Adult male Wistar rats (n=56; aged 7 weeks; weighing 180–220 g) were obtained from the Experimental Animal Center of Wuhan University of Science and Technology. All rats were housed in a room with a temperature-controlled (23±2°C) environment with a normal 12-h light-dark cycle and allowed access to normal rat chow ad libitum.

All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (the US National Institutes of Health Publication no. 85–23, revised 1996) and approved by the Committee on the Ethics of Animal Experiments of Wuhan University of Science and Technology, Wuhan, China.

The male Wistar rats were randomly divided into 2 groups (n=28) as follows: the normal salt diet group administered 0.3% NaCl (NS group) as a control, the high-salt diet group administered 8% NaCl (HS group) in their food for 8 weeks to induce hypertension, as previously described (5,6). After 8 weeks, the rats in the NS and HS group were respectively administered ALA (60 mg/kg) dissolved in the vehicle (0.9% saline) or an equal volume of the vehicle daily by gastric perfusion for 8 weeks, as previously described (17). Thus, there were now 4 groups of rats (HS + vehicle, HS + ALA, NS + vehicle and NS + ALA) with 14 rats in each group.

In the rats from all the chronic feeding groups, arterial pressure was measured non-invasively using a tail-cuff instrument and a recording system (Kent Scientific Corp., Torrington, CT, USA), as previously described (18). Briefly, unanesthetized rats from each group were warmed to an ambient temperature of 30°C by placing them in a holding device mounted on a thermostatically controlled warming plate. Tail cuffs were placed on the animals, and each rat was allowed to become accustomed to the cuff for 10 min prior to performing the BP measurements. All measurements were taken within the same 2-h time window each day. Each session consisted of 30 cycles. BP was measured on 5 consecutive days each week, and values were averaged from ≥6 consecutive cycles. BP was measured at baseline (7 weeks of age) and then weekly until the end of either chronic study period.

Each group of rats (n=14) was anesthetized with a ketamine (90 mg/kg) and xylazine (10 mg/kg) mixture via intraperitoneal (i.p.) injection. From each group, 7 rats were perfused with 4% paraformaldehyde for immunofluorescence and immunohistochemistry. The remaining rats were decapitated and trunk blood was collected for high performance liquid chromatography (HPLC) fresh tissue was obtained for western blot analysis, ELISA and other experiments. The RVLM tissue was isolated following Palkovits' microdissection procedure as previously described (19). Plasma and tissue samples were stored at −80°C until analysis.

The rats were anesthetized and perfused through the heart with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4). The brains were dehydrated in graded sucrose, and OCT-embedded. The RVLM was identified as the region extending caudally 500–700 µm from the caudal pole of the facial nucleus. Serial coronal sections (14-µm-thick) were cut and mounted on glass slides, which were stored at −80°C until use for measurements, as previously described (20).

Immunohistochemical and immunofluorescence staining was carried out on brain sections as described previously to identify NAD(P)H oxidase (NOX2 and NOX4) expression in the RVLM using respective antibodies [NOX2, sc-20782, 1:200; NOX4, sc-5827, 1:200; and copper/zinc (Cu/Zn)-superoxide dismutase (SOD), sc-11407, 1:200; all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA] (19). The brain sections were washed in PBS, permeabilized in 0.5% Triton, blocked using 5% normal goat serum and incubated with the primary antibodies in blocking buffer at 4°C overnight. Following incubation with the primary antibodies (anti-NOX4 and anti-Cu/Zn-SOD antibodies), the sections were incubated with secondary antibodies for immunofluorescence [Alexa 488-labeled anti-rabbit (1:200, green fluorescence) or Alexa 594-labeled anti-rabbit (1:200, red fluorescence); Invitrogen Life Technologies, Carlsbad, CA, USA] for 60 min at 37°C.

For immunohistochemistry, the brain sections were incubated with anti-NOX2 primary antibody and then with anti-rabbit secondary antibody from a Histostain™-Plus kit (SP-9001; ZSGB-Bio, Beijing, China) for 60 min. Antibody binding was visualized using a 3,3′-diaminobenzidine (DAB) kit (AR-1002; Boster Bio-Engineering, Wuhan, China) according to the manufacturer's instructions. Follwing a 10-min wash in tap water, the slices were stained in Harris' hematoxylin solution for 8 min and then differentiated in 1% acid alcohol for 30 sec. Processing was terminated with H₂O and the sections were imaged using a Nikon camera (Tokyo, Japan), as previously described (21).

Superoxide anion levels in the RVLM were determined by fluorescent-labeled dihydroethidium (DHE; Molecular Probes, Eugene, OR, USA) staining. The coronal sections (14-µm-thick) were incubated with 1 µmol/l DHE at 37°C for 10 min as previously described (20).

Protein extracted from the RVLM tissues was prepared as previously described (21). For NOX2, NOX4 and Cu/Zn-SOD detection, protein extracts (5 µl) from the RVLM were resolved by 10–15% SDS-polyacrylamide gels, and electroblotted onto nitrocellulose membranes (Immobilon-P; EMD Millipore, Billerica, MA, USA) that were blocked in Tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% bovine serum albumin for 1 h at room temperature (20). The blots were incubated overnight at 4°C with the primary antibodies to NOX2 (sc-20782, 1:400), NOX4 (sc-5827, 1:400) and Cu/Zn-SOD (sc-11407, 1:200) (all form Santa Cruz Biotechnology, Inc.) to determine the relative expression levels in the RVLM. After washing with wash buffer (1X TBS, 0.1% Tween-20), the blots were then incubated for 1 h with the secondary antibody (1:10,000 dilution; Santa Cruz Biotechnology) labeled with horseradish peroxidase. Protein loading was controlled by probing all blots with β-actin antibody (Thermo Fisher Scientific, Waltham, MA, USA) and normalizing their protein intensities to those of β-actin. The immuno-complexes were visualized using an ECL-immunoblotting detection kit (PerkinElmer, Inc., Waltham, MA, USA). Band densities were analyzed using NIH ImageJ software, as previously described (21).

Mitochondrial matrix (stroma) was prepared by applying the method described as follows: brain tissues were rapidly removed and washed with 0.86% cold normal saline, then chopped into small sections, and placed into ice-cold isolation buffer for mitochondria (10 mM Tris-HCl, pH 7.4, 250 mM sucrose, 0.5 M methylene diamine tetra-acetic acid (EDTA), and 0.5 % bovine serum albumin). After being homogenized, the homogenate was centrifuged at 750 × g for 10 min. The supernatant was then centrifuged at 10,000 rpm for 10 min at 4°C. Mitochondrial pellets were washed twice with isolation buffer and then resuspended in the same buffer solution. The mitochondrial matrix was extracted from freshly prepared mitochondria by freezing and defrosting with repeated homogenization in order to burst mitochondria. Following centrifugation at 10,000 rpm for 10 min, the supernatant was the source of SOD, glutathione (GSH) and malondialdehyde (MDA), as previously described (22).

Lipid peroxidation product in the RVLM was determined by measuring the MDA content in tissue homogenates according to the method of Begue and Aust spec-trophotometrically at 532 nm (23). Values were expressed as mm/g protein. SOD activity was determined by following the method of Kono at 550 nm (24). Values were expressed as U/mg protein. The level of reduced GSH was measured as protein-free sulfhydryl content by the method of Sedlak and Lindsay at 412 nm and values were expressed as µm/g protein (25).

According to the manufacturer's instructions, the standards or sample diluents were added to the appropriate well of a microtiter plate pre-coated with specific antibodies and incubated. Conjugate was added followed by incubation at 37°C for 1 h and then washing. The reactions were terminated with stop solution and read at 450 nm for MDA, GSH and SOD measurements using a microtiter plate reader (MK3; Thermo Fisher Scientific).

Plasma NE levels were measured by HPLC as described previously with minor modifications in plasma sample preparation Plasma samples were prepared by adding activated alumina, Tris buffer, EDTA, and internal standard 3,4-dihydrobenzylamine, along with 0.5 ml of rat plasma. The samples were centrifuged, and supernatant was separated, rinsed 2 times in ultrapure water, and filtered through a Millipore filter (Ultrafree MC UFC30GV00; Millipore). The composition of the mobile phase was as follows: monochloroacetic acid (14.14 g/l), sodium hydroxide (4.675 g/l), octanesulfonic acid disodium salt (0.3 g/l), ethylenedi-aminetetraacetic acid (0.25 g/l), acetonitrile (3.5%) and tetrahydrofuran (1.4%). The mobile phase was carried out in pyrogen-free water and then filtered and degassed through the Millipore filter and pumped at a flow rate of 1.8 ml/min. The sensitivity of the detector was 1 nA full scale, and the potential of the working electrode was 0.65 V. The column oven maintained the temperature of the column at 37°C. At the time of HPLC analysis, tissue samples were homogenized in 150 µl of 0.1 M HClO₄ using a micro-ultrasonic cell disruptor (Kontes, Vineland, NJ, USA) and centrifuged at 10,000 × g for 10 min. 50 µl of the supernatant along with 25 µl of the internal standard (0.05 M dihydroxybenzylamine) were injected into the HPLC system (26,27).

All data are expressed as the means ± standard error of the mean (SEM). The significance of differences between mean values was analyzed by ANOVA followed by Tukey's test. A value of P<0.05 was considered to indicate a statistically significant difference.

A high-salt diet induced a significant increase in MAP compared with the control rats after 8 weeks prior to the supplementation of ALA (Fig. 1A). Fig. 1B presents the MAP trends for each group of rats treated with ALA for 8 weeks. The MAP of rats fed a high-salt diet was significantly higher compared to that of the control animals (NS group). The supplementation of ALA decreased the MAP in the rats with high salt-induced hypertension.

Imunohistochemisty and immunofluorescence staining revealed that the high-salt diet induced a significant increase in the expression of NOX2 and NOX4 in the RVLM compare to the control rats. The supplementation of ALA decreased the number of NOX2- and NOX4-positive neurons in the hypertensive rats (Fig. 2).

Immunofluorescence staining revealed that the high-salt diet induced a significant decrease in Cu/Zn-SOD levels, and an increase in fluorescence-labeled DHE compared with the control rats. The supplementation of ALA decreased the DHE fluorescence intensity and increased the number of Cu/Zn-SOD-positive neurons in the hypertensive rats (Fig. 3).

The results of western blot analysis indicated that the rats fed a high-salt diet exhibited significantly increased levels of NOX2 and NOX4, and decreased expression levels of Cu/Zn-SOD in the RVLM compared with the control rats. The supplementation of ALA decreased the levels of NOX2 and NOX4, and increased the Cu/Zn-SOD expression levels in the hypertensive rats (Fig. 4).

The MDA levels in the RVLM mitochondria were significantly higher in the rats fed a high-salt diet than in those in the normal control group. The supplementation of ALA decreased the levels of MDA as compared with the respective control group (HS + vehicle; Fig. 5A). On the other hand, the results revealed that the levels of SOD and GSH in the RVLM mitochondria were decreased in the rats fed the high-salt diet. The supplementation of ALA increased the levels of SOD and GSH (Fig. 5B and C). The GSH level in the ALA-treated rats was similar to that in the control groups (Fig. 5C).

Whole heart weight/body weight (WHW/BW) ratio, WHW/tibia length (TL) ratio and left-ventricular weight (LVW)/TL ratio were measured as indicators of cardiac hypertrophy. Plasma NE presents the activity of the sympathetic nervous system. The rats fed a high-salt diet exhibited increased cardiac hypertrophy as indicated by the increased WHW/BW ratio, WHW/TL ratio, and LVW/TL ratio, which were decreased by ALA supplementation (Fig. 6A–C). In addition, the plasma NE levels in the rats fed a high-salt diet were higher than those in the control group. The supplemenation of ALA decreased the levels of plasma NE in the hypertensive rats (Fig. 6D).