C57BL/6J mice (6− to 8-week-old), bought from Janvier Laboratories (Le Genest-Saint-Isle, France) and Charles River Laboratories (Sulzfeld, Germany), were bred at the Laboratory for Experimental Biomedicine (Sahlgrenska Academy, University of Gothenburg, Sweden). The animals were kept in a 12 h light-dark cycle with ad libitum access to standard laboratory chow (B&K, Solna, Sweden) and water. All the animal experiments were approved by the Gothenburg Animal Ethics Committee. In all the experiments, animals from at least three litters were used, and pups within each litter were equally distributed among the treatment groups, including distribution of sex of pups whenever possible.

The day of birth was considered post-natal day (PND) 0. On PND 8, mice were randomly selected for injection with either lipopolysaccharide (LPS) (O55:B5; i.p.; 0.3 mg/kg in endotoxin-free saline) or an equivalent volume of endotoxin-free saline (SAL). In a separate cohort, PND 8 pups were administered LPS or SAL as above and 14 h after injection (i.e., on PND 9) the pups were also exposed to hypoxia-ischemia (HI) using the modified Rice-Vanucci model as described before (Wang et al., 2009). Briefly, the left common carotid artery was ligated, and the mice were exposed to 10% O₂ for 30 min in a humidified incubator maintained between 35.5 and 36.5°C.

N-acetyl cysteine or vehicle (VEH, i.e., endotoxin-free saline) was administered at the time of LPS injection and immediately before HI in animals subjected to the combination of LPS and HI. The dose of NAC (200 mg/kg in endotoxin-free saline; i.p.; Sigma-Aldrich, St. Louis, MO, United States) was selected based on our previous study on neonatal rats (Wang et al., 2007).

Mice were deeply anesthetized via intraperitoneal administration of pentobarbital (Pentacour) and intracardially perfused with 0.9% saline. Brain tissue was collected on a cold plate and frozen in dry ice followed by storage at −80°C. The tissue samples were homogenized using a lysis buffer containing Tris-HCl (25 mM, pH 7.9), NaCl (100 mM), EDTA (5 mM), NP40 (1%), sodium butyrate (0.1 mM), Nam (5 mM), protease inhibitor (1%), phosphatase inhibitor, and PBS (pH 7.4). The lysate was analyzed by Western blotting and quantitative real-time PCR. The fraction for Western blotting was centrifuged at 15,000 g for 5 min at 4°C, and the supernatant was stored at −80°C. The remaining fractions were directly stored at −80°C. Protein concentration in the lysates was measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA, United States).

Total RNA was extracted from the brain samples using the RNeasy mini kit (Qiagen, Solna, Sweden) and measured in a spectrophotometer at 260-nm absorbance (NanoDrop 2000/2000c; Thermo Fisher Scientific, Rockford, IL, United States). Total RNA (500 ng) from each sample was used for first-strand cDNA synthesis according to the instructions of the manufacturer (QuantiTect reverse transcription kit; Qiagen, Solna, Sweden). In order to determine mRNA expression, the cDNA samples were further processed by quantitative real-time (qRT)-PCR. Each PCR reaction (20 μl) contained 10 ng of cDNA in a 5-μl volume, 10 μl of QuantiFast SYBR Green PCR master mix (Qiagen, Solna, Sweden), 2 μl of PCR primers, and 3 μl of H2O, to make a final reaction volume of 20 μl. The PCR primer was Mm_Sirt6_1_SG QuantiTect Primer Assay QT00112700 (Qiagen, Solna, Sweden). The amplification protocol comprised an initial 5-min denaturation at 95°C, followed by 40 cycles of denaturation for 10 s at 95°C and annealing/extension for 30 s at 60°C on LightCycler 480 (Roche, Stockholm, Sweden). Melting-curve analysis was performed to ensure that only one PCR product was obtained. For quantification and estimation of amplification efficiency, a standard curve was created using increasing concentrations of cDNA. The amplification transcripts were quantified with the relative standard curve and normalized to the concentration of cDNA in each RT sample measured using the Quant-iTTM 338 OliGreen ssDNA Assay Kit 339 (Invitrogen, Waltham, MA, United States) in accordance with (26). Data were normalized to geomean of Hprt1 and Ywhaz genes.

Tissue lysates were mixed with 4× Laemmli Sample buffer (Bio-Rad Laboratories, Hercules, CA, United States) and 10% β-Mercaptoethanol (Merck, Darmstadt, Germany), and diluted using PBS to obtain same protein concentration across all samples. The samples were heated at 95°C for 5 min before 10–20 μg of each was loaded on a 4–20% reducing gel (Criterion^TM TGX Stain-Free^TM Precast Gels; Bio-Rad Laboratories, Hercules, CA, United States), and transferred to a 0.2-μm nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, United States). Stain-free blots were imaged using a Gel Doc XR Plus system (Bio-Rad Laboratories, Hercules, CA, United States), and images were saved for normalization of immunoreactive bands to the total protein. Membranes were blocked with TBS-Tween (TBS-T) buffer (30 mM/L Tris-HCl, 100 mM/L NaCl, and 0.1% Tween: pH 7.45) containing 5% nonfat milk for 1 h at room temperature. After washing with TBS-T, the membranes were incubated overnight at 4°C with the following primary antibodies: sirt1 mouse monoclonal antibody (1:500, CST 8469; Cell Signaling, Leiden, WZ, Netherlands) and Sirt6 rabbit monoclonal antibody (1:500, CST 12486; Cell Signaling, Leiden, WZ, Netherlands). The membranes were washed with TBS-T and incubated with appropriate peroxidase-labelled secondary antibody (1:5,000; Vector Laboratories, Burlingame, CA, United States) in 5% nonfat milk for 1 h. Immunoreactive bands were visualized using an ECL substrate (Bio-Rad Laboratories, Hercules CA, United States) and a Gel Doc XR Plus system (Bio-Rad Laboratories, Hercules, CA, United States), and quantified using the Image Lab^TM software after normalizing to total protein (Bio-Rad Laboratories, Hercules CA, United States).

Cytokines and chemokines in lysates from hippocampus were detected with a cytometric bead array using a Bio-Plex Pro mouse cytokine 9-plex assay (Bio-Rad Laboratories, Hercules CA, United States) and analyzed with a Luminex 200 system (Bio-Rad Laboratories, Hercules CA, United States) according to the instructions of the manufacturer and as previously described (Lai et al., 2020).

The oxidation of plasma albumin was measured as previously described (Lim et al., 2020) with the following modifications: blood samples were collected by heart puncture and applied onto a PerkinElmer 226 Spot Saver RUO Card containing polyethylene glycol maleimide. Cards were stored with silica gel desiccant for transport to the University of Western Australia. Albumin was extracted into 0.05% Tween 20 in 20 mM phosphate with further binding to Cibacron Blue F3GA agarose. Albumin was eluted with 25 μl of 1.4 M NaCl in 20 mM phosphate buffer, pH 7.4. Gel electrophoresis, imaging, and calculation of total albumin oxidation were performed as previously described (Lim et al., 2020).

The mice were deeply anaesthetized via intraperitoneal administration of pentobarbital (Pentacour) (60 mg/ml). The brains were removed from the skull and immersed in 6% buffered formaldehyde (Histofix; Histolab products AB; Västra Frölunda, Sweden) and stored at 4°C for 1 week. At the time of tissue processing, the brains were placed in a cryoprotective solution containing 30% (w.v1) sucrose for 48 h followed by placement on copper blocks for freezing in cold isopentane (Sigma Aldrich, Darmstadt, Germany). Free-floating 60-μm thick sections were cut coronally on a cryostat (Leica CM 3050 S; Leica, Wetzlar, Germany) and selected based on a systematic sampling principle and a section-sampling fraction of 1/6 (Gundersen, 2002) by selecting the first section of each series randomly using a random table.

The free-floating 60-μm thick sections were washed in phosphate-buffered saline (PBS, 0.1 M, pH 7.2, 30 min) followed by blocking with endogenous peroxidase (3% H₂O₂ in PBS, 30 min). Antigen retrieval was performed using 0.01 M citrate buffer (40 min at 85°C). Thereafter, the sections were rinsed in PBS (2 min × 15 min) and placed in 4% goat serum in PBS for 1 h. In the next step, the sections were incubated with a monoclonal rabbit anti-HMGB1 and anti-sirt6 (1:350, Ref# ab79823; 1:500, D8D12 CST; Abcam, Cambridge, United Kingdom) overnight at 4°C, followed by washing in PBS, incubation in polyclonal secondary goat-anti-rabbit biotinylated (1:200;Vector Laboratories, Olean, NY, United States) for 2 h, and washing in PBS (2 min × 15 min) and ABC elite (avidin-biotin-complex; Vector Laboratory, Burlingame, CA, United States) for 1 h at room temperature. Immunolabeling was performed using a 3,3-diaminobenzidine solution (Acros Organics, Geel, Belgium). Finally, the sections were mounted on gelatine-coated slides, dried, re-hydrated in demineralized water, dehydrated through a graded series of alcohol (95% and 99%), cleared in xylene, and coverslipped.

Quantification of the area in hippocampus that exhibited HMGB1 translocation and release was performed on the HMGB1-stained sections by point counting using the Cavalieri estimator (Anzabi et al., 2018) and using a 10× objective lens under light microscope modified for stereology with a digital camera (Leica DFC 295; Leica, Wetzlar, Germany) and the newCAST^TM software (Visopharm, Hørsholm, Denmark) (Figure 5). The following formula was used to calculate the volume of the HMGB1-positive area:

where ΣP is the total number of the points hitting the region of interest per animal, (a/p) is the area per test point; T is the section thickness (60 μm), and SSF is the section sampling fraction (1/6). The size of the HMGB1-positive area was presented as % of the size of the hippocampus.

Data are presented as mean ± SD. All the statistical analyses were conducted using the SPSS statistical software (SPSS Statistics 21; IBM, Armonk, NY, United States). Graphs were created using Prism 8 (GraphPad Software Inc., San Diego, CA, United States). Prior to statistical tests, normal distribution of data was checked by making a Q–Q plot of the data. The variance homogeneity of data was also examined by Levene’s test. Data with normal distribution were compared between two independent groups (Saline/HI and LPS/HI) by independent t-test, and those with non-normal distribution were analyzed by Mann–Whitney U test. For analysis between ipsilateral and contralateral sides, the data were considered as paired. In such analysis, normally distributed data were analyzed by paired t-test, and non-normally distributed data were analyzed by paired Wilcoxon signed-rank test. P < 0.05 was considered statistically significant.

In order to understand if LPS by itself affects sirtuin-6 expression, we measured the sirt6 mRNA and SIRT6 protein expression in the brain 12 h after LPS injection. There was no difference in mRNA expression (Supplementary Figure 1A, p > 0.05) or in protein expression (Supplementary Figure 1B, p > 0.05) in the brain after LPS.

To analyze temporal changes in the expression of SIRT1 and SIRT6 proteins in response to an injurious event, we collected hippocampal tissues at 1, 2, 6, and 12 h after SAL + HI and LPS + HI (n = 5–6 per group and time point). The hippocampus was analyzed, as it is the most sensitive brain region in infection-induced sensitization of HI injury in neonatal mice (Lai et al., 2020). SIRT1 protein levels significantly decreased in the ipsilateral hippocampus compared with the contralateral side at 1, 2, and 6 h in the LPS + HI group (Figure 1A). There were no significant changes in SIRT1 levels between hemispheres following SAL + HI (Figure 1A). Analysis of SIRT1 protein levels in the ipsilateral hippocampus in SAL + HI and LPS + HI, within each time-point, showed no difference between the two groups (Figure 1A).

Sirtuin 6 protein expression was significantly decreased in the ipsilateral hippocampus compared with the contralateral side at all time points (1–12 h) after LPS + HI, while SIRT6 protein expression in the hippocampus was reduced at 2–12 h after SAL + HI (Figure 1B). Analysis of SIRT6 protein levels between the ipsilateral hippocampus in SAL + HI and LPS + HI within each time point showed significantly lower level of SIRT6 protein at 12 h after LPS + HI, while no changes at other time points were observed (Figure 1B).

Immunohistochemical staining for SIRT6 was evident in neurons in the contralateral CA1 region of the hippocampus, while staining was markedly reduced in the ipsilateral hippocampus 6 h after LPS + HI (Figure 1C).

As both SIRT1 and SIRT6 protein expressions changed already 1–2 h after LPS + HI, we next used a cytometric bead array to measure a panel of cytokine and chemokine proteins in the hippocampus 2 h following SAL + HI and LPS + HI. Cytokines IL-1β, IL-6, IL-12 (p40), IL-12 (p70), GM-CSF, TNF-α, and the chemokine CCL2 were unchanged in the ipsilateral compared with contralateral hippocampi in the SAL + HI and LPS + HI groups (Figure 2, n = 5–6/group, p > 0.05). In contrast, G-CSF and CXCL1 were increased in the ipsilateral hippocampi compared with the contralateral hippocampi following LPS + HI (Figure 2, n = 5–6/group, p = 0.026 and p = 0.002, respectively). Analysis of cytokine and chemokine proteins in the ipsilateral hippocampus following SAL + HI and LPS + HI showed significant difference in IL-1β, G-CSF, and CXCL1 between the two groups (Figure 2).

To investigate the effects of an antioxidant/anti-inflammatory compound on SIRT6 and HMGB1 in our model, we used NAC, as we have previously shown this to be protective in neonatal rats following inflammation-induced HI injury (Wang et al., 2007; Mottahedin et al., 2020). First, we confirmed the antioxidant effect of NAC and show reduced thiol oxidation in blood 30 min following NAC compared with VEH-treated LPS + HI animals (Figure 3A, n = 7/group, p = 0.007). Inspection of brain tissue 12 h after LPS + HI in VEH- and NAC-treated animals showed clear reduction in tissue pallor (indicative of reduced injury) in the NAC-treated animals (Figure 3B).

To determine the effects of NAC on the expression of SIRT6 protein, hippocampi were collected from NAC- and VEH-treated LPS + HI mice 12 h after HI. SIRT6 levels were reduced in the ipsilateral hippocampus compared with the contralateral side in both VEH- and NAC-treated animals (Figure 4, n = 10–12/group, p = 0.001, p = 0.001, respectively). The reduction in SIRT6 in the ipsilateral hippocampus was significantly less in the NAC-treated animals compared with the SAL-treated mice (Figure 4, n = 10–12, p = 0.009).

Sirtuin 6 has been suggested as a regulator of HMGB1 release. Thus, we investigated the immunohistochemical expression of extracellular HMGB1 in the hippocampus after LPS-HI with or without NAC treatment. Extracellular HMGB1 positive areas, as determined by unbiased stereological method, were apparent in the ipsilateral hemisphere in both groups (Figures 5A,B). We found that the area of extracellular HMGB1 staining in the ipsilateral hippocampus was significantly smaller in the NAC group compared with the VEH group (Figure 5C, n = 6–8/group, p = 0.041).