Sixteen male or female Beagle dogs aged 10–12 months and weighing 9.4–11.6 kg were provided and housed by the Laboratory Animal Center of the Soochow University (Jiangsu Province, China). Procedures were performed under the guidance of professional researchers in a clean laminar flow operating room. Perioperative feeding and care of the animals were carried out by professional staffs. Neurogenic hypertension was generated in all dogs, which were randomly divided into two groups, including the RDN group (n = 12) receiving L-RDN and the sham group (n = 4) receiving similar procedures except for radiofrequency ablation.
Hypertension model induction
The neurogenic hypertension model was established via carotid artery as previously described by Jannetta et al.In all surgeries, anesthesia was induced with 3% pentobarbital sodium injection (30 mg/kg) and endotracheal intubation. If the operation last more than 2 hours, 3mg/kg pentobarbital sodium should be added every half hour. After successful anesthesia and conventional disinfection, a vertical cervical incision was performed to the left side of the midline to allow sequential separation of the skin, muscles, and connective tissue layers. After full exposure of the left common carotid artery (LCA) and isolation of the vagal trunk, 4-0 enteric chromium surgical suture was cut into 1-2mm fragments and placed lightly around the vagus nerves. The vagus nerve and surrounding connective tissue were fixed to form cross compression between the vagus nerve and the LCA, and the regular pulse of the carotid artery formed continuous compression on the vagus nerve.
After successful anesthesia, each animal in the RDN group was placed in a supine position with head and limbs secured on an operating table. The abdominal skin was prepared by shaving and disinfection. A 1-cm midline abdominal incision was made at 3 cm below the umbilicus. A double-channel trocar of 10-mm in diameter was inserted, through which an operative laparoscope (Karl Storz, Tuttlingen, Germany) was introduced. Carbon dioxide pneumoperitoneum of 10-12 mmHg was generated. Two additional 5-mm trocars were inserted through incisions in both midaxillary lines at 15 cm above the anterior iliac crest. Another two 5-mm trocars were inserted at the middle points between the laparoscope trocar and the anterior iliac crest trocars. The laparoscopic hook electrode, suction and elastic separating plier were inserted as appropriate. A combination of blunt and sharp dissection of the renal fascia was applied until the renal hilus was visualized clearly. The radiofrequency ablation catheter (7F IBI radiofrequency ablation catheter; St. Jude Medical) was inserted through the side port of the double-channel trocar towards the renal artery and connected to a radiofrequency ablation device (IBI, St. Jude Medical, Inc., St. Paul, MN, USA) (figure 1A, 1B). Six to eight ablation sites were selected from bilateral trunks of renal arteries through branches. Each spot was ablated for 60 sec, with a power limit of eight W, until the vascular adventitial temperature reached 55°C. During the procedure, we ablated bilateral renal arteries from the proximal end to the distal end of the renal arterial trunk. After ablation of all points, laparoscopic devices were removed from the abdominal cavity and incisions were closed layer by layer with sutures. Each dog was given 1.6 x106 units of penicillin i.m. postoperatively for three consecutive days. Dogs in the sham group underwent similar laparoscopic surgery except for radiofrequency ablation.
Blood pressure monitoring
Non-invasive blood pressure (NIBP) was measured using a blood pressure cuff around one thigh of the hind limbs and connected to a multifunctional blood pressure monitor. NIBP were monitored before hypertension induction (defined as the baseline) and 2, 4, 6, and 8 weeks after hypertension induction. At the eighth week after hypertension induction, L-RDN and sham surgeries were performed in the RDN group and sham group, respectively, and changes in NIBP were recorded at 2, 4, 6, and 8 weeks after the procedures.
To measure invasive blood pressure (IBP), animals were anesthetized and the right femoral area was disinfected. After successful puncture with a syringe, a pressure transducer (TruWave PX260, Edwards Lifesciences, USA) was attached for temporal IBP measurement using a PHILIPS IntelliVue X2 System. IBP was monitored before hypertension induction (defined as the baseline), at 8 weeks after hypertension induction, and at 8 weeks after RDN or sham procedures.
Renal function and norepinephrine levels determination
The renal functions and norepinephrine (NE) levels before hypertension induction (defined as the baseline), at 8 weeks after hypertension induction, and at 8 weeks after RDN or sham procedures were measured at the Institute of Neuroscience and Laboratory Animal Center of Soochow University. All Beagle dogs were humanely killed by intravenous injection of potassium chloride solution（2mg/kg）under anesthesia at 8 weeks after RDN.
Histological analysis and immunofluorescence staining
All experimental animals were humanely killed 8 weeks after RDN to isolate the complete renal arteries and kidneys for gross specimen examination for evidence of stenosis, dissection, and perforation in renal arteries. Tissue specimens were fixed in 4% paraformaldehyde, followed by paraffin-embedding, tissue sectioning, and hematoxylin and eosin (HE) staining using a commercial kit (C0105, Beyotime, Shanghai, China). Light microscopy was used to evaluate the histomorphological changes of the renal arteries in different groups.
Transverse sections of the renal artery(15μm, Leica Cryostat CM3050) were collected directly in glass slides (4–8slides/tissue, 2–4 slices/slide) and incubated with a blocking buffer containing 10% goat serum, 1% bovine serum albumin (BSA), and 0.3% Triton X-100 for 1 h. The sections were then incubated with anti-VEGF antibody (1:1000, Abcam, USA) at 4°C overnight. After washing with PBS for 3 times, the sections were incubated with fluorescent-conjugated anti-rabbit secondary antibodies (1:1000, Jackson ImmunoResesrch Laboratories, PA, USA) and counterstained with DAPI for 1 h. All slides were examined under a fluorescence microscope (AXIO SCOPE A1, ZEISS, Germany).
Statistical analysis was performed using SPSS software (version 22, SPSS Inc., Chicago, Illinois, USA). Continuous variables with normal and skewed distribution were expressed as means with standard deviations or medians with interquartile ranges. Categorical variables were presented as numbers and percentages. Changes in BP were expressed as mean (95% CI). Independent samples t-test and Mann-Whitney U test were used to compare the differences in continuous variables with normal and skewed distribution between RDN and sham groups. Comparison of continuous variables with normal and skewed distribution at different time points in the same group was performed using paired t-test and Mann-Whitney U test, respectively. Categorical data between the groups were compared using the Chi-square test. Homogeneity-of-variance was determined by Levene’s test of equality of variances. A two-way repeated measures ANOVA (blood pressure × time after the procedure) adjusted for baseline blood pressure was performed using the BP reduction as dependent variables. Sphericity was determined by Mauchly’s test, and interactions between variables were estimated. Variables were compared using the Greenhouse-Geisser adjustment method when sphericity was absent. Otherwise, comparisons were performed using the simple effects tests if interactions were present, or using the tests of within-subjects effects if no significant interactions were observed. Bootstrapping (1000 replications) with a simple percentile 95% CI was used to validate the results. A p-value <0.05 was considered as statistically significant.