.
Non-fluorescent staining for Iba1
Microglia were stained using an anti-Iba1 antibody (ab107159, Abcam). After rehydration, slides were processed with hydrogen peroxide for 15 min followed by antigen retrieval using citric acid buffer (pH-6). Next, slides were blocked with rabbit serum for 1 h at room temperature and then incubated with the primary antibody (1:1000) overnight. On the next day, samples were processed using the anti-goat VectaSTAIN ABC Kit and developed with DAB.
Fluorescent staining
GFAP/Iba1/CD68/P-SYK
Fluorescent staining was performed using the following antibodies: Iba1 (ab107159, Abcam), GFAP (7857983, Aves Labs), CD68 (ab125212, Abcam) and phosphorylated spleen tyrosine kinase P-SYK (Tyr525/526) (2710S, Cell Signaling). Following rehydration, antigen retrieval was performed by heating slides in citric acid buffer for 7 min in a microwave oven. Next, slides were washed with PBS and transferred to a Sudan Black solution for 15 min to prevent autofluorescence. Slides were then blocked for 1 h with 10% donkey serum solution in PBS, and primary antibodies for Iba1 (1:300), GFAP (1:1000), CD68 (1:500) and P-SYK (1:200) were applied overnight. On the next day, secondary antibodies AlexaFluor488 (A21202, Life Technologies), AlexaFluor647 (A21449, Life Technologies), AlexaFluor568 (ab175477, Abcam), were applied for P-SYK, GFAP, and Iba1/CD68, respectively. Slides were mounted with ProLong Gold Antifade 4′,6-diamidino-2-phenylindole (DAPI) Mount. Each marker was stained separately except for the double staining for Iba1/P-SYK that was performed to evaluate colocalization of the two. Fluorescent imaging was performed using a confocal microscope (LSM 800 Zeiss) at 20× and 63× magnification. Z-stacks were recorded for every image and orthogonal projections were obtained to enable a 3D representation of the picture.
Immunohistochemical quantification
Imaging of non-fluorescent samples stained for GFAP/Iba1 was performed on an Olympus DP72 microscope at 10× magnification. Further analysis of the images included quantification of GFAP and Iba1 signal using ImageJ. Images were separated into individual color channels (hematoxylin counterstain and DAB chromogen) using the color deconvolution algorithm. Three nonoverlapping regions of interest (ROI) of 100 μm2 per image were then selected for the hippocampus, the body of the corpus callosum and the cortex. A coverage area (\(\%\)) per ROI was calculated and the mean value for each animal was used for further statistical analysis.
MesoScale discovery (MSD) multi-spot assay
A tau profile was assessed in cortical mouse homogenates using MSD protocol using the Phospho (Thr231)/Total Tau kit (V-PLEX K15121D) following manufacturer’s instructions. First, the calibration solution Tau441 was prepared according to the kit protocol. Samples and controls were diluted twofold in Tau441. Then, the plate was blocked with Blocker A for 1 h followed by adding the samples and calibrators into the wells. After 1 h of incubation, the plate was incubated for 1 h with detection antibodies (SULFO-TAG), and the Read Buffer T was added to analyze the plate. The plate was washed with the Tris Wash Buffer after each step. The plate analysis was conducted on the MSD instrument (MESO Quick Plex SQ120).
Statistical analysis
All experimental data were analyzed using JMP 12 and GraphPad Prism 6 software. The data were checked for normality using Skewness-Kurtosis and Goodness of Fit. If normal, parametric method one-way ANOVA was applied to calculate the significance in the tested groups (p values less than 0.05 were considered significant). If significant, post-hoc analysis was applied using Turkey’s multiple comparison test/Honest Significant Difference (HSD). The Turkey’s test compares all possible pairs of means between different treatment groups and was considered significant if \(p< 0.05\). The Shapiro–Wilk test was used if data were not normally distributed. All data were transformed to logarithm or square root, when required, to reach normality before further analysis. Repeated-measure analysis of variance (MANOVA) was used to analyze continuous performance of mice in the Barnes Maze and Rotarod (\(p<0.05\) is significant). Error bars represent the standard error of the mean.