Background
Microglia are the resident macrophages of the brain, with important roles in development, homeostasis, and disease [
1,
2]. Under physiologic conditions, microglia are primarily found in the resting state (M0), but are activated into two phenotypes, the “classically activated” M1 and the “alternative activated” M2 phenotypes, following an imbalance to normal physiological conditions [
2,
3]. M1 microglia secrete various pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)α, which are induced by lipopolysaccharide (LPS) and/or interferon-γ (IFN-γ) [
2,
3]. Conversely, M2 microglia produce anti-inflammatory cytokines, such as IL-10 and TGFβ, and are induced by IL-4 and/or IL-13 [
2]. After cerebral ischemia, microglia/macrophages are activated: M2 microglia/macrophages promote brain restorative processes, including neurogenesis, axonal regeneration, angiogenesis, oligodendrogenesis, and remyelination; while M1 microglia/macrophages impair neurogenesis and aggravate neurological deficits [
2]. Recent evidence suggests that a shift from the M1 phenotype to the M2 phenotype is beneficial for recovery after stroke, and thus may provide novel therapeutic approaches to aide stroke victims [
2,
3].
Salidroside (SLDS) is a phenylpropanoid glycoside extracted from the root of
Rhodiola rosea L and is one of the main active ingredients of this plant.
Rhodiola rosea grows in high altitudes and cold regions and has been used as a medicine in many European countries and China [
4,
5]. Beneficial roles of SLDS have also been reported in aging [
5], cancer [
6], inflammation [
7,
8], oxidative stress [
4,
7], and several central nervous system (CNS) diseases, including Alzheimer’s disease [
9] and stroke [
10,
11]. Recently, SLDS was shown to ameliorate activation of both a microglial [
12] and a macrophage cell line [
13]. However, to date, the role of SLDS in microglial polarization remains unknown.
The goal of this study was to gain new insight into the medicinal value of SLDS after stroke. The optimal dose of SLDS following middle cerebral artery occlusion (MCAO) in mice was found and the ability of SLDS to regulate microglial polarization was explored both in vivo and in vitro. In addition, the effects of SLDS on primary microglia-mediated inflammation, phagocytosis, oligodendrocyte differentiation, and neuronal death were also investigated. These data provide evidence that SLDS induces neuroprotection by modulating the conversion of M1 microglia to M2 microglia.
Methods
Animal model and drug administration
All animal experiments were approved by the Institutional Animal Care and Use Committee of Capital Medical University and in accordance with the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Transient focal ischemia was induced in male C57/BL6 mice weighing 21–23 g using the intraluminal vascular occlusion method as previously described [
14]. Mice underwent MCAO for 1 h and then were reperfused. The mice were randomly assigned to sham-operated, vehicle, and SLDS groups with different doses. Regional cerebral blood flow was measured using laser Doppler flowmetry (PeriFlux System 5000, Perimed, Stockholm, Sweden). Rectal temperature was maintained at 37.0 °C during and after surgery via a temperature-regulated heating pad. SLDS (43866, Sigma, St. Louis, MO, USA) was dissolved in phosphate buffer saline (PBS) for use in animals. Two experimental procedures were initiated:
Experiment 1: To select the optimal dose, SLDS, at 2.5, 5, 10, and 20 mg/kg/day (or PBS) was administered daily via the caudal vein after cerebral ischemia. The first dose of SLDS was given immediately after reperfusion and mice were sacrificed 3 days after MCAO.
Experiment 2: To detect the role of SLDS in microglial polarization after stroke, SLDS was administered once a day for 5 days via the caudal vein. The first dose of SLDS was injected immediately after reperfusion.
Infarct volume and brain loss analysis
Infarct volume was determined using 2, 3, 5-triphenyltetrazolium chloride (TTC) as previously described [
15]. Hematoxylin and eosin (H & E) staining was performed to detect brain loss. The brain loss was measured by subtracting the nonlesioned area of the ipsilateral hemisphere from that of the contralateral hemisphere. The volume of tissue loss was calculated from the lesioned areas in six sections.
Neurological functional test
To evaluate neurological functional deficits, neurological severity scores were performed at 3 days after MCAO as previously described, by investigators who were blinded to the experimental group assignments [
16‐
18]. The modified neurological severity score is a composite of motor and sensory test. Motor tests were assessed by raising the animal by the tail (normal: 0; flexion of forelimb: 1; flexion of hindlimb: 1; head moved > 10° to vertical axis within 30 s: 1; maximum: 3) and placing the animal on the floor (normal: 0; inability to walk straight: 1; circling toward the paretic side: 2; falling to the paretic side: 3). Sensory tests included tactile response (normal: 0; slowed reaction: 1; no reaction: 2) and proprioceptive response (normal: 0; slowed reaction: 1; no reaction: 2). Tactile response was evaluated by touching the palmar area of forepaw with a sharp needle and proprioceptive response was assessed by pressing a cotton swab against the side of the neck. The overall neuroscore was determined by an investigator blinded to the treatment of the animals.
Rotarod test
A rotarod test was performed with the Rotamex 5 apparatus (Columbus Instruments, Columbus, OH, USA) as previously described [
19]. Briefly, mice were placed on an accelerating rotating rod at an accelerating speed (acceleration from 4 to 40 rpm within 5 min) until the mouse fell onto the platform below, or until the 5 min had elapsed. Each animal underwent three trials daily with an inter-trial interval of 20 min.
RT-PCR
Total RNA was extracted from microglia or brain tissues using Trizol (Qiagen, Hilden, Germany) according to the manufacturer’s protocol, after which RNA was reverse transcribed into cDNA using Superscript III First-Strand Synthesis SuperMix (Invitrogen, Carlsbad, CA, USA). The resulting cDNA was used for PCR using SYBR GREEN FAST mastermix (Qiagen) in triplicate. The expression of CD16 and CD206 were detected by RT-PCR using the primers: CD16, Forward: 5′-TCAAATCACTTTCTGCCTGCT-3′, Reverse: 5′-CTATTGCTCTCCTCATCCCAT-3′; CD206, Forward: 5′-AGTGATGGTTCTCCTGTTTCC-3′, Reverse: 5′-GGTGTAGGCTCGGGTAGTAGT3′. All other primers for RT-PCR were used as previously described [
14,
20‐
23]. Data collection was performed on the RT-PCR System (Bio-Rad, Hercules, CA, USA). GAPDH was used as an internal control. The relative quantitation value for each gene was performed using the comparative cycle threshold method [
24].
Immunofluorescence staining
Immunofluorescence staining was performed on free-floating sections (25 μm) for tissues or glass coverslips for cell cultures in 24-well plates. Primary microglia, neurons, and oligodendrocytes grown in 24-well plates were fixed with 4% paraformaldehyde. Slides or glass coverslips were washed in PBS and immersed in monkey serum (Jackson Immuno Research Laboratories Inc., West Grove, PA, USA) for 30 min. Primary antibodies included the following: rabbit anti-MAP2 (sc-20172, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rat anti-CD16/32 (553142, BD, Franklin Lakes, NJ, USA), goat anti-CD206 (AF2535, R & D Systems, Minneapolis, USA), rabbit anti-inducible nitric oxide synthase (iNOS, ab15323, Abcam, San Francisco, CA, USA), goat anti-Arg1 (sc-18351, Santa Cruz Biotechnology), rabbit anti-Iba1 (019-19741, Wako, Osaka, Japan), mouse anti-NG2 (MAB5384, Millipore, Billerica, MA, USA), and rabbit anti-MBP (ab40390, Abcam). The nuclei of cells were stained with DAPI (4′6-diamidino-2-phenylindole; Invitrogen) before taking images. Sections or cells were observed under a fluorescence microscope (Carl Zeiss, Jena, Germany) or confocal microscopy (Leica, Wetzlar, Germany).
Primary culture of microglia, oligodendrocytes, and neurons
Primary rat-enriched microglia were isolated from the whole brains of 1-day-old pups and cultured as previously described [
25]. Microglia were shaken off, collected, and reseeded 10 days after initial seeding. Microglia were incubated in DMEM/F12 (Gibco, Life Technologies, Gaithersburg, MD, USA) with 10% fetal bovine serum (Gibco, Life Technologies), and 100 U/ml penicillin/streptomycin (Life Technologies). After microglial collection, oligodendrocytes were shaken off overnight, collected, and incubated in basal chemically defined medium as previously described [
26]. NG2 and MBP double immunostaining was performed to identify the stages of oligodendrocyte maturity. All cells were maintained at 37 °C and 5% CO
2. The M1 phenotype was induced using a combination of LPS (L4391, 100 ng/ml, Sigma) and rat IFN-γ (20 ng/ml, Peprotech, Rocky Hill, NJ, USA) and the M2 phenotype was induced using a combination of rat IL-4 (20 ng/ml, Peprotech) and rat IL-13 (20 ng/ml, Peprotech). Microglia were collected 48 h after initial seeding for mRNA analysis.
Primary cortical neurons were isolated from the brains of E18 rat embryos and incubated in neurobasal medium (Life Technologies) supplemented with 2% B27 (Life Technologies), 2 mM glutamine (Life Technologies) and 100 U/ml penicillin/streptomycin (Life Technologies). The medium was changed every 3 days by replacing two thirds of the medium. Ten days after initial seeding, the purity of neurons was assessed by MAP2 immunostaining (requirement of ≥ 95% purity). At least three independent replicates were performed for all experiments.
Lactate dehydrogenase (LDH) assay
LDH release was measured using Pierce LDH cytotoxicity kit (Thermo Scientific, Pittsburgh, PA, USA) at 24 h after treatment. Absorbance was read at 450 μm using a Varioskan Flash Reader (Thermo Scientific, Waltham, MA, USA).
Phagocytosis assay
Microglia were plated in 24-well (3 × 10
5 cells/well) or 96-well (8 × 10
4 cells/well) D-lysine (Sigma) coated plates and incubated with different treatments for 48 h. Nile red fluorescent microspheres (Invitrogen) were then added to the cultures for 3 h at a concentration of 0.02% solids. Microsphere counts were performed at 3 h after the addition of the fluorescent microspheres as previously described [
14]. Cultures were stained with AlexaFluor488 phalloidin (Invitrogen) and absorbance was read using a Varioskan Flash Reader (Thermo Scientific).
Assay for pro-inflammatory factors in culture media
Supernatants were collected from microglia with the various treatments stated above at 48 h after treatment. Concentrations of IL-1β, IL-2, IL-6, IL-8, and TNFα were measured with a commercial enzyme-linked immunosorbent assay (ELISA) kit (Neobioscience, Shanghai, China) according to the manufacturer’s instructions. Absorbance was read at 450 μm using a Varioskan Flash Reader (Thermo Scientific).
Neuron-microglia cocultures
The following two coculture systems were employed: (1) a transwell contact-independent neuron-microglial system and (2) a direct-contact neuron-microglial system; both of which were performed as previously described [
14]. To assess neuronal survival in the transwell contact-independent system, neurons were seeded in six-well plates at a density of 8 × 10
5 per well. Ten days after initial seeding of neurons, activated microglia (4 × 10
5/well) were added into inserts directly above the neuronal cultures (both cultures shared the same medium). To assess neuronal survival in the direct-contact system, neurons cultured for 10 days in 96-well plates (8 × 10
4/well) were subjected to either normal conditions or oxygen glucose deprivation (OGD) for 1 h. Primary microglia (4 × 10
4/well) were then seeded and cultured together with neurons in the presence of different agent combinations in defined culture medium (minimum essential medium containing 10% fetal bovine serum and 10% horse serum). For immunostaining, neurons cultured for 10 days at a density of 2 × 10
5 per well in 24-well plates were directly cocultured with primary microglia (1 × 10
5/well).
Microglia-oligodendrocyte cocultures
Using the transwell coculture system, microglia (1 × 105/well) seeded in inserts were activated by different agent combinations for 48 h, after which the inserts were gently washed twice and added directly above oligodendrocytes (2 × 105/well) cultured in 24-well plates. After 3 days of coculture, microglial inserts were replaced by new 48 h-activated microglial inserts.
Quantitative MAP2 ELISA
Neurons cultured in six-well plates in the transwell system were collected at 48 h after treatment and lysed. A MAP2 ELISA kit was used (Cusabio, Wuhan, China) to detect the expression of MAP2 as previously described [
14].
Statistics
GraphPad Prism 7.03 software (GraphPad Software Inc., La Jolla, CA, USA) was used for statistical analyses. All data were presented as mean ± standard error of mean (SEM). The Shapiro-Wilk normality test was used to confirm the values derived from a Gaussian distribution. Statistical power was calculated using Gpower 3. Assumptions of equal variance were tested with Brown-Forsythe tests. Significant differences were assessed by Student’s t test for two-group comparison and one-way analysis of variance (ANOVA) followed by Tukey’s test or two-way ANOVA followed by Sidak’s test for multiple comparisons. Statistical significance was set at P < 0.05.
Discussion
SLDS is known to protect against stroke and other neurological diseases [
5,
9,
10,
27], the effect of SLDS on microglial polarization status after stroke has not previously been investigated. To our knowledge, this is the first study to describe a beneficial effect of SLDS on microglial polarization after stroke.
Cell quantification revealed that there was a delayed peak at 14 days for CD16
+/Iba1
+ M1 cells and at 7 days for CD206
+/Iba1
+ M2 cells after stroke in mice [
28]. Therefore, brain samples at 14 days after ischemia were chosen to define the role of SLDS in microglial polarization in the present work. Our data suggested that SLDS treatment caused a reduction in M1 macrophage/microglia polarization and an increase in M2 macrophage/microglia polarization in both the cortex and striatum of MCAO mice compared to controls. In addition, results from in vitro experiments were in agreement with the above results. Taken together, the findings of this study indicate a role for SLDS in driving M2 polarization. Consistent with our in vivo findings, a previous study reported that SLDS inhibited activation of BV2, a murine microglia cell line [
12]. In addition, another study indicated that M1 microglia exhibited reduced phagocytosis and produced pro-inflammatory cytokines, while M2 microglia increased phagocytosis and secreted anti-inflammatory mediators and neurotrophic factors [
14,
29]. As has been documented, inflammation is considered to be a vital determinant of outcome following cerebral ischemia injury, which depends partly upon pro-inflammatory factors [
29,
30]. Classically, three pro-inflammatory cytokines, IL-1β, IL-6, and TNFα are associated with the inflammatory response following ischemic stroke [
31]. Previous studies indicated that SLDS exhibited anti-inflammatory activities in stroke and other diseases [
11,
32,
33]. Here, we confirmed that SLDS inhibits secretion of the pro-inflammatory cytokines in M1 microglia. In addition, M1 microglia treated with SLDS showed increased phagocytosis, similar to levels found in both M0 and M2 microglia, and thus may facilitate brain recovery after stroke.
A previous study showed that SLDS modulated NF-κB and MAPK signaling in LPS-induced BV2 microglial cells [
12]. The current results, combined with the fact there are many similarities in microglial differentiation and polarization between humans and rodents [
34], point to a potential role for SLDS in the treatment of human patients following stroke, although further clinical research would be needed to confirm SLDS as a treatment option for stroke patients.
Oligodendrocytes and neurons are highly susceptible to ischemic injury, and damage to these cells leads to myelin loss, axonal injury, and neuronal death. Substantial evidence shows that M2 macrophage/microglia drive oligodendrocyte differentiation during central nervous system remyelination, which may promote neurologic recovery [
35]. It has been shown that SLDS attenuates arthritis-induced and beta amyloid-induced cognitive deficits [
7,
36]. Indeed, our data showed that M1 microglia treated with SLDS promoted oligodendrocyte differentiation via a shift from the M1 to the M2 phenotype, which suggest that SLDS may promote remyelination following neurologic diseases. Previous studies showed that SLDS protected neurons by inhibiting autophagy [
37], apoptosis [
38] and oxidative stress [
39]; which is in agreement with our results whereby SLDS treatment improved the survival of OGD-conditioned neurons cultured with or without microglia.
Our study has several limitations. First, the effects of SLDS on oligodendrocyte differentiation via microglial polarization were limited to normal conditions, rather than in OGD conditions. Moreover, it was not determined whether SLDS-mediated microglial polarization reduced ischemia-induced loss of oligodendrocytes. Second, we did not confirm whether SLDS promoted white matter integrity and long-term functional recovery of white matter after MCAO. Third, the underlying protective mechanism influencing the regulation of microglial polarization induced by SLDS was not explored. The mechanisms of microglial polarization have been investigated extensively [
2], but there are no studies defining the role of SLDS on M1/M2 polarization, and its role remains controversial [
40]. Further investigation is needed in order to elucidate all of the aforementioned points.
Acknowledgements
The authors thank Dr. Xiaoming Hu and Dr. Jun Chen (Pittsburgh University Medical Center) for their informative advice.