Animals. The APPswe/PSEN1dE9 double- transgenic (APP/PS1) and their littermate wild-type (WT) mice were obtained from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). These mice were housed in specific pathogen free (SPF) environment with a 12-hour light/12-hour dark cycle. Mouse was caged singly and allowed to have food and water ad libitum. All experimental animal procedures were approved by the Institutional Animal Care and Use Committee (Approval No.: IACUC-20180918-010) and the Laboratory Animal Ethics Committee of Jinan University.
Light irradiation treatment. At the age of 8.5 months, APP/PS1 mice were randomly distributed to the following groups: sham treatment group, the visible light (λ = 500 nm) treatment group (VIS light-treated APP/PS1 mice), the near infrared light (λ = 800 nm) treatment group (NIR light-treated APP/PS1 mice), and the far infrared light (the wavelength range was from 3 to 25 μm) treatment group (FIR light-treated APP/PS1 mice). A WT mouse group was also included. Mice in all groups were allowed free feeding during light irradiation that was 60 min per day for 1.5 months.
Morris water maze. After 5-week treatment with different light (VIS, NIR and FIR), all mice began to be subjected to Morris water maze behavior test under SPF environment. The Morris water maze experiment apparatus consisted of a circular pool with a diameter of 120 cm and a height of 50 cm, and water was injected into the pool. The camera was located just above the tank (connected to the computer), which was used to record the movement of mice. An appropriate amount of non-toxic protein powder was added into water to facilitate this recording. The pool temperature was maintained at 20 - 22°C, and the water surface was divided into four quadrants (from one to four) and twelve zones (from one to twelve).
The water maze experiment was divided into two parts: the first is the training trial section (navigation stage of water maze), and the second is the spatial probe test (space memory stage). In the training trial, the target platform (8 cm in diameter, 20 cm in height) was located in the center between zone 2 and zone 6 in the second quadrant, and the water surface was 1 cm higher than the target platform, so that the mice could not see the platform. The experimental parameters were as follows: swimming time (60 s), residence time on target platform (3 s). During the water maze training trials, mice were placed into the water in accordance with the east and west directions. After mouse facing the pool wall into the water, the time required to find the target platform was recorded, namely escape latency. Mice were allowed to stand on the target platform for 10 s when climbing up to the target platform. However, if mice were unable to find the target platform within 60 s, they were manually guided to find the target platform, and allowed to stay on it for 15 s. Each training cycle was done for 4 times and for 7 continuous days. In the spatial probe test that was 24 h after the last training trial, the target platform was removed. Mice were placed into the water again and observed for a period of time (60 s). The number of entry into effective zones to find the target platform was recorded to reflect the spatial memory of mice.
Immunostaining. After the behavioral tests, the brain tissues of the mice were isolated and bisected longitudinally. The left hemisphere was frozen at -80°C for further biochemical study. The right hemisphere was soaked in 4% paraformaldehyde solution for 24 h. Subsequently, they were immersed in 10%, 20% and 30% sucrose solutions each for 24 h and then stored in Tissue-Tek OCT compound at -20°C. Coronal 30-μm thick sections from the right hemisphere were cut by a cryostat. The sections were washed with phosphate buffered saline (PBS).
For immunohistochemistry, the activity of endoperoxidase was blocked by 3% H2O2. Antigen retrieval with citric acid buffer (pH 6.0) was performed at 95°C for 10 min. After washed with PBS, sections were incubated with 0.3% triton X-100 and 5% donkey serum for 60 min at room temperature. The sections were then incubated with mouse anti-Aβ antibody (1:10000, Sig-39300, Biolegend) overnight at 4°C. Sections were then washed with PBS containing 0.3% triton x-100 and incubated with goat anti-mouse/rabbit IgG conjugated with horseradish peroxidase (Universal kit, PV-600, Zhongshan Jinqiao, Beijing, China) at room temperature for 30 min. After washed with PBS, sections were incubated with hydrogen peroxide (DAB kit, Zhongshan Jinqiao) and co-stained with hematoxylin [E803FA0003, Sangon Biotech (Shanghai) Co., Ltd, China] at room temperature for 5 min. Images were captured using a Nikon microscope (Nikon Ni-U)
For immunofluorescent staining, after blocking with 0.3% triton X-100 and 5% donkey serum at room temperature for 60 min, sections were then incubated at 4°C overnight with the following primary antibodies: mouse monoclonal anti-Aβ (6E10) antibody (1:10000, Sig-39300, Biolegend), rabbit polyclonal anti-ionized calcium binding adapter molecule 1 (Iba1) (1:1000, 019-19741, Wako) or rat polyclonal anti-cluster of differentiation 68 (CD68) (1:2000, ab53444, Abcam). Sections were rinsed with PBS containing 0.3% Triton X-100. The sections were incubated at room temperature for 2 h with fluorescent secondary antibodies as follows: donkey anti-mouse IgG antibody conjugated with Alexa Fluor 488 (1:200, A21202, Invitrogen), donkey anti-rat IgG antibody conjugated with Alexa Fluor 488 (1:200, A21208, Invitrogen), donkey anti-rabbit IgG antibody conjugated with Alexa Fluor 555 (1:200, A31572, Invitrogen) or donkey anti-mouse IgG antibody conjugated with Alexa Fluor 647 (1:200, A31571, Invitrogen). Afterwards, the sections were incubated with 4’, 6-diamidino-2-phenylindole (DAPI, C0065, Solarbio) for 10 min to stain the nuclei. Images were acquired using ZEISS microscope (ZEISS Imager A2) or ZEISS confocal microscope (ZEISS LSM 800 with airyscan).
Aβ plaque-associated microglial analysis. The quantification of Aβ plaque-associated microglia was similar to the previously reported method with minor modification. Within 20 μm range from the edge of an Aβ plaque, the number of Iba1 staining positive cells was manually counted. At least 100 plaques in the cerebral cortex region and 40 plaques in the hippocampus per group were quantified. For further analysis, plaques were divided into several groups according to their sizes (< 300, 300 to 600, 600 to 1200 and > 1200 µm2). The percentage of microglial CD68+ area was expressed as the co-staining area of Iba1+ and CD68+ divided by Iba1+ area. To further analyze the proximal interaction of microglia with Aβ plaque, the percentage of the co-staining area of 6E10+ and Iba1+ in total 6E10+ area was calculated. The co-staining area of 6E10+, CD68+ and Iba1+ divided by the total 6E10+ area was calculated to analyze the proximal interaction of CD68+ cells with Aβ plaque.
Primary microglial culture. Primary microglial cultures were performed as previously described with slight modifications[21, 22]. Briefly, C57BL/6 mouse at postnatal 0 to 3 days was quickly immersed in 75% ethanol, and then the entire brain tissue was carefully removed with a sterile spatula. The olfactory bulbs, cerebellum and hind brain were dissected away, and the meninges were also removed. Subsequently, the cortical tissues were harvested, transferred to a 35-mm petri dish containing 2 mL Hanks' Balanced Salt Solution (HBSS) and minced into approximately 1 mm pieces by using sterile surgical scissors. The minced tissues were mechanically dissociated into suspension with a 1 mL pipette, and then filtered into a 50 mL conical tube by using a 40-μm mesh filter. The filtrated cell suspension was then transferred into a poly-L-lysine-coated T75 flask and cultured with Dulbecco’s modified Eagle’s medium (DMEM) (C1995500BT, Gibco) supplemented with 10% fetal bovine serum (FBS) (SFBE, Natocor). After 3 days, medium was changed to that composed of 25 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) (415-ML-010, R&D system) and 10% FBS. After cultured for 12 - 14 days, microglial cells on top of the mixed glial cell layer were harvested by shaking the culture flask and were used for further experiment when their purity evaluated by Iba1 staining was over 95%.
Phagocytosis assay. The microglial phagocytosis of Aβ1-42 was analyzed similarly to a previously reported method with slight modifications. Briefly, fluorescein amidite-labeled Aβ1-42 (FAM-Aβ1-42) (AS-23525, Anaspec) was first aggregated at 37°C for 24 h with agitation. Primary microglial cultures were seeded in the poly-L-lysine-coated 6-well plates at a density of 1 x 105 cells/cm2 and then cultured overnight. After the cells were treated with FIR light for 1 h, the aggregated FAM-Aβ1-42 was added into the culture medium with a final concentration of 0.8 μg/mL. The primary microglial cultures were continuously treated with FIR light for additional 2 h. FAM-Aβ1-42-containing cultured medium was removed. Cultures were washed three times with PBS. Subsequently, the fluorescent images were randomly captured in three fields for each well by using an OLYMPUS microscope (OLYMPUS, IX71). For fluorescent intensity detection by flow cytometry, primary microglial cultures were digested with 0.25% trypsin-EDTA (25200056, Gibco), and then were centrifuged at 1500 g for 5 min at 4°C and washed with PBS for three times. For the microscopic images, the green fluorescent intensity of internalized FAM-Aβ1-42 in each well, which was captured under a green fluorescent protein channel, was quantified based on the mean of fluorescent intensity from three random fields by using ImageJ 1.52n. The results of three images were averaged to reflect the phagocytosis activity of the cells in the well. In the case of detection by flow cytometry, the green fluorescent intensity of internalized FAM-Aβ1-42 from 10000 cells each sample was recorded under a FITC channel and then was expressed as the Geo mean for each sample. These measurements were performed by a person who was blind to group assignments. Finally, the green fluorescent intensity of treated samples was normalized to the mean fluorescent intensity of the control group without FIR light treatment.
Elisa assay. The protein amount of interleukin (IL)-1β and IL-6 in the cerebral cortex were determined by using ELISA kits (catalog No: E-EL-M0037c for IL-1β, Catalog No: E-EL-M0044c, Elabscience) according to the manufacture’s instruction. Briefly, small pieces of the cerebral cortex of mice were weighted. The tissue was then homogenized in PBS on ice with protease inhibitor cocktail for general use (P1005, Beyotime) [tissue weight (g): PBS (mL) volume=1:9]. Subsequently, the homogenates were centrifuged at 5000 g for 8 min at 4°C. The amount of IL-1β and IL-6 in the supernatant was then determined. The final level of IL-1β and IL-6 was then normalized to its protein content determined by a BCA protein assay kit (P0010, Beyotime).
Western blot. The level of synaptic proteins postsynaptic density-95 protein (PSD-95) and synaptophysin in the hippocampus were determined by using Western blotting as previously described with slight modifications[24, 25]. The hippocampal tissues were homogenized in the RIPA lysis buffer (CW23335, CWBIOTECH) by using 1 mL insulin syringe on ice. Homogenates were centrifuged at 12000 g at 4°C for 15 min. The supernatant was used for Western blotting. The protein content of each sample was determined by the enhanced BCA protein assay kit (P0010, Beyotime). Twenty microgram protein per lane was electrophoresed on a 10% polyacrylamide gel and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked by 5% nonfat milk at room temperature for 60 min and then incubated at 4°C over night with the following primary antibodies: mouse monoclonal anti-PSD-95 antibody (1:1000, 75-028, NeuroMab), mouse monoclonal anti-synaptophysin (1:1000, MABN1193, EMD Millipore), or rabbit monoclonal anti-β-actin (1:1000, 8457s, Cell Signaling). After washed with Tris Buffered Saline Tween (Catalog No: T1081, Solarbio), the membranes were then incubated with corresponding secondary antibodies at room temperature for 2 h. The signal of protein band was visualized using NcmECL Ultra (Catalog No: P10200, Ncmbiotech). The protein band intensity of PSD-95 and synaptophysin was normalized to the corresponding band intensity of β-actin from the same sample.
Statistical analysis. All data were expressed as means ± S.E.M. The statistical analysis of results was performed by using GraphPad Prism version 7.0. The significant difference was assessed by unpaired t test, one-way or two-way ANOVA followed by Turkey’s or Dunett multiple comparisons test. The data of training sessions in the Morris water maze test was analyzed by two-way repeated measures ANOVA. A p value < 0.05 was considered statistically significant difference.