Animals and study design
Male C57bl/6j mice (16-week-old, weighing 25 to 35 grams, Bio-Lasco Taiwan Co., Ltd) were used for all the experiments. They were placed under controlled temperature (22 ± 1°C) and humidity (55 ± 10%) with a 12 h light/dark cycle (lights on at 07:00 h). Food and water were given as libitum to all mice throughout the experiments. Animal care and experimental procedures in this study had been approved and were in accordance with the guidelines for the Care and Use of Laboratory Animals from Ethics Committee of Taipei Medical University.
The mice groups were divided into two parts: part one was before HMGB1 CRISPR/Cas9 KO plasmid injection, and part two was after the injection. For part one, the mice were randomly grouped as sham-control (n=12) and BCCAO (n=12). For part two, the mice were randomly grouped as sham-CRISPR control, sham-CRISPR HMGB1 KO, BCCAO-CRISPR control, and BCCAO-CRIPSR HMGB1 KO. Each group in part two had six mice (Fig. 1). This study followed the ARRIVE guidelines.
Model establishment of CCH by modified bilateral common carotid artery occlusion (BCCAO) surgery
The modified-BCCAO was performed by ligating both the common carotid artery (CCA) but with some modification from the previous procedure (17, 18). Due to mice were dead soon after both of the CCA were ligated simultaneously, hence one of the CCA was ligated transiently for 30 minutes. The animals were anesthetized with isoflurane 2%. Cerebral blood flow (CBF) was measured while doing the surgery. A sagital midline incision (~1 cm length) was performed to expose the parietal skull. The skin was carefully dissected and a fiberoptic probe of laser doppler flowmetry (LDF) was put directly to the skull 2 mm caudal and 5 mm lateral from bregma to measure the CBF. A cervical midline neck incision (~1 cm length) was made. Both salivatory glands were carefully separated and mobilized to visualize the underlying common carotid artery (CCA). Both CCA were carefully separated from the respective vagal nerves and accompanying veins without harming these structures. A tight, double 5-0 silk suture loop (proximal & distal) was made around right CCA. Re-measured the CBF, there must be a reduction of CBF by 80-90% from the baseline. Finally, we closed the wound by suturing.
One week later, the procedure was repeated for left CCA. The steps were similar except there was transient ligation of left CCA. A small polyethylene tubing (diameter 0,58 mm) was inserted in between the left CCA and the silk sutures. This tubing was used as a splinting of the left CCA in order to avoid damaging of the arterial walls when the sutures were tightened. Left CCA was occluded for 30 minutes by tightening the silk sutures. Then, we re-measured the CBF and there must be a reduction of CBF by 80-90% from the baseline. After 30 minutes, released the ligation of left CCA and removed the polyethylene tubing. After the whole procedure, mice were put in a heating pad for 30 minutes while waiting them awake. After awaking, they were put back into their cages. The procedure of sham-surgery was similar with BCCAO excluding the ligation of both CCA.
Injection of HMGB1 CRISPR/Cas9-KO plasmid
The HMG-1 Crispr/Cas9 KO plasmid (sc-400735) and HMG-1 HDR plasmid (sc-400735-HDR) were purchased from Santa Cruz Biotechnology, Inc. HMG-1 Crispr/Cas9 KO plasmid consisted of a pool of three plasmids each encoding the Cas9 nuclease and a HMGB-1-specific 20 nt guide RNA (gRNA) designed for maximum knockout efficiency. gRNA sequences were derived from the GeCKO (v2) library and direct the Cas9 protein to induce a site-specific double strand break (DSB) in the genomic DNA. HMG-1 HDR plasmid consisted of a pool of 2-3 plasmids, each containing a homology-directed DNA repair (HDR) template corresponding to the cut sites generated by the HMG-1 Crispr/Cas9 KO plasmid. Each HDR template contained two 800 bp homology arm designed to specifically bind to the genomic DNA surrounding the corresponding Cas9-induced double-strand DNA break site.
We injected HMG-1 Crispr/Cas9 KO plasmid and HMG-1 HDR plasmid mixed with jetSITM 10mM transfection reagent according to manufacturer’s recommendations. A total volume of 4 mL of the mixture was injected into each hippocampus (anteroposterior [AP] – 2 mm, mediolateral [ML] – 1.5 mm, and dorsoventral [DV] – 2 mm related to bregma), at a rate of 0.4 mL/min by a 26-gauge Hamilton syringe under isoflurane anesthesia. For CRISPR control group, we injected a vehicle with the same volume and procedure. The injection was conducted at 1 month after BCCAO/sham surgery. Two months after injection, the mice had an MRI examination to evaluate the brain pathology and hippocampal atrophy as well as a novel object recognition (NOR) test.
Novel object recognition (NOR)
Mice tend to interact more with a novel object than with a familiar one. This tendency has been used by behavioral pharmacologists and neuroscientists to study learning and memory. A popular protocol for such research is the object recognition task (19). The procedure consisted of three phases: habituation phase, sample phase, and test phase. We perform this procedure with modifications (20). During habituation phase, each mouse was allowed to explore the field in the absence of objects for 10 min for two consecutive days in order to make them become familiar with the field. On the third day during the sample phase, two objects were placed symmetrically onto the arena. The mice were placed at the mid-point of the wall opposite the sample objects with its body parallel to the side walls and its nose pointing away from the objects, allowed to freely explore for 5 min. Time spent exploring the objects was recorded. During the test phase, one of the two objects used in sample phase was randomly replaced by a novel one, then the mice were re-introduced to the arena for 5 min exploration after a 4-hours delay. Between each mouse, any feces were cleared, the arena and objects were cleaned with 70% ethyl alcohol. The video tracking system was used to collect behavioral performances automatically. The time spent exploring both the novel and the familiar objects was recorded (TN, TF). Object discrimination was evaluated by the recognition index (RI): RI = TN/(TN + TF).
Motor function test
Beam walking test: Further subtle motor coordination and balance were assessed using a modified balance beam (beam walking) test. This procedure was based on modified protocol describe in Luong et al (2011) (21). The beam apparatus consisted of 1-meter beams with a round-rough surface of 12 mm (or 6 mm width) resting 50 cm above the table top on two poles. A black box was placed at the end of the beam as the finish point. Food was placed in the black box to attract the mouse to the finish point. A lamp (with 60-watt light bulb) was used to shine light above the start point and serves as an aversive stimulus. The time to cross the center 80 cm was measured manually: one at 0 cm that started a timer and one at 80 cm that stopped the timer. A video camera was set on a tripod to record the performance. This test took place over 3 consecutive days: 2 days of training and 1 day of testing. On training days, each mouse crossed the 12 mm (or 6 mm) beam 3 times. On the test day, time to cross each beam were recorded. Two successful trials in which the mouse did not stall on the beam were averaged. Video recordings could be used for finer analysis of slipping and other observable motor deficits.
Rotarod: Briefly, gross motor control was measured using the rotarod (IITC Life Science, CA, USA). This procedure was based on the protocol described in Tung et al. (2014) with some modification (22). For this test, each mouse was placed on a cylindrical dowel (69.5 mm in diameter) raised 27 cm above the floor of a landing platform. Mice were placed on the dowels for 5 min to allow them to acclimatize to the test apparatus. Once initiated, the cylindrical dowels began rotating and accelerated from 5 rpm to a final speed of 44 rpm over 60 s. During this time, mice were required to walk in a forward direction on the rotating dowels for as long as possible. When the mice were no longer able to walk on the rotating dowels, they fell onto the landing platform below. This triggered the end of the trial for an animal and the measurements of time to fall (TTF) were collected. Passive rotations where mice clung to, and consequently rotated with the dowel were also used to define the end of the trial. Mice were then returned to their cages with access to food and water for 10 min. This procedure took 3 days; day 1 and 2 were for training (each day consisted of 2-3 trials), and day 3 was for testing (consisted of 2 trials). The trials from testing day was used for analysis.
MRI experiments to monitor the brain condition (e.g. atrophy and white matter lesion) after BCCAO were performed with a 7T horizontal MRI scanner (Bruker PharmaScan 70/16, USA), with a 7T/40cm magnet (Biospect Bruker console) and a surface coil. The mice were initially anesthetized with 3.0% isoflurane (Escain, Mylan Japan, Tokyo, Japan) and then with 1.5% to 2.0% isoflurane and 1:5 oxygen/room-air mixture during the MRI experiments. Rectal temperature was continuously monitored using an optical thermometer (FOT-M, FISO, Quebec, QC, Canada) and maintained at 37.0 °C ± 0.5 °C using a heating pad (Temperature control unit, Rapid Biomedical), and warm air was provided by a homemade automatic heating system based on an electric temperature controller (E5CN, Omron, Kyoto, Japan) throughout the MRI experiments. During MRI scanning, the mice laid in a prone position on an MRI-compatible cradle and were held in place by handmade ear bars. The first imaging slices were carefully set at the rhinal fissure, with reference to a mouse brain atlas. The modality of MRI performed was T2-weighted spin echo.
Trans-axial T2-weighted images were acquired using rapid acquisition with a relaxation enhancement (RARE) sequence as follows: TR/TE = 2500/33 ms, slice number/thickness = 16/0.75 mm, matrix = 256 × 256, FOV = 16 × 16 mm2, average = 8, RARE factor = 8, flip angel = 90, scan time = 10 min 40s. Volumes of the hippocampus and lateral ventricles were measured using MRIcron software (NITRC 2016).
For amyloid-PET scanning, the mice were anesthetized with isoflurane (1.5%, delivered via a mask at 3.5 L/min in oxygen) and received bolus injection of 18.5 MBq/0.2 cc of 18F-AV45 (Eli Lily, IN, USA) via the tail vein with a catheter. Following placement in the scanner (Inveon, Siemens Medical Solutions, Germany), a 10 min transmission scan was obtained using a rotating 57Co point source, followed by a single frame emission recording for the interval 40–60 min post-injection. The PET image reconstruction procedure consisted of a 3-dimensional ordered subset expectation maximization (OSEM) with four iterations and 12 subsets followed by a maximum a posteriori (MAP) algorithm. Scatter and attenuation correction were performed and a decay correction for 18F was applied. With a zoom factor of 1.0 and a 128 x 128 x 159 matrix, a final voxel dimension of 0.78 x 0.78 x 0.80 mm was obtained. The image analysis was performed by using PMOD (PMOD version 3.7, Technologies Ltd, Switzerland). A manual rigid-body re-alignment of individual 18F-AV45 images on a 18F-AV45 template was performed using the PMOD fusion tool. The normalized PET images were co-registered to a magnetic resonance imaging brain template for region of interest delineation. Using the predefined mice brain MRI template of PMOD and manual ROI of frontal lobe and hippocampus fused with co-registered AV-45 PET/CT image, the AV-45 signal in the region of interests were determined, respectively.
Mice were anesthetized with isoflurane 2% then decapitated. Brain were collected from the skull and separated into 3 regions in each hemisphere: cortex, hippocampus, and striatum. Since hippocampal tissue was very tiny, we combined right and left hippocampus for western blot experiment. Protein samples of each region were extracted in the following manner. Individual tissue samples were homogenized in lysis buffer (50 mmol/L Tris, pH 7.4, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 4 lg/mL aprotinin and leupeptin, 1% sodium dodecyl sulfate), protease inhibitor cocktail solution and phosphatase inhibitor cocktail solution (GenDEPOT, Barker, TX, USA). The homogenates were then centrifuged at 13.400 × g for 30 minutes at 4°C, and the supernatants were harvested, snap-frozen, and stored at −80°C. The protein concentration of the supernatants was determined using the bradford assay. Equal amounts of protein (20 μg) were then separated via SDS-PAGE and transferred to a PVDF membrane, which were subsequently incubated in a primary antibody (Ab) against HMGB1 (mouse monoclonal antibody, 1:1000, GeneTex, USA), IL-1β (rabbit polyclonal antibody, 1:1000, GeneTex, USA), TNF-a (rabbit polyclonal antibody, 1:1000, GeneTex, USA) and IL-6 (rabbit polyclonal antibody, 1:1000, GeneTex, USA). Following incubation in the primary Ab, the membranes were incubated in horseradish peroxidase (HRP)-conjugated secondary Ab (Cell Signaling, MA, USA) and then detected using an ECL system (Thermo Scientific, MA, USA) with a UVP Biospectrum AC system (Fisher Scientific, USA). Densitometry is performed for specific markers normalized to b-actin (1:1000, clone C4, Millipore Corporation, USA) using Image J (v1.37) software.
On 3 months after the BCCAO or sham surgery, the mice were anesthetized and intracardially perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde. The brains were removed, post-fixed 4 hours in 4% paraformaldehyde at 4 °C and stored in 30% sucrose in 0.1 M PBS (pH 7.4). Serial coronal-cryopreserved sections cut at 30 μm thickness that spanned from the anterior of the corpus callosum (bregma 0.26 mm) to the anterior of the hippocampus (bregma 0.94 mm) (adjusted according to the mouse brain atlas) (23) using a cryostat. The slides then were permeabilized with 0.5% Triton X-100 in PBS for 2 hours. After being blocked with BlockPRO (VISUAL PROTEIN) at room temperature for 1 h, the brain slides were incubated with the following primary antibodies: mouse monoclonal anti-HMGB1 antibody (1:100, GeneTex, USA), rabbit polyclonal anti-TNF-α (1:100, GeneTex, USA), rabbit polyclonal anti-IL-6 (1:100, GeneTex, USA) and rabbit polyclonal anti-IL-1β (1:100, GeneTex, USA) at 4 °C overnight. The brain slides were then incubated with secondary antibodies conjugated to Alexa 488 (1:1000; Thermo Fisher Scientific, MA, USA) or Alexa 594 (1:500; Thermo Fisher Scientific, MA, USA) for 2-h at RT. Brain sections were then washed and counterstained with 2 μg/mL DAPI (Life Technologies) for 20 min at RT. Fluorescence images were obtained using a Tissue FAXS system (TISSUE GNOSTICS) with a ×25 objective lens. For the triple stained, tissue slides were incubated with primary antibodies: mouse monoclonal anti-HMGB1 antibody (1:100, GeneTex, USA) and rabbit polyclonal anti-NeuN (1:300, Cell Signaling, USA). The images were obtained by using Confocal Microscope with ×63 oil objective lens. All images were taken at 1024 × 1024-pixel resolution.
One-way ANOVA followed by Sidak post-hoc test was used to analyze western blot results, IF, and CBF measurement. NOR results were analyzed by using two-way ANOVA. Student unpaired t-test was used to analyze motor function MRI results. All analyses were performed by using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA). The statistically significance was considered when the p value was less than 0.05.