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.

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 animals were maintained under specific closed pathogen-free conditions in a temperature-controlled room at 24 &#177; 1 &#176;C with a 12h light/dark cycle and free access to standard rodent chow and water ad libitum.
1. Preparation of the operation field
<ol>
	<li>Disinfect the operation table with 70% ethanol. Adjust the heating pad temperature to 37 &#176;C.</li>
	<li>Ensure that all surgical instruments are sterilized before surgery at 121 &#176;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.</li>
</ol>
2. Angiotensin II induced hypertension
<ol>
	<li>Provide meloxicam (0.5 mg/kg, SC) to the C57BL/6 mice shortly before anesthesia induction. Then, anesthetize the 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.</li>
	<li>Remove the hair on the back with a shaver. Apply vet ointment to the eyes to prevent dryness while under anesthesia.</li>
	<li>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.</li>
	<li>Make a 1 cm incision using a sterile scalpel blade, perpendicular to the tail, behind the ear over the shoulder blade of the front leg.</li>
	<li>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.</li>
	<li>Suture the muscle with interrupted 6-0 Vicryl sutures and close the skin with interrupted 4-0 nylon sutures. Swab and wipe the wound site with povidone-iodine. Perform the same surgery with equal volume of saline for the control group.</li>
	<li>Place all the surgical instruments into a sterilizer for 10 s and replace the sterile gloves between surgeries. Monitor all mice until fully recovered.</li>
	<li>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.</li>
	<li>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 &#177; 2 &#176;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.</li>
</ol>
3. 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 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 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.</li>
	<li>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. 
	&#8203;NOTE: The phenol solution is stable in plastic tubes but not in glass vials. Therefore, the solution must be freshly prepared for every experiment18.</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>
4. 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>

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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+recent+clinical+trials+and+hypertension+guidelines+on+stroke+and+future+cerebrovascular+research.">Implications of recent clinical trials and hypertension guidelines on stroke and future cerebrovascular research.</a> Stroke. 49 (3), 772-779 (2018).</li><li>Rossignol, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+double+challenge+of+resistant+hypertension+and+chronic+kidney+disease.">The double challenge of resistant hypertension and chronic kidney disease.</a> The Lancet. 386 (10003), 1588-1598 (2015).</li><li>Du, X., Patel, A., Anderson, C. S., Dong, J., Ma, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+cardiovascular+disease+in+China+and+opportunities+for+improvement.">Epidemiology of cardiovascular disease in China and opportunities for improvement.</a> JACC International. Journal of the American College of Cardiology. 73 (24), 3135-3147 (2019).</li><li>Valenzuela, P. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+controlled+trial+of+renal+denervation+for+resistant+hypertension.">A controlled trial of renal denervation for resistant hypertension.</a> The New England Journal of Medicine. 370 (15), 1393-1401 (2014).</li><li>Kjeldsen, S. E., Narkiewicz, K., Burnier, M., Oparil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Blood Press. 27 (4), 185-187 (2018).</li><li>B&#246;hm, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+catheter-based+renal+denervation+in+the+absence+of+antihypertensive+medications+(SPYRAL+HTN-OFF+MED+Pivotal):+a+multicentre,+randomised,+sham-controlled+trial.">Efficacy of catheter-based renal denervation in the absence of antihypertensive medications (SPYRAL HTN-OFF MED Pivotal): a multicentre, randomised, sham-controlled trial.</a> The Lancet. 395 (10234), 1444-1451 (2020).</li><li>Azizi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endovascular+ultrasound+renal+denervation+to+treat+hypertension+(RADIANCE-HTN+SOLO):+a+multicentre,+international,+single-blind,+randomised,+sham-controlled+trial.">Endovascular ultrasound renal denervation to treat hypertension (RADIANCE-HTN SOLO): a multicentre, international, single-blind, randomised, sham-controlled trial.</a> The Lancet. 391 (10137), 2335-2345 (2018).</li><li>Kandzari, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> The Lancet. 391 (10137), 2346-2355 (2018).</li><li>Townsend, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> The Lancet. 390 (10108), 2160-2170 (2017).</li><li>Sun, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+denervation+restrains+the+inflammatory+response+in+myocardial+ischemia-reperfusion+injury.">Renal denervation restrains the inflammatory response in myocardial ischemia-reperfusion injury.</a> Basic Research in Cardiology. 115 (2), 15 (2020).</li><li>Sharp, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+denervation+prevents+heart+failure+progression+via+inhibition+of+the+renin-angiotensin+system.">Renal denervation prevents heart failure progression via inhibition of the renin-angiotensin system.</a> Journal of the American College of Cardiology. 72 (21), 2609-2621 (2018).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+denervation+attenuates+progression+of+atherosclerosis+in+apolipoprotein+E-deficient+mice+independent+of+blood+pressure+lowering.">Renal denervation attenuates progression of atherosclerosis in apolipoprotein E-deficient mice independent of blood pressure lowering.</a> Hypertension. 65 (4), 758-765 (2015).</li><li>Chen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+denervation+mitigates+atherosclerosis+in+ApoE-/-+mice+via+the+suppression+of+inflammation.">Renal denervation mitigates atherosclerosis in ApoE-/- mice via the suppression of inflammation.</a> American Journal of Translational Research. 12 (9), 5362-5380 (2020).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+denervation+promotes+atherosclerosis+in+hypertensive+apolipoprotein+E-deficient+mice+infused+with+Angiotensin+II.">Renal denervation promotes atherosclerosis in hypertensive apolipoprotein E-deficient mice infused with Angiotensin II.</a> Frontiers in Physiology. 8, 215 (2017).</li><li>Eriguchi, M., Tsuruya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+sympathetic+denervation+in+rats.">Renal sympathetic denervation in rats.</a> Methods in Molecular Biology. 1397, 45-52 (2016).</li><li>Thukkani, A. 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There are no conflicts of interest, financial or otherwise, as declared by the authors.

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</...

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.

<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>Anti-Tyrosine Hydroxylase antibody</td>
			<td>Abcam</td>
			<td>ab137869</td>
			<td>To evaluate the expression of TH of renal nerves</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>Norepinephrine ELISA Kit</td>
			<td>Abcam</td>
			<td>ab287789</td>
			<td>to measure renal norepinephrine levels</td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Sangon Biotech</td>
			<td>CAS:108-95-2</td>
			<td>Damage the renal sympathetic nerve</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.&#160;</td>
		</tr>
	</tbody>
</table>

improved renal denervation mitigated hypertension induced by angiotensin ii infusion

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&#39;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&#38;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.

Statistics 
All data are expressed as mean &#177; 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 (...

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Improved Renal Denervation Mitigated Hypertension Induced by Angiotensin II Infusion

Here, we present a protocol for renal sympathetic denervation (RDN) in mice with hypertension induced by Angiotensin II infusion. The procedure is repeatable, convenient and allows to study the regulatory mechanisms of RDN on hypertension and cardiac hypertrophy.

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 ...

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2.9K Views.  Huadong Hospital Affiliated to Fudan University. Почечная симпатическая денервация является потенциальным методом лечения гипертонии, что было доказано многими клиническими испытаниями. Данная методика денервирует свободные почечные нервы без повреждения почечной артерии. Более того, это простая, критическая, восп...

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A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also underscores the importance of ethical issues associated with animal use in research.

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is ...

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

Osmotic pumps continuously deliver compounds at a constant rate into small animals. This article introduces a standard protocol used to induce aortic aneurysms via subcutaneous infusion of angiotensin II (AngII) from implanted osmotic pumps. This protocol includes calculation of AngII amount and dissolution, osmotic pump filling, implantation of osmotic pumps subcutaneously, observation after pump implantation, and harvest of aortas to visualize aortic aneurysms in mice. Subcutaneous infusion of AngII through osmotic pumps following this protocol is a reliable and reproducible technique to induce both abdominal and thoracic aortic aneurysms in mice. Infusion durations range from a few days to several months based on the purpose of the study. AngII 1,000 ng/kg/min is sufficient to provide maximal effects on abdominal aortic aneurysmal formation in male hypercholesterolemic mouse models such as apolipoprotein E deficient or low-density lipoprotein receptor deficient mice. Incidence of abdominal aortic aneurysms induced by AngII infusion via osmotic pumps is 5 - 10 times lower in female hypercholesterolemic mice and also lower in both genders of normocholesterolemic mice. In contrast, AngII-induced thoracic aortic aneurysms in mice are not hypercholesterolemia or gender-dependent. Importantly, multiple features of this mouse model recapitulate those of human aortic aneurysms.

Subcutaneous implantation of osmotic pumps provides a convenient approach for prolonged and consistent delivery of compounds. This approach has been used extensively to study both abdominal and thoracic aortic aneurysms in mice.

Osmotic pumps continuously deliver compounds at a constant rate into small animals.  This article introduces a standard protocol used to induce aortic ...

Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity (RSNA) is of great interest in many areas of research such as chronic kidney disease, hypertension, heart failure, diabetes and obesity. Unequivocal assessment of the role of the sympathetic nervous system is thus imperative for proper interpretation of experimental results and understanding of disease processes. RSNA has been traditionally measured in anesthetized rodents, including mice. However, mice usually exhibit very low systemic blood pressure and hemodynamic instability for several hours during anesthesia and surgery. Meaningful interpretation of RSNA is confounded by this non-physiological state, given the intimate relationship between sympathetic nervous tone and cardiovascular status. To address this limitation of traditional approaches, we developed a new method for measuring RSNA in conscious, freely-moving mice. Mice were chronically instrumented with radio-telemeters for continuous monitoring of blood pressure as well as a jugular venous infusion catheter and custom-designed bipolar electrode for direct recording of RSNA. Following a 48-72 hour recovery period, survival rate was 100% and all mice behaved normally. At this time-point, RSNA was successfully recorded in 80% of mice, with viable signals acquired up to 4 and 5 days post-surgery in 70% and 50% of mice, respectively. Physiological blood pressures were recorded in all mice (116&plusmn;2 mmHg; n=10). Recorded RSNA increased with eating and grooming, as well-established in the literature. Furthermore, RSNA was validated by ganglionic blockade and modulation of blood pressure with pharmacological agents. Herein, an effective and manageable method for clear recording of RSNA in conscious, freely-moving mice is described.

Anesthetized mice exhibit non-physiological systemic blood pressure, which precludes meaningful assessment of autonomic tone given the intimate relationship between blood pressure and the autonomic nervous system. Thus, a novel method to simultaneously record renal sympathetic nerve activity and blood pressure with intravenous infusion in conscious mice is outlined.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity ...

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt ...

Improved Registration of 3D CT Angiography with X-ray Fluoroscopy for Image Fusion During Transcatheter Aortic Valve Implantation

The fusion of 3D anatomical models derived from high-fidelity pre-interventional computed tomography angiography (CTA), and x-ray (XR) fluoroscopy to facilitate anatomical guidance is of huge interest for complex cardiac interventions like TAVI procedures with cerebral protection. Co-registration of CTA and XR has been introduced either based on additional intraoperative non-/contrast-enhanced cone-beam computed tomography (CBCT) or two separate aortograms. With the related increase of radiation exposure and/or contrast agent (CA) dose, a potential additional risk for the patient is introduced. Here, we propose a modified co-registration approach making use of arteriograms of the iliofemoral arteries, routinely performed during the femoral puncture and sheath introduction. On-the-fly refinement of the co-registration during the on-going procedure enables accurate co-registration without any additional angiograms, thus reducing CA, XR dose and procedure time, while simultaneously improving operator confidence and procedure safety.

The aim of this study was to improve the co-registration for image fusion (IF) of pre-interventional CT data with real-time x-ray (XR) fluoroscopy during transfemoral transcatheter aortic valve implantation (TAVI).

The fusion of 3D anatomical models derived from high-fidelity pre-interventional computed tomography angiography (CTA), and x-ray (XR) fluoroscopy to ...

Left Atrial Stenosis Induced Pulmonary Venous Arterialization and Group 2 Pulmonary Hypertension in Rat

The mechanism of mitral stenosis-induced pulmonary venous arterialization and group 2 pulmonary hypertension (PH) is unclear. There is no rodent model of group 2 PH, due to mitral stenosis (MS), to facilitate the investigation of disease mechanisms and potential therapeutic strategies. We present a novel rat model of pulmonary venous congestion-induced pulmonary venous arterialization and group 2 PH caused by left atrial stenosis (LAS). LAS is achieved by constricting the left atrium using a half-closed titanium clip. After the LAS surgery, a rat model with a transmitral inflow velocity greater than or equal to 2.0 m/s on echocardiography gradually develops pulmonary venous arterialization and group 2 PH over an 8- to 10-week period. In this protocol, we provide the step-by-step procedure of how to perform the LAS surgery. The presented LAS rat model mimics MS in humans and is useful for studying the underlying molecular mechanism of pulmonary venous arterialization and for the preclinical evaluation of therapies for group 2 PH.

Left atrial stenosis (LAS) is a novel surgical technique used for studying group 2 pulmonary hypertension (PH) and mechanisms underlying pulmonary venous arterialization. Here, we present a protocol to constrict the left atrium using a titanium clip to cause pulmonary venous arterialization and moderate PH in a rat.

The mechanism of mitral stenosis-induced pulmonary venous arterialization and group 2 pulmonary hypertension (PH) is unclear. There is no rodent model of ...