Molecular Hydrogen Mediates Neurorestorative Effects After Stroke in Diabetic Rats: the TLR4/NF-κB Inflammatory Pathway

Animals

One hundred fifty-six male Sprague–Dawley (SD) rats weighing 60–80 g were obtained from the Laboratory Animal Department of Harbin Medical University. The rats were reared on a 12 h light–dark cycle under controlled temperature conditions (21–24 °C), and they were provided free access to food and water. The rats were randomly divided into 7 groups: Sham group (group S, n = 30), MCAO group (group M, n = 30), Non-diabetic MCAO group (group N, n = 18), H2 daytime treatment group (group H, n = 30), LPS + H2 group (group L, n = 18), TAK242 + H2 group (group T, n = 18) and H2 night-time treatment group (group HN, n = 12) (Fig. 1). According to the different monitoring indicators, this study was divided into two programs. (1) The rats were studied at 28 days postoperatively and nerve function, survival rate and weight change percentages were measured; (2) rats were sacrificed 48 h after injury and samples were harvested for subsequent measurements, including measurements of the infarct volume, brain water content, inflammatory cells and factors, catecholamine levels, acetylcholine levels, immunohistochemical staining, and western blotting.

Fig. 1figure 1

Flow chart. Timeline of the experimental protocol

Diabetic Rat Model

We fed rats a high-fat diet consisting of 20% lard, 20% sucrose, 2.5% cholesterol and 57.5% standard diet for 4 weeks to induce diabetes (DM) (Reed et al. 2000; Duca et al. 2015). Then, the rats were allowed to drink freely overnight and STZ (streptozotocin) dissolved in citric acid-citrate buffer (pH 4.5; intraperitoneal injection, 35 mg/kg; Sigma–Aldrich (Shanghai) Trading Co., Ltd., Shanghai, China) Zotobacter) was injected. Seven days later, blood glucose levels were randomly detected. Animals with a level ≥ 11.1 mmol/l were selected as diabetic rats. The blood glucose level of diabetic rats in this study was 15–25 mmol/l.

MCAO Rat Model

We established a rat model of middle cerebral artery occlusion (MCAO) (Belayev et al. 1996). Experimental rats were administered 5% sevoflurane (Baxter, USA) through a face mask for the induction of anaesthesia, 2–3% sevoflurane was used for anaesthesia maintenance, and spontaneous breathing was retained. After preparation and disinfection of the skin in the operation area and local infiltration anaesthesia with 1% lidocaine, a midpoint incision of approximately 0.5 cm was made at the midpoint of the craniopia of both ears to identify the fontanelle, where the anterior fontanelle was defined as zero. The cerebral blood flow probe was fixed on the left side of the fontanelle at 4 mm and 1.5 mm, and the cerebral blood flow (CBF) was monitored with a laser Doppler blood flow metre (TF5000; PRIMED AB; Stockholm; Sweden). The animal was slowly turned over, and the neck skin was prepared and disinfected. Local infiltration anaesthesia was induced with 1% lidocaine. An incision was created 3 mm parallel to the midline, micro tweezers were used to bluntly separate the subcutaneous tissue, the left sternocleidomastoid muscle was exposed, and the first sternocleidomastoid muscle was gradually freed. The left carotid sheath can be seen between the second abdominal muscles, sternocleidomastoid muscle and scapula-thyroid muscle. The common carotid artery, external carotid artery and internal carotid artery were bluntly separated. Electrocoagulation disconnected the communication branch between the external carotid artery and the internal carotid artery and the accessory branch of the external carotid artery, the left external carotid artery was ligated and disconnected, the proximal end of the common carotid artery and the distal heart of the internal carotid artery was clamped with an arterial clip, and a suture (Doccol #4035, #4037, and #4039, USA) was inserted from the stump of the external carotid artery. The cerebral blood flow suddenly decreased to a value greater than 70% of the basal level. The incision was sutured. The plug insertion time and cerebral blood flow value were recorded. For rats with a Longa score of 2 (Belayev et al. 1996), the thread plug was removed after 90 min of embolization. After the operation, the animals were sent to the PACU to rouse from anaesthesia and remain warm.

Molecular Hydrogen Preparation

Hydrogen/oxygen mixture therapy was delivered using a novel device, Hydrogen/Oxygen Generator with Nebulizer (AMS-H-01, Shanghai Asclepius Meditech Co., Ltd., China), at a flow rate of 3.0 L/min and hydrogen/oxygen volume ratio of 2:1. In addition, the gas ratio was 42% H2-21% O2-37% N2. The mixed gas was output to an explosion-proof box. The explosion-proof box contained a gas absorption part, an inhalation and exhalation gas circuit, and an exhaust gas absorption device. It was also equipped with a safety alarm and a hydrogen concentration detection metre.

Molecular Hydrogen Therapy

Rats inhaled molecular hydrogen for 60 min beginning 20 min after MCAO modelling and daily after MCAO/R. The treatment time of the H2 daytime treatment group was 9:00–11:30, and the treatment time of the H2 night-time treatment group was 16:30–19:00.

Measurement of Blood Sugar Levels

Thirty minutes before the establishment of the MCAO model in diabetic rats and 15 min after MCAO/R, blood was collected from the tail vein of the rats, and the blood glucose level was measured and recorded.

Assessment of Neurological Outcomes, Survival Rate, and Weight Change Percentage

Neurological function was measured in rats on the 1st, 3rd, 5th, 7th, 14th, and 28th days after surgery with the modified nervous system severity score (mNSS) (Chen et al. 2001) and the Garcia test, and the 28-day survival rate was recorded. All rats received two adaptation training sessions before surgery. The evaluation was conducted by the same researcher and supervised by the same person under mutual blinding conditions. Body weight was measured daily for 28 days, and the percentage of weight change relative to the baseline body weight was determined (n = 6 rats/group).

Infarct Volume Measurement

After 48 h of reperfusion, the rats were sacrificed with a lethal dose of sodium pentobarbital. The rat brain was quickly removed, cut into 2 mm coronal sections, stained with 1% 2,3,5-triphenyltetrazolium chloride (TTC, Biotosharp, Beijing, China) at 37 °C for 20 min, and then incubated at 4 °C overnight with 1% paraformaldehyde (Chi et al. 2018; Li et al. 2019). ImageJ software was used to measure the infarct and hemisphere areas. The ratio of hemisphere infarction was calculated as (area of the contralateral hemisphere -area of the ipsilateral hemisphere without infarction)/area of the contralateral hemisphere (n = 6 rats/group).

Measurement of the Brain Water Content

After 48 h of reperfusion, we randomly selected 6 rats from each group and estimated the brain water content (Li et al. 2020). Briefly, we separated the cerebral hemisphere on the ischaemic side from other parts and recorded the weight (wet weight). Next, each specimen was dried in an electric furnace at 80 °C for 48 h, and the weight (dry weight) was recorded. We used the following formula to calculate brain water content: brain water content = (wet weight-dry weight)/wet weight × 100%.

Measurement of Inflammatory Cytokine, Catecholamines and Acetylcholine Levels

The rats were anaesthetized with pentobarbital, and the femoral artery was punctured to collect blood. The blood sample was placed in a heparin-coated tube and centrifuged (3500 rpm, 4 °C, 20 min). The plasma levels of IL-6, IL-1β, TNF-α, epinephrine, norepinephrine, dopamine and acetylcholine were measured 48 h after reperfusion. We used enzyme-linked immunosorbent assay (ELISA) kits to analyse the levels of each protein (#rat IL-6, #rat IL-1β and #rat TNF-α ELISA kit, Boster, China; # Rat Adrenaline, # Rat Norepinephrine, # Rat Dopamine and # Rat Acetylcholine, Langton, China) according to the manufacturer’s instructions (n = 6 rats/group).

Immunofluorescence Measurement

After 48 h of reperfusion, the rats were euthanized with a lethal dose of sodium pentobarbital and perfused transcardiacally with normal saline and 4% paraformaldehyde. The brain was postfixed with 4% paraformaldehyde at 4 °C for 24 h. The infarct region was cut into 10 mm transverse frozen sections. Sections were washed with 0.1% Triton X-100 for 20 min and then blocked with 3% BSA for 30 min. The sections were incubated with primary antibodies against TLR4 (1:100; BS3489, Bioworld Technologies) and NF-κB (1:200; BMS-33117 M, Bioss) at 4 °C overnight and then incubated with FITC-conjugated secondary antibodies for 50 min. Finally, the sections were incubated with DAPI for 10 min and then visualized with an optical microscope (Nikon Eclipse Ti-SR, Japan) at 400 × magnification. Merged images were captured under the same contrast settings: TLR4 is shown in green, NF-κB is shown in red, and the nucleus is shown in blue (n = 6 rats/group).

Western Blot Assay

After 48 h of reperfusion, the cerebral cortex was homogenized and centrifuged at 14,000 rpm for 20 min at 4 °C. We collected the supernatant and tested the protein concentration with the BCA test kit. Then, we separated the protein samples on 10% SDS–PAGE gels and transferred them to a nitrocellulose membrane. The membrane was blocked with 5% milk powder in 0.1% TBS-T, and primary antibodies against NF-κB p65 (1:1000; #48,498, SAB), β-Actin (1:400; PR-0255, ZSGB Bio), and phospho-NF-κB p65 (phospho Ser536; 1:2000; YP0191, Immunoway) were incubated with the membrane at 4 °C overnight. Subsequently, we washed the membrane with TBS-T and incubated it with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5000; ZB-2301, ZSGB Bio) for one hour. An electrochemiluminescence kit was used to detect the protein bands, and a gel imaging system was used for data collection and analysis (n = 6 rats/group).

Statistical Analysis

The sample size was based on our previously reported research. No data were missing, and no data were excluded from the analysis. We used SPSS (version 21.0) statistical software and GraphPad Prism 9 software to analyse the data. Data are presented as the means ± standard errors of the means (SEM). The normality of the distribution was assessed using the Shapiro–Wilk normality test, and a smaller sample size distribution was considered normal. Two groups were compared using t tests or Mann–Whitney U tests. One-way analysis of variance (ANOVA) was used to compare multiple groups, and then the Student-Newman–Keuls test was used. Nonparametric tests were used to compare the differences between the tested groups. Kaplan–Meier survival curves were compared using the log-rank test. Repeated measures analysis of variance was used for comparisons within the same group. A p value < 0.05 was considered statistically significant.

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