Far infrared light irradiation enhances Aβ clearance via increased exocytotic microglial ATP and ameliorates cognitive deficit in Alzheimer’s disease-like mice

The APP_swe/PSEN1dE9 double-transgenic (amyloid precursor protein (APP)/presenilin 1 (PS1)) AD mice 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 environment with a 12-h light/12-h 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-20180918-07) and the Laboratory Animal Ethics Committee of Jinan University.

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 (λ = 3 to 25 μm) treatment group (FIR light-treated APP/PS1 mice). The mice in the VIS and NIR light treatment groups were irradiated with customized light-emitting diode (LED) based illuminating lamps (SD, Xuzhou Aijia Electronic Technology Co., Ltd, China). The two lamps contained the LEDs with the specific wavelength of 500 ± 10 nm and 800 ± 10 nm, respectively. The mice in the FIR light treatment group were irradiated with the FIR light emitter (JF-802, Guangdong Junfeng BFS Technology Co., Ltd, China), a ceramic FIR light generator that can emit infrared light with the wavelength range from 3 to 25 μm. A wild-type (WT) mouse group was also included. Mice in all groups were allowed free feeding during light irradiation that was for 60 min at 0.13 mW/cm² per day for 1.5 months.

After 5-week treatment with different lights (VIS, NIR and FIR), all mice were subjected to Morris water maze behavior test under specific pathogen-free environment. The Morris water maze 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 that connected to a computer was located just above the tank, 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 and twelve zones. The water maze test 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 of zone 2, zone 6 and zone 10 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) and 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 was placed 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. 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 60 s. The number of entries into effective zones to find the target platform was recorded to reflect the spatial memory of mice.

After the behavioral tests, the brains of the mice were harvested 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 (1:10,000, 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 being 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 being blocked 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 (1:10,000, 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 (CD) 68 (1:2000, ab53444, Abcam). After rinsed with PBS containing 0.3% Triton X-100, the sections were incubated at room temperature for 2 h with fluorescent secondary antibodies: 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 a ZEISS microscope (ZEISS Imager A2) or ZEISS confocal microscope (ZEISS LSM 800 with airyscan).

The protein amount of interleukin (IL)-1β, IL-6, Aβ_1-40 and Aβ_1-42 in the cerebral cortex were determined by using enzyme-linked immunosorbent assay kits (catalog No. E-EL-M0037c for IL-1β, catalog No. E-EL-M0044c for IL-6, catalog No. E-EL-H0542c for Aβ_1-40, catalog No. E-EL-H0543c for Aβ_1-42, Elabscience) according to the manufacturer’s instruction. Briefly, small pieces of the cerebral cortex of mice were collected. The tissue was then homogenized in PBS on ice with protease inhibitor cocktail for general use (P1005, Beyotime). Subsequently, the homogenates were centrifuged at 5000g for 8 min at 4 °C. The amount of IL-1β, IL-6, Aβ_1-40 and Aβ_1-42 in the supernatant was then determined. For the Aβ_1-40 and Aβ_1-42, after the PBS soluble fraction in the supernatant were collected, the pellets were further resuspended with guanidine hydrochloride (5 M) to extract the PBS-insoluble Aβ fraction as previously reported [36]. The final level of IL-1β, IL-6, Aβ_1-40 and Aβ_1-42 was then normalized to its protein content determined by a BCA protein assay kit (P0010, Beyotime).

The level of synaptic proteins from the hippocampus and key molecules involved in Aβ production from cerebral cortex were determined by using Western blot as previously described with slight modifications [37, 38]. The tissues were homogenized in the radio immunoprecipitation assay lysis buffer (CW23335, CWBIOTECH) by using 1-mL insulin syringe on ice with protease inhibitor cocktail for general use. Homogenates were centrifuged at 12,000g at 4 °C for 15 min. The supernatant was used for Western blot. The protein content of each sample was firstly determined, and twenty microgram protein per lane was electrophoresed on a 10% polyacrylamide gel and then transferred to a polyvinylidene fluoride membrane. The membrane was blocked by 5% nonfat milk at room temperature for 60 min and then incubated at 4 °C overnight with the following primary antibodies: mouse monoclonal anti-postsynaptic density protein-95 (PSD-95) antibody (1:1000, 75-028, NeuroMab), mouse monoclonal anti-synaptophysin (1:1000, MABN1193, EMD Millipore), rabbit polyclonal anti-APP (1:1000, AF6219, Beyotime), mouse monoclonal anti-Aβ (6E10) antibody that also recognized soluble APPα (sAPPα) (1:10,000, Sig-39300, Biolegend), rabbit polyclonal anti-nicastrin (1:1000, 14071-1-AP, Proteintech), rabbit polyclonal anti-beta-site-APP cleaving enzyme 1 (BACE1) (1:1000, AF6273, Beyotime), rabbit polyclonal anti-PS1 (1:1000, 16163-1-AP, Proteintech), or rabbit monoclonal anti-β-actin (1:1000, 8457 s, Cell Signaling Technology). After being 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 these proteins was normalized to the corresponding band intensity of β-actin from the same sample.

The quantification of Aβ plaque-associated microglia was similar to the previously reported method with minor modification [39]. Within 20 μm range from the edge of an Aβ plaque, the number of Iba1 staining positive cells was manually counted, which were performed by a person who was blind to group assignments. 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 the total 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 cultures were performed as previously described with slight modifications [40, 41]. Briefly, C57BL/6 mouse at postnatal 0 to 3 days was quickly immersed in 75% ethanol for disinfection process, 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 and minced into approximately 1 × 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 (C1995500BT, Gibco) supplemented with 10% fetal bovine serum (SFBE, Natocor). After 3 days, medium was changed to that composed of 25 ng/mL granulocyte–macrophage colony-stimulating factor (415-ML-010, R&D system) and 10% fetal bovine serum. After being 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% (Additional file 1: Fig. S1A and S1B).

The microglial phagocytosis of Aβ was analyzed similarly to a previously reported method with slight modifications [42]. Briefly, fluorescein amidite-labeled Aβ_1-42 (FAM-Aβ_1-42) (AS-23525, Anaspec) was first aggregated at 37 °C for 24 h with agitation. Microglia were seeded in the 6-well plates at a density of 1 × 10⁵ cells/cm² (primary cultured microglia) or 5 × 10⁴ cells/cm² (BV2 cells) and then cultured in medium containing 25 mM or 0.1 mM glucose overnight. In some experiments, primary cultured microglia were pre-incubated with 2-deoxy-D-glucose (2-DG) (5 mM, 2 h), antimycin A (0.5 μM, 2 h), suramin (100 μM, 30 min), apyrase (2 U/mL, 2 h), wortmannin (500 nM, 30 min) or rapamycin (100 nM, 30 min). After the cells were pretreated with FIR light (the same FIR light emitter used in the animal irradiation experiments mentioned above) at 0.13 mW/cm² for 1 h, the aggregated FAM-Aβ_1-42 was added into the culture medium with a final concentration of 0.8 μg/mL. The primary cultured microglia were continuously treated with FIR light at 0.13 mW/cm² for an additional 3 h (irradiation of 1 h and then free-irradiation of 10 min, three cycles). As a negative control, the phagocytosis inhibitor cytochalasin D (10 µM, 11330, Cayman) was added for 30 min before the addition of FAM-Aβ_1-42. Then FAM-Aβ_1-42-containing cultured medium was removed and cultures were washed three times with PBS. Subsequently, the fluorescent images were randomly captured in five fields for each well by using an OLYMPUS microscope (OLYMPUS, IX71). For fluorescent intensity detection by flow cytometry, primary cultured microglia were digested with 0.25% trypsin-ethylenediaminetetraacetic acid (25200056, Gibco), centrifuged at 500g for 5 min at 4 °C and washed with PBS for three times. For the microscopic images, the green fluorescent intensity of internalized FAM-Aβ_1-42 in each well, which was captured under a green fluorescent protein channel, was quantified based on the mean of fluorescent intensity from five random fields by using ImageJ 1.52n. The results of five 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-Aβ_1-42 from 10,000 cells in each sample was recorded under a fluorescein isothiocyanate (FITC) channel and then was expressed as the FITC 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.

Primary cultured microglia (3 × 10⁵) or mouse microglial cells line BV2 (1.5 × 10⁵) suspended in 1 mL medium containing 25 mM or 0.1 mM glucose were added to the 12-well plate overnight. In some experiments, the cells were pre-incubated with 2-DG (5 mM, 2 h), antimycin A (0.5 μM, 2 h), or apyrase (2 U/mL, 2 h). The medium was replaced with that containing vehicle or Aβ_1-42 (0.1642 μM). Then the cell cultures were irradiated with FIR light at 0.13 mW/cm² for 1 h or 3 h (irradiation of 1 h and then free-irradiation of 10 min, three cycles). For the intracellular adenosine triphosphate (ATP) measurements, the medium was removed and then the cells were washed with PBS. 100 uL ATP lysis solution (S0026-4, Beyotime) was added to the cells for 15 min under shaking condition. The lysis solution was collected and centrifuged at 12,000g for 5 min at 4 °C, and then the supernatants were collected for ATP measurement by using an ATP assay kit (S0026, Beyotime). For the extracellular ATP measurements, the medium was collected and centrifuged at 500g for 5 min at 4 °C. The supernatants were collected for ATP measurement by using an enhanced ATP assay kit (S0027, Beyotime). The final level of intracellular and extracellular ATP was then normalized to their cellular protein content.

Lactate dehydrogenase (LDH) activity was determined by using LDH cytotoxicity assay kit (C0017, Beyotime) as done previously with slight modification [37]. Briefly, primary cultured microglia suspended in 0.5 mL were added to the 24-well plate overnight. The culture medium was replaced with that containing vehicle or Aβ_1-42 (0.1642 μM), followed by treatment with FIR light at 0.13 mW/cm² for 1 h. The medium collected from 24-well plates at the end of experiments were centrifuged at 500g for 5 min at 4 °C and 120 μL of cell-free supernatant was transferred to 96-well plate. After removal of the medium from 24-well plates, 1% Triton X-100 lysing solution was added to the remaining cells and incubating for 15 min under shaking condition. Then the 1% Triton X-100 lysing solution was centrifuged at 12,000g for 5 min at 4 °C and 120 μL supernatant was transferred to 96-well plate for LDH activity measurement. The final percentage of LDH released to the medium was calculated as follows: spontaneously released LDH in the medium/(spontaneously released LDH in the medium + intracellular LDH released by 1% Triton X-100) × 100.

The total RNA was isolated from primary cultured microglia treated as indicated in the text using an RNA-Quick Purification kit (RN001, Yishan). Total RNA at 500 ng was reversely transcribed into the cDNA using Hifair® III 1st Strand cDNA Synthesis SuperMix for quantitative polymerase chain reaction (qPCR) (gDNA digester plus) kit (11141ES60, Yeasen). Real-time qPCR was performed using a Hieff UNICON® qPCR SYBR Green Master Mix kit (11198ES08, Yeasen) and achieved on 480 LightCycler (Roche). The primer sequences are listed in Additional file: Table S1. The mRNA level of these genes was normalized to that of β-actin from the same sample. 1

The intracellular lactate content was measured using a lactate colorimetric assay kit (E-BC-K044-M, Elabscience) according to the manufacturer’s instruction. Briefly, 5 × 10⁵ primary cultured microglia treated with or without FIR light at 0.13 mW/cm² for 1 h were collected. The medium was removed and the cells were washed with PBS. PBS at 200 μL was added and cells were homogenized on ice. After that, the homogenates were centrifuged at 10,000g at 4 °C for 10 min. The supernatant was used for the lactate measurement. The lactate content was finally normalized to its protein content.

Microglial mitochondrial respiratory chain complex I (BC0515, Solarbio), II (BC3235, Solarbio), III (BC3245, Solarbio), IV (BC0945, Solarbio) and ATP synthase (BC1445, Solarbio) activities were performed using corresponding assay kits according to the manufacturer’s instruction with slight adjustments. Briefly, at least 1.2 × 10⁷ BV2 cells treated with or without FIR light at 0.13 mW/cm² for 1 h were collected. After the medium were removed, 1 mL extract buffer was added to each sample, and homogenized on ice with a homogenizer. Subsequently, the homogenates were centrifuged at 500g for 5 min at 4 °C. The supernatants were transferred to another centrifugal tube, and centrifuged at 11,000g for 10 min at 4 °C. After the supernatants were removed, 400 μL extract buffer were added and these homogenates were then subjected to the ultrasonic lysis (power 52 W, ultrasonic 5 s, interval 10 s, for 15 times). The supernatant is used for mitochondrial respiratory chain complex activity determination. Finally, the enzymatic activity of these complexes was normalized to its protein content.

The oxidized nicotinamide adenine dinucleotide (NAD⁺) content and the ratio of NAD⁺/reduced NAD (NADH) were measured using NAD⁺/NADH assay kit with WST-8 (S0175, Beyotime) according to the manufacturer’s instruction. Briefly, 1 × 10⁶ BV2 cells treated with or without FIR light at 0.13 mW/cm² for 1 h were collected. The medium was removed and the cells were washed with PBS. NAD⁺/NADH extract buffer at 200 μL was added and gently pipetting to promote cell lysis. After that, the cell lysate was collected and centrifuged at 12,000g at 4 °C for 5 min, and the supernatant was used for the NAD⁺ content and the ratio of NAD⁺/NADH determination. The NAD⁺ content was finally normalized to its protein content.

All data were expressed as means ± standard error of the mean (SEM). The statistical analysis of results was performed by using GraphPad Prism version 8.0. The significant difference was assessed by unpaired t test, one-way or two-way analysis of variance (ANOVA) followed by Turkey’s or Dunnett multiple comparisons test. The data of training sessions in the Morris water maze test, Aβ phagocytosis of microglia or intracellular ATP level of microglia treated with FIR light and cytochalasin D, or cultured in the medium containing antimycin A and different concentrations of glucose were analyzed by two-way repeated measures ANOVA. A p value < 0.05 was considered statistically significantly different.

The time-course of the in vivo experiments is shown in Fig. 1A. The Morris water maze test consisted of two stages: training trials and spatial probe test (Fig. 1B). WT mice took a shorter time than APP/PS1 mice to find the platform in the training trials of the test (Fig. 1C). This impaired learning in the APP/PS1 mice was improved by the treatment of various wavelengths of light [F (4, 203) = 4.113, p = 0.0032]. Interestingly, mice in different groups during training stage had an increase in escape latency on one day compared to that on the prior day. For instance, VIS and NIR light-treated APP/PS1 mice had an increased escape latency on day 3 compared with that on day 2, and FIR light-treated APP/PS1 mice had an increase in escape latency on day 4 compared with that on day 3. This phenomenon was found in other studies [43–45]. Although this phenomenon can result in statistical difference in the performance of mice between different treatment groups, there was no significant difference (p = 0.1837) in the escape latency on day 7 between NIR and FIR light-treated mice. Compared to APP/PS1 mice without treatments, FIR light-treated but not VIS light- or NIR light-treated APP/PS1 mice had more entries into the zones around the platform in the spatial probe test (Fig. 1D). Notably, there was no difference in swimming speed among WT mice and APP/PS1 mice treated with or without various lights (Fig. 1E). These results suggested that FIR light improved the spatial memory of AD mice and later studies were thereby focused on the effects of FIR light on AD mouse brain.

Aβ pathology is one of the most distinctive hallmarks of AD, and these Aβ accumulation and deposition in the brain would result in a series of subsequent pathological events, including neuroinflammation, impaired neuronal function and cognitive dysfunction for AD progression [6, 46]. We next determined whether FIR had an beneficial effect on the alleviation of Aβ burden in the brain of AD mice. Compared to WT mouse, APP/PS1 mouse exhibits abundant Aβ plaque in both cerebral cortex and hippocampus (Fig. 2A). A significant reduction of Aβ plaque in these brain areas of the FIR light-treated APP/PS1-mice was found compared to the APP/PS1 mice (Fig. 2B–E). The levels of Aβ_1-40 and Aβ_1-42 in the PBS and guanidine hydrochloride fractions from cerebral cortex were markedly lower upon the FIR light treatment (Fig. 2F and G). These results indicate that FIR light significantly reduces Aβ level, resulting in decreased Aβ load in the brain of APP/PS1 mice.

We next determined whether FIR light alleviated neuroinflammation in the AD mice. As shown in Additional file 1: Fig. S2A and S2B, the proinflammatory factors including IL-1β and IL-6 were significantly higher in the APP/PS1 mice compared to WT mice. However, they were markedly reduced in the FIR light-treated APP/PS1 mice. In addition, we examined the expression of pre- and post-synaptic proteins including synaptophysin and PSD-95 due to their pivotal role in regulating synaptic plasticity, which closely correlates with neuronal function and cognitive functions [47, 48]. Western blot results showed that the level of PSD-95 did not exhibit significant changes in APP/PS1 mice compared with WT mice (Additional file 1: Figs. S3A and S3B). However, the level of synaptophysin was significantly decreased in the APP/PS1 mice compared to the WT mice, but this decrease was attenuated in the APP/PS1 mice treated with FIR light (Additional file 1: Figs. S3A and S3C). These results suggest that FIR light effectively reduces Aβ burden and neuroinflammation and restore the expression of synaptophysin in the brain of AD mice.

The Aβ accumulation and aggregation are mainly resulted from the imbalance of Aβ production and clearance [5]. Given the finding that FIR light was able to reduce the Aβ burden in the brain of AD mice, we asked whether FIR light could decrease Aβ production or increase Aβ clearance. We first examined a series of key molecules that are implied in the production of Aβ. The Western blot results showed that there was no significant change in the protein expression of amyloid precursor protein [APP full length (APPfl)], and APP processing secretases, including BACE1, and γ-secretase complex composed of nicastrin and PS1, between control and FIR light-treated APP/PS1 mice (Fig. 3A–F), suggesting that FIR light did not influence the process of Aβ production. Accordingly, we speculated that FIR light reduced Aβ burden through the clearance pathway. As the resident immune cells in the brain of AD, microglia are responsible for the clearance of Aβ [49]. In the present study, numerous microglia were recruited to the Aβ plaques (Fig. 3G). In the cortical region, FIR light enhanced the recruitment of microglia surrounding the Aβ plaques (Fig. 3H and Additional file 1: Fig. S4A). The number of microglia surrounding the Aβ plaques was increased with an increased size of Aβ plaque and FIR light increased the number of microglia no matter which size of Aβ plaque was considered (Additional file 1: Fig. S4B). Similar results were observed in the hippocampus (Additional file 1: Fig. S4C–S4E).

To investigate whether the microglia surrounding the plaques could engulf Aβ to achieve Aβ clearance, co-immunostaining of Iba1, CD68 and Aβ was carried out, and the CD68⁺ microglial phagosomes and internalized Aβ were quantified. The results showed that the microglia around the Aβ plaque expressed the phagocytic marker CD68 (Fig. 3I). Compared to the microglia in the control APP/PS1 mice, the microglia in the FIR light-treated APP/PS1 mice had a significant increase in CD68 expression (Fig. 3J). In addition, the interaction between microglia and Aβ was enhanced due to the increase in their contact area and phagocytic area in the FIR light-treated APP/PS1 mice (Additional file 1: Figs. S4F and 3 K), suggesting that FIR light enhanced microglial Aβ phagocytosis and clearance.

To further confirm whether FIR light could enhance microglial Aβ phagocytosis, mouse primary cultured microglia were used to assess FAM-Aβ_1-42 uptake. The co-immunostaining results showed that microglia could engulf FAM-Aβ_1-42 (Fig. 3L), and FIR light markedly enhanced the microglial capacity to engulf FAM-Aβ_1-42 (Fig. 3M), which was accompanied by an increased mRNA level of putative microglial phagocytic receptors, including triggering receptor expressed on myeloid cells 2 (Trem2), Toll-like receptor (TLR) 2, TLR4, CD14, integrin alpha M (Itgam), scavenger receptor class B type 1 (Scarb1) and Mer receptor tyrosine kinase (MerTK) (Additional file 1: Fig. S4G). However, this enhanced phagocytic effect was almost abolished in the present of the phagocytic inhibitor cytochalasin D (Fig. 3M), which was verified by measurements with the flow cytometry (Additional file 1: Figs. S4H and S4I). These results were in agreement with the findings that FIR light was able to significantly enhance Aβ phagocytosis of microglia in the APP/PS1 mice.

Since microglial phagocytosis requires dynamic reorganization of the actin cytoskeleton, a substantial amount energy is necessary [50]. We observed that the decreased intracellular ATP level in the microglia cultured in glucose-free medium or pretreated with the glycolytic inhibitor 2-DG was accompanied by decreased microglial Aβ phagocytosis (Additional file 1: Fig. S5A–S5D), indicating that intracellular ATP may play a pivotal role in the microglial Aβ phagocytosis. Recent studies on other types of cells treated with FIR light show improvement in mitochondrial function [32–34], which can promote energy metabolism. Accordingly, we asked whether FIR light could promote microglial energy metabolism and consequently enhanced microglial Aβ phagocytosis. As shown in Fig. 4A, we observed that intracellular ATP was markedly increased in microglia upon Aβ stress. Intracellular ATP level in the Aβ-induced microglia was further elevated with FIR light treatment (Fig. 4A). We found that FIR light could increase the intracellular ATP even without Aβ stress (Fig. 4B).

Tricarboxylic acid (TCA) cycle coupling with mitochondrial oxidative phosphorylation (OXPHOS) was the major pathway to produce ATP. To further investigate the relationship between the increase in the intracellular ATP and the enhancement in the microglial Aβ phagocytosis after FIR light treatment, the mitochondrial OXPHOS inhibitor antimycin A was used to reduce ATP production (Fig. 4C). This reduced ATP production was accompanied by the compromised FIR light-enhanced microglial Aβ phagocytosis (Fig. 4D) and the decreased intracellular ATP even under Aβ stress (Fig. 4E). Since glucose concentration in cultured medium influences the ATP production in cells [51, 52], we tried to change intracellular ATP in the microglia by culturing them in medium containing different glucose concentrations to further investigate the relationship between intracellular ATP and microglial Aβ phagocytosis under the condition without FIR light treatment. The results showed that increased intracellular ATP in microglia cultured in the medium containing 0.1 mM glucose was accompanied by enhanced microglial Aβ phagocytosis, as compared to that of microglia cultured in the medium containing 25 mM glucose (Fig. 4F and G). Decreased intracellular ATP was found in microglia cultured in the medium containing antimycin A, which was associated with decreased microglial Aβ phagocytosis (Fig. 4F and G). This intracellular ATP change was not altered even under Aβ stress (Fig. 4H). Interestingly, upon the antimycin A pretreatment, the intracellular ATP was further decreased in the microglia cultured in the medium containing 0.1 mM than 25 mM glucose, which was associated with more compromised Aβ phagocytosis by microglia (Fig. 4F–H). These results clearly suggest the important role of intracellular ATP in the microglial Aβ phagocytosis. These findings also suggest that FIR light promotes ATP production and such an increase in ATP was involved in the enhanced Aβ phagocytosis of microglia treated with FIR light.

Previous study has shown that microglia would release ATP responding to the Aβ stress [53]. ATP has been recently reported to modulate microglial Aβ phagocytosis [54]. In the present study, microglia showed increased extracellular ATP level when stimulated by Aβ (Fig. 5A), and this Aβ-induced increase was further elevated when microglia was exposed to the FIR light (Fig. 5A). To rule out the possibility that ATP release caused by Aβ or Aβ plus FIR light was due to microglial lysis, medium was analyzed for LDH release at the end of experiments. The results exhibited that Aβ or Aβ plus FIR light did not significantly cause microglial LDH release (Additional file 1: Fig. S6A), suggesting that this increased ATP release was not caused by microglial lysis.

We then asked whether increased ATP production would spontaneously promote extracellular ATP release. To answer this question, extracellular ATP level was analyzed after increasing and decreasing the ATP production of microglia induced by 0.1 mM glucose and antimycin A, respectively, as described above (Fig. 4F). The results exhibited that 0.1 mM glucose or antimycin A did not significantly change extracellular ATP level as compared to the level from microglia cultured in the medium containing 25 mM glucose and without antimycin A pretreatment, respectively (Fig. 5B and 5C). However, upon the treatment with Aβ, extracellular ATP levels were significantly increased and decreased, respectively (Fig. 5D and E). Moreover, this antimycin A-induced decrease in extracellular ATP level was unchanged even with the FIR light treatment (Additional file 1: Fig. S6B), suggesting that ATP production influences extracellular ATP release for the microglia stimulated by Aβ. Interestingly, microglia pretreated by antimycin A reduced extracellular ATP release when they were exposed to FIR light (Fig. 5F). Next, we asked whether FIR light could induce extracellular ATP release just like Aβ stress. The results exhibited that FIR light promoted microglial extracellular ATP release without cell lysis, which was verified by LDH assay (Fig. 5G and Additional file 1: Fig. S6C). Nevertheless, Aβ induced additional microglial ATP release upon the FIR light treatment (Additional file 1: Fig. S6D). Therefore, elevated ATP production of microglia treated with FIR light was beneficial to the extracellular ATP release of microglia stimulated by Aβ.

In addition, we observed that FIR light changed the mRNA levels of microglial ATP-related receptors, including P2X1, P2X5, P2X7, P2Y2, P2Y6, P2Y13, P2Y14 (Additional file 1: Fig. S6E and S6F), upon the Aβ stimulation, indicating that the increase in Aβ-induced extracellular ATP release of microglia treated with FIR light influences the ATP-related signal transduction. We blocked these ATP-related receptors by using suramin, a broad-spectrum antagonist of P2 purinoceptors [55], to investigate the role of extracellular ATP in the enhanced Aβ phagocytosis of microglia treated with FIR light. The results showed that suramin compromised the Aβ phagocytosis of microglia in the presence or absence of FIR light (Fig. 5H and Additional file 1: Fig. S6G), suggesting that extracellular ATP is involved in the FIR light-enhanced microglial Aβ phagocytosis. However, suramin did not stop the enhancement in Aβ phagocytosis of microglia treated with FIR light (Fig. 5I), which might be due to a limited ability of suramin to inhibit these ATP-related receptors. Thus, we next directly decreased the extracellular ATP level through the pretreatment with apyrase (Additional file 1: Fig. S6H to S6J), an enzyme that hydrolyzes extracellular ATP [56]. The results exhibited that addition of apyrase markedly decreased the Aβ phagocytosis of microglia treated with FIR light (Fig. 5J) or cultured in medium containing 25 mM or 0.1 mM glucose (Additional file 1: Figs. S6K and S6L). Notably, apyrase effectively abrogated the ability of FIR light to enhance microglial Aβ phagocytosis (Fig. 5K), including the microglia cultured in the medium containing 0.1 mM glucose (Additional file 1: Fig. S6M).

Additionally, it has been reported that phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) pathways can be activated by ATP and affect the endocytosis of microglia [54, 57]. To investigate whether PI3K and mTOR pathway was involved in the FIR light-induced enhanced microglial Aβ phagocytosis, their corresponding inhibitors wortmannin and rapamycin were used to block these pathways. As shown in Fig. 5L, M, Additional file 1: Fig. S6N and S6O, wortmannin and rapamycin decreased the Aβ phagocytosis of microglia treated with or without FIR light. Moreover, rapamycin but not wortmannin was capable of suppressing the enhancement in Aβ phagocytosis of microglia treated with FIR light (Fig. 5N and O), suggesting that PI3K and mTOR pathways are involved in the FIR light-enhanced microglial Aβ phagocytosis.

Given that increased ATP production was critical in the enhanced Aβ phagocytosis of microglia exposed to FIR light and that glycolysis and TCA cycle coupling with OXPHOS in the mitochondria are the two major pathways to produce ATP, the effects of FIR light on microglial energy metabolism pathway were investigated. 2-DG and antimycin A were used to block glycolysis and OXPHOS pathway, respectively, and then to monitor the Aβ phagocytosis of microglia treated with FIR light. The results exhibited that 2-DG or antimycin A did not suppress the enhancement in Aβ phagocytosis of microglia treated with FIR light, although both were able to decrease the microglial Aβ phagocytosis under the condition of FIR light treatment (Fig. 6A, B, Additional file 1: Fig. S7A and 4D). However, 2-DG plus antimycin A effectively abrogated the ability of FIR light to enhance microglial Aβ phagocytosis (Fig. 6C). Interestingly, compared with antimycin A pretreatment, FIR light had a greater positive effect on Aβ phagocytosis (increase of 13.9% and 8.0% in the microglia pretreated with 2-DG and antimycin A, respectively) in 2-DG-pretreated microglia (Fig. 6A and B), suggesting that OXPHOS pathway effectively affects the enhanced Aβ phagocytosis of microglia treated with FIR light. Thus, increased ATP production of microglia treated with FIR light might be mainly through the OXPHOS pathway.

We further examined the production of lactate and the enzymatic activity of the mitochondrial respiratory chain complexes (complex I, II, III, IV and ATP synthase), which are closely associated with the glycolysis and OXPHOS pathways, respectively. As shown in Fig. 6D, lactate level was not significantly increased in the microglia treated with FIR light. In the present study, microglial BV2 cell line was used to investigate the effect of FIR light on the enzyme activity of these mitochondrial complexes due to their ability to generate adequate cells for enzymatic activity assay. BV2 cells had a increased ATP production and enhanced Aβ phagocytosis when exposed to FIR light (Additional file 1: Figs. S7B to S7D), which was similar to that of mouse primary cultured microglia. The results showed that FIR light markedly improved the enzyme activity of complex I but had no significant effect on the enzyme activity of complex II, III, IV and ATP synthase (Fig. 6F–I). Since complex I in the mitochondria converts NADH to NAD⁺, we monitored the NAD⁺ level and the ratio of NAD⁺ to NADH. The results showed that the NAD⁺ level and the ratio of NAD⁺ to NADH were significantly increased in BV2 treated with FIR light (Fig. 6J and K), which was in agreement with the increased enzyme activity of the complex I in BV2 following FIR light treatment.

All animal studies were performed with the approval by the Institutional Animal Care and Use Committee and the Laboratory Animal Ethics Committee of Jinan University, Guangzhou, Guangdong, China.

Not applicable.

The authors declare that they have no competing interests.

The datasets generated during and/or analyzed during current study are available from the corresponding authors on a reasonable request.