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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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 animals9,10,11,12,13,14,15,16,17. 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 &#176;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.

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 (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 &#177; 5.43 vs 196.67 &#177; 14.26 mmHg, p &#60; 0.01). There was no difference between Sham group and RDN group (113.33 &#177; 9.35 vs 113.17 &#177; 8.47 mmHg, p &#62; 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&#38;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&#38;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 &#177; 6.91 vs 180.76 &#177; 11.47 ng/g, p &#60; 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% &#177; 0.28 vs 4.53% &#177; 0.32, p &#60; 0.01) and cardiomyocyte size (348.39 &#177; 31.56 vs 322.21 &#177; 22.26 &#181;m, p = 0.37; Figure 6).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63719/63719fig01v2.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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63719/63719fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63719/63719fig02.jpg" /> 
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 &#60; 0.05), ** indicates statistical significance (p &#60; 0.01). Values are represented as mean &#177; 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63719/63719fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63719/63719fig03.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63719/63719fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63719/63719fig04.jpg" /> 
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 &#181;m. (B) Quantification of TH expression in renal nerves. ** indicates statistical significance (p &#60; 0.01), ns indicates not significant. Values are mean &#177; 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63719/63719fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63719/63719fig05.jpg" /> 
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 &#60; 0.01), ns indicates not significant. Values are mean &#177; 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63719/63719fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63719/63719fig06.jpg" /> 
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&#38;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 &#181;m. N = 6 in each group; RDN + Ang II indicates renal denervation operated on 1 week after Ang II infusion in C57BL/6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63719/63719fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Improved Renal Denervation Mitigated Hypertension Induced by Angiotensin II Infusion at JoVE.com

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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3.0K 조회수.  Huadong Hospital Affiliated to Fudan University. 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.

비디오: Improved Renal Denervation Mitigated Hypertension Induced by Angiotensin II Infusion

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure&gt;150 mmHg), proteinuria (&gt; 50 mg/24 hr) and mild uremia (&gt;10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (&lt;10%). Follow-up until 12 weeks after SNX shows further progression of CKD.

A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions.

높은 소금 다이어트와 루이스 쥐에서 만성 신장 질환을 유발하는 산화 질소 합성 효소 억제와 함께 5/6th의 절제

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

연구

의학

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.

비주얼 결함의 특성을 만성 안구 고혈압의 레이저 유도 마우스 모델

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;

경골 신경 절개 - 마우스의 탈 신경에 의한 골격근의 위축에 대한 표준 모델

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.

만성 신장 질환 환자에서 혈관 기능의 평가

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.

쥐에서 초음파 기술을 사용하여 약물 유발 신장 혈역학 적 부전의 조기 발견

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.

쥐 신장 이식 기술

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.

삼투압을 이용하여 피하 안지오텐신 II 주입 쥐에서 유도 대동맥 동맥류 펌프

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

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).

간 대동맥 밸브 주입 하는 동안 이미지 퓨전에 대 한 x 선 Fluoroscopy와 3D CT 혈관의 등록 향상

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.

왼쪽된 심 방 협 유발 쥐에서 그룹 2 폐 동맥 고혈압과 폐 정 맥 Arterialization

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

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

면역 표지된 신장 조직의 광학 클리어링 및 화상 진찰

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...