Animals

All animal experiments were performed according to the approved guidelines by the Catholic Kwandong University animal committee, International Saint Mary’s Hospital (No. CKU 2020-012). 5xFAD transgenic mice (B6SJL-Tg (APPSwF1Lon, PSEN1 × M146L × L286V) 6799Vas/Mmjax) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and inbred by crossing with C57BL/6 mice. All mice were genotyped using polymerase chain reaction (PCR) from genomic DNA extracted from the mouse tail biopsy. Animals were housed with 24-h food and water with 12-h dark/light cycles at a constant temperature of 23 °C.

Surgical destabilization of the medial meniscus (DMM)

OA was induced by DMM as described previously [11, 12]. Animals were anesthetized under 3% isoflurane, and the right knee joint was exposed through a medial parapatellar approach, making a 1-cm longitudinal medial parapatellar incision. The medial meniscus, located between the medial condyle of the femur and the medial plateau of the tibia, was identified and severed using a No. 10 surgical blade. The surgeries were either a sham, performed by just opening the joint capsule and DMM, in which the menisco-tibial ligament was cut to destabilize the medial meniscus. The knee was flushed with saline, and the joint capsule incision was closed with a No. 6 absorbable suture.

Y-maze test

A Y-maze test was performed to measure short-term spatial memory in mice. Mice were habituated to the testing room for 30 min. A mouse was placed in an arm of the Y-maze and allowed to freely explore the arms for 10 min. Spontaneous alterations were recorded and analyzed by a tracking software (EthoVision XT, Noldus, Wageningen, the Netherlands) connected to a video recording camera. The spontaneous alteration was calculated as a percentage by dividing the number of alterations by the total number of alterations.

Limb harvesting and micro-CT scanning

Following behavioral tests, both hind limbs were dissected immediately and fixed in 4% paraformaldehyde for 24 h after skin and extra tissue removal. Mouse hind limbs were scanned using a desktop micro-CT (SkyScan 1176, Bruker, MicroCT, Kontich, Belgium). The organs were scanned at 35–60 kV, 200 µA, 0.5 mm aluminum filter, 360° rotation, and a voxel size range of 1.65–6.56 µm. Micro-CT projections were back-reconstructed using the NRecon software (NReconServer64bit, Bruker, MicroCT, Kontich, Belgium) and volume-rendered and visualized in 3D with the CTVox software (Bruker, MicroCT, Kontich, Belgium).

Immunofluorescence staining of brain tissues

Mice were anesthetized and perfused intracardially with 0.9% saline. Knee and brain samples were collected and post-fixed for 12 h in 4% paraformaldehyde. Brain tissue sections of 20 μm thickness were acquired with a CM3050S microtome (Leica, Wetzlar, Germany). Brain tissue sections were permeabilized in 0.3% TritonX-100 containing phosphate-buffered saline (PBST), followed by blocking with 0.3% normal donkey serum and 1% bovine serum albumin for 60 min at room temperature. The sections were then incubated with ionized calcium binding adaptor molecule 1 (Iba1, microglia marker, 1:200, goat, Novus Biologicals, NB100-1028), β-amyloid (amyloid plaques marker, 1:200, mouse, Santa Cruz, sc-28365), or Neu-N (neuron marker, 1:1000, rabbit, Millipore, ABN78) as described previously [13]. Sections were mounted with DAPI (Molecular Probes, Life Technologies).

Microscopy and quantification

Fluorescent signals were captured with a Carl Zeiss Axio Imager Z1 fluorescence microscope equipped with ApoTome (Carl Zeiss MicroImaging, Inc.) using the same exposure time for each image. Images were analyzed using ImageJ software (NIH), converted to grayscale, and used the same post-acquisition threshold for analysis. Manual counting of Iba1+ cells or amyloid plaques was performed with ImageJ software, using the ‘analyze’ grid, by analyzers blind to mouse genotype and treatment groups. Five to 10 images from one brain slice were analyzed for quantification of the number and area (μm2) of Iba1 cells and amyloid plaques.

Quantitative RT-PCR analysis

Total RNA was extracted from mouse brain frontal cortex tissue by using TRIZOL (Invitrogen, Carlsbad, CA, USA). Total RNA (2 μg) was used to synthesize cDNA using the Superscript II reverse transcriptase (Invitrogen) and an oligo (dT) primer. Conventional PCR was performed with the specific primer sets in Table 1 using a T100 Thermal Cycler (Bio-Rad, Richmond, CA, USA). PCR products were electrophoresed on a 2% agarose gel. PCR bands were imaged under ultraviolet light and quantified by ImageJ. Quantitative real-time PCR experiments were carried out using the One Step SYBR PrimeScript RT-PCR Kit (Takara Bio, Otsu, Shiga, Japan), and the ABI Prism 7000 Sequence Detection System (Applied Biosystems, California, CA, USA). As an internal reference gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or cyclo were used. All experiments were performed at least in triplicate, and relative expression was calculated using the comparative threshold cycle method.

Table 1 PCR conditions and sequence information for the primersWestern blot analysis

Proteins were extracted with RIPA lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, and 1% NP-40). Equal amounts of protein were resolved on a 10% SDS–polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, California, USA). The blots were blocked with 4% skim milk in PBS with 0.1% Tween-20 and then incubated with primary antibodies, GFAP (1:1000, Rabbit, DAKO, Z0334), MBP (1:1000, Rabbit, Sigma, M3821), and GAPDH (1:2000, Rabbit, Cell Signaling, #2118) overnight at 4 °C. Specific bands for the primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (1:2000) using an ECL compound solution (SuperSignal West Femto; Thermo Fisher, Franklin, MA, USA).

Statistical analysis

Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test or unpaired t-tests, assuming equal variances were used to compare the groups of interest. Results are presented as the mean ± standard error of the mean. A p-value < 0.05 was considered significant for all tests. All analyses were carried out using GraphPad Prism version 8.00 (GraphPad Software, San Diego, CA, USA).