sympathetic denervation of renal arteries in an angiotensin ii-induced hypertension mouse model

Sympathetic Denervation of Renal Arteries in an Angiotensin II-Induced Hypertension Mouse Model

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 3 wipes with 70% ethanol. After making a 2 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 2 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.

sympathetic-denervation-renal-arteries-an-angiotensin-ii-induced

Nanyang Technological University

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JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Cerebrovascular Diseases

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Bilateral renal denervation
<ol>
	<li>Select the mice with elevated blood pressure (BP) &#8805;140/90mmHg or 25% increase in systolic BP/diastolic BP, 1 week after the Ang II infusion.</li>
	<li>Record animal weight before surgery and choose animals with a minimum weight of 24 g for renal denervation surgery.</li>
	<li>Anesthetize the mice using sodium pentobarbital. Confirm anesthesia depth with a negative toe pinch reflex.</li>
	<li>Remove the hair on the abdomen with a shaver. Perform this procedure carefully and thoroughly to avoid any surgical contamination.</li>
	<li>Place the mice on the operating table, keeping the abdomen up, and fix its limbs with tape. Disinfect the abdominal skin with povidone-iodine, followed by three wipes with 70% ethanol.</li>
	<li>Make a 2 cm ventral midline abdominal incision using a scalpel blade. Pull back the intestine with gauze soaked in 37 &#176;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).</li>
	<li>Cut the weighing paper into a rectangle 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 to try to cut several pieces of the weighing paper at a time to keep the same shape.</li>
	<li>Dip the weighing paper in a 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, keeping it for 2 min (Figure 1D). Use gauze to protect the surrounding tissues to prevent the weighing paper from touching the surrounding kidney and intestine. 
	&#8203;NOTE: The phenol solution is stable in plastic tubes but not glass vials. Therefore, the solution must be freshly prepared for every experiment.</li>
	<li>Perform the same procedure for the right renal artery. Perform the sham surgery with weighing paper immersed in saline.</li>
	<li>Reposition muscles into their initial position and close the peritoneum with 6-0 Vicryl sutures in an interrupted suture. Then, close the skin with interrupted 4-0 nylon sutures. Monitor all mice until fully recovered.</li>
</ol>
2. Post-operative care
<ol>
	<li>Apply povidone-iodine to the incision and place the animal in a warmed electric blanket for recovery and post-operative monitoring.</li>
	<li>Monitor the mice twice a day to assess for redness, swelling, and pain or abdominal infection. Provide meloxicam (0.5 mg/kg, SC) to all mice about 1 h before and 24 h after the RDN procedure.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Angiotensin II</td>
			<td>Sangon Biotech</td>
			<td>CAS:4474-91-3</td>
			<td>To make a hypertensive animol model</td>
		</tr>
		<tr>
			<td>Blood Pressure Analysis</td>
			<td>Visitech Systems</td>
			<td>BP-2000</td>
			<td>Measure the blood pressure of mice</td>
		</tr>
		<tr>
			<td>Mini-osmotic pump</td>
			<td>DURECT Corporation</td>
			<td>CA 95014</td>
			<td>To fill with Angiotensin II</td>
		</tr>
		<tr>
			<td>Weighing paper</td>
			<td>Sangon Biotech</td>
			<td>F512112</td>
			<td>To destroy renal nerve with weighing paper immersed with phenol; https://www.sangon.com/productDetail?productInfo.code=F512112.</td>
		</tr>
	</tbody>
</table>

Source: Wang, M., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63719">Improved Renal Denervation Mitigated Hypertension Induced by Angiotensin II Infusion.</a> J. Vis. Exp. (2022)
This video demonstrates a procedure for renal artery sympathetic denervation in an angiotensin II-induced hypertensive mouse model, using phenol to disrupt sympathetic nerve function and reduce blood pressure.

<img alt="Figure 1" src="/files/ftp_upload/23199/23199_Figure1.jpg" /> 
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.

Source: Wang, M., et al., Improved Renal Denervation Mitigated Hypertension Induced by Angiotensin II Infusion. J. Vis. Exp. (2022)
This video ...

Secure an anesthetized adult mouse with a shaved abdomen.
This mouse exhibits elevated blood pressure (hypertension) following infusion of angiotensin II.
Disinfect the abdomen and make a midline incision.
Gently pull out the intestine with saline-soaked gauze to expose the left renal artery.
Carefully remove the fat surrounding the renal artery.
Cover and wrap the renal artery with equally sized phenol-ethanol solution-soaked weighing papers. Place gauze around the surrounding organs to protect them from the solution&#39;s effects.&#160;
When applied to the renal artery, the phenol-ethanol solution penetrates and destroys the sympathetic nerve fibers surrounding the artery, causing denervation.
Repeat the denervation procedure for the right renal artery.
Next, retract the intestines and abdominal muscles.
Suture the surgical incision and allow the mouse to recover.
Renal denervation disrupts sympathetic nerve signals, reducing the kidneys&#39; influence on blood pressure regulation and significantly lowering overall blood pressure.

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Video: Sympathetic Denervation of Renal Arteries in an Angiotensin II-Induced Hypertension Mouse Model - Experiment

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM.
The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.

This manuscript describes the use of transgenic reporter mice and different administration routes of fluorescent dyes in angiotensin II-induced hypertension using intravital video microscopy of blood vessels to evaluate the activation of immune cells and their ability to roll and adhere to the endothelium.

Visualizzazione del leucocita rotolamento e adesione in angiotensina II-infuso topi: tecniche e insidie

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...

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Immunologia e infezioni

Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies have suggested that nerves may also modulate skin disorders including atopic dermatitis, psoriasis and cancer. Here, we describe protocols for testing the requirement for nerves in maintaining a cutaneous mechanosensory organ, the touch dome (TD). Specifically, we discuss methods for genetically labeling, harvesting and visualizing TDs by whole-mount staining, and for performing unilateral surgical denervation on mouse dorsal back skin. Together, these approaches can be used to directly compare TD morphology and gene expression in denervated as well as sham-operated skin from the same animal. These methods can also be readily adapted to examine the requirement for nerves in mouse models of skin pathology. Finally, the ability to repeatedly sample the skin provides an opportunity to monitor disease progression at different stages and times after initiation.

This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount &#946;-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

Cutanea denervazione chirurgica: Un metodo per il test il requisito per i nervi in ​​modelli murini di malattia della pelle

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies ...

Medicina

Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are limited resources directing scientists in the techniques for measuring and analyzing these variables. This protocol describes the methods for measuring RSNA and mean arterial pressure in anesthetized rats. The protocol also includes the approaches for accessing the brain during RSNA recordings for central nervous system (CNS) manipulations. The RSNA recording technique is compatible with pharmacologic, optogenetic, or electrical stimulation of the CNS. The approach is useful when an investigator will measure short-term (min to h) autonomic responses in non-survival experiments to correlate anatomically with CNS nuclei. The approach is not intended to be used to obtain chronic (survival) recordings of RSNA in rats. Discharges in RSNA, averaged rectified RSNA, and mean arterial pressure can be quantified and analyzed further using parametric statistical tests. Methods for obtaining venous access, recording mean arterial pressure telemetrically, and brain fixation for future histological analysis are also described in the article.

Methods for measuring sympathetic and cardiovascular responses to central nervous system (CNS) manipulations are important for advancing neuroscience. This protocol was developed to assist scientists with measuring and quantifying acute changes in renal sympathetic nerve activity (RSNA) in anesthetized rats (non-survival).

Quantificare i cambiamenti acuti nell'attività comprensiva renale del nervo in risposta a manipolazioni del sistema nervoso centrale in ratti anestetizzati

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are ...

Sympathetic Denervation of Renal Arteries in an Angiotensin II-Induced Hypertension Mouse Model

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Sympathetic Denervation of Renal Arteries in an Angiotensin II-Induced Hypertension Mouse Model