Name: a murine model of vertical sleeve gastrectomy
Uploaded: 2017-12-18T13:00:00.000+00:00
Duration: 6 min 47 s
Description: Watch this Scientific Journal Video about A Murine Model of Vertical Sleeve Gastrectomy at JoVE.com

这项研究得到了 NIH/R21CA195002-01A1, 康奈尔妇女协会会长和美国纽约州立大学研究生多样性奖学金的支持。Dr. 卡明斯实验室也在项目期间从康奈尔比较癌症生物训练计划和礼礼和公司获得了资金。

所有的实验方案都得到了康奈尔大学机构动物护理和使用委员会的批准。
1. 手术前准备
注意: 研究小鼠通常是 C57BL 饮食诱发的肥胖背景, 使研究翻译与人类肥胖和胰岛素抵抗有关。在随后的步骤中, 可以对雄性和雌性小鼠进行研究。
<ol>
	<li>在2月的年龄, 将老鼠放在45% 或60% 高脂肪的饮食中2月 (参见材料表)。 注: 老鼠被喂食高脂肪饮食, 以创造一个肥胖和胰岛素抵抗的表型。更短或更长时期的高脂饮食喂养, 可根据该研究的目标使用。</li>
	<li>继续喂养小鼠ad 随意直到手术。如果需要基线测量, 经过7周的高脂饮食喂养, 快速小鼠6小时或一夜, 并采取基线血液样本测量的利益代谢物 (例如,葡萄糖)。</li>
	<li>继续吃高脂肪的小鼠, 允许小鼠在进行假手术或 VSG 前一周的血液回收。在手术前4天将老鼠放在液体饮食上 (见材料表)。 注意: 小鼠在手术前后保持液体饮食, 以清除可能损害愈合反应的微粒物质的消化道。</li>
	<li>在手术前对下列物品进行消毒: 箔、纱布、棉签 (cta) 和手术器械 (止血、操作剪刀、虹膜剪刀、cep、锯齿褐色-Adson cep、胃针、针驱动、弹簧剪刀).</li>
</ol>
2. 垂直袖胃切除术和假手术
<ol>
	<li>在手术当天, 称小鼠为基线体重测量。</li>
	<li>为手术室配备麻醉系统、温水浴和加热垫。将水浴设置为37° c, 用它加热0.9% 盐水溶液进行灌溉。用70% 乙醇清洁手术场, 使用无菌技术打开蒸压的手术包和工具。 注意: 在手术和麻醉恢复过程中, 加热垫用于保持动物的体温。</li>
	<li>使用感应室, 麻醉5% 异氟醚和 O2的流量为1升/分, 确认鼠标在适当的麻醉平面上执行脚趾捏。一旦达到了一致的麻醉平面, 保持1-3% 异氟醚和 O2流量0.6 升/分钟的鼠标。</li>
	<li>一旦鼠标被麻醉, 眼睛药膏 (见材料表) 在其眼中, 以保持他们在手术中潮湿。在这个时候也管理镇痛剂, 例如昔 (2 毫克/千克皮下)。修剪头发从肚脐到腋保持皮肤轻轻地提供紧张。
	<ol>
		<li>由于鼠标的皮肤是薄而细腻的, 注意不要引起皮肤损伤时修剪头发。用聚维酮碘和酒精清洁皮肤。</li>
		<li>不育手套和准备无菌的外科领域和仪器。使用无菌技术进行其余的过程。在老鼠的两侧放置不育的手术窗帘, 准备无菌的外科手术场。使用蒸压锡箔为鼠标创建一个外科悬垂。</li>
		<li>在锡纸上切一个小洞, 允许进入老鼠的腹部。</li>
	</ol>
	</li>
	<li>用虹膜剪刀将皮肤从 mid-abdomen (脐) 切开到剑软骨的水平。识别式阿尔巴, 并使用虹膜剪刀削减沿式阿尔巴的身体壁。用 cta 轻轻地将胃从腹部抬起, 然后直接解剖大网膜, 使其更大的曲率。</li>
	<li>结扎用7-0 单丝可吸收缝合线放置两个连字, 在胃底和脾之间运行的短胃动脉。用弹簧剪刀剪下两个连字。一旦动脉断, 完全取出胃从腹腔。将纱布置于胃下, 用盐水浸湿, 保持组织湿润。</li>
	<li>假程序
	<ol>
		<li>对于假手术, 采用6-0 单丝可吸收缝线和锥形针, 放置一个松散的简单连续缝合方式。从食道右侧 (外科医生右侧) 开始2毫米。将缝合线沿腹侧胃壁放置, 然后沿胃背壁继续。 注: 对于所有的胃操作, 使用单丝可吸收缝合, 以减少感染的风险。此外, 使用针针缝合所有的胃操作, 因为使用切割针头增加缝合的风险 "拉过" 导致胃组织损伤和伤口裂开。</li>
		<li>将针完全通过一个胃壁, 同时放置缝合。确保缝线是平的, 但不能限制胃。用3-4 投掷轻轻地扎下缝合线, 跳到步骤2.9。</li>
		<li>对于 VSG 过程, 请跳过步骤2.7 并继续步骤2.8。</li>
	</ol>
	</li>
	<li>VSG 做法
	<ol>
		<li>对于 VSG 程序, 结扎7-0 单丝可吸收缝线与锥针使用3投掷每个结的胃动脉和静脉突出分支。将连字放在下面 (即, 对胃的较小的曲率) 所预期的横断线。 注意: 预期的横断线起始于 ~ 2 毫米以上 (意思是朝向更大的胃的曲率) 胃的心脏切口和至少2毫米在玛歌 pilcatus 之下并且延伸到胰腺的右叶的近端。通常, 每胃壁有4血管需要结扎;然而, 这可能是不同的小鼠。</li>
		<li>为防止胃切除术中的胃液溢出, 将一条简单的连续缝合线通过6-0 单丝可吸收缝线与锥形针一起穿过两个胃壁。开始缝合线, 以外科医生的右食管和在玛歌 pilcatus 和结束略高于胰腺。</li>
		<li>在缝合线上方放置细尖的止血, 用弹簧剪刀在缝合线和止血之间切割。从不育的外科领域取出断的胃组织。</li></ol></li></ol>使用 cta 清洁血液和糜的胃。
		<li>用一个简单的间断模式, 用6-0 单丝可吸收缝线和锥形针加强胃壁的并列。 注意: 建议采用一种简单的间断缝合模式, 因为它比简单的连续缝合模式提供更安全的闭合。</li>
		<li>在整个手术过程中用盐水冲洗残胃, 以保持组织的湿润和清洁。使用一个20G 胃针连接到一个20毫升的注射器执行洗胃的盐水。使用这种大小, 以确保有足够的压力, 而不会危及胃组织损伤过度的压力或意外的胃组织损害使用常规尖针。</li>
		<li>使用最少20节, 以安全地关闭胃;注意沿食管一侧闭合, 因为这一侧更难进入, 因此往往是一个站点的开裂。</li>
		<li>用 cta 轻轻按压胃部, 确保无渗漏。如果发现泄漏, 在泄漏区域放置额外的简单不连续节点, 然后再次进行泄漏测试。泄漏测试, 直到未检测到泄漏。</li>
		<li>用至少60毫升的盐水做最后彻底的胃灌洗, 以确保没有任何传染性的颗粒物被留下。</li>
	
	
	<li>用 CTA 将胃放回肝脏下的腹腔。将 cta 放在腹腔的背侧, 吸收所有多余的液体。使用钝的18G 针, 注入林格林格的解决方案 (0.5 毫升/-20 毫克/千克恩诺) 直接进入腹腔, 只是在关闭之前。 注意: 这取代了在手术过程中经历的体液损失, 并提供了一种直接应用抗生素到外科手术部位以帮助恢复的方法。</li>
	<li>用6-0 单丝可吸收缝线与锥形针在一个简单的间断模式下闭合腹肌层。然后用6-0 单丝可吸收缝合线在一个简单的连续模式下闭合皮肤层。</li>
	<li>将组织粘合剂贴在皮肤上, 将皮肤折叠在缝合线上, 以掩埋缝线, 使小鼠不能扰乱伤口闭合术。</li>
	<li>关闭异氟醚, 让鼠标恢复在加热垫 10-15 minbefore 返回它的家庭笼。不要让动物无人看管, 直到它恢复知觉并能够移动。</li>
	<li>将动物单独安置, 直到从手术中完全恢复。 注意: 如果在整个研究过程中都能获得准确的食物摄入量, 老鼠应该继续在整个研究中单独居住。有外国身体阻塞的危险, 从吃家庭笼床上用品。动物应该被安置在笼子里没有寝具, 除了嵌套材料提供丰富 (参见材料表)。</li>

3. 操作后鼠标护理和测量
<ol>
	<li>将鼠标返回其主笼, 并将其放在加热垫上。在手术后至少7天内维持小鼠的液体饮食。在手术后2天内施行镇痛药, 如昔 (2 毫克/千克皮下)。 注: 抗生素, 如恩诺 (20 毫克/千克), 可在手术后7天内给予。抗生素被用来减少手术过程中感染的风险, 并且可能或可能不需要, 这取决于整个过程中保持的不育程度。然而, 如果使用抗生素, 必须在同一项研究中对所有小鼠进行管理, 以保持一致性。</li>
	<li>在术后期间评估体重, 食物摄入量, 排便, 活动水平和处置至少每天, 以确保适当的愈合和恢复。术后14天进行术后监测。 注: 手术失败通常在术后或术三天内立即出现。在手术后第二天不进食或饮酒的动物必须严密监测手术并发症。</li>
	<li>术后成功完成后, 定期测量体重和食物摄入量, 确保适当的表型保持正常。</li>
	<li>进行口服葡萄糖耐受试验 (OGTT), 以评估葡萄糖耐受, 葡萄糖刺激胰岛素分泌, 和肠道激素分泌。
	<ol>
		<li>执行 OGTT 在小鼠禁食后6小时, 然后胃小鼠50% 葡萄糖溶液在1克/千克剂量。</li>
		<li>收集尾血样本在 0, 2.5, 5, 15, 30, 60, 120 分钟后, 胃。在这些时间点测量血糖与血糖。在每个时间点收集血清样本, 以测量各种激素, 如胰岛素和 GLP-1。</li>
		<li>安乐死前6小时的快速小鼠。做最后的血样和葡萄糖测量。将最后的葡萄糖测量与基线测量进行比较。安乐小鼠腹腔注射戊巴比妥200毫克/千克剂量。</li>
		<li>使用基线和最终血清样本来测量额外的分析, 并比较术前和手术前后的价值。</li>
	</ol>
	</li>
</ol>

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作者声明没有利益冲突, 除非 Dr. 卡明斯收到了 Eli 和公司的资助。

减肥手术是最有效的长期治疗肥胖和结果的其他健康的好处, 如高率2型糖尿病和高血压缓解1,9,15。小鼠减肥手术模型提供了一个强有力的工具, 以确定的机制, 减肥手术导致迅速和明显的改善肥胖并存。此外, 小鼠减肥手术模型提供了一个新的范式, 研究的基础生物学肠道调节各种过程, 如新陈代谢, 心血管功能, 并致癌<sup class="x...

随着肥胖流行病的持续增长, 世界范围的减肥手术越来越受欢迎, 因为它是最有效的长期治疗肥胖症的1。不幸的是, 节食和运动造成的减肥很难实现, 而且在长期的23中相对无效。减肥手术, 如垂直袖胃切除术 (VSG), 是指为减轻体重的目的而操作胃肠道1,4。虽然减肥是减肥手术的显著结果, 减肥手术提供了其他的健康好处, 如改善肥胖并存和延长寿命5。例如, 减肥手术导致2型糖尿病和高血压的高缓解率, 并减少某些类型的癌症的发展的终生风险1,6,7。值得注意的是, 减肥手术导致2型糖尿病和高血压缓解的效果往往是在手术后和体重减轻之前,8,9。这凸显了一个概念, 即有独立于体重的机制, 有助于在手术后观察到的健康益处。动物模型的减肥手术已经开发, 并被用来研究的机制, 这些健康的好处发生10,11,12。
我们已经验证了一个小鼠模型的 VSG, 我们已经应用于各种基因改良的小鼠模型, 以研究减肥手术改善肥胖并存, 如2型糖尿病, 高血压, 和结直肠癌的机制10,11,12. 啮齿类动物模型允许更多的实验控制和执行基因或药物操作的能力来定义特定基因的作用或信号通路的兴趣。我们主要关注 VSG, 因为 VSG 是美国临床上最常执行的减肥手术13。此外, VSG 是一个简单的手术模型, 较少解剖修改相比, 其他程序, 如糊-en-Y 胃旁路或胰分流。
我们的小鼠模型 VSG 重述的减肥手术观察到的以下影响: 体重下降, 减少食物摄入量, 改善葡萄糖调节, 改善胰岛功能, 增加 post-prandial 胰高血糖素样 peptide-1 (GLP-1) 分泌物,降低动脉血压, 增加循环胆汁酸浓度10,11,12,13,14,15。因此, 这是一个理想的模型来研究体重依赖性和独立的机制, VSG 改善或解决肥胖并存。此外, 它是一个可靠的模型, 可以结合其他手术程序, 允许调查的影响 VSG 在各种疾病的情况下与更大的机械洞察力12。

<table><tbody><tr><td>45% high fat diet</td><td>Research Diets</td><td>D12451</td><td></td></tr><tr><td>60% high fat diet</td><td>Research Diets</td><td>D12492</td><td></td></tr><tr><td>Boost</td><td>Nestle</td><td>160-67538</td><td>rich chocolate flavor</td></tr><tr><td>6-0 Suture</td><td>Ethicon</td><td>Z432</td><td>monofilament absorbable/taper</td></tr><tr><td>7-0 Suture</td><td>Covidien</td><td>8866127-01</td><td>monofilament absorbable/taper</td></tr><tr><td>Cotton Swabs</td><td>Fisherbrand</td><td>23-400-118</td><td>small</td></tr><tr><td>Cotton Swabs</td><td>Fisherbrand</td><td>233-400-101</td><td>large</td></tr><tr><td>Gauze</td><td>Various</td><td></td><td>4x4 4 ply and 2x2 4 ply</td></tr><tr><td>Foil</td><td>Various</td><td></td><td></td></tr><tr><td>Surgery drape</td><td>Various</td><td></td><td></td></tr><tr><td>0.9% saline solution</td><td>Various</td><td></td><td></td></tr><tr><td>LRS</td><td>Hospira</td><td>170RX</td><td></td></tr><tr><td>Betadine</td><td>Various</td><td></td><td></td></tr><tr><td>Alcohol</td><td>Various</td><td></td><td></td></tr><tr><td>Eye Ointment</td><td>Paralube&amp;reg; Vet Ointment</td><td>17033-211-38</td><td></td></tr><tr><td>Tissue Adhesive</td><td>Vetbond</td><td>1469SB</td><td></td></tr><tr><td>Meloxicam (Metacam)</td><td>Boehringer Ingelheim</td><td>141-213</td><td>5 mg/ml</td></tr><tr><td>Enrofloxacin</td><td>Baytril</td><td>08713254-186599</td><td>22.7 mg/ml</td></tr><tr><td>Thin tipped hemostats</td><td>Fine Science Tools</td><td>13021-12</td><td></td></tr><tr><td>Metzenbaum Scissor</td><td>Fine Science Tools</td><td>14018-18</td><td></td></tr><tr><td>Iris Scissors</td><td>Fine Science Tools</td><td>14058-09</td><td></td></tr><tr><td>Dumont Forcep</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Serrated Forcep</td><td>Fine Science Tools</td><td>11020-12</td><td></td></tr><tr><td>Gavage needle</td><td>Fine Science Tools</td><td>18060-20</td><td></td></tr><tr><td>Microneedle driver</td><td>Fine Science Tools</td><td>12075-14</td><td></td></tr><tr><td>Spring Scissor</td><td>Fine Science Tools</td><td>15396-00</td><td></td></tr><tr><td>Insulin syringe</td><td>Various</td><td></td><td></td></tr><tr><td>1mL syringe</td><td>Various</td><td></td><td></td></tr><tr><td>20mL syringe</td><td>Various</td><td></td><td></td></tr><tr><td>Glucometer (one touch ultra mini)</td><td>Lifescan</td><td>70021208</td><td></td></tr><tr><td>Multiplex insulin and GLP-1 kit</td><td>Meso Scale Discovery</td><td>K15171C-1</td><td></td></tr><tr><td>GraphPad Prism 6.00</td><td>GraphPad Software</td><td></td><td></td></tr><tr><td>Nestlets</td><td>Ancare</td><td>NES3600</td><td></td></tr></tbody></table>

a murine model of vertical sleeve gastrectomy

减肥手术, 如垂直袖胃切除术 (VSG), 是一个手术的胃肠道, 是为了减肥的目的。减肥手术是目前最有效的长期治疗肥胖症。除了减肥, 减肥手术还会产生额外的健康好处, 如2型糖尿病缓解, 高血压缓解, 并减少罹患某些类型的癌症的风险。这些好处的减重机制仍未完全定义。因此, 动物模型的减肥手术正在开发和验证, 以确定机制, 导致这些好处, 目的是提高对胃肠生理的认识和确定新的治疗目标。VSG 已成为最常见的减肥手术在美国诊所, 因为它是非常有效的, 以产生体重下降和新陈代谢改善, 并比其他减肥程序更简单。因此, 我们已经开发和验证了一个小鼠模型的 VSG。这种小鼠 VSG 模型重述了许多 VSG 在人类中的作用, 其中包括改善葡萄糖和血压调节。该方法是根据胃的分离, 结扎胃血管, 并去除70% 的胃由 transecting 沿更大的曲率的胃。我们已成功地将这一手术协议应用于各种基因修饰的小鼠品系, 以确定机械贡献者对 VSG 的益处。此外, 这种小鼠 VSG 模型已与其他手术技术结合, 以获得更深的机械洞察力。因此, 这是一个简单而多用途的模型, 研究胃肠生理和减肥手术的健康效益。

在图 1中描述了假的和 VSG 的过程。图 1A显示缝合线在假手术过程中沿胃壁放置的位置。在 VSG 手术中, 这一区域是胃切开的地方。图 1B显示了 VSG 的表现后左胃的管状残留。
统计和数据分析 数据被显示为平均± SEM. 通过方差分析与 Tukey 的后测试?...

Watch this Scientific Journal Video about A Murine Model of Vertical Sleeve Gastrectomy at JoVE.com

A Murine Model of Vertical Sleeve Gastrectomy

下面介绍了垂直袖状胃切除术在小鼠中的表现。这是一种减肥手术, 包括切除约70% 的胃。

小鼠垂直套胃切除术的动物模型

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. ...

a-murine-model-of-vertical-sleeve-gastrectomy

Research

JoVE Journal

Biology

11.1K 次观看.  Cornell University. 下面介绍了垂直袖状胃切除术在小鼠中的表现。这是一种减肥手术, 包括切除约70% 的胃。

视频: A Murine Model of Vertical Sleeve Gastrectomy

Murine Pancreatic Islet Isolation

Hi, I am Greg Zott. I'm with the UCSF Diabetes Center in the Bluestone Laboratory, and today I'm gonna show you the process for isolating mouse eyelets. The procedure involves the injection of a collagenase solution into the pancreas of a C3 H donor mouse, where we will then remove that pancreas, digest it away, purify the eyelets on a fial gradient, and then handpick them for ultimate transplant into the kidney capsule of a diabetic NOD mouse, Which will be our recipient.Before we start this procedure, we diluted our ATE solution in a modified Hanks buffer and we have adjusted the concentration specific for the strain of mouse that we're using the C3 H.We found digests better at a 0.8 mgs per mil collagenase solution, and we will use that to inject into the common bile duct to distend the pancreas.Performing. The procedure today Will be Pavo codre of the diabetes center. He will inject, open up the mouse, expose the the intestines, and then inject into the common bile duct.We will spray down the mouse with ethanol to keep the hair down, and then we will also verify that the mouse has been sacrificed properly with a toe pinch. Pavo will now open up the mouse and exposing the intestine and the pancreas with a midline incision. The diaphragm will also be cut to verify that the mouse will be sacrificed.Before we get started, I'd like to point out some of the important structures. The stomach is lying right here with the pancreas attached underneath into the duodenum and above. This is the liver where we'll find the gallbladder and the common bile duct, which you can see right here.So what Pavo will do next is he will isolate the small intestine, find where the common bile duct enters the small intestine. Once the intestine is exposed, we clamp on either side of the common bile duct with mosquito clamps. We find that this allows more collagenates to enter the pancreas while other groups try to direct the clamp on top of the common bile duct.We find that this is a better technique in allowing enzyme to enter the pancreas. This is a gentle procedure. You don't want to tear the bowel.You wanna allow the clamps to lie gently on either side of the common bile duct. Now we will find a gallbladder, and once the gallbladder's exposed, we will use that as a guide to injecting the collagenase into the common bile duct. Powell's using a 30 gauge needle with a five cc syringe containing three mills of collagenase solution.As he's entering the common bile duct, he's distending that duct with collagenase solution and then he travels down the common bile duct till he reaches near the intestine. And as he's going down, the enzyme solutions being pumped into the injected into the pancreas. Now that the pancreas is inflated with collagenase solution, Powell's gonna remove the hemostats and then remove the pancreas from the the mouse carefully tearing it away from the intestine.He removes it from the stomach. Okay, and then using the spleen as a handle, he lifts up the pancreas and then slowly snips it away in the common bile duct, and that's it. Pancreas is laid out and then the spleen is removed, and there he's removing the meso enteric tissue, and then the pancreas is placed in a two mill collagenase solution and a sterilized glass vial and it's placed back on Ice.The isolated pancreas that we put into the glass vials will now be placed into a 37 degree water bath for approximately 17 minutes. We placed them into a 50 mil conical rack so that they don't float up. Once during the bath, the timer starts and the digestion begins.Once the digestion time is completed, vials are removed, and then they are shaken and disrupted gently. And once the tissue starts falling apart, the vial is then placed on ice. A sterile screen is placed over a 400 mil beaker that contains washing buffer.The pancreas digest is then carefully poured over the screen into the wash buffer. For demonstration purposes, the hood has been turned off and the glass of the hood has been raised so we could see what we're doing. Once the pancreas digest is poured into the beaker, the vials are rinsed with the same washing buffer and the wash buffer placed over the screen.We have a syringe filled with washing buffer. We will use that to forcefully wash the tissue through the screen. The idea is to release any loose eyelets that are unattached to the matrix, but leaving the membranes and the undigested tissue on the screen.And when you're finished washing a tissue, this is what you usually See at the end of the procedure. The Pancreas tissue that's in the bottom of the tube will now be transferred to two 50 mil conical, and the tissue will be washed, the tissue's mixed evenly, and then distributed evenly so that we have a equal pellet volume in each tube. The beaker is now rinsed with more washing buffer.The sides are rinse so that you get all tissue that is entered the beaker again. Then it's equally distributed between each two, both 50 mil. Conical then are topped off with wash buffer.The tissue is inverted several times and then placed into a centrifuge. This is a quick spin. The centrifuge is allowed to reach a thousand RPM and then is turned off.The tissue at this time is very S eyelets are very sensitive, and you don't want to over compact them. The tubes are removed and we will do this wash step three more times. This is the end of the third wash.The components of the 50 mil conical tube are transferred to the 15 conical tube and centrifuge for a thousand RP M and then stopped. The supernat will be removed and the first part of the FI Call will be started. We are at the step where we Have our pelleted di pancreas digest.We'll resuspend that tissue so it's nice and loose to the pellets. We'll add the first density gradient, which is 1 1 0 8. We'll add five mils to each tube.The pellet is then mixed thoroughly in each of the gradients. It's important that you get good tissue distribution in your heaviest gradient. Now we will layer two mils of 1 0 9 6 density.We will then on top of that, layer two mils of 1 0 6 9 density, and then on top of that 1 0 3 7 density. It's important while you're layering the densities that you don't mix. Then all the layers have been added to the tube.And you can see this is the top of 1 1 0 8, the top of 9 1 0 9 6, the top of 1 0 6 9 and the top of 1 0 3 7. And you could see the tissue is already starting to move to its proper density even without centri fusion. The fial gradients are brought up to speed at 1800 RPM for 15 minutes, and remember to keep the brake off after the tubes are removed from the centrifuge.You can see between the 0 0 9 6 and 0 0 7 layer, there's a nice band of eyelet tissue Pablo's. Now removing the 0 3 7 layer to remove any tissue debris and DNA that might have been released during the processing. Using a sterile plastic pasture pipette, he will now remove the islet layer and transfer them to a 50 mil conical containing 25 mils of wash buffer.You want do this step quickly because fol is toxic to eyelets. We actually removed the entire 0 6 9 and 0 9 6 layer. Pavo cuts the top of the plastic pasture pipette and rinses it with wash buffer.The eyelets are then centrifuge like we centrifuge previously. Centrifuge is brought up to a Thousand RPM and then we stop it and We aspirate off. The supernatant eyelets will be washed three times with wash media.So we just completed the eyelid Isolation process we took from the digest to the fol gradient to the purified eyelets after our third wash with wash buffer, we resuspended the eyelets in our RPMI media and we played it in a tissue culture plate. These eyelets are now ready for in vitro assays or in vivo preps like eyelet Transplant.

小鼠胰岛隔离

科研

生物学

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

肢体缺血小鼠模型

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

小鼠肾移植程序

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

帕金森氏病的小鼠模型：从6 - OH多巴胺病变行为测试

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

法援署结扎：有心肌梗死的小鼠模型

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

Techniques of Sleeve Gastrectomy and Modified Roux-en-Y Gastric Bypass in Mice

Obesity is a major public health issue, with a prevalence of 4 to 28% for men and 6.2 to 36.5% for women in Europe (from 2003 to 2008). Morbid obesity is frequently associated with metabolic complications, such as type 2 diabetes, hypertension, and dyslipidemia, reducing life expectancy and quality. In the absence of any effective noninvasive treatments, bariatric surgery is a valuable therapeutic option for patients with morbid obesity (body mass index (BMI) &#62;40 kg/m2), leading to long-term, sustained weight loss and improvements in metabolic complications. However, the underlying cellular and molecular mechanisms sustaining the beneficial effects of bariatric surgery are not yet fully understood. Due to the numerous genetically-modified strains available, the mouse model is the most convenient animal model to explore the molecular mechanisms behind the pleiotropic beneficial effects of bariatric surgeries. Here, we detailed the optimized healthcare methods and surgical protocols in mice for the two most widely-used bariatric surgeries: the sleeve gastrectomy and the modified Roux-en-Y gastric bypass. Deciphering the molecular mechanisms underlying the therapeutic effects of bariatric surgeries offers the promise of identifying new therapeutics targets.

Bariatric surgery is the most efficient way to reduce body weight and the deadly metabolic complications (diabetes, obesity, and dyslipidemia) frequently associated with morbid obesity. Mouse models of bariatric surgery represent a unique asset for deciphering molecular mechanisms behind the beneficial effects of these surgeries on diabetes, hypertension, and dyslipidemia.

袖胃切除术的技术和改良的小鼠空肠Roux-en-Y胃旁路

Obesity is a major public health issue, with a prevalence of 4 to 28% for men and 6.2 to 36.5% for women in Europe (from 2003 to 2008). Morbid obesity is ...

医学

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Sleeve Gastrectomy in Mice using Surgical Clips

The number of people who are overweight and obese is continually increasing both in the adult and adolescent populations. This coincides with the increased universal phenomenon of type 2 diabetes (T2D) and other metabolic problems. Bariatric surgery, such as SG, is currently one of the most effective and commonly used long-term treatment for obesity and T2D, but the association between them is not completely explored yet. The mechanisms underlying the outcomes seen after bariatric surgery in humans can be investigated based on preclinical animal studies. The SG reduces body weight, glucose levels and many metabolic parameters, and is easy to perform with a low incidence of complications. The goal of this work is to provide a simple method and an uncomplicated preclinical model of bariatric surgery in animals for researchers.

The prevalence of diabetes and obesity is continuously increasing worldwide. The mechanisms between diabetes, obesity and their associated mortality and co-morbidities need to be further investigated. Here, we present a protocol for sleeve gastrectomy (SG) in animals as an uncomplicated preclinical model of bariatric surgery.

使用手术夹的小鼠的套管胃切除术

The number of people who are overweight and obese is continually increasing both in the adult and adolescent populations. This coincides with the ...

Single-Anastomosis Duodeno-Ileal Bypass with Sleeve Gastrectomy Model in Mice

Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes mellitus, dyslipidemia, non-alcoholic steatohepatitis, cardiovascular events, and cancers) through restrictive and malabsorptive mechanisms. Understanding the mechanisms by which these procedures allow such improvements often require their transposition into animals, especially in mice, because of the ease of generating genetically modified animals. Recently, the single-anastomosis duodeno-ileal bypass with sleeve gastrectomy (SADI-S) has emerged as a procedure that uses both restrictive and malabsorptive effects, which is being used as an alternative to gastric bypass in case of major obesity. Thus far, this procedure has been associated with strong metabolic improvements, which has led to a marked increase in its use in daily clinical practice. However, the mechanisms underlying these metabolic effects have been poorly studied as a result of a lack of animal models. In this article, we present a reliable and reproducible model of SADI-S in mice, with a special focus on perioperative management. The description and use of this new rodent model will be helpful for the scientific community to better understand the molecular, metabolic, and structural changes induced by the SADI-S and to better define the surgical indications for clinical practice.

Single-anastomosis duodeno-ileal bypass (SADI-S) is an emerging bariatric procedure with important metabolic effects. In this article, we present a reliable and reproducible model of SADI-S in mice.

小鼠单吻合十二指肠回肠旁路与袖状胃切除术模型

Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes ...

Murine Vertical Sleeve Gastrectomy: A Surgical Procedure to Downsize Stomach in Experimental Mouse Models

Source: Garibay, D., Cummings, B. P. <a href="https://www.jove.com/video/56534" target="_blank">A Murine Model of Vertical Sleeve Gastrectomy.</a> J. Vis. Exp. (2017)
In this video, a vertical sleeve gastrectomy procedure is performed on an experimental mouse model. This procedure helps remove a significant portion of the stomach along its greater curvature.

小鼠垂直袖状胃切除术:一种在实验小鼠模型中缩小胃的外科手术

Source: Garibay, D., Cummings, B. P. A Murine Model of Vertical Sleeve Gastrectomy. J. Vis. Exp. (2017)
In this video, a vertical sleeve gastrectomy ...