Animals
Male 12-week-old C57BL/6J mice were purchased from Japan Charles River (Yokohama, Japan). α2AP-deficient (α2AP−/−) mice were generated by homologous recombination using embryonic stem cells, as described previously [24, 25], and were repeatedly backcrossed to C57BL/6J mice for more than 10 generations. All experiments were approved by the institutional animal care and use committee of Kyoto Pharmaceutical University (permit number: 18-16-017) and were performed in accordance with the institutional guidelines. All efforts were made to minimize suffering.
Y-maze test
The Y-maze apparatus consisted of three arms, the walls of which had different markings. The mice were placed into the center and allowed to explore the apparatus for 8 min, while being monitored by a video-tracking system (Ethovision XT; Noldus Company, Wageningen, The Netherlands). The alteration of their behavior was calculated as the ratio of the number of alterations to the total number of arm entries minus 2.
Morris water maze (MWM) test
Mice received visible platform pre-training on the first day, followed by hidden platform training for two days. In the hidden platform training, two sessions consisting of four trials per session were performed on two days. Mice were placed into the pool from four different directions in each of the four trials, and the escape latency was measured by a video-tracking system (SMART; Panlab, Spain). The second day of training was performed the following day, followed by a 60-s probe test without a platform. The time in each quadrant was analyzed by a video-tracking system (SMART). To evaluate long-term memory, the probe tests were performed 1 and 3 months after hidden platform training.
Intracerebroventricular injection
After the first day of MWM training, mice were anesthetized with 1.8–1.9% isoflurane, and then were injected 200 nM of α2AP (Calbiochem, MA, USA) or 0.1 μg/μL of an anti-α2AP neutralizing goat antibody (AF1470, R&D System, MN, USA) as well as saline or normal goat IgG control, respectively (AB-108-C, R&D System) in a total volume of 20 μL into the lateral ventricle (0.5 mm caudally and 1.0 mm laterally to the bregma and 2.0 mm vertically from the skull surface) with a two-step needle (Star needle; Seiseido, Tokyo, Japan) attached to a glass syringe (As one, Osaka, Japan). After injection, the needle was held at the site for 1 min to prevent reverse flow.
In vivo BrdU labeling
5-Bromo-2′-deoxyuridine (BrdU) (Nacalai Tesque Inc, Kyoto, Japan) was dissolved in saline, and injected intraperitoneally at 24-h intervals for 7 days at 50 mg/kg. The mice were perfused with PBS and 4% paraformaldehyde (PFA) in PBS, and the brains were then fixed in 4% PFA for 48 h, then soaked in 30% sucrose for 5 days. Thirty-micrometer-thick frozen coronal sections were prepared and stained with anti-BrdU mouse antibody (555627, BD Biosciences, Franklin Lakes, NJ, USA; diluted 1:200 with blocking solution) and anti-Ki67 rabbit antibody (ab16667, Abcam, Cambridge, UK; diluted 1:500 with blocking solution) after 2 N HCl-treatment at 37℃ for 30 min, neutralization with 0.1 M of boric acid pH 8.5 at room temperature for 10 min, and blocking with a Mouse on Mouse blocking kit (Vector Laboratories, CA, USA) and blocking solution (0.3% Triton X-100 and 10% normal goat serum (Vector Laboratories) in PBS). The sections were then incubated with anti-BrdU and anti-Ki67 antibodies at room temperature at 4 °C overnight. After washing with PBS, the sections were treated with Alexa 488-conjugated goat anti-mouse IgG (A11001, Invitrogen; diluted 1:1000 with blocking solution) and Alexa 546-conjugated goat anti-rabbit IgG (A11010, Invitrogen, CA, USA; diluted 1:1000 with blocking solution), and then coverslipped in Prolong Gold™ antifade reagent (Invitrogen). The specimens were observed using a confocal laser microscope NIKON A1R (Nikon, Tokyo, Japan).
Immunostaining of doublecortin
Thirty-micrometer-thick frozen coronal sections were treated with 0.3% H2O2 in methanol at room temperature for 10 min. The sections in Retrievagen A (pH 6.0) (BD Biosciences) were autoclaved and washed in PBS, and then incubated with blocking solution at room temperature for 1 h, and treated with anti-doublecortin (Dcx) mouse antibody (ab18723, Abcam; diluted 1:200 with blocking solution) at 4 °C overnight. After washing with PBS, they were incubated with biotinylated anti-rabbit IgG antibody (BA-1000, Vector Laboratories; diluted 1:200 with blocking solution) at room temperature for 30 min. The detection of antibody-antigen complexes was accomplished using a Vectastain Elite ABC kit (Vector Laboratories) and Metal-Enhanced DAB Substrate kit (Thermo Scientific, IL, USA). The immunostained sections were photographed using a microscope with a digital camera (model IX71; Olympus, Tokyo, Japan). Images were taken at full resolution with a single image dimension set at 1360 × 1024 pixels.
Immunoblotting
After perfusion with PBS, the hippocampi and cerebral cortexes from the mice were homogenized and sonicated in lysis buffer: 10 mM Tris–HCl buffer (pH 7.5) containing 1% SDS, 1% Triton X-100, and a protease inhibitor cocktail (Roche, Mannheim, Germany). The protein concentration in each lysate was measured using a BCA protein assay kit (Pierce, IL, USA). Lysates containing equal amounts of protein were subjected to SDS–polyacrylamide gel electrophoresis on a 10% acrylamide gel. Proteins were transferred onto PVDF or nitrocellulose membranes. After blocking with 3% skim milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T), the membranes were incubated with anti-α2AP goat antibody (AF1239, R&D System; diluted 1:500 with blocking solution), anti-hexanoyl-lysine (HEL) mouse antibody (MHL-021P, Japan Institute for the Control of Ageing, Shizuoka, Japan; diluted 1:500 with blocking solution), or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mouse antibody (016-25523, Wako Pure Chemical Industries; diluted 1:4000 with blocking solution) at 4 °C overnight. After washing with TBS-T, the membranes were incubated with horseradish peroxidase-conjugated rabbit anti-goat IgG (P0449, Dako, Glostrup, Denmark), or goat anti-mouse IgG (A108PS, American Qualex, CA, USA; diluted 1:2500 with TBS-T or 0.3% skim milk in TBS-T) for 1 h. After washing again, immunoreactive bands were detected using Chemi-Lumi One Super (Nacalai Tesque) with an LAS-3000 mini-image analysis system (Fujifilm, Tokyo, Japan). The band intensities were quantified using the ImageJ software program. The same sample as a loading control was included in each Western blot analysis, and the band intensities were normalized.
Enzyme-linked immunosorbent assay
The α2AP levels in the cerebrospinal fluid and plasma were measured with a mouse α2AP ELISA kit (Innovative Research, MI, USA). The cerebrospinal fluid was collected as detailed below. Briefly, a guide cannula (PEG-4; Eicom, Kyoto, Japan) was implanted into the lateral right ventricle (0.2 mm caudally and 1.1 mm laterally to the bregma; and 1.4 mm vertically from the brain surface), fixed to the skull with dental cement (Unifast III; Corp., Tokyo, Japan), and was then occluded with a dummy cannula (PED-4; Eicom). The mice were returned to their home cage and allowed to recover for 2 days. A microdialysis probe (PEP-4-01; Eicom) was inserted into the lateral right ventricle through the guide cannula under anesthesia with 1.5% isoflurane. The probe was perfused continuously at a flow rate of 10 μL/min with artificial cerebrospinal fluid (ACSF) containing 147 mM NaCl, 4 mM KCl and 3 mM CaCl2. The outflow fraction for the first 3 h was discarded, and then the dialysis sample perfused with ACSF at a flow rate of 1 μL/min was collected for 1 h under anesthesia with 1.5% isoflurane.
Extraction of RNA and real-time PCR
Total RNA was isolated from the hippocampi and cerebral cortexes using TRIsure (Bioline, London, UK). After the addition of CHCl3, centrifugation was performed at 15,000×g for 15 min. The resultant supernatants were each mixed with an equal volume of 2-propanol. After centrifugation, the pellets were rinsed with 75% ethanol/diethylpyrocarbonate (DEPC)-treated water and then dried. The pellets were each dissolved in an appropriate volume of DEPC-treated water as total RNA fractions. RNA from each sample (1 µg) was transcribed using ReverTra Ace-α (Toyobo, Osaka, Japan) according to the manufacturer’s protocol. Quantitative PCR was performed to analyze the murine IL-6, TNF-α and IL-1β mRNA expression relative to the GAPDH mRNA expression using a MiniOpticon real-time PCR system (Bio-Rad Laboratories, CA, USA). We used the following primers: IL-6, 5′-GTTCTCTGGGAAATCGTGGA-3′ (sense) and 5′-GGAAATTGGGGTAGGAAGGA-3′ (antisense); TNF-α, 5′-AAATGGGCTTTCCGAATTCA-3′ (sense) and 5′-CAGGGAAGAATCTGGAAAGGT-3′ (antisense); IL-1β, 5′-CAAATCTCGCAGCAGCACA-3′ (sense) and 5′-TCATGTCCTCATCCTGGAAGG-3′ (antisense); and GAPDH, 5′-TGTGTCCGTCGTGGATCTGA-3′ (sense) and 5′-TTGCTGTTGAAGTCGCAGGAG-3′ (antisense). The fold-change in the expression levels of IL-6, TNF-α and IL-1β relative to the GAPDH expression as an endogenous control gene were determined by the − ∆Ct method.
Statistical analysis
Data are reported as the mean ± standard error of the mean (SE). Differences among mean values were analyzed using a one-way analysis of variance (ANOVA) followed by an LSD post-hoc test or Student’s t-test. P values of < 0.05 were considered to indicate statistical significance.