The authors acknowledge the funding support from the Ministry of Education, Singapore, grant number MOE2010-IF-1-025.

所有的动物实验是由应用科学学院，淡马锡理工学院的动物护理和使用委员会批准。 1. DMN的制备<ol><li>吸取200μl的DMN（1微克/毫升原液），并添加19.8毫升PBS中以制备用于注射10毫克/毫升DMN溶液。 注:DMN是致癌物质。使用通风橱准备DMN和执行动物注射。 </li></ol> 2. DMN腹腔注射<ol><li>使用雄性Wistar大鼠，平均体重为150 - 100克在实验开始前7天 - 适应大鼠4。 </li><li>在22±1℃的室温下保持大鼠，12小时光照和12小时黑暗的周期。提供大鼠食物和水随意 。 </li><li>每天测量食物和水的摄入量。测量人体体重每周。 </li><li>计算DMN的量为基于剂量为10毫克/公斤体重各大鼠。用1ml注射器用25号针头吨Ø绘制DMN溶液放入注射器中计算的体积。 </li><li>为抑制腹腔注射21大鼠。 </li><li>与大鼠适当抑制，引入针到腹部的右下象限，抽回注射器（没有应该吸气）的柱塞腹部空间内检查针的正确位置，并缓慢地注入DMN溶液。 </li><li>当DMN的剂量已被施用，慢慢抽出针弃置于尖锐件箱。 </li><li>执行DMN的腹膜内注射，连续3天，每周4周。每天在同一时间施用的注射剂。重新计算基于所述体重在注射每一周的开始的剂量。 </li><li>从尾静脉每周采集血液测定生化指标:谷丙转氨酶（ALT）和天冬氨酸氨基转移酶（AST）22。 </li><li>测定血清ALT和AST使用商用兽医测试化学分析仪。 </li></ol> 3.肉眼检查和肝脏的收获<ol><li>安乐死大鼠戊巴比妥腹膜内注射，剂量为100毫克/公斤体重。通过检查缺乏心跳确保死亡。 </li><li>制备为后验和如帕金森等人 23将死老鼠背斜卧与腹部朝上描述到解剖板评估动物的状况。 </li><li>消毒和滋润用70％乙醇的皮肤。 </li><li>使用剪刀，切开皮肤从肛门到下巴的ventrum的全长，和反映皮肤。用剪刀，切割从肛门腹壁剑突软骨，暴露腹腔脏器。 </li><li>检查腹部器官原位检查任何异常。注意在颜色，大小和任何变化，器官的位置的流体的腹腔内的存在。检查器官和一致性注意任何气味的存在。 </li><li>用剪刀和镊子，解剖整个肝脏免费的韧带和附件。 <ol><li>开始在其被连接至该膜片的门区和工作以释放附件的所有肝叶。小心切割所有韧带和血管。 </li></ol></li><li>转移肝脏到培养皿中，并称重肝脏。 </li><li>使用手术刀，切开2 - 5 mm产品肝叶厚的部分。为了保持一致性，总是从同一个肝叶样本。取一个楔直径约5毫米的厚度，从右侧侧叶24从lobe.Immediately处的边缘约1cm到10％缓冲的福尔马林固定。确保组织福尔马林体积为1:10或更多。 </li><li>如果需要其他研究收获，并立即在这一点上冻结部分（用液氮）肝叶。作为（3.8），从相同的肝叶一致性样品。 </li></ol><ol><li>固定在10％缓冲福尔马林的肝样品最少24小时。 </li><li>固定后，修剪肝，放入磁带并将其加载到自动组织处理器的篮子里。 </li><li>放置带盒在其中含有10％缓冲的福尔马林中，第一站篮筐。确保纸盒完全淹没。他们可以留在这个房间长达12小时，直到周期开始。 </li><li>编程组织处理器旋转盒的每个1小时，通过含有以下的溶液连续工位:乙醇以50％，70％，90％，100％（2站），二甲苯（2站）和液体石蜡的浓度（ 2站）。 </li><li>转移肝脏样品放入嵌入站的蜡浴。保证嵌入机器的至少一个小时前接通。 </li><li>使用预热钳（65°C），来自W删除的每个磁带斧头浴缸和放置到包埋机的温暖板（65°C）。分配液体石蜡的足量到不锈钢母模具（预加热至65℃），直到半满。从盒转移肝脏的部分到模具中。确保在试样被置于与切割表面（待检查显微镜）平放在模具的底部。 </li><li>用液体石蜡完全填充模具，安装在顶部的空盒和模具放置到包埋机的4°C板来冷却至少30分钟。 </li><li>检查该蜡已经冷却并固化，从模具中取出的石蜡块。块应该很容易蹦出来，而不是固守或裂纹。如果发生这种情况，熔块，并重复此步骤。一旦它固化的石蜡块可切片。块可在室温下保存了很多年。 </li><li>放置蜡块放入切片和部分在10微米厚，直到蜡层被充分除去嵌入的肝组织可见。 </li><li>变化对切片机的设置在5微米厚的削减。使用镊子，通过在边缘保持它捡起一个井切断部，并仔细漂浮在水浴部分在40℃的温度。 </li><li>轻轻地点下的浮动部分干净的载玻片上，然后将其在玻璃载片表面。放置滑动到滑动，在37℃过夜温暖。 </li></ol> 5. Masson染色<ol><li>施加污渍之前，治疗的幻灯片以除去蜡和再水合的组织如下: <ol><li>沉浸在二甲苯中的部分5分钟。重复2倍。 </li><li>浸泡在100％的乙醇的章节3分钟。重复1X。 </li><li>浸入部分的每个中的乙醇以下解决方案1分钟:90％，80％和70％。 </li></ol></li><li>冲洗自来水下滑动删除在幻灯片上的任何现有的乙醇。 </li><li>沉浸在铁苏木载玻片10分钟进行染色细胞核。 </li><li>冲洗载玻片用水从幻灯片除去过量的污渍。 </li><li>沉浸在比布列希猩红-酸性品红幻灯片2分钟进行染色细胞质中。 </li><li>用水冲洗的幻灯片。 </li><li>放置在磷钨/磷钼酸溶液的载玻片10分钟，以促进苯胺蓝的摄取。经过2轮的使用改变磷钨/磷钼酸溶液。 </li><li>放置在苯胺蓝溶液的载玻片10分钟，进行染色胶原纤维。 </li><li>冲洗过量的染色与水，并放置在1％乙酸溶液的幻灯片1分钟，以允许分化发生呈现幻灯片的颜色更细腻和透明。 </li><li>用水冲洗的幻灯片，并继续脱水组织切片。脱水步骤如下: </li><li>沉浸载玻片依次对每个时间10秒，在以下conce乙醇ntrations:70％，80％，95％和100％。 </li><li>浸入的幻灯片在100％乙醇30秒。重复1X。 </li><li>冲洗通过二甲苯的3个站的幻灯片，每次5分钟，以除去乙醇。 </li><li>挂载盖玻片上有安装介质（ 如 DPX封固）试样并晾干。 注意:DPX是干得很快，蜜饯染色和保护组织部分的合成树脂。 </li></ol> 6.纤维化评分对肝脏Masson的腺毛染色切片<ol><li>有兽医病理学家根据纤维化得分25评估纤维化的严重性。如果纤维化得分是由两个或三个独立地病理学家以盲方式进行这将是最佳的。在这种情况下，获得讨论和小组审核后的最终得分进行梳理得分的任何差异。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>得分了</td><td>描述</td></tr><tr><td> 0 </td><td>无纤维化</td></tr><tr><td> 1 </td><td>一些门户区域扩张的纤维，带或不带短纤维隔</td></tr><tr><td> 2 </td><td>大多数门户区纤维状扩大，有或无短纤维隔</td></tr><tr><td> 3 </td><td>大多数门户区纤维状扩大，偶尔门户门户网站（PP）桥接</td></tr><tr><td> 4 </td><td>门脉区纤维状扩张与标桥接（门户网站，门户网站（PP）以及门户中心（PC） </td></tr><tr><td>五</td><td>标志着桥接（PP和/或PC）偶尔结节（不完全肝硬化） </td></tr><tr><td> 6 </td><td>肝硬化，可能或肯定</td></tr></tbody></table> 表1:纤维化评分用于读取马森的腺毛彩绘肝脏切片 25。

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作者宣称，他们没有竞争的经济利益。

我们已经描述了使肝纤维化的动物模型，并评估肝纤维化的严重性的方法。它提供DMN正确的剂量，坚持每周腹腔注射的日程安排是非常重要的。随着实验的进行，它是衡量老鼠和DMN注射每星期开始重新计算剂量的关键。请记住，DMN是有毒的，需要在通风柜进行处理。我们在通风柜进行腹腔注射为好。随着需要反复注射，注射的正确的克制和技术是重要的，可以避免污染物引入和内脏损害。我们很?...

DMN是一种强效的肝毒素特异性。报道了马吉1其代谢，组织分布，并造成伤害到大鼠的肝脏能力，和由Pritchard的和巴特勒2中记载的细胞凋亡的肝细胞损伤和细胞死亡的机制。该化合物的间歇给药报道以诱导在狗和大鼠3,4-肝纤维化。 该机制与肝纤维化的形态学变化，采用这种模式被广泛研究。在使用大鼠早期研究中，DMN 3周的治疗产生小叶中央出血性坏死后肝硬化结节无脂肪变性5。它表明，在早期纤维化，所形成的胶原是更多的交叉与III型挂钩比I型6更为突出。在与其他原因常见，DMN诱发纤维性变化用的增加Kupffer细胞相关联;驻留在我的肝巨噬细胞n个血窦。这些细胞演变成肌成纤维细胞，并产生细胞外基质这是纤维化7的首要问题过量。 在细胞信号方面，Nakamura等人证明了TGF-β在肝纤维化8的进展中起关键作用。他们用表达截短的II型TGF-β受体的腺病毒，以专门抑制TGF-β信号传导。在这些大鼠肝纤维化与对照组比较时，被壮观DMN治疗期间停止。其他研究已经证实，TGF-β抑制导致肝纤维化的发展9,10缓解。该模型也被用于在一个全局基因表达谱研究，以确定其中可以作为药物靶点抗纤维化疗法11其他纤维化标记物和蛋白质。 其他化学试剂用于诱导肝纤维化包括硫代乙酰胺（TAA）和c阿尔邦四氯化碳（CCL 4）。 TAA首次使用大鼠，后来在小鼠12。这种模式的优点包括:易于管理化学品的饮用水，以特色小结节性肝硬化生化变化模型的重复性。缺点包括:3个月肝纤维化的长时间周期来开发和缺乏对肝纤维化的诱导分子机制的理解。至于四氯化碳 ，它的使用有所下降，原因如下:它不模仿人类肝脏疾病，具有对臭氧层有害的影响，引起疼痛和痛苦动物，是非常对人体有毒，并要求其处理额外的预防措施和处置13,14。 长时间胆道梗阻（通过外科手术），作为肝硬化实验模型最早由Kountouras 等 15日报道。这种方法是无毒的人类和动物。 HH但是，开发所需的肝纤维化的时间变化。由马克斯等人 16 30份报告进行审查，发现花了由七天到四个星期后手术治疗肝纤维化的发展。所描述的病理变化模仿那些人慢性胆道纤维化和模型会更适合有意在这一领域的研究人员。 总之，DMN的大鼠在4周内恒定剂量的间歇给药产生肝纤维化模仿人类疾病。给药的大鼠显示肝损伤和肝纤维化实质的4,17逐步发展。疾病进展和严重性可以通过血样或处死动物在特定时间点进行监测和效果是高度可再现18。因此该模型已被广泛用于研究肝纤维化和肝硬化的机制，以及筛选潜在抗纤维化剂10,19,20。

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			<td>Dimethylnitrosamine</td>
			<td>Wako</td>
			<td>147-03781</td>
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			<td></td>
			<td></td>
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			<td>Formalin</td>
			<td>Sinopharm chemicals</td>
			<td>F63257009</td>
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			<td>Ethanol</td>
			<td>Sigma</td>
			<td>64-17-5</td>
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			<td>Xylene</td>
			<td>Fisher</td>
			<td>1330-20-7</td>
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			<td></td>
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			<td>Masson trichome stains</td>
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			<td>Aniline Blue</td>
			<td>Electron Microscopy Sciences</td>
			<td>#42755</td>
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			<td>Electron Microscopy Sciences</td>
			<td>#26905</td>
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			<td>Phosphotungstic Acid Hydrate</td>
			<td>ALFA AESAR</td>
			<td>ALFA40116.4</td>
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			<td>Phosphomolybdic Acid Hydrate</td>
			<td>Sigma SG</td>
			<td>221856-25G</td>
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			<td>Weigert&#39;s Iron Hematoxilyn</td>
			<td>Merck</td>
			<td>1.15973.0002</td>
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			<td>DPX Mounting Medium</td>
			<td>Merck</td>
			<td>HX066873</td>
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			<td>Tissue processor</td>
			<td>Leica</td>
			<td>Leica TP 1020</td>
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			<td>Embedding machine</td>
			<td>Sakura</td>
			<td>&#160;Sakura Tissue Tek TEC5 Embedding System</td>
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			<td>Microtome</td>
			<td>Leica</td>
			<td>Leica RM 2235</td>
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			<td>Vet Test Analyzer</td>
			<td>Idexx</td>
			<td>Vet Test 8008</td>
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the dimethylnitrosamine induced liver fibrosis model in the rat

四至六周龄，雄性Wistar大鼠用于产生肝纤维化动物模型。该方法需要四个星期10毫克/公斤的二甲基亚硝胺（DMN）施用，每周连续三天腹膜内给予。作为DMN是一个已知hepatoxin和致癌物质腹膜内注射是在通风柜进行。该模型具有若干优点。首先，肝变化可依次或在感兴趣的特定阶段的研究。其次，肝脏疾病的阶段可通过血清丙氨酸转氨酶（ALT）和天冬氨酸转氨酶（AST）的酶测定进行监测。第三，在不同阶段的肝损伤的严重程度可以通过在指定的时间点处死动物来确认，随后Masson的腺毛染色的肝组织的组织学检查。 4周DMN给药后，典型的纤维化评分是对伊沙克比例5至6。该模型可以一致地被再现，并已广泛用于评估潜在的抗纤维化剂的功效。

DMN组大鼠减肥，成为竖起被毛缺乏活力。有一个在DMN治疗的大鼠的平均体重显著的损失;第一可检测2周后DMN的治疗，并且这种差异仍然通过DMN处理（ 图1a）周后3和4。由于老鼠收到DMN在连续数周，对肝脏的损害使之变得更小。肝指数;这是在最终体重肝重的百分比是该DMN治疗的大鼠（ 图1b）显著低。 <img alt="图1" src="/files/ftp_uploa...

Watch this Scientific Journal Video about The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat at JoVE.com

The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

We describe a method to produce an animal model of liver fibrosis in the rat, and assess the degree of fibrosis by histological examination of the liver. The model can be used to study the development of liver disease as well as to test the efficacy of potential anti-fibrotic agents.

该二甲基亚硝胺诱发肝纤维化模型大鼠

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg ...

the-dimethylnitrosamine-induced-liver-fibrosis-model-in-the-rat

Research

JoVE Journal

Medicine

11.9K 次观看.  Temasek Polytechnic. We describe a method to produce an animal model of liver fibrosis in the rat, and assess the degree of fibrosis by histological examination of the liver. The model can be used to study the development of liver disease as well as to test the efficacy of potential anti-fibrotic agents.

视频: The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

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  ...

科研

医学

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;

胫骨神经切断术 - 一个标准化的模型失神经诱导小鼠骨骼肌

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

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

直接节段性肝内交付使用大鼠肝门部夹具模型方法

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

肝分型与门静脉结扎大鼠分期肝切除 (ALPPS) 手术模型的建立

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

静脉大鼠部分肝移植的体内血管定向70% 肝切除术

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

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.

左心房狭窄致大鼠肺静脉动脉化及第2组肺动脉高压

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

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

矫形肝移植大鼠模型中动脉重联后并发症减少

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

毛利精密切肝片作为肝脏生物学的Ex Vivo模型

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

器官缺血-输液损伤模拟大鼠肝脏移植模型的血动力学变化

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...