All procedures were approved by the Medicine Ethics Review Committee for animal experiments of the China Academy of Chinese Medical Sciences, and all efforts were made to minimize suffering of rats. Sprague-Dawley rats (specific pathogen-free grade, certificate No. 2010-0034), weighing 300 ± 20 g, 8 weeks old, were used for the study, which were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Male rats were used in the initial study because estrogen was known to protect against ischemic injury35,36,37. However, future studies with female rats will have to be conducted to assess potential sex-dependent effects on the inflammatory response after MCAO. The animal experiment was carried out in Clean Grade Animal Center of Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. The rats were housed in a controlled environment (21 ± 1 °C temperature, 55 ± 10% relative humidity) with a 12/12-h light/dark cycle and free access to water and standard diet. The sample size was calculated by a power analysis and previous inflammation studies of the MCAO model38,39,40. The rats were allowed to acclimate for 7 days before the experiment. Sixty rats were randomly divided into six groups with ten in each one, that was Sham group, Sham+40 μg LPS group, Sham+80 μg LPS group, MCAO group, MCAO+40 μg LPS group, and MCAO+80 μg LPS group.
Transient focal cerebral ischemia
Transient focal cerebral ischemia was induced using the intraluminal filament model of MCAO, which was firstly described by Koizumi12 and Longa13 in 1980s. Briefly, rats were anesthetized with 1.5-2.0 % isoflurane (Beijing zhongshidichuang Technology Development Co., Ltd., Beijing, China) using respiratory anesthesia machine (ZS-MV, Beijing zhongshidichuang Technology Development Co., Ltd., Beijing, China), and fixed on homeothermic electric blanket (37 ± 0.5 °C) throughout surgery until coming around. Nylon filament (tip diameter 0.38 ± 0.02 mm, polylysine coated) was inserted into the right external carotid artery (ECA), and advanced through the internal carotid artery (ICA) until it obstructed the MCA. Reperfusion was performed after 90 min occlusion in anesthetic state. Sham surgery was performed exactly the same as above, but the filament was immediately withdrawn after reaching the origin of the MCA.
Following MCAO, rats were placed in temperature-controlled (37 ± 1.0 °C) recovery cages for 2 h to prevent post-surgery hypothermia. The order in which rats from different groups were subjected to MCAO was randomized.
Neurological function assessment
Rats were evaluated for neurologic deficits 24 h after reperfusion (ischemia 90 min reperfusion for 24 h, I/R 90min/24h) according to Longa 5 scores by a fixed investigator who was blind to the groups. The scoring criteria are as follows:
0=no deficit; 1=failure to fully extend left forepaw, mild neurological deficit; 2=circling to the left, moderate neurological deficit; 3= falling to the left, severe neurological deficit; 4= unable to walk spontaneously, conscious loss. This method is suitable for early stage of MCAO, within 7 days after surgery. The rats with 0 value in MCAO or MCAO+LPS group were eliminated and euthanized with intraperitoneal injection of 3 % pentobarbital sodium salt (Sigma, USA) at 0.5 ml/100g.
Measurement of infarct volume and edema degree
Rats were anaesthetized with I.P. of 1 % pentobarbital sodium salt. Brains were frozen on dry ice and serially sectioned into six coronal slices (2 mm) with brain mold. The brain slices were stained with 2 % triphenyl tetrazolium chloride (TTC) at 37 °C for 15 min in the dark and fixed with 4 % paraformaldehyde overnight. The infarct volume corrected for swelling, and edema degree were quantified using Image ProPlus Software by a fixed investigator who was blind to the groups, using the following formula41 ,42 , 43 : Infarct rate%= CoV-IpV 100%; Edema rate%= IpV+InV-CoV 100%; (CoV:2CoV 2CoV contralateral hemisphere volume; IpV: ipsilateral no infarct volume; InV: infarct volume) as shown in Fig.2.
Real-time cerebral blood perfusion
Cerebral blood perfusion (CBP) was dynamically and instantly monitored using Pericam Perfusion Speckle Image (PeriCam PSI) system by a fixed investigator who was blind to the groups, which could display the image and blood flow curve at the same time44,45. The rats lied prone on an homeothermic electric blanket (37 ± 0.5 °C) under anesthesia. Head median incision was made to expose the entire parietal bone and scrape the skull with a scalpel. The laser spot is located 2 mm behind the anterior fontanelle and 6 mm beside the middle line. Keep the skull moist with 37 °C physiological saline throughout monitoring. Region of interest (ROI) 1 delineated the healthy side, ROI 2 infarcted side and ROI 3 the whole brain, corresponding to blue line, red line and green line respectively in the blood flow curve. Time of interest (TOI) delineated the relatively stable recording range to calculate the corresponding CBP, TOI 1 delineated the blood flow curve before surgery, TOI 2 during surgery and TOI 3 after surgery. CBP of two hemispheres and the whole brain was recorded before, during and after surgery at least 3 min each time. Reduced rate of CBP %= T1IS-T2IS 100% (T1IS:T1IS TOI 1 of infarcted side; T2IS: TOI 2 of infarcted side).
Systemic inflammatory challenge with Lipopolysaccharide (LPS)
Lipopolysaccharide (LPS, serotype: 055: B5, Sigma L2880) was administered intraperitoneally at doses of 40 μg/ 300g rat (134 μg/kg) or 80 μg/ 300g rat (268 μg/kg) immediately after MCAO surgery14,15,16. No rats died or needed to be terminated due to LPS injection.
Measurement of IL-6, TNF-a, IL-1b in plasma and brain homogenates by ELISA
5 mL blood was taken from inferior vena cava after rats subjected to I/R 90min/3h, then centrifuged at 3500rpm, 4 °C for 10 min, and the plasma was stored in -80 °C refrigerator for later use. The rat was decapitated and the infarcted hemisphere was rapidly freezed with liquid nitrogen and stored in -80 °C refrigerator for later use. After balance to room temperature, the infarcted hemisphere was grinded with high throughput tissue lapping instrument (CK1000D, Thmorganh). 500 μL PMSF: RIPA (1:100) lysis buffer and 1 μL protease inhibitor was added to 100 mg rats brain homogenates. The mixture was re-grinded to thoroughly blended, and then centrifuged at 14000rpm, 4 °C for 10 min, and the supernatant was used for determination of protein concentration by BCA Protein Assays kit (Thermo Fisher Scientific, USA) according to the manufactures protocol by a fixed investigator who was blind to the groups. Interleukin 6 (IL-6), Tumor necrosis factor a (TNF-a), Interleukin 1b (IL-1b) in plasma and brain homogenates was measured by ELISA kit (Invitrogen, Carlsbad, CA, USA) according to the manufactures protocol.
RT2 ProfilerTM PCR array
Sixteen rats were divided into four groups with four in each one, that was Sham group, Sham+80 μg LPS group, MCAO group, and MCAO+80 μg LPS group. The rat subjected to I/R 90min/3h was decapitated, then the infarcted hemisphere was rapidly washed with RNase-free water and loaded into RNase-free EP tubes, and freezed with liquid nitrogen. Investigator was required to operate the whole process quickly to avoid RNA enzyme contamination. We employed the Toll-Like Receptor Signaling Pathway PCR Array (QIAGEN, German, PARN-018Z) to detect 84 genes known to be involved in the pathway. RNA isolation, DNase treatment, and RNA clean-up were performed according to the manufacturer’s protocol (Qiagen, Hilden, Germany). The isolated RNA was reverse transcribed into cDNA using the RT2 First Strand Kit (Invitrogen, Carlsbad, CA, USA). PCR was performed using the RT2 SYBR Green qPCR Master Mix (Invitrogen, Carlsbad, CA, USA) on an ABI PRISM7700 instrument (Applied Biosystems, Foster City, CA). Data normalization (ΔCt) was based on correcting all Ct values for the average C’t values of several stable expressed housekeeping genes present on the array containing the gene-specific primer sets. [ΔCt1 (group 1) = average Ct – average of HK genes’ C’t for group 1 array; ΔCt2 (group 2) = average Ct – average of HK genes’ C’t for group 2 array]. The fold change between two groups was expressed as 2^-(ΔCt1-ΔCt2), that is 2^-ΔΔCt46. All the procedures were conducted by a fixed investigator who was blind to the groups. Every group had four biological repeats.
Quantitative real-time polymerase chain reaction (qRT-PCR) analysis
The mRNA level of CXCL10 (IP-10) in the brain tissue was quantitated by real-time PCR. Total RNA was extracted manually from brain tissue using TRIZOL (Invitrogen, Carlsbad, CA, USA), then RNA was reverse-transcribed to cDNA using SuperScript. III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). The primers for CXCL10 designed by software Primer 5.0 were as follows: 5' AGCCAACCTTCCAGAAGCACCA 3' (sense) and 5’ TCATGGAAGTCGATGCAGGTGC3’ (antisense); for GAPDH used as internal control were as follows: 5’ GCTCTCTGCTCCTCCCTGTTCTA3' (sense) and 5’ TGGTAACCAGGCGTCCGATA3’ (antisense). The cycling programs were as follows: 95 °C for 10 min for 1 cycle, then 95 °C for 10 s, 60 °C for 60 s, and 95 °C for 15 s for 40 cycles. The quantitative real-time PCR was performed using ViiA 7 Real-time PCR System (Applied Biosystems, Carlsbad, CA, USA) with 2X PCR master mix (Arraystar, USA) according to the manufacturer’s protocol. The gene CXCL10 concentration of each sample is corrected by its housekeeping gene GAPDH. Relative quantification was processed by the standard curve method. All the procedures were conducted by a fixed investigator who was blind to the groups.
Measurement of CXCL10 production in brain homogenates and plasma by ELISA
The production of CXCL10 in brain homogenates and plasma was measured by ELISA kit (Cusabio biotech co., Ltd, WuHan, China) according to the manufactures protocol by a fixed investigator who was blind to the groups.
GO enrichment analysis
To explore the gene function of the different genes, we employ the GO analysis for functional annotation. The 27 different genes were imported into DAVID Bioinformatics Resources 6.7 (https://david-d.ncifcrf.gov/), which supply a high-throughput and integrated data-mining environment. The results were downloaded from the internet.
PPI network construction
To explore the protein-protein interaction (PPI) correlation, the different expression genes were mapped to the Search Tool for the Retrieval of Interacting Genes (STRING, https://string-db.org/) database. In the process of analysis, species were limited to "Rattus norvegicus" and the minimum interaction threshold was set to "medium confidence" 0.4. The other parameters were set by default, and the target with weak correlation was removed. Based on the above results of PPI analysis, Cytoscape v3.6.1 software was employed to describe the interaction relationship. The Network Analyzer was used to analyze the topological properties, and the targets with degree ³ 2-fold Median were selected to construct the PPI network graph.
Data were analyzed using Student’s t test for single comparisons and one-way ANOVA followed by Student’s t test with Bonferroni’s correction or Dunnett’s test for multiple comparisons. The criterion for statistical significance was p<0.05. Data were expressed as mean ± standard error of the mean (SEM).