Animals
C57BL6 mice were purchased from the Laboratory Animal Center of Southern Medical University and were maintained under standard laboratory conditions, with the temperature controlled at 24°C and with free access to a standard diet and sterile water. All animal procedures were performed in accordance with the guidelines and approval of the Animal Ethics Committee of Southern Medical University.
Middle cerebral artery occlusion (MCAO) and post-stroke infection model
Mice weighing 20–22 g (aged 7–8 weeks) were allowed free access to water but were fasted for 12 h to standardize glycaemic state. MCAO was performed under anaesthesia induced by intraperitoneal injection of pentobarbital (100 mg/kg). Body temperature was maintained at 37°C ± 0.5°C using a heating pad (RWD Life Science, Shenzhen, China). To induce MCAO, a 6 − 0 nylon suture (Covidien, Mansfield, MA, USA) with a round tip and silicon coating was inserted from the left external carotid artery into the middle cerebral artery. The success of the surgery was verified by monitoring surface cerebral blood flow using a laser Doppler flowmeter (Moor Instruments, Devon, UK). After 1 h, the occluding filament was gently withdrawn back into the common carotid artery to allow reperfusion. Mice in the sham group underwent a sham operation without suture insertion.
E. coli were cultured as previously described[18] and stored in 30% glycerol at − 80°C until use. E. coli were grown in Luria-Bertani (LB) medium (10 g/l tryptone, 5 g/l yeast extract, and 171.1 mM NaCl). Growth was determined by measuring the optical density at 620 nm (OD620) or by plating the cells on LB plates and counting viable cells. For infection, age-matched male mice were intravenously injected with 107 colony forming units (CFU) of E. coli resuspended in 500 µl phosphate-buffered saline (PBS) immediately after sham or MCAO operation.
To determine the degree of infection, the mouse liver, lung, and brain were removed and homogenized in distilled water with 0.01% Triton X-100. The number of viable E. coli cells was counted after plating serial dilutions of organ homogenates and blood on LB plates and culturing overnight at 37°C.
Assessment of neurological function and measurement of cerebral infarct area
Neurological function was determined based on the Modified Neurological Severity Score (mNSS). The test was carried out by a blinded investigator before and 3 days after MCAO, as previously described[19]. The infarct areas of different experimental groups were measured in photomicrographs of methylthioninium chloride-stained tissue sections (5 sections/animal). Experiments were repeated five times.
Hematoxylin and eosin (HE) staining, immunohistochemistry, immunofluorescence analysis and flow cytometry analysis
At 24 h after MCAO or post-stroke infection, mice were anesthetized and transcardially perfused with 20 ml cold PBS and 20 ml of 4% paraformaldehyde in 0.1 M PBS. The brain, lung, liver, and spleen were removed, post-fixed, and embedded in paraffin. The tissue blocks were cut into 5-mm sections that were deparaffinized and stained with HE according to standard protocols.
CD8+ T cells and NK cells in the brain and spleen were identified by immunofluorescence analysis and immunohistochemistry, as previously described. For the latter, brain and spleen tissue sections were incubated overnight at 4°C with primary antibodies against CD8 (ab25117) and natural cytotoxicity receptor (NCR) (ab199128),Iba1(ab5076),CD68(ab125212)(Abcam,Cambridge,MA,USA) respectively followed by processing with avidin-biotin-peroxidase (BosterBio, Wuhan, China). The sections were stained with diaminobenzidine, and nuclei were counterstained with hematoxylin.
For immunofluorescence, the specimens were first treated with anti-CD8 or -NCR antibody, followed by Alexa Fluor 594-conjugated secondary antibody (A0453; Beyotime Institute of Biotechnology, Shanghai, China). Immunofluorescence images were acquired with a confocal laser scanning microscope (TCS SP2; Leica Microsystems, Wetzlar, Germany).
Blood biochemical analysis
Mouse blood was collected via the angular vein under anaesthesia into an anticoagulant-containing tube. Biochemical analyses were performed at Southern Medical University Huayin Laboratory.
Enzyme-linked immunosorbent assay (ELISA)
Plasma was isolated by centrifugation of blood samples at 1500 rpm for 20 min. TNF-α, IL-6, IL-10 in the plasma were detected with ELISA kits (Cusabio, Wuhan, China) according to the manufacturer’s instructions. Briefly, 100 µl of plasma was added to each well of a 96-well plate. After incubation for 2 h at 37°C, the plasma was removed, and the plates were sequentially incubated with biotin-conjugated primary antibody followed by horseradish peroxidase-conjugated secondary antibody for 1 h at 37°C, with three washes between each step. After adding the chromogenic substrate, the plates were incubated in the dark for 30 min at 37°C. The reaction was terminated, and the OD450 was measured using an iMark microplate reader (Bio-Rad, Hercules, CA, USA).
Co-culture of bacteria and ucMSC
Platelets alone or in combination with ucMSC were inoculated with E. coli for 1, 2, 4, or 6 h. Bacterial growth was determined by measuring the OD620.
Statistical analysis
Statistical analysis was performed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). Data are presented as the mean ± SD. The significance of differences between means was examined by Student's t-test or one-way analysis of variance. Results with P < 0.05 were considered significant.