Twelve-week-old male spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were purchased from Charles River Laboratories. The rats were housed in a climate-controlled room with a 12 h light-dark cycle and allowed access to standard rat chow and tap water ad libitum. All study protocols were approved by the Institutional Animal Care and Use Committee, Xi’an university. The design conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication No. 85 − 23, revised 1996).
Adenovirus-associated virus preparation
Adenoassociated virus (AAV) is an efficient and safe vector for gene transfer in vivo. Thus, the fragment comprising the entire protein-coding region of rat CTRP9 was cloned into the AAV vector (pHBAAV-CMV-MCS-3flag-EF1-ZsGreen), which was driven by a cytomegalovirus promoter and harbored a GFP region. Recombinant AAV was plaque-purified, and the titer measured by a plaque assay on cells in culture at 1×1012 viral genomes. After we obtained the denovirus-associated virus, the AAV would be subpackaged (200 µl/tube) and store at -80°C. Before bilateral paraventricular nucleus injection of AAV-CMV-CTRP9 or AAV-CMV-GFP, the vectors should be dissolved on ice. All procedures were undertaken by Hanbio Biotechnology Co. Ltd (Shanghai, China,).
General experimental protocol
One week after habituation to their new environment, rats were divided randomly into four groups: (1) SHR + PVN AAV-CTRP9, (2) SHR + PVN AAV-GFP, (3) WKY + PVN AAV-CTRP9, (4) WKY + PVN AAV-GFP. Rats were anaesthetized by intraperitoneal injection with a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg), and their heads were placed in a stereotaxic instrument. Each group of rats were administered with bilateral PVN microinjection of the AAV-CMV-CTRP9 or AAV-CMV-GFP. The microinjector (5 µl) with vectors (either AAV-CMV-CTRP9 or AAV-CMV-GFP) were installed in the microinjection pump. Vectors were bilaterally injected into the PVN (coordinates:±0.4 mm from midline, -1.8 mm posterior to bregma, and − 7.9 mm ventral to dura according to the Paxinos and Watson rat brain atlas) using 1 µl volume each side at the rate of 0.1 µl/min[22–24]. At the end of the study, after rats were anesthetized, blood was collected from the abdominal aortic vein and centrifuged at 3,000 r/min to obtain the plasma. The PVN tissue was isolated following Palkovits’s microdissection procedure as previously described[25, 26]. One part of fresh brain tissues was put into 4% paraformaldehyde for 3 days and then moved into 30% sucrose for dehydration. Plasma and other brain tissues were stored at -80°C for future analysis.
Measurement of mean arterial pressure
During the 18 days experimental period, mean arterial pressure (MAP) was measured two days in the SHR and WKY rats. All rats were habituated to the blood pressure measuring system and to the holders daily for two days prior to the initiation of experimental measurements. Each rat was allowed to accommodate the cuff for 10 minutes before the blood pressure measurement. Blood pressure values were averaged from five consecutive cycles per day obtained from each rat.
The methods for immunofluorescence and immunohistochemistry studies were performed as described previously to immunolocalize gp91phox, gp47phox, IL-1β and IL-6 expressions in the PVN. The primary antibodies for gp91phox (ab280952, a Polyclonal Rabbit Anti-Rat) and gp47phox (ab133303, a Polyclonal Rabbit Anti-Rat) were purchased from Abcam. The primary antibodies for IL-1β (sc-1251, a Polyclonal Goat Anti-Rat) and IL-6 (sc-1265, a Polyclonal Goat Anti-Rat) were purchased from Santa Cruz Biotechnology.
Dihydroethidium (DHE) staining was used to determine the superoxide generation and the staining protocol was performed as described. Briefly, free-floating sections containing PVN were incubated with DHE (0.05 mM) at 37°C for 30 min, then sections were rinsed in 0.01 M PBS for three times. Sections were imaged using a Nikon epifluorescence microscope.
Western blotting was used for measurement of MCP-1, TNF-α, IL-6 and IL-1β expressions in the PVN. The methods for Western blotting studies were performed as described previously[28, 29]. Protein loading was controlled by probing all blots with β-actin antibody (Thermo Scientific, USA) and normalizing their protein intensities to that of β-actin. Band densities were analyzed with NIH Image J software.
Total RNA was extracted from microdissected PVN using TRIzol reagent (Invitrogen) and reverse transcribed using oligo (dT) with conditions at 23℃ for 10 min, 37℃ for 60 min, and 95℃ for 5 min. The cDNA used for real-time PCR with specific primers for CTRP9, TNF-α, IL-1β, IL-6, MCP-1, gp91phox, gp47phox and GAPDH were shown in Table 1[30–32]. The quantitative fold changes in mRNA expression were determined relative to GAPDH mRNA levels in each corresponding group.
ELISA measurement of CTRP9 in the PVN and plasma NE
The levels of serum CTRP9 and plasma NE were quantified using commercially available rat ELISA kits (Invitrogen) according to the manufacturer's instructions.
All data were expressed as mean ± SEM. Statistical analysis was done by one-way ANOVA followed by a Tukey post-hoc test. Blood pressure data were analyzed by repeated measures ANOVA. A probability value of P < 0.05 was considered to be statistically significant.