Male Sprague–Dawley rats were housed under diurnal lighting conditions and were allowed access to food and tap water ad libitum. All animal studies were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (NIH, USA 2013) and performed in accordance with ARRIVE guidelines (http://www.nc3rs.org/ARRIVE). The animal protocol used in this study was reviewed and approved by the INHA University-Institutional Animal Care and Use Committee (INHA-IACUC) with respect to ethicality (Approval Number INHA-141124-337). MCAO was carried out as previously described19. In brief, male Sprague–Dawley rats (250–300 g) were anesthetized with 5% isoflurane in 30% oxygen/70% nitrous oxide and maintained during surgery using 0.5% isoflurane in the same gas mixture. Occlusion of the right middle carotid artery was induced for 1 h by advancing a nylon suture (4-0; AILEE, Busan, South Korea) with a heat-induced bulb at its tip (0.3 mm in diameter) along the internal carotid artery for 20–22 mm from the bifurcation of the external carotid artery. Reperfusion was allowed for up to 2 days. A thermoregulated heating pad and heating lamp were used to maintain a rectal temperature of 37 ± 0.5 °C during surgery. Animals were randomly allocated to sham (n = 12), MCAO (n = 38), LPS-treated (n = 10), MCAO+LPS-treated (24 h post-MCAO) (n = 28), MCAO+LPS-treated (24/28 h post-MCAO) (n = 4) groups, MCAO+LPS+A box-treated (n = 11), MCAO+LPS+HPep1-treated (n = 14), MCAO+A box-treated groups (n = 3), MCAO+LPS-RS-treated (n = 3), and MCAO+LPS+LPS-RS-treated groups (n = 8). Animals allocated to the sham group underwent an identical procedure but the MCA was not occluded. In general, mortality was not observed during surgery, but mortality after surgery was 6.9% (9/130).
LPS (from Escherichia coli (E. coli), Salmonella enterica (SE), Salmonella typhosa (ST)) (100 μg/kg; Sigma Aldrich, St. Louis, MO, USA; L6529, L7770, L6143) was administered i.p. at 24 or 28 h post-MCAO. Similarly, LPS-RS (LPS from Rhodobacter sphaeroides; TLR4 antagonist) (Invivogen, San Diego, CA, USA; tlrl-rslps) was administered intraperitoneally at 24 h post-MCAO. Solution concentrations were adjusted to allow an injected volume of 0.3 ml in PBS. For the preincubation experiment, LPS (100 μg/kg) was preincubated with 5 μg/kg of HMGB1 A box (HMGbiotech, Milano, Italy; HM-012) and then administered intraperitoneally.
Intranasal administrations were carried out as previously described by Kim et al49. Briefly, at 21 h post-MCAO, rats were anesthetized with an intramuscular injection of a mixture of ketamine (3.75 mg/100 g body weight) and xylazine hydrochloride (0.5 mg/100 g body weight). A nose drop containing 1 or 5 μg/kg of HMGB1 A box or HPep1 in PBS (20 μl) was carefully placed in each nostril of anesthetized animals (supine 90° angle) using a sterile yellow tip. The procedure was repeated until the entire dosage had been administered with 2-min intervals between applications.
Coronally sectioned (2 mm) brain slices were immediately stained with 2% 2,3,5-triphenyl tetrazolium chloride (TTC) (37 °C for 15 min) and fixed in 4% paraformaldehyde. Infarcted tissue areas were measured using the Scion Image program (Scion Corporation, Frederick, MD, USA). To correct for brain edema following ischemia, measured infarct areas were adjusted with respect to areas in contralateral hemispheres. Infarct volumes were calculated (in mm3) by summing infarct sizes on adjacent tissue sections.
Neurological deficits were evaluated using mNSS at 2 days post-MCAO. The mNSS system is based on the results of four tests, namely, motor, sensory, balance, and reflex tests, all of which are graded using a 0 to 18 scale (normal: 0; maximal deficit: 18)50. Motor scores were determined by: (1) suspending a rat by its tail and awarding a score of zero or one for each of the following (total score 0–3); forelimb flexion, hindlimb flexion, head movement by >10° with respect to the vertical axis within 30 s; and by (2) placing a rat on the floor and awarding scores from 0 to 3 for each of the following: normal walking, 0; inability to walk straight, 1; circling toward the paretic side, 2; or falling on the paretic side, 3. Sensory tests included a placing test (score 0–1) and a proprioceptive test (score 0–1). The beam balance test was used to test balance and scores from 0 to 6 were allocated as follows: balancing with a steady posture, 0; grasping the side of the beam, 1; hugging the beam with one limb off the beam, 2; hugging the beam with two limbs off the beam or rotating around the beam for over 60 s, 3; attempting to balance on the beam but falling off within 20 to 40 s, 4; attempting to balance on the beam but falling off within 20 s, 5; or making no attempt to balance or hang onto the beam, 6. Reflex test scores were determined by awarding scores to the following four items (maximum possible score of 4): pinna reflex, 0–1; corneal reflex, 0–1; startle reflex, 0–1; seizures, myoclonus or myodystony, 0–1.
At 24 h before MCAO, rats were conditioned on a rotarod unit at a constant 3 rpm until they were able to remain on the rotating spindle for 180 s. One day after MCAO, each rat was subjected to a test trial on the rotarod at 10 rpm, and residence times on the rotarod were measured at 1, 2, 4, 6, and 10 days post-MCAO.
At 24 h before MCAO, rats were conditioned to cross the horizontal ladder. At 1, 2, 4, 6, and 10 days post-MCAO, each rat was placed on the grid and the number of foot-fault errors were monitored and recorded until the rats crossed the horizontal ladder.
For sampling serum, rats were placed in a supine position, a needle was inserted into the heart, and blood samples were collected. Samples were centrifuged at 3000 rpm for 15 min and stored at −80 °C. For CSF sampling, rats were placed on a stereotaxic apparatus. The skin was incised, and a 27 G needle was inserted into the cisterna magna. When the tip of the needle was inserted 1–1.5 mm, reflux of the CSF was observed. Approximately 100 μl of CSF was withdrawn.
RNA preparation and real-time PCR were performed as described previously19. Total RNA was purified using TRI reagent (Sigma, St. Louis, MO, USA; T9424), according to the manufacturer’s instructions. First-strand cDNA was synthesized using a Takara RNA PCR Kit (Doctor Protein, Seoul, Korea; DR01612) in a total volume of 20 μl containing 1 μg of total RNA. Real-time PCR was performed in a final volume of 20 μl containing 10 μl of 2× SYBR Green supermix (Takara Bio, Otsu, Japan; RR420), forward and reverse primers (1 μl each of 5 pmol/μl of both), and 5 μl of cDNA (50 ng; 1/100 dilution) using a Mini-Opticon Real-Time PCR System Detector (Bio-Rad, Richmond, CA, USA). PCR was performed as follows: 5 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, 30 s at 57 °C, and 30 s at 72 °C. Specificity of amplification was determined by DNA melting-curve analysis using built-in software. Differences in amplification fold were calculated by real-time PCR amplification of the target gene using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal standard using the built-in Gene Expression Analysis software in the iCycler iQ Real-Time RCR Detection System (Bio-Rad, Richmond, CA, USA). The following primer sets were used: 5′-CACCACGCTCTTCTGTCTACTG-3′ (forward) and 5′-GTACTTGGGCAGATTGACCTC-3′ (reverse) for TNF-α; 5′-GGAGAAGCTGTGGCAGCTA -3′ (forward) and 5′-GCTGATGTACCAGTTGGGGA-3′ (reverse) for IL-1β; 5′-GCTGTACAAGCAGTGGCAAA-3′ (forward) and 5′-GTCTGGAGTGGGAGGCACT-3′ (reverse) for cyclooxygenase-2; 5′-TCATTGACCTCAACTACATGGT-3′ (forward) and 5′-CTAAGCAGTTGGTGGTGCAG-3′ (reverse) for GAPDH; and 5′-GCATCCCAAGTACGAGTGGT-3′ (forward) and 5′-CCATGATGGTCACATTCTGC-3′ (reverse) for iNOS. Real-time PCR was performed in quadruplicate.
Immunoblotting and co-immunoprecipitation studies were carried out as previously described19. Briefly, blood serum samples (30 μg) were prepared from animals in seven groups mentioned in a previous section (Sham, LPS, MCAO, MCAO+LPS, and MCAO+LPS/A box, MCAO+LPS/HPep1, MCAO+LPS+LPS-RS) and separated in 10% sodium dodecyl sulfate-polyacrylamide gels. After blocking membranes with 5% non-fat milk for 1 h, they were incubated with primary antibodies for anti-HMGB1 (1:2000; Abcam, Cambridge, UK; ab67281), anti-albumin (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA; SC-374670), and anti-α-tubulin (1:5000; Cell Signaling, Danvers, MA, USA; #2144) overnight at 4 °C. The next day, blots were detected using anti-rabbit or anti-mouse horse radish peroxidase-conjugated secondary antibody (1:2000; Santa Cruz Biotechnology; SC-2004 or SC-2005) and a Chemiluminescence Kit (Roche, Basel, Switzerland). Blood sera containing 500 μg of protein were immunoprecipitated with 2 μl of anti-LPS antibody (Abcam, Cambridge, UK; ab35654) overnight at 4℃. Pre-equilibrated protein A PLUS-Agarose beads (Pierce Biotechnology, Rockford, IL, USA; #20365) were then added and incubated for 2 h at 4 °C on a rotating wheel. Beads were washed three times with radioimmunoprecipitation assay buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Serum was incubated with biotin-IAM (1 μg/ml; Sigma Aldrich, St. Louis, MO, USA; B2059) for 18 h. Mixtures were incubated with 20 μl (50% slurry) of streptavidin-agarose beads (Pierce, Rockford, IL, USA; #20349) for 1 h at 4℃ with rotation and centrifuged at 8000 rpm for 1 min. The pellets contained reduced-HMGB1, while the supernatants contained disulfide-HMGB1. The supernatants were incubated with biotin-M (1 μg/ml; Sigma Aldrich, St. Louis, MO, USA; B1267) in the presence of dithiothreitol (10 mM) for 6 h and then with streptavidin beads for 1 h and then centrifuged at 8000 rpm. The pellets were washed three times and analyzed by immunoblotting. To show that HMGB1 binds to LPS, serum was preincubated with HMGB1 A box peptide (0.5 or 1 μg/ml; HMGbiotech, Milano, Italy; HM-012) for 6 h and then incubated with biotin-LPS (5 μg/ml; Invivogen, San Diego, CA, USA; tlrl-bblps) for 24 h. The mixture was precipitated using streptavidin-agarose beads for 1 h at 4℃ with rotation and analyzed by immunoblotting with anti-HMGB1 antibody (Abcam, Cambridge, UK; ab67281).
To detect IL-1β or TNF-α in blood serum, we used a solid-phase sandwich ELISA (enzyme-linked immunosorbent assay) Kit (eBioscience, San Diego, CA, USA; BMS630 or BMS622). Sera were prepared at the indicated times, and standard samples were diluted with distilled water and applied to ELISA plates. IL-1β or TNF-α concentrations were determined according to the manufacturer’s protocol. Absorbance levels were measured at 450 nm using an ELISA reader.
Two-sample comparisons were performed using Student’s t test and multiple comparisons using one-way or two-way analysis of variance. Analysis was performed using PRISM software 5.0 (GraphPad Software). Results are presented as the means ± SEM and statistical difference was accepted at the 5% level.
