All experimental protocols were approved by the ethical committee of the Animal Care and Experimental Committee of Shanghai Jiao Tong University School of Medicine and were performed according to the guidelines from the EU Directive 2010/63/EU. Efforts were made to minimize suffering due to surgery and to reduce the overall number of animals we used.
Ten-week-old male SD rats of Specific pathogen-free (SPF) grade were provided by the Shanghai SIPPR-BK Laboratory Animal Co., Ltd. Standard housing of temperature (18-22℃) and humidity (55-65%) with a 12-h light/dark cycle and free access to food and water was maintained during the experimental period. 32 rats were randomly housed 2 per cage and divided into four groups: control, CP, CP plus CTS and CTS group. After anesthesia (intraperitoneal injection, 0.8ml 5% chloral hydrate per 100g weight), the ligature of maxillary first molars in the CP group and CP plus CTS group was administrated. In addition, the rats in the CP plus CTS group were injected with CTS (5mg/kg, i.p.) 1 hour in advance. The control group was given an equivalent volume of saline. The amount of CTS was calculated based on earlier studies . Three days after the last administration, behavioral tests were performed to evaluate the cognitive function of the rats. Timeline and intraoral photographs could be found in Supplementary Figure 1-2.
The 0.25mm ligature wire was provided by department of orthodontics, Shanghai Ninth People’s Hospital. CTS was purchased from Abmole (USA) and dissolved according to the instructions from Selleck (Shanghai, China) (2% DMSO+30% PEG300+5% Tween 80+ddH2O).
2.2 Measurement of Alveolar Bone Resorptionby Micro-Computed Tomography (CT)
The maxillae of rats were obtained to detect bone parameters by micro-CT. Fixed in 4% paraformaldehyde, the bone morphometry was assessed using Skyscan1172 (Bruker, Kontich, Belgium) with an accuracy of 18μm. Parameters including bone volume percentage (bone volume/total volume, BV/TV), Bone surface/volume ratio (BS/BV) and Bone mineral density (BMD) were calculated.
2.3 Assessment of periodontal inflammation responses by Histologic Staining Assay (HE staining)
The maxillae of rats were immersed with EDTA decalcifying solution (10% EDTA-2Na) and replaced every 5 days for 4-6 weeks for further histologic staining assay. After decalcification, hematoxylin-eosin (HE) staining assay was used to detect the inflammation response. Samples were then dehydrated through a serial alcohol gradient and embedded in paraffin wax blocks. Before immunostaining, 5-μm-thick tissue sections were dewaxed in xylene, rehydrated through decreasing concentrations of ethanol, and washed in PBS. Then sections were stained with hematoxylin and eosin (H&E). After that, sections were dehydrated through increasing concentrations of ethanol and xylene.
2.4 Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
Peripheral blood from rats (5 ml per rat) was collected from abdominal aorta in the presence of heparin as the anticoagulant. 3 ml of the whole blood was diluted with sterile PBS of the same volume and gently resuspended. 6 ml of the diluted whole blood fraction was overlaid onto 3 ml of the Ficoll-Paque Plus (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) and then subjected to 800g for 20 mins at RT with the centrifuge brake “off”. Then PBMCs layers were washed twice with RPMI 1640 media by centrifugation at 1200rpm for 5 mins at 4℃. After isolation, all samples were dissolved in Trizol reagent (Takara, Kusatsu, Shiga, Japan) for lysis of cells to extract RNA.
2.5 RNA extraction and RT-PCR analysis
The RNA was extracted from PBMCs and homogenization of cortex using Trizol reagent (Takara, Kusatsu, Shiga, Japan) and the Total RNA Kit (Omega Bio-Tek, Inc., Norcross, GA, USA) respectively. The purity and concentration of RNA, as well as the cDNA synthesis, were conducted according to Li et al. . Briefly, the purity and concentration were measured by a spectrophotometer (NanoDrop ND-1000; NanoDrop Technologies, Wilmington, DE, USA). The ratios of the absorbances at wavelengths of 260 and 280 nm were1.8–2.0. Subsequently, an RT-PCR assay was performed using SYBR Premix Ex TaqTM (Takara, Kusatsu, Shiga, Japan) on a Roche LightCycler 480 Real-Time PCR Detection System (Roche, Basel, Switzerland) according to the manufacturer’s protocol. Data were then processed using the 2-ΔΔCT method. All results were based on at least three independent tests, and the final results were expressed as normalized fold values relative to the control group. The sequences of genes including Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), interleukin-1β (IL-1β), IL-6, IL-6R, IL-8, IL-21, IL-21R, APP, amyloid precursor-like protein 1 (APLP1), APLP2, a disintegrin and metalloproteinase 10 (ADAM10), ADAM17, β-site APP cleaving enzyme 1 (BACE1), presenilin 1 (PS1), PS2 and their primer pairs were listed in table 1.
2.6 Protein levels in plasma and cortex measured by ELISA
Of the approximately 5ml of blood collected from rats, 2ml of blood were collected in heparinized tubes for the measurement of plasma cytokines. After centrifuging (4℃, 2500rpm*15min), plasma was immediately aliquoted into 1.5 milliliter cryogenic tubes and frozen at −80°C until use. For tissue, radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Beijing, China), 1% protease inhibitor cocktail (Sigma, St. Louis, MO, USA), and 1% PMSF (Beyotime, Beijing, China) were used to homogenize samples of the cerebral cortex. Protein qualification was performed by BCA Protein Assay Kit (Beyotime, Beijing, China). All ELISA kits were rat specific. Equal amounts of protein were used in ELISA to measure levels of IL-1β (detection limits<0.1pg/ml; UBI, Sunnyvale, CA, USA), IL-6 (detection limits<0.1pg/ml; UBI, Sunnyvale, CA, USA), IL-8 (detection limits<0.1pg/ml; UBI, Sunnyvale, CA, USA), IL-21 (detection limits<0.1pg/ml; UBI, Sunnyvale, CA, USA), total Aβ (detection limits<1.0ng/ml; UBI, Sunnyvale, CA, USA), Aβ1-40 (detection limits<1.0ng/ml; Enzyme-linked Biotechnology, Shanghai, China) and Aβ1-42 (detection limits<0.1ng/ml; Enzyme-linked Biotechnology, Shanghai, China) both in the cortex and plasma according to the manufacturer’s instructions.
2.7 Open filed test
The open field in the present study consisted of a rectangular arena (530mm×478mm), enclosed by a black wall, 590mm in height (Mobile Datum, Shanghai, China). The test was initiated by gently placing a single rat in the middle of the arena, allowing the animal to move freely for 5 minutes while being recorded.
2.8 Morris water maze (MWM) test
The MWM test was conducted in a round pool 160 cm in diameter and 55 cm in depth (Mobile Datum, Shanghai, China). The pool was filled with water made opaque with white non-toxic water-based tempura paint. The water temperature was controlled to remain with a range equivalent to that of room temperature (22±1°C). The platform was placed in the center of one quadrant of the pool and submerged 2.5 cm beneath the water surface; it remained in the same position throughout the learning trials and was removed from the pool during the probe test. A video-tracking system (Shanghai Jiliang Software Technology Co., Ltd.) was used to monitor and record the swimming activity of the rats. The rats should have learned to use the visual tips around the pool to find the hidden platform within 90s, otherwise it would be gently guided to the platform and allowed to re-orient for an additional 10s. Each rat was trained four times per day with a 30s of rest per training interval. To examine spatial reference memory, a probe test was carried out on the sixth day when the platform was removed from the pool and each rat was placed into the water at the two quadrants furthest from the platform used on days 1–5, being allowed to navigate freely for 60s.
2.9 Western blot
The samples of the cerebral cortex of rats in four groups were homogenized and lysed by RIPA containing 1% protease inhibitor cocktail and 1% PMSF (Beyotime, Shanghai, China). Equal amounts of protein were separated by SDS polyacrylamide gel electrophoresis and transferred onto PVDF membrane blocked with 5% skimmed milk as previously described . A pre-stained protein marker (Thermo Fisher Scientific, MA, USA) was run in parallel to detect the molecular weight of proteins. GAPDH was used as a protein loading control according to its high and constant expression in most tissues and cell types, earning the gene and its protein housekeeping status. Proteins were probed with appropriate antibodies including anti-JAK2 (Rabbit mAb, 1:500, 3230T; Cell Signaling Technology, USA), anti-STAT3 (Rabbit mAb, 1:1000, 4904T; Cell Signaling Technology, USA), anti-phosphor STAT3-Tyr705 (Rabbit mAb, 1:1000, 9145S; Cell Signaling Technology, USA) and anti-GAPDH (Rabbit mAb, 1:1000, AB-P-R001, Goodhere Biotechnology Co., Hangzhou, China). The data were quantified by Image J 1.51j8.
Rats were anesthetized with 10% chloral hydrate and perfused with cool PBS before removal of the brain. One hemisphere was placed in 4% paraformaldehyde overnight at 4°C, after which paraffin sections were prepared. This procedure is consistent with the previous study . Briefly, brain sections were incubated with 3% H2O2 in methanol, blocked with 10% goat serum and incubated overnight at 4 °C with the following primary antibodies: Iba1 (Goat pAb, 1:400, ARG63338; Arigo Biolaboratories, Hsinchu City, Taiwan, China) and GFAP (Rabbit pAb, 1:400, ab7260; Abcam) to label microglia and astrocytes. After being washed, sections were incubated with biotinylated goat anti-rabbit or goat secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA). After being rinsed with PBS, streptavidin-labeled peroxidase was added and left for 30 min. This was followed by further rinsing, after which newly prepared 3,3′-diaminobenzidine (DAB) solution was added, and the mixture was left for the reaction to develop. The sections were dyed with hematoxylin and dipped in 1% hydrochloric acid in alcohol for differentiation. They were then washed in ammonia and stained blue, after which they were rinsed with water.
For cell counting, images were obtained with a Leica camera. The numbers of Iba1-positive or GFAP-positive cells were determined by counting positive cells in two areas of each section in every tenth serial coronal section. At least three sections were analyzed per rat, and the average of the individual measurements was used to calculate group means.
2.11 Statistical analysis
All data are presented as the mean ± standard error of the mean (SEM). P values were calculated with one-way ANOVA and two-way ANOVA with the GraphPad Prism software 7.0. An analysis of variance was performed using Turkey’s post hoc multiple comparison test. A value of p<0.05 was indicative of statistical significance.