Renal Denervation by Noninvasive Stereotactic Radiotherapy Induces Persistent Reduction of Sympathetic Activity in a Hypertensive Swine Model

Figures

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  Figure 1. Screenshot of external beam planning system. Axial (left top panel), sagittal (left lower panel), and coronal (right lower panel) views of contouring outcome and radiation delivery route (yellow arc in left top panel) of 25 Gy to radioablate renal nerves. Dose volume histogram (right top panel) demonstrates the designated dose concentrates on the planning target areas around the left (red) and right (green) renal arteries with a rapid dose falloff to adjacent critical structures, including left (yellow) and right (pink) kidneys, renal vein (orange), aorta (brown), spinal cord (dark blue), and bowel (light blue).

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  Figure 2. Blood pressure. Systolic blood pressure (SBP) (A) and diastolic blood pressure (DBP) (B) at baseline and 1‐ and 6‐month follow‐up in control and 6‐month ablation groups (mean±SD; n=5; unpaired Student t test).

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  Figure 3. Chronological evolution of renal nerve injury. A, Semiquantitative scores of nerve injury in control and treatment groups (mean±SD; n=4 for 1‐month group and n=5 for the other groups; Kruskal‐Wallis test, followed by all pairwise multiple comparisons). B, Representative images of nerves stained with hematoxylin and eosin at various time points (top) and magnified photomicrographs (bottom, red squares in top). Control nerves were chosen from untreated vessels at 6 months. Nerve fascicle at 1 month shows mild to moderate perineural fibrosis, vacuolization, and digestion chambers. Nerve fascicle at 6 months illustrates mild perineural fibrosis and calcification of necrotic endoneurium.

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  Figure 4. Renal nerve apoptosis. A, Example images of active caspase‐3–positive nerves in control and treatment groups. B, Quantitative analysis of renal nerve apoptosis (mean±SD; n=4; Kruskal‐Wallis test, followed by all pairwise multiple comparisons). DAPI indicates 4′,6‐diamidino‐2‐phenylindole. *P<0.05 vs control.

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  Figure 5. Immunohistochemistry of renal nerves. A, Representative micrographs of untreated normal nerve and ablated nerves at 1 and 6 months postprocedure with hematoxylin and eosin (H&E) and immunohistochemical stains against S‐100, tyrosine hydroxylase (TH), and GAP43 (growth‐associated protein 43). Positive reaction to S‐100 confirms the presence of nerve fascicles. Compared with strong TH staining in normal nerve, decreased expression of TH, which demonstrates functional nerve damage, is continuously observed in treated nerves, whereas GAP43 expression, an indicator for nerve regeneration, remains weak throughout 6 months. Semiquantitative scores of TH (B) and GAP43 (C) in control and treatment groups (mean±SD; n=5; Kruskal‐Wallis test, followed by all pairwise multiple comparisons). *P<0.05 vs control.

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  Figure 6. Radiation‐induced denervative effect in depth. A, Semiquantitative scores of nerve injury in separate regions of <2, 2 to 4, and >4 mm from arterial lumen in control and treatment groups (mean±SD; n=4 for 1‐month group and n=5 for the other groups; 2‐way ANOVA, between group; Bonferroni multiple comparison test). *P<0.05 vs control. B, Treatment plan example of renal nerve radioablation with 25 Gy in axial plane of contrast‐enhanced computed tomography scan, showing colored isodose lines at approximate distances of 2, 4, 6, 8, and 10 mm from renal arterial lumen. The planning target volume is highlighted in cyan; and red, orange, yellow, green, and blue curves indicate the distribution of zones receiving doses of at least 24.5, 23.6, 21.5, 19.0, and 15.8 Gy, respectively. C, Spearman correlation between nerve injury scores at 6 months postprocedure in <2‐, 2‐ to 4‐, and >4‐mm areas and the minimum radiation dosages at isodose lines of 2, 3, and 4 mm from arterial wall.

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  Figure 7. Renal arterial and periarterial tissue injury. (Top) Representative images of renal arteries and circumferential area stained with Movat pentachrome in control and treatment groups. Magnified photomicrographs of renal arteries (middle, red squares in top) and arterioles (bottom, blue squares in top). Renal arteries at 1 month show no apparent medial injury, whereas mild proteoglycan replacement of smooth muscle cell loss, which stains green, is occasionally found at 6 months. Minimal eccentric thickening of neointima (yellow arrow) formed on the internal elastic lamina is observed at 6 months after ablation. Arterioles at 1 month show mild perivascular inflammation and smooth muscle cell loss. Arteriolar occlusion, induced by intimal hyperplasia and thrombosis, is frequently observed at 6 months.