这项研究支持由汤姆皮特森基金会和美国国立卫生研究院授予 R01 HL119780 （耆那教、 MK）。

动物议定书 》 进行了审查和批准机构动物护理和使用委员会在凯斯西储大学医学院和按照国家卫生研究院 (NIH) 指南进行维护和使用的实验室动物 （2011 年第 8 版）。小鼠处死的方法符合美国兽医医疗协会 (AVMA) 准则为动物安乐死 （2013年版）。在本议定书中使用 C57BL/6J，男，8-16 周老小鼠。老鼠被安置在 12 h 黑暗/光照周期环境。
 1.手术前准备 <ol><li>转移小鼠到干净的笼子在手术前 24 小时。让动物免费的水。周食物替换软食物 （凝胶膳食补充剂） 4-6 小时前手术，以减少的小肠手术内容。</li>
	<li>消毒所有的外科手术器械、 一次性使用无菌手术材料准备。清洁手术范围和麻醉的鼻锥，用 70%乙醇。</li>
	<li>成立于解剖显微镜、 异氟醚麻醉汽化、 文书和温度控制小动物外科表体温度的保养。在手术期间组织文书、 缝线和注射器中自由访问的位置。</li>
	<li>设置光源为外科领域提供足够的光线。</li>
	<li>准备在 5 毫升注射器的肠道和腹部清洁无菌 0.9%的生理盐水。</li>
</ol> 2。回肠和吻合 <ol><li>Anesthetize 小鼠与异氟醚 （2-3%) 在小动物的孵化室。确定适当的麻醉用脚趾夹点技术，虽然这种动物是对异氟醚。</li>
	<li>采用脱毛产品脱毛腹部和擦干头发去维持麻醉时使用外科纱布。温度控制小动物在手术台上 ( 图 1) 以维持体温 鼠标置于 37 ° C.维持麻醉与异氟醚 （1-2%) 通过面罩。治疗老鼠的眼睛，用眼药膏。</li>
	<li>清洁皮肤使用聚维酮碘和 70%的酒精和覆盖与无菌手术纱布 ( 图 1 B) 腹部手术区域。</li>
	<li>使使用外科手术刀，一旦麻醉实际上是正中腹部切口。使用棉签来保护肠道和拉鼠标腹直肌与牵开器完全打开并公开 ( 图 1 C) 腹腔。</li>
	<li>定位盲肠。从盲肠，开始小心翼翼地连接的回肠和空肠的一部分从腹腔内 ( 图 1 D). 
	注: 盲肠容易被人识别由于规模庞大禁食后甚至。</li>
	<li>结扎肠系膜上动脉与 7-0 丝线以阻塞血供是切除回肠段上面的分支。回肠颜色变化从粉色到深紫色结扎后。( 图 1 E-F)。</li>
	<li>根据不同目的的实验中，利用剪刀，消费税和删除 50%或 90%的回肠。 
	注: 为假手术，不执行肠系膜上动脉结扎并不删除回肠。</li>
	<li>冲洗的 0.9%生理盐水回肠两端管腔. 
	注: 由于其余完好回肠仍在收集血液供应正常从肠系膜上动脉，极少量的血将会刷新过程。这也表明回肠末端的血液供应是正常的并确保没有缺血过程中吻合 ( 图 1 G)。</li>
	<li>定位在回肠两端直肠系膜。对齐的肠系膜和缝合在一起使用 8-0 缝合 ( 图 1 G-H) 回肠末端。</li>
	<li>缝合回肠保持回肠的对侧侧吻合以自然的方式 ( 图 1 我)。</li>
	<li>缝合上部和下部的双方之间两个原始缝合到彻底联接两个回肠末端在一起 ( 图 1 J)。</li>
	<li>确认是完成三个步骤的缝合后有无渗漏吻合部位 ( 图 1 G-我)。回盲肠和小肠入腹腔的原始的解剖位置。使用钝针的温暖 0.9%生理盐水清洗手术的地方。( 图 1 K)。</li>
	<li>关闭腹部肌肉层与 6-0 缝合的切口。对齐使用产钳的腹部皮肤切口和缝合腹部的皮肤，以便优化伤口愈合 ( 图 1 L)。</li>
</ol> 3。术后护理 <ol><li>转移手术后小鼠对重症监护病房的恢复。他们房子温控加热垫，一夜之间继续手术后恢复的纸-床上用品笼子里。供应小鼠软的食物，除了常规的食物和水。</li>
	<li>管理丁丙诺啡 (0.05-0.1 mg/kg) 与皮下注射每 8-12 h 为镇痛。 
	注: 安乐死老鼠的 CO 2，如果严重生病。</li>
	<li>在终点处，牺牲小鼠使用超剂量异氟醚和收获的样本，根据需要 （第 4 节）。</li>
</ol> 4。评价的回肠致胆汁积滞 <ol><li>权衡和解剖小鼠的 0%(sham)、 50%或 90%的回肠切除术后的一天。</li>
	<li>删除胃肠道和衡量它。计算 GI 重量和身体重量比例来评估的严重性胆盐吸收不良和积滞。</li>
	<li>将消化道转移到 15 毫升锥形管和把它们切成小段，用剪刀。切割后，10 分钟在 3,000 x g 离心机转移 （GI 液含胆汁酸盐） 上清液到清洁管。</li>
	<li>测量的总体积和重量的 GI 流体和计算胃肠道重量比，以便进一步评估胆积滞在胃肠道中的流体的重量。</li>
	<li>确定总胆汁量上清液中的胆汁酸测定在参考 8 所述。</li>
</ol>

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作者没有透露。

为了执行成功的回肠，必须预先结扎肠系膜上动脉来阻止对切除部分的血液供应。缺血性回肠段结扎后会变成暗紫色。然后必须完全切除回肠段。保留的两端必须保证正常的血液供应。这是必须避免出血和不当的去除，这可以很容易地导致手术失败由于缺血性坏死后缝合。期间吻合，它是重要的回肠末端一起加入他们自然的解剖位置。肠系膜被认为是为初始的缝合线，以避免可能发生的扭曲两端?...

在现代生物医学研究中，基因操纵的动物模型被广泛的应用，以收集洞察人类疾病。尤其是，组织或细胞特异性基因的增益损失功能已被用于研究分子调控，以及诱发的生物学效应。尽管操纵目标基因在体内的进步，有挥之不去的限制。第一，许多细胞或组织的特定缺失会影响多个器官。例如上, 皮基因缺失将消除多种组织中上皮细胞的表达。此外，即使删除仅限于为特定的组织，空间的控制是很少可行的。例如，在像肠组织，不同的部分进行十分具体的职能不能操纵与精度的体内。在这些情况下，基因包含组织切除术被认为是更有效的学习方法，以确定组织传播的机械和功能意义。
回肠是克罗恩病的主要是用于的患者和涉及回肠1，，23的炎症性疾病。回肠通常会产生几个能量存储激素样成纤维细胞生长因子 15/19 (FGF15/19)、 肽 (PYY) 和胰高血糖素样肽-1/2 (GLP1/2);这些激素许多生物学功能4，，56中发挥了重要的地方和内分泌作用。这些激素间 FGF15 已被确定为肝脏的胆汁酸合成鲁棒内分泌抑制剂。一旦进入回肠肠吸收，胆汁酸激活核受体法尼酯 X 受体 (FXR) 来刺激Fgf15的表达，随后导致的肝胆汁酸合成7反馈抑制。在最近的一项研究中，我们介绍了小鼠回肠模型为研究回肠 kruppel 样因子 (KLF15) 15-Fgf15信号调节昼夜节律的胆汁酸生产中肝8的轴。最重要的是，我们新的家庭，kruppel 样因子，特别是 KLF15，引入胆汁酸生物学。我们决定通过一种间接的非肝机制，KLF15 上调胆汁酸合成包括回肠手术的功能研究的基础。最后，回肠 KLF15 也被称为Fgf15第一内源性负性调节。
从近端到远端区域降序的肠段是负责吸收不同的营养。回肠是负责胆汁酸和维生素 B12 (VB12) 吸收9的主要部分。早先的研究中采用小鼠模型的近端肠切除术研究短肠综合征;提出了各种切除长度、 饮食和缝合类型，以保持最佳的手术后生存率10。此外，最近的审查表明，回肠切除通常会导致更严重的疾病比其他胃肠道 (GI) 段切除术由于剩余道11的自适应能力下降。本主题获得了集约化利益的基础和临床研究群体，而复苏和有效的治疗方法的了解仍然很有限。
胆汁酸腹泻结果从胆汁酸肝肠循环12，13稳态失衡。它可以是回肠切除术、 胃肠道疾病，或结果特发性胆汁酸吸收不良的后果。已发现了超过 80%的患者，切除回肠切除14后提出与腹泻。回肠有潜力成为重要手术模型为胆汁酸腹泻的调查。在此研究中，回肠切除一系列提供梯度评估 FGF15 不足以及肠胆盐吸收不良、 积滞和毒性损害。

<table width="722">
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	<tbody>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Dissection microscope</td>
			<td style="width:195px;">Olympus</td>
			<td style="width:97px;">SZ61</td>
			<td style="width:183px;">For surgery&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Animal temperature controller</td>
			<td style="width:195px;">Physitemp Instruments, Inc.</td>
			<td style="width:97px;">TCAT-2LV</td>
			<td style="width:183px;">For body temperature control</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Isoflurane anesthetic vaporizer</td>
			<td style="width:195px;">VetEquip</td>
			<td style="width:97px;">&#160;911104</td>
			<td style="width:183px;">For anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Dissection forceps</td>
			<td style="width:195px;">&#160;Fine Science Tools, Inc.</td>
			<td style="width:97px;">11274-20</td>
			<td style="width:183px;">For surgery&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Scissors</td>
			<td style="width:195px;">&#160;Fine Science Tools, Inc.</td>
			<td style="width:97px;">14084-08</td>
			<td style="width:183px;">For surgery&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Needle holder</td>
			<td style="width:195px;">&#160;Roboz Surgical Instrument Co.</td>
			<td style="width:97px;">RS-7882</td>
			<td style="width:183px;">For surgery&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Micro knives-needle blade</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:97px;">10318-14</td>
			<td style="width:183px;">For surgery&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">6-0 monofilament suture</td>
			<td style="width:195px;">Ethicon</td>
			<td style="width:97px;">1698G</td>
			<td style="width:183px;">For abdominal skin closure</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">7-0 silk suture</td>
			<td style="width:195px;">Ethicon</td>
			<td style="width:97px;">766G</td>
			<td style="width:183px;">For ligation</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">8-0 monofilament suture</td>
			<td style="width:195px;">Ethicon</td>
			<td style="width:97px;">1714G</td>
			<td style="width:183px;">For anastomosis</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Surgical sponges</td>
			<td style="width:195px;">Dynarex Corp.</td>
			<td style="width:97px;">3333</td>
			<td style="width:183px;">For surgery&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Small cotton-tipped applicators&#160;</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:97px;">23-400-118</td>
			<td style="width:183px;">For surgery&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Isoflurane</td>
			<td style="width:195px;">Piramal Healthcare Limited</td>
			<td style="width:97px;">66794-013-25</td>
			<td style="width:183px;">For anasthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Buprenorphine hydrochloride</td>
			<td style="width:195px;">Reckitt-Benckiser Pharmaceuticals</td>
			<td style="width:97px;">12496-0757-1</td>
			<td style="width:183px;">For analgesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">0.9% sodium chloride Injection</td>
			<td style="width:195px;">B. Braun Medical Inc.</td>
			<td style="width:97px;">0264-7800-10</td>
			<td style="width:183px;">For washing/injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Povidone iodine prep solution</td>
			<td style="width:195px;">Dynarex Corp.</td>
			<td style="width:97px;">1413</td>
			<td style="width:183px;">For skin preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Puralube vet ointment</td>
			<td style="width:195px;">Dechra Veterinary Products</td>
			<td style="width:97px;">17033-211-38</td>
			<td style="width:183px;">For eye pretection</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Hair remover lotion</td>
			<td style="width:195px;">Church &#38; Dwight Co., Inc.</td>
			<td style="width:97px;"></td>
			<td style="width:183px;">For skin preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Intensive care unit</td>
			<td style="width:195px;">ThermoCare</td>
			<td style="width:97px;">FW-1</td>
			<td style="width:183px;">For post-surgery recovery</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">DietGel recovery</td>
			<td style="width:195px;">ClearH2O</td>
			<td style="width:97px;">72-06-5022</td>
			<td style="width:183px;">For post-surgery recovery</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Aurum total RNA fatty and fibrous tissue kit</td>
			<td style="width:195px;">Bio-Rad</td>
			<td style="width:97px;">7326830</td>
			<td style="width:183px;">For RNA isolation</td>
		</tr>
		<tr height="31">
			<td height="31" style="width:248px;height:31px;">iScript reverse transcription supermix for RT-qPCR</td>
			<td style="width:195px;">Bio-Rad</td>
			<td style="width:97px;">1708841</td>
			<td style="width:183px;">For reverse transcription assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">TaqMan fast advanced master mix</td>
			<td style="width:195px;">Applied Biosystems/Life Technologies</td>
			<td style="width:97px;">4444965</td>
			<td style="width:183px;">For QPCR analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Total bile acid assay kit</td>
			<td style="width:195px;">Genzyme Diagnostic</td>
			<td style="width:97px;">DZ042A-K01</td>
			<td style="width:183px;">For bile acid assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">C57BL/6J&#160;</td>
			<td style="width:195px;">The Jackson Laboratory</td>
			<td style="width:97px;"></td>
			<td style="width:183px;"></td>
		</tr>
	</tbody>
</table>

ileectomy-induced bile overaccumulation in mouse intestine

肠段切除术是常见的治疗方法，为人类的疾病，如肥胖、 炎症性肠病、 克罗恩病和结肠癌，往往导致严重的短肠综合征样不良反应包括胆汁酸腹泻脱水、 电解质紊乱、 营养吸收不良。在这里我们介绍一种小鼠回肠切除模型，称为回肠，评价组织通信和系统稳态的维持。回肠切除术后血液循环是永久没有回肠特定内分泌激素成纤维细胞生长因子 15 (FGF15)，释放出肝脏的胆汁酸合成及其内分泌抑制。与增加的生产和胆汁酸后删除回肠的废除的吸收相结合，接受手术的老鼠患胆盐积滞在肠和相关性的腹泻、 发病率和死亡率。小说使用的这项研究中引入的手术模型可能洞察机械和功能回肠控制的系统性代谢调控的生理和疾病。

回肠的程序如图 1所示。第一步包括制备小鼠腹部皮肤，使腹部的切口，并利用牵开器充分暴露肠 (图 1A C)。接下来，鼠标盲肠是位于 (图 1D);给出了它的大小和形状使其很容易辨认的一个里程碑。盲肠、 回肠与远端空肠的一部分被曝光并移除从腹腔内 (图 1<s...

Watch this Scientific Journal Video about Ileectomy-induced Bile Overaccumulation in Mouse Intestine at JoVE.com

Ileectomy-induced Bile Overaccumulation in Mouse Intestine

小肠依赖胆汁酸的重吸收和反馈抑制肝胆汁酸合成系统稳态和健康至关重要。在此研究中，我们描述为回肠切除回肠致胆汁吸收不良，积滞，和毒性小鼠肠道中的小鼠模型。

回肠致胆汁积滞在小鼠肠道中

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer ...

ileectomy-induced-bile-overaccumulation-in-mouse-intestine

Research

JoVE Journal

Medicine

9.4K 次观看.  Case Western Reserve University. 小肠依赖胆汁酸的重吸收和反馈抑制肝胆汁酸合成系统稳态和健康至关重要。在此研究中，我们描述为回肠切除回肠致胆汁吸收不良，积滞，和毒性小鼠肠道中的小鼠模型。

视频: Ileectomy-induced Bile Overaccumulation in Mouse Intestine

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

在自觉的，奔放的小鼠正常血糖高胰岛素 - 夹钳

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

科研

医学

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.
A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure1,2, alterations in brain vasculature of a Alzheimer's disease mouse model3, portal vein abnormalities in portal hypertensive and cirrhotic mice4, developmental steps in rat lymphatic maturation between immature and adult lungs5, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury6.
Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

树脂浇铸和3维成像的小鼠门静脉或肝内胆管系统

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

一个简单的方法小鼠肺插管

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

在的鼠标Aortocaval瘘的技术方面

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

胆管结扎小鼠:感应炎性肝损伤和纤维化的阻塞性胆汁淤积

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

宫内遥测来衡量鼠标收缩压力<em&gt;在体内</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

一个简单的设备，迅速准备小鼠肠的全坐骑

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

分离和剖析人力胆汁含外来体的MicroRNA-

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

使用多氟化胆汁酸和<em&gt;在体内</em&gt;磁共振成像来衡量胆汁酸运输

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

利用隔离的完美大鼠小肠进行肠道激素分泌的机制

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...

回肠致胆汁积滞在小鼠肠道中

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