Animals and treatment
Male C57BL/6 mice (aged 10–12 weeks, weighing 23–25 g) were purchased from the Animal Core Facility of Nanjing Medical University. All mice were housed in five per cage groups in a standardized light-dark cycle at 22 °C and given free access to food and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University. All procedures were designed to minimize suffering and reduce the number of animals used.
MCAO/R model
MCAO/R mice were established as previously described [17]. For the transient model, reperfusion was produced by withdrawal of the 6-0 nylon filament (Doccol Corp., Readlands, CA, USA) 60 min after the occlusion. In the sham group, arteries were visualized but not ligated. After the surgery, body temperature was controlled with a heating pad and kept at 37 ° C until mice wholly recovered from anesthesia. The mice were then returned to their home cages.
Grouping and treatment
After 24 h of reperfusion, the model mice were subjected to neurological function evaluation based on the neurological severity scores (NSS) system. Mice with NSS scores of 4 to 10 were randomly divided into 4 groups and received intraperitoneal injections with saline, minocycline 10 mg/kg, 20 mg/kg, or 50 mg/kg. They were then sacrificed on days 3, 7, or 14 after reperfusion according to different experimental purposes. Minocycline (Sigma-Aldrich, St. Louis, MO, USA) was freshly diluted to the concentrations needed with saline.
Neurobehavioral function evaluation
Using the revised NSS system [20], the neurobehavioral evaluation of mice was examined by two trained observers who were blinded to the drug treatment group. According to the NSS presented in Table 1, mice's neurological status was examined on day 1 and day 14 following MCAO/R (Fig. 1a). The 1-day assessment was used to verify the ischemic state of MCAO/R groups. The 14-day assessment was used to evaluate motor and behavioral impairments in recovery after induction of MCAO/R.
Rotarod test
We carried out the rotarod test to calculate mice's motor ability as previously described [21]. All mice were acclimatized to the rotarod by undergoing a 3-day training program before MCAO/R surgery experiments. Only mice that can remain on the rotarod for 180 s were used for the experiments. On day 14 following MCAO/R, mice performed three trials on the rotarod, with an interval of 5 min. The mean of the three trials was used to show the functional recovery of mice.
Corner turning test
The corner test was performed as previously described [22]. Briefly, a mouse was left in the test device consisting of two vertical boards at a 30° angle. When the mouse entered the corner, the direction in which it contacted the board with the vibrissae was recorded. The non-ischemic mouse turned left or right with equal frequency, but the mouse suffered from MCAO/R preferentially turned toward the impaired (right side in our experiment). The number of turns in each direction was summed from 10 trials. The following formula was used to calculate right-biased turning percentage: right-biased turning percentage = right-biased turning / total turning × 100%.
TTC assay
As previously described [21], the cerebral infarct volume of nine mice in each group was measured with 1% TTC (dissolved in saline) on day 7 following MCAO/R. The infarcted regions were acquired by a digital camera and quantified using ImageJ software (NIH, Bethesda, MD, USA). The following formula was used to calculate infarct percentage: Infarct percentage = infarct volume/volume of the contralateral hemisphere × 100%.
Immunohistochemistry and cell counting
On day 3 or day 7 following MCAO/R, six mice were anesthetized with pentobarbital sodium (80 mg/kg) and then perfused with saline and 4% paraformaldehyde as previously described [21]. Their brains were fixed overnight in 4% paraformaldehyde at 4 °C, followed by dehydration in 30% w/v sucrose at 4 °C. The frozen coronal sections of 30 μm were cut with a vibrating microtome (Leica CM1950, Nussloch, Germany).
For immunohistochemistry, the sections were incubated with primary antibody against NeuN (1:1000, #MAB377; Millipore, Billerica, MA, USA), GFAP (1:1000, #ab53554; Cambridge, MA, USA), and Iba-1 (1:500, #019-19741; Wako, Osaka, Japan) at 4 °C overnight. After incubated with corresponding secondary antibodies for one hour at room temperature, the sections were incubated with 3,3′-diaminobenzidine (DAB) staining (Beyotime, Shanghai, China). The total numbers of NeuN+, GFAP+, and Iba-1+ cells were estimated with an optical dissector (Stereo Investigator, MicroBright Field. Inc., Williston, VT, USA) as described previously [23].
For immunofluorescence, the sections were incubated with primary antibody against Iba-1, CD16/32 (1:50, #553142BD; Biosciences Pharmingen, San Jose, CA, USA), and CD206 (1:50, #AF2535; R&D Systems, Minneapolis, MN, USA). After incubation with corresponding Alexa Fluor secondary antibodies, the sections were mounted with ProLong Gold antifade reagent (#P36930, Life Technologies, Carlsbad, CA, USA). Quantitative assessment of co-localization between Iba-1 and CD16/32 or CD206 was performed by calculating the positive cells in every 6th section containing ten preassigned fields in the penumbra using a fluorescence microscope (AX10, Carl Zeiss, Thornwood, NY, USA).
Quantitative real-time polymerase chain reaction
Quantitative real-time polymerase chain reaction (qRT-PCR) was performed as previously described [24]. Briefly, total RNA from penumbral tissue or culture cells was purified using the Trizol extraction method. Real-time qPCR of cDNA was carried out using SYBR Green detection on the ABI 9600 (Applied Biosystems, Foster City, CA, USA). The qRT-PCR primers for IL-1β (#MQP092978), IL-6 (#MQP088375), iNOS (#MQP088374), TNF-α (#MQP096302), Arg1 (#MQP026580), IL-10 (#MQP092952), TGF-β (#MQP088616), Ym1 (#MQP091480), and GAPDH (#MQP027158) were purchased from GeneCopoeia, Inc. (Rockville, MD, USA).
Enzyme-linked immunosorbent assay
The protein levels of IL-1β (#CSB-E08054m, Cusabio Biotech Co., Wuhan, Hubei, China), TNF-α (#CSB-E04741m, Cusabio Biotech Co., Wuhan, Hubei, China), IL-10 (#CSB-E04594m, Cusabio Biotech Co., Wuhan, Hubei, China), and TGF-β (#CSB-E04726m, Cusabio Biotech Co., Wuhan, Hubei, China) from tissues or culture cells were quantitated by a specific enzyme-linked immunosorbent assay (ELISA) kit following the manufacturer's instructions.
Cell culture and treatment
Primary microglia from cerebral cortex of newborn mice were cultured and isolated as previously described [25]. At days in vitro (DIV) 10, microglia cells were seeded on 6-well plates and incubated with 100 ng/mL LPS (Sigma-Aldrich, St. Louis, MO, USA) plus 20 ng/mL IFN-γ (PeproTech, Suzhou, China) followed by 50 μM minocycline or saline for 24 h.
Western blot analysis
The cells were lysed in lysis buffer (50 mM Tris‐HCl, 150 mM NaCl, 1% Nonidet P-40, 1 % sodium orthovanadate, 5 mM EDTA, pH 7.4,) containing protease inhibitors and phosphatase inhibitors (Roche Applied Science, Indianapolis, IN, USA). The proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes with the electrophoretic transfer system (Bio-Rad Laboratories, Hercules, CA, USA). Nonspecific blots were blocked with 5 % skimmed milk in Tris-buffered saline with 0.1 % Tween 20 (TBST) for 1 h. The membranes were then incubated with the following primary antibodies: iNOS (1:100, #ab178945; Abcam Inc., Cambridge, UK), p-P65 (1:1000, #3033; Cell Signaling Technology Inc., Danvers, MA, USA), P65 (1:2000, #8242; Cell Signaling Technology Inc., Danvers, MA, USA), p-STAT1 (1:1000, #7649; Cell Signaling Technology Inc., Danvers, MA, USA), STAT1 (1:2000, #14995; Cell Signaling Technology Inc., Danvers, MA, USA), Arg1 (1:1000, #ab124917; Abcam Inc., Cambridge, UK), p-STAT6 (1:2000, #9364; Cell Signaling Technology Inc., Danvers, MA, USA), STAT6 (1:2000, #5397; Cell Signaling Technology Inc., Danvers, MA, USA), or β-actin (1:10000, #YFMA0052; Yifeixue Biotech, Nanjing, China) overnight at 4°C. The membranes were washed and incubated with corresponding secondary antibodies for 1 h at room temperature. Finally, the membrane was observed and analyzed with Tanon 5200 (Tanon Science and Technology Co. Ltd, Shanghai, China).
Neuron‐microglia co‐culture
Primary microglia and cortical neurons were co-cultured in a two-layer system (Transwell, Corning, Corning, NY, USA). Briefly, microglia on Transwell inserts were treated with 100 ng/mL LPS plus 20 ng/mL IFN-γ followed by 50 μM minocycline or saline for 24 h. Then, the medium was removed and washed with fresh medium three times. Primary cortical neurons were cultured and subjected to oxygen-glucose deprivation/reperfusion (OGD/R) for 1 h, as previously described [24]. When the medium was replaced with a normal medium, the microglia inserts were added on top of neuronal cultures to generate a neuron‐microglia co‐cultures. After 24 h of co-culture, neuronal cell death and survival were measured by thiazolyl blue tetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) assay and lactate dehydrogenase (LDH) assays (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocol.
Statistical analysis
All data were expressed as mean ± S.E.M. Statistical analysis was performed using GraphPad Prism software (San Diego, CA, USA). A log-rank test was used to compare the survival rate of the MCAO/R model. A non-parametric Kruskal-Wallis H-test was used to compare the NSS scores of the MCAO/R model. Other parametric data were analyzed via one-way analysis of variance (ANOVA) followed by Tukey's test or a two-sample t-test. P < 0.05 was considered statistically significant.