All 36 pigs underwent surgery. Of these animals, 18 underwent bilateral laparoscopic-based perivascular RDN, and the remaining pigs underwent sham operations. No side effects were observed and no pigs died unexpectedly during the experiment. Each artery in the RDN group was ablated at 6 points longitudinally and rotationally, the ablation points were uniformly distributed in the main renal artery, and the ablation time of every point was 120 s. The mean energy delivered to the tissues was 7.9 ± 0.43 W, the temperature was 43.95 ± 1.45 °C and the impedance was 210.78 ± 4.71 Ω.
Body weight, serum total cholesterol, triglyceride, creatinine, cystatin C and neutrophil gelatinase-associated lipocalin levels
After the pigs consumed the high-fat diet, body weight and serum TC and TG levels were significantly increased. The body weight of the Bama pigs increased significantly from 21.69 ± 0.78 kg at baseline to 64.15 ± 3.12 kg (P < 0.001) at day 180 in the RDN group, but the difference was not significant compared with the sham group. Serum TC and TG levels were increased from 2.56 ± 0.14 mmol/l and 1.07 ± 0.10 mmol/l at baseline to 3.64 ± 0.29 mmol/l (P = 0.004) and 1.73 ± 0.13 mmol/l (P = 0.001), respectively, at day 180 in the RDN group, but were not significantly different from the values of the sham group. The serum creatinine, cystatin C and neutrophil gelatinase-associated lipocalin (NGAL) levels were not significantly different between the two groups (Fig. 2).
Changes in blood pressure at the 4 time points
Before the surgery and high-fat diet feeding, the baseline systolic blood pressure was 127.67 ± 2.67 mmHg in the RDN group and 128.78 ± 2.08 mmHg in the sham group (P = 0.743), while diastolic blood pressure was 75.61 ± 1.70 mmHg in the RDN group and 74.50 ± 2.87 mmHg in the sham group, and the differences in SBP and DBP were not significant between the two groups (P = 0.678).
Two days after the surgery, SBP and ΔSBP were significantly lower in the RDN group than in sham group (113.83 ± 3.26 mmHg vs 129.67 ± 3.32 mmHg, P = 0.011, and − 15.00 ± 3.77 mmHg vs 3.33 ± 2.68 mmHg, P = 0.005, respectively), while non-significant differences in both DBP and ΔDBP (70.83 ± 2.54 mmHg vs 73.50 ± 2.95 mmHg, P = 0.254, and − 4.83 ± 2.22 mmHg vs − 0.50 ± 2.28 mmHg, P = 0.102, respectively) were observed.
At day 90, the values of SBP, ΔSBP, DBP and ΔDBP in the RDN group were significantly lower than in the sham group (116.83 ± 3.88 mmHg vs 145.00 ± 4.22 mmHg, P = 0.001, − 7.17 ± 4.28 mmHg vs 11.67 ± 3.61 mmHg, P = 0.012, 72.17 ± 2.7 mmHg vs 81.50 ± 2.22 mmHg, P = 0.037, and − 3.00 ± 3.33 vs 7.5 ± 3.08 mmHg, P = 0.022, respectively).
At day 180, significantly lower SBP, ΔSBP, DBP and ΔDBP were observed in the RDN group than in the sham group (129.33 ± 2.87 mmHg vs 168.57 ± 2.86 mmHg, P < 0.001, − 2.33 ± 2.19 mmHg vs 42.00 ± 4.34 mmHg, P < 0.001, 76.83 ± 2.75 mmHg vs 86.33 ± 2.22 mmHg, P = 0.021, and 0.833 ± 3.33 mmHg vs 10.83 ± 3.66 mmHg, P = 0.035, respectively) (Fig. 3).
Arteriography, optical coherence tomography and pathological evaluation of the atrial lumen and arterial wall
Arteriography is the gold standard for identifying vessel narrowing of the vascular lumen, while OCT, which has a high axial resolution of 10–20 μm, accurately visualizes arterial wall lesions. All renal arteries were assessed using arteriography and OCT scanning. Spasms were immediately observed after laparoscopic-based perivascular RDN (Fig. 4), but no spasms were observed during subsequent assessments on days 2, 90 and 180. No aneurysmal changes, thrombi or other abnormalities were noted in the lumen or arterial wall in the 180-day study. HE staining did not reveal thrombi, dissections, aneurysms, perforations, hematomas, neointimal formation, or negative remodeling. Representative images of the arterial lumen at different time points are shown in Fig. 5.
Pathological and immunohistochemical evaluations of nerve fascicles
Two days after laparoscopic-based perivascular RDN, HE staining of the affected arterial section indicated that nerve fascicles surrounding renal arteries exhibited vacuolization and nuclear pyknosis, and the endoneurium became disorganized. In contrast, the tissues of the sham group were intact.
The S100 protein is a marker of Schwann cells (SCs), which wrap around the axons of nerve cells. TH is the rate-limiting enzyme involved in catecholamine synthesis within the postganglionic nerve terminals, and TH expression has also been used as a functional marker of the activity of renal sympathetic nerve fascicles. Immunohistochemical staining for the S100 protein and TH protein revealed no significant differences between the sham group and RDN group 2 days after laparoscopic-based perivascular RDN.
At day 90, the affected nerve epineurium resembled a thick layer, the nerve bundles had atrophied, and endoneurial and perineural fibrosis were observed. Stained sections from the RDN group showed a marked decrease in levels of the TH protein but an increase in levels of the S100 protein compared with the sham group.
At day 180, the thick layer, endoneurial and perineural fibrosis were also observed. In addition, some disordered nerve regrowth mixed with connective tissue was observed at the site of radiofrequency ablation, along with a decrease in immunohistochemical staining for the S100 protein and TH protein compared with sham group but a slight increase compared with day 90. Representative images of the sympathetic nerve fascicles at different time points are shown in Fig. 6.
Expression of the TH and S100 proteins in renal arteries, and norepinephrine concentration in the renal tissue
Two days after surgery, the expression of both the TH and S100 proteins was not significantly different between the RDN group and sham group. Significantly lower TH expression was observed in the RDN group than in the sham group at 90 days and 180 days after surgery, while the expression of the S100 protein in the atria was increased at day 90 and day 180 after RDN compared with the sham group. The NE concentration in renal tissues from RDN group was significantly lower than in the sham group (112.02 ± 17.34 ng/g vs 268.48 ± 20.61 ng/g, P < 0.001, 152.15 ± 16.61 ng/g vs 318.97 ± 24.84 ng/g, P < 0.001, and 190.56 ± 19.78 ng/g vs 355.58 ± 27.65 ng/g, P = 0.001, respectively) (Fig. 7).