我们感谢克丽斯塔：书房Ouden为她出色的技术援助。这项技术是由荷兰肾脏基金会，授予C06.2174财政支持。支持MCV是由荷兰科学研究组织（NWO）VIDI补助016.096.359。

所有的实验都根据动物实验的伦理导向线的乌得勒支的实验动物委员会执行。该协议是作者的机构的动物护理和使用委员会的指导和批准下进行。 CKD是诱发雄性近交系Lewis大鼠（查尔斯河，Sulzfeld，德国）在8周龄。大鼠被安置在标准条件下，光，温度和湿度控制的环境中。 1。术前准备<ol><li>消毒手术器械： <ul><li> 1学生组织钳1-2齿12厘米</li><li> 1学生标准模式钳</li><li> 2 Semken钳</li><li> 1梅奥剪刀</li><li> 1学生虹膜剪刀</li><li>奥尔森有解用剪刀持针器</li><li>毯子</li></ul></li><li>检查库存清单： <ul><li>手术台变暖垫和灯</li><li> Ø <suB&gt; 2，异氟醚</li><li>组织</li><li>剃须刀</li><li>秤</li><li>无菌操作集</li><li>无菌纱布5X5和10x10 </li><li> 70％酒精</li><li> 0.9％NaCl溶液</li><li> 1ml注射器</li><li>针（25G） </li><li>凝胶海绵垫：spongostan </li><li>薇乔4.0和5.0 </li><li>丁丙诺啡0.03毫克/公斤（1:10稀释在生理盐） </li><li>清洁笼大鼠手术后</li></ul></li></ol> 2。右侧单侧肾切除<ol><li>在表中，用70％的酒精消毒。 </li><li>将大鼠感应盒，用4％的异氟醚和1升流量的O 2诱导麻醉。注：这取决于所用的大鼠株，前和围手术期镇痛所规定，这需要与当地的兽医讨论。 </li><li>转入大鼠表和地方鼻锥。大鼠和鼻锥体被放置在一个加热垫维持大鼠的体温。鼠我Ş通风与2％异氟醚和1升O 2的混合物中。 </li><li>剃须大鼠的侧面，并用酒精消毒。手清洗和消毒，穿无菌手术手套。戴头帽和口罩。 1 - 1.5厘米的切口平行于肋骨解剖钳和剪刀钝。背部肌肉钝性分离，暴露肾脏。 </li><li>肾下极可见，小心地用小钝钳，夹住肾周脂肪组织。外部肾肾周脂肪用钳子轻轻拉动。在此过程中不干扰肾上腺照顾。肾上腺可以容易地除去，通过将上面的内侧部位在脂肪组织中的钳子，并轻轻地向上移动，肾上腺和图2中所示的肾之间。 </li><li>上轻轻将肾脏周围的脂肪和结缔组织的纱布清晰。确定肾动脉和静脉，放置结扎（5.0薇乔）与血管周围单结，但不打领带结。 </li><li>仔细将松散的结约0.5厘米，沿着朝向主动脉血管创造空间之间的肾脏和结。这样做是为了防止从切割后脱落结扎。 </li><li>两个双结打结。如果停止灌注，肾的颜色会立即变为褐色。不要切断结扎线的两端。 </li><li>切靠近肾脏的肾血管和肾脏排除。轻轻拉出长端结扎检查肾血管出血。结扎应该留在地方上的船只。切长两端结扎。请注意，残余肾血管会收缩，立即进入腹腔。 </li><li>吸干切除肾脏干燥和称重。 </li><li>关闭骨骼肌切开与薇乔5.0运行缝线。注射0.10毫升（0.03毫克/千克）丁丙诺啡IM后肢。关闭皮肤与薇乔4.0与皮内缝合。这可以防止将大鼠从开放的伤口。注：由于呼吸抑制Lewis大鼠，我们只给手术后的镇痛之前关闭异氟醚。 </li><li>房子在一个干净的食物和水容易进入孤笼大鼠。将笼变暖垫O / N的一半，第二天仔细检查老鼠。当将伤口闭合和大鼠是积极的，这通常是在手术后6和12小时之间，大鼠可以被放置成组壳体的。 </li></ol> 3。左侧肾切除<ol><li>七天后，相同的制剂为从2.1至2.5所描述的右侧的左侧。在手术前，需要被移除对应约分之二的重量右肾的肾组织中的量的计算方法。术前准备小块泡沫凝胶（Spongostan，强生公司，新泽西州）。 </lI&gt; <li>左肾上极锋利的剪刀周围放置在一个冲程（剪刀的位置，参见图3），切除上极。立即盖上一块泡沫凝胶，并用无菌纱布施加温和的压力。 </li><li>重复3.3肾脏下极。 </li><li>提起肾脏皮肤上，以防止凝血。保持温和的压力，用无菌纱布都凝胶海绵垫，直到出血停止。当出血仍然存在，添加另一个泡沫垫。 </li><li>将残肾腹部内贴壁凝胶海绵垫。关闭的肌肉和皮肤，并按照2.9-2.12。 </li></ol> 4。假手术<ol><li>假手术对照经过同样的过程，以揭露肾脏。而是肾脏摘除或切割的两极，一个星期后，两个肾脏被解封间隔一个星期，照顾不来打扰的肾上腺。伤口缝合手术后的护理是ID2.11及2.12 entical。 </li></ol>

<ol>
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作者什么都没有透露。

手术切除5/6th Lewis大鼠肾的质量，再加上高盐饮食和临时NOS抑制剂导致的模型类似于人类CKD CKD，CKD的治疗干预的因果机制和疗效，并允许研究。 的5/6th肾切除模型是一个著名的和广泛的描述模型，用于CKD。然而，简单地删除5/6th肾质量不会导致立即肾功能衰竭大鼠品系。我们使用Lewis大鼠的影响进行研究基于细胞的疗法，在CKD的可用性GFP + Lewis大鼠21允许nonGFP跟踪管理?...

由于CKD的先进性，随后终末期肾脏疾病及相关心血管疾病的发病率和死亡率，是一个日益严重的公共卫生问题 。 CKD进展缓慢是一个主要的健康优先。由于慢性肾脏病的特点是复杂的动态平衡紊乱，综合性的动物模型是必需的研究开发和CKD恶化。肾脏由范围广泛的不同类型的细胞，彼此交互。这种复杂性，不能在体外模仿。 要研究新的治疗干预CKD中，我们使用的5/6th切除术烧蚀模型，一个著名的实验模型，进行性肾疾病，类似几个方面的人CKD 2,3。肾质量的毛利减少，导致渐进性肾小球和肾小管间质损伤，损失残肾和发展的系统性和肾小球内高压。这是与程序控制essive肾内毛细血管亏损4，炎症和肾小球硬化。危险因素CKD不约而同地影响内皮功能5。我们使用大鼠株（刘易斯），是比较耐CKD发展，因此，我们结合5/6th去除肾质量与一氧化氮（NO）枯竭6，7，8，高盐饮食9。抵达和驯化后，动物收到一氧化氮合酶抑制剂（L-NNA）一个为期4周的补充饮用水（20毫克/升），其次为右侧单侧肾切除（UNX）延续L-NNA两天后。一个星期后，在左侧肾切除（SNX）， 即去除肾质量分之二。 SNX后，动物被允许恢复为2天，然后再由20 mg / L的LNNA饮用水中的4个星期为一个周期。高盐饮食（6％），辅以地面州城（见时间线图1），继续在整个实验过程。 Ť他理由进行UNX的右侧，并在左侧的SNX是，肾血管长度的左侧，这使得它更易于访问未经拉伸的船只太多时身体外面的露出肾脏的肾。在文献中，模型是描述了其中的磁极的左肾首先被清除，UNX的右肾一个星期后10,11,12。在我们的手中，这个模型显示出更快速的发展，肾功能衰竭，也有更大的变化，肾功能丧失。通过测量血浆尿素，收缩压和蛋白尿，肾功能衰竭的进度之后随着时间的推移。通过SNX 6周后，肾功能衰竭发展，其特征在于由肾小球滤过率显着减少（69％）和有效肾血浆流量（62％）13高压（收缩压&gt; 150毫米汞柱），蛋白尿（&gt; 50毫克小时），轻度尿毒症（&gt; 10毫米）。病理特征包括tubul的邻间质损害反映炎症，肾小管萎缩，纤维化和局灶性肾小球硬化，导致残余人口健康肾小球内大量减少（&lt;10％）。 SNX显示随访，直到12周后进一步CKD进展，评价治疗干预的机会提供了一个窗口。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>L-NNA</td>
			<td>Sigma-aldrich</td>
			<td>N5501</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spongostan dental: gel foam pads 1x1x1 cm</td>
			<td>Johnson&amp;Johnson</td>
			<td>Ms0005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethicon Vicryl FS-2S naald 4/0 V392H p/36</td>
			<td>Ethicon</td>
			<td>V303H</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethicon Vicryl RB-1+ naald 5/0 V303H p/36</td>
			<td>Ethicon</td>
			<td>V392H</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Buprenorphine (0.3 mg/ml)</td>
			<td>Via local pharmacist ordered by Reckitt Benckiser pharmaceuticals</td>
			<td>unknown</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Student Tissue Forceps - 1x2 Teeth 12 cm</td>
			<td>Fine Science Tools (FST)</td>
			<td>91121-12</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Student Standard Pattern Forceps</td>
			<td>FST</td>
			<td>91100-12</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mayo Scissors</td>
			<td>FST</td>
			<td>14010-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2X Semken Forceps</td>
			<td>FST</td>
			<td>11008-13</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Student Iris Scissors</td>
			<td>FST</td>
			<td>91460-11</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olsen-Hegar Needle Holder</td>
			<td>FST</td>
			<td>12002-14</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

5/6th nephrectomy in combination with high salt diet and nitric oxide synthase inhibition to induce chronic kidney disease in the lewis rat

慢性肾脏病（CKD）是一个全球性的问题。 CKD进展缓慢是一个主要的健康优先。由于慢性肾脏病的特点是复杂的动态平衡紊乱，综合性的动物模型是必需的研究开发和CKD恶化。要研究开发在CKD CKD和新的治疗干预，使用5/6th切除术，消融模型，一个众所周知的实验模型，进行性肾疾病，类似人类CKD的几个方面。肾质量的毛利减少，导致渐进性肾小球和肾小管间质损伤，损失残肾和发展的系统性和肾小球内高压。它也与渐进肾内毛细管损失，炎症和肾小球硬化。 CKD总是危险因素对血管内皮功能的影响。为了模仿这一点，我们结合5/6th去除肾质量与一氧化氮（NO）的枯竭和高盐饮食。抵达和驯化后，动物重新ceive一氧化氮合酶抑制剂（NG-硝基-L-精氨酸）（L-NNA）补充饮用水（20毫克/升），一个为期4周，随后右侧单侧肾切除。一个星期后，一个肾切除（SNX）的左侧。 SNX后，动物被允许恢复两天LNNA饮用水中（20毫克/升）为进一步为期4周其次。高盐饮食（6％），辅以地面州城（见时间线图1），继续在整个实验过程。通过测量血浆尿素，收缩压和蛋白尿，肾功能衰竭的进度之后随着时间的推移。通过SNX 6周后，肾功能衰竭发展。使用&#39;黄金标准&#39;菊粉和对氨基马尿酸（PAH）的间隙技术测定肾功能。这种模式的特点是CKD的减少，肾小球滤过率（GFR）和有效肾血浆流量（ERPF），高血压（收缩压&gt; 150毫米汞柱），蛋白尿（&gt; 50毫克小时）和轻度尿毒症（&gt; 10毫米）。组织学特征包括炎症，肾小管萎缩，纤维化和局灶性肾小球硬化，导致残余人口健康肾小球内大量减少（&lt;10％）反映了肾小管间质损害。后续SNX显示，直到12周后进一步CKD恶化。

肾切除后，约六分之一肾总质量被留下。 图4显示了与前两次实验的平均值和标准偏差右肾切除部分的重量。每个人都应该记住，在UNX一周后，左肾肥大发生，这表明的重量，计算上在5/6th去除小于右侧肾脏的重量的结果总是需要被移除。然而，由于它是不可能的左肾在手术过程中确定权重，这是最准确的方法，以消除约5/6th的原肾质量。 随着时间的推移，CKD大鼠...

Watch this Scientific Journal Video about 5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat at JoVE.com

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

两阶段方法建立Lewis大鼠慢性肾脏病（CKD），通过手术取出肾质量5/6th描述。结合的外科手术过程中，一氧化氮合酶抑制和高盐饮食导致类似人类的CKD的模型，允许因果机制的研究和发展新的治疗干预。

5/6th联合切除术，高盐饮食和一氧化氮合酶抑制剂诱导Lewis大鼠慢性肾脏病

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

56th-nephrectomy-combination-with-high-salt-diet-nitric-oxide

Research

JoVE Journal

Medicine

23.4K 次观看.  University Medical Center Utrecht. 两阶段方法建立Lewis大鼠慢性肾脏病（CKD），通过手术取出肾质量5/6th描述。结合的外科手术过程中，一氧化氮合酶抑制和高盐饮食导致类似人类的CKD的模型，允许因果机制的研究和发展新的治疗干预。

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

Analytical Techniques for Assaying Nitric Oxide Bioactivity

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO2-) and nitrate (NO3-) may only represent part of NO's effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography.

The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.

为含量测定一氧化氮生物活性分析技术

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

科研

医学

Catheter Ablation in Combination With Left Atrial Appendage Closure for Atrial Fibrillation

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting millions of individuals worldwide 1-3. The rapid, irregular, and disordered electrical activity in the atria gives rise to palpitations, fatigue, dyspnea, chest pain and dizziness with or without syncope 4, 5. Patients with AF have a five-fold higher risk of stroke 6.
Oral anticoagulation (OAC) with warfarin is commonly used for stroke prevention in patients with AF and has been shown to reduce the risk of stroke by 64% 7. Warfarin therapy has several major disadvantages, however, including bleeding, non-tolerance, interactions with other medications and foods, non-compliance and a narrow therapeutic range 8-11. These issues, together with poor appreciation of the risk-benefit ratio, unawareness of guidelines, or absence of an OAC monitoring outpatient clinic may explain why only 30-60% of patients with AF are prescribed this drug 8.
The problems associated with warfarin, combined with the limited efficacy and/or serious side effects associated with other medications used for AF 12,13, highlight the need for effective non-pharmacological approaches to treatment. One such approach is catheter ablation (CA), a procedure in which a radiofrequency electrical current is applied to regions of the heart to create small ablation lesions that electrically isolate potential AF triggers 4. CA is a well-established treatment for AF symptoms 14, 15, that may also decrease the risk of stroke. Recent data showed a significant decrease in the relative risk of stroke and transient ischemic attack events among patients who underwent ablation compared with those undergoing antiarrhythmic drug therapy 16.
Since the left atrial appendage (LAA) is the source of thrombi in more than 90% of patients with non-valvular atrial fibrillation 17, another approach to stroke prevention is to physically block clots from exiting the LAA. One method for occluding the LAA is via percutaneous placement of the WATCHMAN LAA closure device. The WATCHMAN device resembles a small parachute. It consists of a nitinol frame covered by fabric polyethyl terephthalate that prevents emboli, but not blood, from exiting during the healing process. Fixation anchors around the perimeter secure the device in the LAA (Figure 1). To date, the WATCHMAN is the only implanted percutaneous device for which a randomized clinical trial has been reported. In this study, implantation of the WATCHMAN was found to be at least as effective as warfarin in preventing stroke (all-causes) and death (all-causes) 18. This device received the Conformit&eacute; Europ&eacute;enne (CE) mark for use in the European Union for warfarin eligible patients and in those who have a contraindication to anticoagulation therapy 19.
Given the proven effectiveness of CA to alleviate AF symptoms and the promising data with regard to reduction of thromboembolic events with both CA and WATCHMAN implantation, combining the two procedures is hoped to further reduce the incidence of stroke in high-risk patients while simultaneously relieving symptoms. The combined procedure may eventually enable patients to undergo implantation of the WATCHMAN device without subsequent warfarin treatment, since the CA procedure itself reduces thromboembolic events. This would present an avenue of treatment previously unavailable to patients ineligible for warfarin treatment because of recurrent bleeding 20 or other warfarin-associated problems.
The combined procedure is performed under general anesthesia with biplane fluoroscopy and TEE guidance. Catheter ablation is followed by implantation of the WATCHMAN LAA closure device. Data from a non-randomized trial with 10 patients demonstrates that this procedure can be safely performed in patients with a CHADS2 score of greater than 1 21. Further studies to examine the effectiveness of the combined procedure in reducing symptoms from AF and associated stroke are therefore warranted.

Catheter ablation is combined with placement of the WATCHMAN Left Atrial Appendage Closure Device to prevent ischemic stroke in a patient with non-valvular atrial fibrillation.

结合消融房颤左心耳封堵

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting millions of individuals worldwide 1-3. The rapid, irregular, and ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

光谱核型多囊肾病的研究在人类和小鼠的染色体异常

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

在大鼠模型慢性营养输液

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

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.

血管功能在慢性肾脏疾病的评估

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

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

通过40W的Elvax缓释给药的大鼠视网膜：对慢性疾病的治疗

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct biomarker isolated from donor mice. This technique allows for identification of donor-derived hematopoietic cells within the recipient mice, and can be used to isolate and characterize donor cells using various biochemical techniques. BMT typically relies on myeloablative conditioning with total body irradiation to generate niche space within the BM compartment of recipient mice for donor cell engraftment. The protocol we describe here uses myelosuppressive conditioning with the chemotherapeutic agent busulfan. Unlike irradiation, which requires the use of specialized facilities, busulfan conditioning is performed using intraperitoneal injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been administered. Moreover, myeloablative irradiation can have toxic side effects and requires successful engraftment of donor cells for survival of recipient mice. In contrast, busulfan conditioning using these doses is generally well tolerated and mice survive without donor cell support. Donor BM cells are isolated from the femurs and tibiae of mice ubiquitously expressing green fluorescent protein (GFP), and injected into the lateral tail vein of conditioned recipient mice. BM chimerism is estimated by quantifying the number of GFP+ cells within the peripheral blood following BMT. Levels of chimerism&nbsp;>80% are typically observed in the peripheral blood 3-4 weeks post-transplant and remain established for at least 1 year. As with irradiation, conditioning with busulfan and BMT allows for the accumulation of donor BM-derived cells within the central nervous system (CNS), particularly in mouse models of neurodegeneration. This busulfan-mediated CNS accumulation may be more physiological than total body irradiation, as the busulfan treatment is less toxic and CNS inflammation appears to be less extensive. We hypothesize that these cells can be genetically engineered to deliver therapeutics to the CNS.

We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

白消安作为骨髓抑制剂在小鼠生成稳定的高水平骨髓嵌合体

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

生物血管内送货到大鼠肾脏

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

小鼠肾小球疾病模型肾脏功能的评价

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

板基检测血液中一氧化氮的 vasp 磷酸化

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...