Improved Renal Denervation Mitigated Hypertension Induced by Angiotensin II Infusion

Renal sympathetic denervation is a potential treatment for hypertension, which have been proven by many clinical trials. This technique denervates free flow renal nerves without renal artery damage. Moreover, it's a simple, critical, repeatable, and standardized model.

Demonstrating the procedure will be Ming Wang, a lab assistant from my laboratory. Begin by disinfecting the operation table with 70%ethanol, then adjust the heating pad temperature to 37 degrees Celsius. Before surgery, sterilize all surgical instruments, including microsurgical scissors, fine straight forceps, fine curved forceps, hemostatic forceps, sterile gauzes, and weighing paper at 121 degrees Celsius for 30 minutes.

After anesthetizing a 14-week-old male C57BKL/6 mouse, remove the hair on the back with a shaver, then apply vet ointment to the eyes to prevent dryness. Place the mouse on an operating table in the dorsal position. Swipe and wipe the shaved area with povidone-iodine, followed by three wipes with 70%ethanol.

Using a sterile scalpel blade, make a one centimeter incision perpendicular to the tail behind the ear over the shoulder blade of the front leg. Next, use a sterile hemostat to make a subcutaneous tunnel under the skin and create a pocket for the pump. Gently insert an osmotic pump filled with angiotensin II into the pocket, ensuring enough free space to suture the wound without stretching the skin.

Suture the muscle with interrupted 6-0 Vicryl sutures and close the skin with interrupted 4-0 nylon sutures. then swab and wipe the wound site with povidone-iodine. Place all the surgical instruments into a sterilizer for 10 seconds and replace the sterile gloves between surgeries.

Monitor the mouse until it has fully recovered. Closely monitor and observe wound healing at least twice a day during the first week and once every day subsequently, including redness, swelling, and infection. Perform dissection immediately if the mouse dies during angiotensin II infusion.

Measure blood pressure at baseline and every week after angiotensin II infusion with the tail-cuff plethysmography method in conscious mice. One week after angiotensin II infusion, select the mice with elevated blood pressure and record their weight. Choose animals with a minimum weight of 24 grams for renal denervation surgery.

Next, remove the hairs on the abdomen of an anesthetized mouse with a shaver, then place the mouse on the operating table. Keep the abdomen up and fix the limbs with tape. Disinfect the abdominal skin with povidone-iodine, followed by three wipes with 70%ethanol.

After making a two centimeter ventral midline abdominal incision, pull back the intestine with gauze soaked in 37 degrees Celsius saline to expose the left renal artery. Carefully, but bluntly, dissect the fat away from the renal artery using curved tweezers. Using sterile sharp scissors, cut the weighing paper into a rectangle of the same size as the renal artery.

Cut several pieces of the weighing paper at a time to keep the shape same. Dip the weighing paper in 10%phenol ethanol solution for at least 30 seconds. Cover the surface of the left renal artery and wrap the vessel with the weighing paper for two minutes.

Use gauze to protect the surrounding tissues to avoid the weighing paper from touching the kidney and intestine. Repeat the same procedure for the right renal artery. After surgery, reposition the muscles into their initial position and close the peritoneum with 6-0 Vicryl sutures in an interrupted suture pattern, then close the skin with interrupted 4-0 nylon sutures.

Monitor all mice until fully recovered. A significant increase in systolic blood pressure was observed at one week after angiotensin II infusion. The renal sympathetic denervation, or RDN, angiotensin II group showed a significant reduction in systolic blood pressure compared to the sham angiotensin II group at 21 days after the RDN procedure.

No significant difference was observed between sham and RDN groups at two weeks after the RDN procedure. Hematoxylin and eosin staining of renal sympathetic nerve and renal artery is shown here. No thickening of the intima layer of the renal artery was observed in the four groups.

Compared to shams group, fragmented, and pyknotic nuclei, digestion and swelling of endoneurial tissue were observed in both RDN groups. Immunohistochemistry of the nerve bundles revealed that the expression of tyrosine hydroxylase was significantly decreased in RDN and RDN angiotensin II group compared to both shams group. Renal cortical norepinephrine content in the denervated kidneys in both normotensive and the hypertensive group was significantly reduced compared to the innervated kidney.

Masson staining showed no remarkable increase in intima media of the abdominal aorta among the groups. Representative images of the myocardium in different groups are shown here. Angiotensin II infusion-induced cardiac hypertrophy was improved by RDN treatment by the decrease in interstitial fibrosis and cardiomyocyte size.

During the procedure, do not touch the phenol to the surrounding tissue except to the renal artery because it may cause intestinal obstruction, abdominal infection, and the renal artery stenosis. This method can establish a standardized RDN model to help study the mechanisms that control hypertension. Future applications of this technique can contribute to expanding the pathways that underline the process of hypertension and cardiac hypertrophy.