The benefits of renal sympathetic denervation (RDN) on blood pressure have been proved in a large number of clinical trials in recent years. However, the regulatory mechanism of RDN on hypertension remains elusive. Thus, it's essential to establish a simpler RDN model in mice. In this study, osmotic mini pumps filled with Angiotensin II were implanted in 14-week-old C57BL/6 mice. One week after the implantation of the mini-osmotic pump, a modified RDN procedure was performed on bilateral renal arteries of the mice using phenol. Age-sex-matched mice were given saline and served as sham group. Blood pressure was measured at baseline and every week subsequently for 21 days. Then, renal artery, abdominal aorta and heart were collected for histological examination using H&E and Masson staining. In this study, we present a simple, practical, repeatable, and standardized RDN model, which can control hypertension and alleviate cardiac hypertrophy. The technique can denervate peripheral renal sympathetic nerves without renal artery damage. Compared to previous models, the modified RDN facilitates the study of the pathobiology and pathophysiology of hypertension.
Hypertension is a major chronic cardiovascular disease around the world. Uncontrolled hypertension could damage target organs and contribute to heart failure, stroke, and chronic kidney diseases1,2,3. The prevalence of hypertension has increased from 20% to 31% between 1991 and 2007 in China. The number of adults with hypertension in China could double following a recent revision of the diagnostic criteria for hypertension (130/80 mmHg)4. Hypertension can be controlled by medicine, however, approximately 20% of patients are unable to control their hypertension, even when receiving at least three antihypertensive drugs (including one diuretic) at maximally tolerated dose, which may lead to the development of drug-resistant hypertension5.
Renal sympathetic denervation (RDN) has been proven to be a potential treatment for hypertension. In 2009, Krum and colleagues reported resistant hypertension treatment using RDN for the first time. It was found that percutaneous renal artery ablation can effectively cause persistent blood pressure reduction in patients6. However, the failure of the Symplicity Hypertension 3 (HTN-3) trial impeded the application of RDN7, turning RDN into a controversial therapy. Nevertheless, the prospect of RDN have not yet been ruled out. Recent clinical trials, including RADIANCE-HTN SOLO, SPYRAL HTN-OFF MED/ON MED, and SPYRAL HTN-OFF MED Pivotal have confirmed the efficacy of RDN on hypertension8,9,10,11,12. Thus, more detailed mechanistic research needs to be performed to explore the effects of RDN.
The overall purpose of this study is to demonstrate how RDN in mice can be modified to produce a simpler and more stable surgery. A large number of experiments have studied various approaches of RDN, such as intravascular cryoablation, extracorporeal ultrasound and local application of a chemical or neurotoxin in different animal models13,14,15,16,17. The RDN model generated using chemical ablation with phenol is a well-established experimental model to study the pathogenesis of sympathetic activation on hypertension. This model is generated by chemical corrosion of the renal sympathetic nerves with 10% phenol/ethanol solution using a cotton swab18. On one hand, the conventional RDN potentially inhibit renal sympathetic activity, which then decreases renin secretion and sodium reabsorption, and increases renal blood flow. On the other hand, it suppresses renin-angiotensin-aldosterone system19. Thereby, RDN has a beneficial effect on hypertension. However, the chemical ablation generated RDN model lacks ablation criteria and ablation time and the details of the experimental procedure are yet unclear. Also, there are no technical reports available. In this report, we describe a surgical protocol for the generation of RDN model with phenol using weigh paper in Angiotensin II (Ang II) induced hypertension in C57BL/6 mice. We wrap the renal artery with weighing paper containing phenol and unify the ablation time, which helps to establish a more reproducible, reliable RDN model. This experimental model is aimed to evaluate the effect of RDN on hypertension.
All animal experimental procedures complied with the relevant ethical Guide for the Care and Use of Laboratory Animals (NIH Publication no. 85-23, revised 2011) and were approved by the committees on animal research of Huadong Hospital affiliated to Fudan University. Fourteen-week-old male C57BL/6 mice (28-30g) were randomly divided into four groups: Sham group, Sham+Ang II group, RDN group, RDN+Ang II group, n = 6 in each group. All the animals were maintained in a temperature-controlled room with a 12 h light/dark cycle and were given free access to tap water and standard rat chow.
1. Preparation of the operation field
- Disinfect the operation table with 70% ethanol. Adjust the heating pad temperature to 37 °C.
- Ensure that all surgical instruments are sterilized before surgery at 121 °C for 30 min or by other methods. This procedure requires micro surgical scissors, two fine straight forceps, two fine curved forceps, hemostatic forceps, sterile gauzes, and weighing papers.
2. Angiotensin II induced hypertension
- Anesthetize C57BL/6 mice using sodium pentobarbital injection as previously described20,21. Isoflurane can also be used, if preferred. Confirm anesthesia depth with a negative toe pinch reflex.
- Remove the hair on the back with a shaver. Apply vet ointment to the eyes to preventdryness while under anesthesia.
- Place the animal on an operating table in the dorsal position. Swab and wipe the shaved area with povidone-iodine followed by three wipes with 70% ethanol.
- Make a 1 cm incision using sterile micro surgical scissors, perpendicular to the tail, behind the ear over the shoulder blade of the front leg.
- Use a sterile hemostat to make a subcutaneous tunnel under the skin and create a pocket for the pump22. Insert an osmotic pump filled with Angiotensin II (1,000 ng/kg/min) into the pocket gently. Ensure that there is enough free space to suture the wound without stretching the skin.
- Suture the incision with a 4-0 cotton silk. Swab and wipe the wound site with povidone-iodine. Perform the same surgery with equal volume of saline for the control group.
- Place all the surgical instruments into a sterilizer for 10 s and replace the sterile gloves between surgeries. Monitor all mice until fully recovered.
- Closely monitor and observe wound healing in mice at least twice a day during the first week and once every day subsequently, including redness, swelling, and infection. Perform dissection immediately if the mice die during Ang II infusion.
- Measure blood pressure at baseline and every week after Ang II infusion with the tail-cuff plethysmography method23 in conscious mice. Ensure that the blood pressure measurement experiments are conducted in a quiet area, at 22 ± 2 °C, where mice are acclimatized for 1 h before the experiment begins. Habituate mice for at least 5 consecutive days before baseline blood pressure measurements23,24.
3. Bilateral renal denervation
- Select the mice with elevated blood pressure (BP) ≥140/90mmHg or 25% increase in systolic BP/diastolic BP, 1 week after the Ang II infusion.
- Fast the animals and stop water supply 12 h before the surgery. Record animal weight before surgery and choose animals with a minimum weight of 24 g for renal denervation surgery.
- Anesthetize the mice using sodium pentobarbital. Confirm anesthesia depth with a negative toe pinch reflex.
- Remove the hair on the abdomen with a shaver. Perform this procedure carefully and thoroughly to avoid any surgical contamination.
- Place the mice on the operating table, keeping the abdomen up and fix its limbs with tape. Disinfect abdominal skin with povidone-iodine.
- Make a 2 cm ventral midline abdominal incision using microsurgical scissors. Pull back the intestine with gauze soaked in 37 °C saline to expose the left renal artery. Carefully yet bluntly dissect the fat away from the renal artery using curved tweezers. (Figure 1A-C).
- Cut the weighing paper into a rectangle of the same size as the renal artery with sterile sharp scissors. For reference, cut the weighing paper in the same size as shown by the dotted line in Figure 1C.
NOTE: It is a critical part of the surgery, try to cut several pieces of the weighing paper at a time to keep the same shape.
- Dip the weighing paper in 10% phenol/ethanol solution for at least 30 s. Cover the surface of the left renal artery and wrap the vessel with the weighing paper, keep for 2 min (Figure 1D). Use gauze to protect the surrounding tissues to avoid the weighing paper from touching the surrounding kidney and intestine.
NOTE: The phenol solution is stable in plastic tubes but not in glass vials. Therefore, the solution must be freshly prepared for every experiment18.
- Perform the same procedure for the right renal artery. Perform the sham surgery with weighing paper immersed in saline.
- Reposition muscles into their initial position and close the skin with a 6-0 cotton silk in an interrupted suture pattern. Close the skin with 4-0 cotton silk with interrupted sutures. Monitor all mice until fully recovered.
4. Post-operative care
- Apply povidone-iodine to the incision and inject ketoprofen intraperitoneally (5 mg/kg). Place the animal in a warmed electric blanket for recovery and post-operative monitoring.
- Monitor the mice twice a day to assess for redness, swelling, and pain or abdominal infection. Provide meloxicam (5 mg/kg, SC) to all mice about 1 h before and 24 h after the RDN procedure.
All data are expressed as mean ± standard deviation. One-way ANOVA was used for experiments with three or more conditions followed by Bonferroni posthoc tests for comparisons between individual groups. Consider a p-value equal or less than 0.05 as significant. A commercial software was used to perform all statistical analysis.
Increase in blood pressure induced by Ang II was attenuated after RDN
Significant increase in systolic BP (SBP) was observed at 1 week after Ang II infusion. The RDN + Ang II group showed significant reduction in SBP when compared with the Sham + Ang II group at 21 days after RDN procedure (143.50 ± 5.43 vs 196.67 ± 14.26 mmHg, p < 0.01). There was no difference between Sham group and RDN group (113.33 ± 9.35 vs 113.17 ± 8.47 mmHg, p > 0.05) at 2 weeks after RDN (Figure 2).
Confirmation of RDN and damage of renal artery
After 21 days of Ang II infusion, the animals were euthanized with intraperitoneal injection of sodium pentobarbital (250 mg/kg). Heart and kidney were collected. H&E staining was performed to detect the damage to the renal nerve and renal arteries. The results showed that there was no obvious thickening of the renal vascular intimal layer in each group (Figure 3A-D). H&E staining of renal nerves showed a large number of pyknotic nuclei, digestion chambers, and swelling nerve nuclei caused by RDN (Figure 3E-H). Immunohistochemistry of the nerve bundles revealed that the expression of tyrosine hydroxylase (TH, 1:500 dilution) was significantly decreased in RDN group and RDN+Ang II group (Figure 4). RDN reduced kidney cortical norepinephrine content in both normotensive and hypertensive group (sham group vs RDN group, 18.60 ± 6.91 vs 180.76 ± 11.47 ng/g, p < 0.01; Figure 5).
RDN treatment mitigated Ang II infusion induced pathological cardiac hypertrophy
Masson staining showed no remarkable increase in intima media of abdominal aorta among these groups. Ang II infusion induced cardiac hypertrophy was improved by RDN treatment as shown by the decrease in interstitial fibrosis (7.45% ± 0.28 vs 4.53% ± 0.32, p < 0.01) and cardiomyocyte size (348.39 ± 31.56 vs 322.21 ± 22.26 µm, p = 0.37; Figure 6).
Figure 1: Procedure of RDN with weighing paper. (A,B) Anatomical images of renal artery from C57BL/6 (ex vivo) mice. (C) The part within the two dotted lines refers to the area covered by the weighing paper. (D) Covering the surface of the bilateral renal artery with the appropriate weighing paper immersed in 10% phenol/ethanol solution. Do not cover the filter paper beyond the dotted line. * indicates the weighing paper. Please click here to view a larger version of this figure.
Figure 2: RDN alleviates hypertension induced by Ang II infusion. Blood pressure was measured by tail-cuff plethysmography method at baseline and every week after Ang II infusion. * indicates statistical significance (p < 0.05), ** indicates statistical significance (p < 0.01). Values are represented as mean ± standard error; N = 6 in each group; RDN + Ang II group indicates renal denervation operated on 1 week after Ang II infusion in C57BL/6 mice. Abbreviations: SBP = systolic blood pressure. Please click here to view a larger version of this figure.
Figure 3: Representative images of renal sympathetic nerve and renal artery. (A-D) No thickening of the intima layer of renal artery was observed in the four groups. Representative images of the damaged renal nerves after RDN. (E-H) Fragmented and pyknotic nuclei, digestion, swelling of endoneural tissue were observed in both RDN and RDN + Ang II group. Please click here to view a larger version of this figure.
Figure 4: Immunostaining of tyrosine hydroxylase in renal sympathetic nerve. (A) Strong positive reaction to TH-antibody staining was observed in sham-operated mice, whereas a weaker reaction was observed in RDN-operated mice. Scale bar = 50 µm. (B) Quantification of TH expression in renal nerves. ** indicates statistical significance (p < 0.01), ns indicates not significant. Values are mean ± standard error; N = 6 in each group; RDN + Ang II group indicates renal denervation operated on 1 week after Ang II infusion in C57BL/6 mice. Please click here to view a larger version of this figure.
Figure 5: Renal cortical tissue norepinephrine levels analyzed by ELISA. Renal cortical norepinephrine content in the denervated kidneys was markedly decreased compared with those from the innervated kidney. ** indicates statistical significance (p < 0.01), ns indicates not significant. Values are mean ± standard error; N = 6 in each group; RDN+Ang II group indicates renal denervation operated on 1 week after Ang II infusion in C57BL/6. Please click here to view a larger version of this figure.
Figure 6: RDN relieves Ang II induced pathological cardiac hypertrophy. (A) Representative pictures of abdominal aorta. No thickening of the intima layer of abdominal aorta was observed in these group (Masson staining). (B,C) Representative images of the myocardium in different groups (H&E, Masson staining). (D) Quantification of percentage of fibrosis in left ventricular area and the analysis of the percentage of fibrosis area (the number of visual fields per mice). Scale bar = 50 µm. N = 6 in each group; RDN + Ang II indicates renal denervation operated on 1 week after Ang II infusion in C57BL/6. Please click here to view a larger version of this figure.
Whether RDN could lower blood pressure has become controversial since the publication of the negative result of the symplicity HTN-3 trial7,25. However, the several clinical trials and animal experiments have demonstrated positive and effective results of RDN on hypertensive humans and animals9,10,11,12,13,14,15,16,17. Phenol is used for destruction of the renal nerve in animals, and the details of ablation remain unknown in previous research, such as the ablation area and ablation time, which might have contributed to different results16.
The conventional methods for RDN, such as using cotton swab with phenol in rats, catheter-based ablation, and stereotactic radiotherapy in swine, cause damage to the renal nerve18,26,27,28. These methods are also not suitable for mice, which weigh only tens of grams, and are more likely to lead to death. Besides, these methods cause renal artery stenosis. In fact, we prepared RDN models and used these methods in our pre-experiment. However, 40/50 mice died. The method using cotton swab with phenol resulted in high mortality.
Thus, in this study, a method that enables the standardized performance of RDN, but requires less surgical skill and reduced operation time was established. We used 10% phenol/ethanol solution-soaked weighing paper, placed for 2 min on the renal artery, which provides a reliable method to corrode the renal sympathetic nerve in mice. Its effectiveness is confirmed by histopathology of the renal nerve. It significantly attenuated the SBP elevation induced by Ang II. Moreover, it also alleviated Ang II-induced cardiac hypertrophy. In addition, the improved procedure has several characteristics, including easy to perform and increased success rate and survival rates when compared with conventional procedures.
The most critical part of the protocol is that the weighing paper with phenol should not touch the surrounding tissues, otherwise, it may cause fatal intestinal obstruction, abdominal infection, and renal artery stenosis. It is advised not to touch the solution to the kidney as only a small amount of phenol can possibly cause renal sympathetic overactivity18. Besides, special attention should be paid when cutting the weighing paper. It is better to tailor it under the microscope with surgical scissors. We do not recommend isolating renal nerves with micro-tweezers, as this may damage renal blood vessels. Usually, the procedure can be safely performed within 20 min, even with slow performance. Furthermore, the melting point of phenol is 40.5 °C.
The major limitation of the improved RDN procedure is that the postoperative follow-up time was only 2 weeks. The effect of long-term RDN on BP and renal nerve regeneration is unclear.
Future application of this model is to produce more standardized denervation animal models that can contribute to expounding the pathways that underlie the process of hypertension and cardiac hypertrophy.
In conclusion, this method is practical and repeatable. Most importantly, it can generate standardized RDN models to study the mechanisms that control hypertension and combat cardiovascular diseases such as cardiac hypertrophy.
There are no conflicts of interest, financial or otherwise, as declared by the authors.
This work was supported by the National Natural Science Foundation of China (81770420), Science and Technology Commission of Shanghai Municipality (20140900600), Shanghai Key Laboratory of Clinical Geriatric Medicine (13dz2260700), Shanghai Municipal Key Clinical Specialty (shslczdzk02801) and Center of geriatric coronary artery disease, Huadong Hospital Affiliated to Fudan University.
|Angiotensin II||Sangon Biotech||CAS:4474-91-3||To make a hypertensive animol model|
|Anti-Tyrosine Hydroxylase antibody||Abcam||ab137869||To evaluate the expression of TH of renal nerves|
|Blood Pressure Analysis||Visitech Systems||BP-2000||Measure the blood pressure of mice|
|Mini-osmotic pump||DURECT Corporation||CA 95014||To fill with Angiotensin II|
|Norepinephrine ELISA Kit||Abcam||ab287789||to measure renal norepinephrine levels|
|Phenol||Sangon Biotech||CAS:108-95-2||Damage the renal sympathetic nerve|
|Weighing paper||Sangon Biotech||F512112||To destroy renal nerve with weighing paper immersed with phenol; https://www.sangon.com/productDetail?productInfo.code=F512112.|
- Messerli, F. H., Rimoldi, S. F., Bangalore, S. The transition from hypertension to heart failure: Contemporary update. JACC Heart Failure. 5, (8), 543-551 (2017).
- Lackland, D. T., et al. Implications of recent clinical trials and hypertension guidelines on stroke and future cerebrovascular research. Stroke. 49, (3), 772-779 (2018).
- Rossignol, P., et al. The double challenge of resistant hypertension and chronic kidney disease. The Lancet. 386, (10003), 1588-1598 (2015).
- Du, X., Patel, A., Anderson, C. S., Dong, J., Ma, C. Epidemiology of cardiovascular disease in China and opportunities for improvement. JACC International. Journal of the American College of Cardiology. 73, (24), 3135-3147 (2019).
- Valenzuela, P. L., et al. Lifestyle interventions for the prevention and treatment of hypertension. Nature Review Cardiology. 18, (4), 251-275 (2021).
- Krum, H., et al. Catheter-based renal sympathetic denervation for resistant hypertension: a multicentre safety and proof-of-principle cohort study. The Lancet. 373, (9671), 1275-1281 (2009).
- Bhatt, D. L., et al. A controlled trial of renal denervation for resistant hypertension. The New England Journal of Medicine. 370, (15), 1393-1401 (2014).
- Kjeldsen, S. E., Narkiewicz, K., Burnier, M., Oparil, S. Renal denervation achieved by endovascular delivery of ultrasound in RADIANCE-HTN SOLO or by radiofrequency energy in SPYRAL HTN-OFF and SPYRAL-ON lowers blood pressure. Blood Press. 27, (4), 185-187 (2018).
- Böhm, M., et al. Efficacy of catheter-based renal denervation in the absence of antihypertensive medications (SPYRAL HTN-OFF MED Pivotal): a multicentre, randomised, sham-controlled trial. The Lancet. 395, (10234), 1444-1451 (2020).
- Azizi, M., et al. Endovascular ultrasound renal denervation to treat hypertension (RADIANCE-HTN SOLO): a multicentre, international, single-blind, randomised, sham-controlled trial. The Lancet. 391, (10137), 2335-2345 (2018).
- Kandzari, D. E., et al. Effect of renal denervation on blood pressure in the presence of antihypertensive drugs: 6-month efficacy and safety results from the SPYRAL HTN-ON MED proof-of-concept randomised trial. The Lancet. 391, (10137), 2346-2355 (2018).
- Townsend, R. R., et al. Catheter-based renal denervation in patients with uncontrolled hypertension in the absence of antihypertensive medications (SPYRAL HTN-OFF MED): a randomised, sham-controlled, proof-of-concept trial. The Lancet. 390, (10108), 2160-2170 (2017).
- Sun, X., et al. Renal denervation restrains the inflammatory response in myocardial ischemia-reperfusion injury. Basic Research in Cardiology. 115, (2), 15 (2020).
- Sharp, T. E., et al. Renal denervation prevents heart failure progression via inhibition of the renin-angiotensin system. Journal of the American College of Cardiology. 72, (21), 2609-2621 (2018).
- Wang, H., et al. Renal denervation attenuates progression of atherosclerosis in apolipoprotein E-deficient mice independent of blood pressure lowering. Hypertension. 65, (4), 758-765 (2015).
- Chen, H., et al. Renal denervation mitigates atherosclerosis in ApoE-/- mice via the suppression of inflammation. American Journal of Translational Research. 12, (9), 5362-5380 (2020).
- Wang, Y., et al. Renal denervation promotes atherosclerosis in hypertensive apolipoprotein E-deficient mice infused with Angiotensin II. Frontiers in Physiology. 8, 215 (2017).
- Eriguchi, M., Tsuruya, K. Renal sympathetic denervation in rats. Methods in Molecular Biology. 1397, 45-52 (2016).
- Thukkani, A. K., Bhatt, D. L. Renal denervation therapy for hypertension. Circulation. 128, (20), 2251-2254 (2013).
- Zhang, Y. J., et al. NAD(+) administration decreases microvascular damage following cardiac ischemia/reperfusion by restoring autophagic flux. Basic Research in Cardiology. 115, (5), 57 (2020).
- Wang, M., et al. Long-term renal sympathetic denervation ameliorates renal fibrosis and delays the onset of hypertension in spontaneously hypertensive rats. American Journal of Translational Research. 10, (12), 4042-4053 (2018).
- Lu, H., et al. Subcutaneous Angiotensin II infusion using osmotic pumps induces aortic aneurysms in mice. Journal of Visualized Experiments: JoVE. (103), e53191 (2015).
- Wilde, E., et al. Tail-cuff technique and its influence on central blood pressure in the mouse. Journal of the American Heart Association. 6, (6), 005204 (2017).
- Daugherty, A., Rateri, D., Hong, L., Balakrishnan, A. Measuring blood pressure in mice using volume pressure recording, a tail-cuff method. Journal of Visualized Experiments: JoVE. (27), e1291 (2009).
- Esler, M. Illusions of truths in the Symplicity HTN-3 trial: generic design strengths but neuroscience failings. Journal of the American Society of Hypertension. 8, (8), 593-598 (2014).
- Han, W., et al. Low-dose sustained-release deoxycorticosterone acetate-induced hypertension in Bama miniature pigs for renal sympathetic nerve denervation. Journal of the American Society of Hypertension. 11, (5), 314-320 (2017).
- Han, W., et al. The safety of renal denervation as assessed by optical coherence tomography: pre- and post-procedure comparison with multi-electrode ablation catheter in animal experiment. Hellenic Journal of Cardiology. 61, (3), 190-196 (2020).
- Cai, X., et al. Noninvasive stereotactic radiotherapy for renal denervation in a swine model. Journal of the American College of Cardiology. 74, (13), 1697-1709 (2019).