Histological evidence supporting the durability of successful radiofrequency renal denervation in a normotensive porcine model

Abstract

Background: Sustained blood pressure reductions after radiofrequency (RF) renal denervation (RDN) have been reported to 3 years in patients with uncontrolled hypertension. However, mechanistic data to support procedural durability are lacking. We aimed to quantify the long-term nerve anatomic and functional effects of RF RDN in a preclinical model.

Methods: Bilateral RF RDN was performed in 20 normotensive swine. Renal tissue samples were obtained in the RDN-treated groups at 7 ( n = 6), 28 ( n = 6), and 180 days ( n = 8) postprocedure for quantification of cortical norepinephrine (NE) levels and renal cortical axon density. Tissue fibrosis, necrosis and downstream nerve fiber atrophy (axonal loss) were also scored for each sample. Three additional untreated groups ( n = 6, n = 6 and n = 8, respectively) served as control.

Results: Pathologic nerve changes were characterized by necrosis in the ablated region at 7 days that partially resolved by 28 days and fully resolved at 180 days. Axonal loss was apparent within and downstream to the ablation regions and was evident at 7, 28 and 180 days in the main vessel and branch vessels. Consequently, renal cortical axon density and corresponding cortical NE levels were significantly reduced at 7 days in the RDN vs. control group and remained suppressed at 180 days.

Conclusions: Reductions in renal NE, cortical axon density and downstream axonal loss caused by axonal destruction persisted through 180 days post-RDN in a normotensive swine model. These results suggest functional nerve regrowth after RF RDN is unlikely and support published clinical evidence that the procedure results in durable blood pressure reduction.

FIGURE 1

Diagram of experimental design. Tissue samples were obtained and analyzed from healthy swine at 7, 28 and 180 days post radiofrequency renal denervation. Identical groups of untreated animals served as controls.

FIGURE 2

Examples of changes in nerve histology pos-tablation. Changes in nerve histology in the ablation region (left) and downstream from the ablation region (right) are shown in full sample view as well as zoomed-in view (black square area). At 7 days post radiofrequency renal denervation, inflammation (white arrow head examples), fibrosis (black arrow) and necrosis (green) are apparent in the ablated region. At 180 days, necrosis was no longer evident but fibrosis remained in the ablated region. Axonal loss (destruction of axonal body) was present within and downstream (∗) to the ablated regions at all timepoints.

FIGURE 3

Schwann Cell proliferation and fibrosis. Chaotic extra-neuronal Schwann cell proliferation (arrow) and fibrotic response at the site of RF treatment at 180 days. Anatomic proliferation was not associated with downstream functional axonal connections. Brown staining indicates Schwann cells. Green staining indicates fibrosis.

FIGURE 4

Tyrosine hydroxylase renal nerve staining. Examples of tyrosine hydroxylase nerve staining of untreated (a), full treated (b,d) and partially denervated (c,e) nerve bundles. Fully denervated nerve bundles do not stain due to absence of neuronal axons at both 7 and 180 days post denervation. Partially denervated nerves, also present at both 7 and 180 days, did not regenerate new axonal connections but remained partially denervated at 180 days.

FIGURE 5

Changes in renal norepinephrine content and cortical axon density. Comparison of changes in renal norepinephrine content (a) and renal cortical axon density (b) between radiofrequency renal denervation at 7, 28 and 180 days post radiofrequency renal denervation with untreated control groups. Both norepinephrine and renal cortical axon density were lower compared to control at all time points. Each animal contributed 2 kidneys for analysis. ∗P < 0.001 for all between group comparisons.

FIGURE 6

Quantification of mean tissue necrosis, fibrosis and nerve body atrophy. Quantification of mean tissue necrosis (a-c), fibrosis (d-f) and nerve body atrophy (axonal loss) (G-I) for each region along the renal artery at 7, 28 and 180 days post radiofrequency renal denervation: Necrosis in the ablated region of the artery was less observeable at 28 days than at 7 days and was not present at 180-days. Fibrosis was apparent at all three time points, but was generally confined to the ablated regions. Axonal loss was present within and beyond the ablated region at all three time points, indicating lack of functional nerve recovery. Gray shaded regions indicate tissue slices located approximately in the ablated region of the vessel, straw colored indicated samples acquired distal to the ablation site. More regions were sampled at 180-days as the model was updated to include branch ablation.

FIGURE 7

Nerve regeneration postradiofrequency renal denervation. Mean nerve regeneration score along the renal artery at 28 and 180-days post radiofrequency renal denervation (scale 0–3). The limited observed neurotamous regeneration was chaotic, with no evidence of functional axonal body formation. Regeneration was not apparent in any samples at 7 days post radiofrequency renal denervation. Regeneration was not consistently evident beyond the approximate ablation region (grey region).