Male ApoE^−/− mice (3 months old) were purchased from the Animal Resources Centre, Perth, Australia. All experiments were conducted in a temperature-controlled animal house (21 ± 1°C) under a 12:12-h light-dark cycle and mice were given standard chow and water ad libitum. All animal protocols conformed to the Guide for the Care and use of Laboratory Animals by the United States National Institutes of Health and the Australian Code of Practice for the Care and Use of Animals for Scientific Purpose (8th Edition, 2013). Institutional ethics approval was obtained from both James Cook University and Federation University Australia.

A preliminary study was carried out to confirm the success of the RDN procedure. Five ApoE^−/− mice underwent sham surgery and seven mice underwent bilateral RDN. Five days later, the mice were euthanized and the right kidney was collected and norepinephrine content determined.

For the main experiment, 18 ApoE^−/− mice underwent bilateral RDN and 20 ApoE^−/− mice underwent sham surgery 1 day after the baseline blood pressure was measured (Figure 1). Five days after RDN or sham surgery, blood pressure was measured and all the mice were then subjected to angiotensin II infusion at a dose of 1 μg/kg/min for the ensuing 28 days. Blood pressure was measured at Day 14 and Day 27 after the angiotensin II infusion commenced. The mice were euthanized at Day 28 (Figure 1) and the right kidney was collected for norepinephrine content measurement, and the aorta was isolated for morphometric analysis. The aortic arch was then used for Sudan IV staining and the thoracic aorta used for RNA extraction.

Bilateral RDN was carried out as previously described (O'Neill et al., 1991; Ye et al., 1997). In brief, after the renal arteries and veins were exposed, all visible nerves around the renal arteries were cut, and connective tissues passing next to and along the course of the renal arteries and veins were dissected and stripped off the adventitia under a dissecting microscope with a 4 × magnification. Then the renal arteries and veins were painted with a solution of 10% phenol in 95% ethanol (O'Neill et al., 1991; Ye et al., 1997). After being washed with saline (0.9% sodium chloride), the abdominal cavity was closed. For the mice in the sham surgery group, the renal arteries were exposed as with the RDN procedure, but the renal nerves were kept intact.

The right kidney was homogenized in 1 mM ethylenediaminetetraacetic acid (EDTA) and 4 mM sodium metabisulfite. The norepinephrine content in the homogenate was measured by phase isocratic high-performance liquid chromatography (HPLC) (Wang et al., 1999) coupled with an electrochemical detector, with an Atlantis C18 column (5 μm particle size, 4.6 × 150 mm) and a mobile phase of 50 mM Na₂HPO₄, 27 μM EDTA, 0.6 mM sodium octane sulfonic acid, and 3.5% acetonitrile (pH 4.0). This method had a good reproducibility with an inter-assay coefficient of variation of 4.7% (n = 10).

Blood pressure was measured using a computerized, non-invasive, tail-cuff system (Kent Scientific, USA) (Seto et al., 2013). Animals were habituated to the device before measuring blood pressure. Good reproducibility of this technique has been established previously (Seto et al., 2013).

Atherosclerosis and aortic aneurysm were induced by subcutaneous infusion of angiotensin II at a dose of 1 μg/kg/min for 28 days (Daugherty et al., 2000). Briefly, under general anesthesia using isoflurane, osmotic minipumps (Model 2004, ALZET, USA) were placed into the subcutaneous space along the dorsal midline to deliver 1 μg/kg/min of angiotensin (Sigma-Aldrich, Castle Hill, Australia) dissolved in distilled water over 28 days.

Atherosclerosis in the aortic arch was quantified by en face staining as described previously (Krishna et al., 2012). Briefly, the aortic arch was opened longitudinally and pinned down on a wax coated petri-dish. Tissue samples were transferred to a 70% ethanol solution and stained with 0.1% Sudan IV dissolved in equal parts of acetone and 70% ethanol for 10 min to identify areas of atherosclerosis. Sudan IV stained areas were quantified using Adobe Photoshop software (version CS5.1) and expressed as a percentage of the total aortic arch luminal surface area. We have previously established that these measurements can be repeated with good reproducibility (Golledge et al., 2010; Krishna et al., 2012).

After the 28-day infusion with angiotensin II, mice were euthanized and aortas were harvested from their origin at the left ventricle to the iliac bifurcation, placed beside a ruler, and digitally photographed. The maximum diameters of the aortic arch, thoracic aorta and the suprarenal aorta were determined using the Adobe Photoshop CS5.1 software (Rush et al., 2009). We have previously established that these measurements can be repeated with good intraobserver reproducibility (Rush et al., 2009).

Six thoracic aortas were randomly selected from each group for RNA extraction. RNA was extracted using the TRI-reagent (Sigma-Aldrich, Castle Hill, Australia) in Eppendorf tubes according to the manufacturer's guidelines. The RNA yield of one sample from the sham surgery group was too low and not sufficient for the experiment, and thus this sample was not used for the subsequent cDNA synthesis and quantitative PCR. RNA was reverse transcribed to cDNA using the High Capacity Reverse Transcription Kit (Life Technologies). Gene expression was assessed by quantitative PCR using SYBR reagents. Primer sets are outlined in Table 1. The cycling conditions were as follows: a hold at 95°C for 2 min, followed by 40 cycles at 95°C for 15 s, 58°C for 20 s, and 72°C for 20 s. Relative gene expression was assessed using the 2^−ΔΔCt method (Livak and Schmittgen, 2001). Gene expression analysis was represented using relative gene expression compared with the control gene eukaryotic translation elongation factor 2 (EEF2) (Kouadjo et al., 2007).

Blood was collected by cardiac puncture at the time of mice sacrifice. The concentration of total cholesterol, low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) cholesterol and high-density lipoprotein (HDL) cholesterol in the plasma were quantified using a commercial available kit (Abcam, San Francisco, CA, USA; catalog number: ab65390) (Wang et al., 2014), according to the manufacturer's instructions.

Continuous numbers were presented as a median and interquartile range (IQR). The difference between two groups was analyzed using Mann-Whitney U-test. Blood pressure was compared between mice that had RDN and controls during the experimental period using a linear mixed effect (LME) model using S Plus (software version 8.2). The difference in survival between the two groups was analyzed using log rank test. The correlation between atherosclerosis severity and renal norepinephrine content was assessed using the correlation analysis function of the GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Differences were considered to be statistically significant at P < 0.05.

The baseline body weight, systolic, diastolic and mean blood pressure, and the heart rate were similar in the sham surgery and the RDN groups (P > 0.05, Table 2).

In a preliminary study, we performed RDN in seven mice and sham surgery in five mice. Five days later, mice were euthanized and norepinephrine content in the kidney was determined. RDN significantly decreased the median norepinephrine content in the kidney by 93.4% (P = 0.003, Figure 2A), which is similar to the previously reported ability of RDN to decrease norepinephrine content by 95% (Nakashima et al., 1996), suggesting that the RDN procedure was successful.

The norepinephrine in the kidney at the end of the main experiment (i.e., at the end of the 28-day angiotensin II infusion) was lower in the RDN group compared with sham surgery group (P = 0.005, Figure 2B). The median norepinephrine content in the RDN group was 71.7% of that in mice from the sham surgery group, suggesting a sustained reduction in renal innervation density.

Baseline systolic, diastolic and mean blood pressure, and heart rate were not significantly different in mice prior to RDN and sham surgery (Figure 3). After RDN and sham surgery systolic blood pressure was significantly lower in mice that had RDN compared to controls (P = 0.017, Figure 3). Diastolic and mean blood pressure were not significantly different in mice having RDN and controls (Figure 3). RDN did not affect heart rate (P > 0.05, Figure 3).

After 28 days of angiotensin II infusion, aortic arch Sudan IV staining area was significantly greater in the RDN group compared to the sham surgery group (P = 0.028, Figures 4A–C). In addition, the norepinephrine content in the kidney at the end of the experiment was negatively correlated with the Sudan IV staining area (Figure 4D).

During the angiotensin II infusion, six mice died in the RDN group and eight died in the sham surgery group due to aortic rupture. There was no difference in the survival rate between the two groups (P > 0.05, Figure 5A). Angiotensin II infusion induced aortic aneurysm formation in the suprarenal and thoracic aorta (Krishna et al., 2015). The maximum diameter of the aortic arch, thoracic aorta and suprarenal aorta were not significantly different in mice receiving RDN and sham surgery (P > 0.05, Figures 5B–D).

mRNA expression of interleulin-6 (IL-6), inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein-1 (MCP-1), nuclear factor-kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α) and matrix metalloproteinase-9 (MMP-9) within the thoracic aorta were similar in mice receiving RDN and sham surgery (Table 3). Mice receiving RDN had higher thoracic aortic mRNA expression of MMP-2 than mice receiving sham surgery (Figure 6).

mRNA expression of angiotensin receptors (both type 1 and 2) and α2 adrenoceptors in the thoracic aorta were similar in mice that received RDN and sham surgery (Table 3).

RDN did not affect plasma levels of LDL/VLDL cholesterol, HDL cholesterol, or total cholesterol (P > 0.05, Figure 7).

This study has several limitations. First, the sample size of the study was small; second, this study employed only one animal model; third, blood pressure was measured by the tail-cuff method rather than the gold standard telemetry method; and fourth, the RDN procedure in mice is different from the clinical practice in which catheter-based methods are used. Finally the plasma concentrations of lipids varied substantially in different mice and the reasons for this are not clear.

Based on our results and other experimental evidence (Murphy et al., 1957; Snyder and Campbell, 1958; Kacem et al., 1997, 2006; Kacem and Sercombe, 2008; Hachani et al., 2010), there is concern that RDN might promote more severe distant atherosclerosis severity. Clinical studies are needed to examine this possibility in patients undergoing RDN.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.