这项研究得到了维也纳市市长的医疗科学基金和Österreichische 德国 Laboratoriumsmedizin 和 Klinische 瓦的支持。

这里描述的所有方法都已得到维也纳医科大学动物护理和使用委员会的批准, 并根据欧洲实验动物科学协会联合会 (FELASA) 进行。请注意, 本议定书所述的所有程序都应在机构和政府批准之后以及在技术上精通的工作人员执行。
1. 风喂养小鼠
注意: 在12小时的光/暗循环中保持所有 C57BL/6J 小鼠, 可以免费获得食物和水。
<ol><li>在6周的年龄, 将老鼠放在风 (40-60% 脂肪卡路里) 上8-12 周以诱发肥胖, 同时喂养精益对照组低脂饮食 (LFD) (10% 脂肪卡路里)。</li>
	<li>每周确定小鼠的体重。重量曲线应显示在两组相似的模式, 在风-美联储组更高的斜率。</li>
</ol>2. OGTT
注: 如果在 OGTT 每15分钟内选择血液取样时间点, 则应同时执行最多15只小鼠的实验, 以便每只老鼠至少有1分钟的处理时间。
<ol><li>OGTT 前一天的准备工作<ol>
<li>将小鼠转移到一个有新鲜床上用品的笼子里, 在测试前一夜将它们快速送到 (14 小时), 同时确保小鼠能够获得饮用水 (例如, 在第二天早上下午6:00 将食物移除, 在上午8:00 开始时间)。 注意: 禁食小鼠过夜是标准的方法, 但较短的快速 (5-6 小时) 是更具生理作用的小鼠 (参见讨论了解详细信息)。</li>
	</ol></li>
	<li>实验当天的准备工作 (但在实验之前)<ol>
<li>准备10毫升的20% 葡萄糖溶液 (溶解 d-(+)-葡萄糖在蒸馏水)。 注: 所有用于动物的试剂必须是药理级和无菌的。</li>
		<li>为等离子收集准备一个96井板, 通过为每个取样时间点和每只老鼠填充一井, 与5µL NaEDTA (0.5 M EDTA, pH 8.0 在0.9% 氯化钠, 存贮在 RT)。在实验中, 把这个盘子储存在冰上。 注意: 有关详细的核对清单, 请参见补充图 1 。</li>
	</ol></li>
	<li>测量所有小鼠的体重并用永久性标记标记它们的尾巴, 以使小鼠易于区分 (例如, 鼠标 1 = 1 破折号, 鼠标 2 = 2 破折号,等)。</li>
	<li>葡萄糖测量和血液取样 (图 2)<ol>
<li>小心地用锋利的剪刀 (图 2中的 "变体 A") 切断 1-2 mm 的尾尖。在服用新的血液样本进行血糖测定之前, 一定要先清除掉第一滴血液以避免溶血或组织液的污染。画一个小的血液样本 (〜3µL) 的测量基础的血糖水平 (= 时间点 0) 与血糖。 注意: 检查并调整血糖上的测试条的电荷数。 注: 作为一种替代采血方法, 尼克在图 2中用锋利的手术刀刀片 ("变体 B") 的鼠标侧尾静脉。侧尾静脉通常是访问大约1/3 沿尾巴长度从尾巴尖端, 向基地的尾巴, 为多个样本。推荐使用局部麻醉膏。在动物被送回笼子之前, 至少三十年代在软组织上应用手指按压来阻止血液流动。</li>
		<li>收集血液样本 (大约30µL) 使用新鲜毛细管 (保持毛细血管管水平)。用吸管把吸管顶端放在毛细管末端的顶部, 并小心地将收集到的血液放入96孔板的井中, 同时避免气泡, 从而清空毛细管管。对所有小鼠重复这个过程-一次一个。 注: 作为血液收集的替代品通过毛细管, 使用的吸管调整到正确的体积 (如, 30 µL) 收集血液, 或收集一滴血从尾部石蜡膜, 并吸管到 EDTA 溶液。严格避免石油果冻与血液或血糖试纸的接触, 因为它可能影响随后的葡萄糖和胰岛素测量。 注意: OGTT 对老鼠很有压力: 瘦老鼠在一夜之间会失去大约15% 的体重。此外, 不同时间点的血液取样会导致大量失血。为了便于采血, 可以用凡士林小心地按摩老鼠尾。 注: 机构指南可限制在规定时间内采集的血液的允许数量。取样量和 timepoints 应调整到不超过允许的最大值。应使用小鼠的体重来计算所允许的总血液提取量。</li>
	</ol></li>
	<li>根据体重计算葡萄糖溶液的体积 (1 克葡萄糖/千克体重; 这可以增加到3克/千克), 由口服胃对每只老鼠进行管理。例如, 一个体重为30克的老鼠需要150µL 20% 葡萄糖溶液来管理30毫克葡萄糖。 注: 以葡萄糖为基础的剂量对鼠标的重量是标准程序。如果身体成份数据是可利用的, 葡萄糖的剂量为 OGTT 应该根据稀薄的身体质量计算 (参见讨论为细节)。</li>
	<li>葡萄糖管理<ol>
<li>准备一切在整个实验期间 (定时器, 实验记录表, 葡萄糖监测和试纸, 毛细血管, 注射器, 葡萄糖溶液, 96-井板, 手术刀, 计算器, 平衡, 永久性标记, 工作台纸,吸管与小费, 和手套)。</li>
		<li>对于葡萄糖的应用, 通过牢牢抓住它的颈背来抑制老鼠。对颈部周围的皮肤施加足够的硬度, 防止老鼠扭出束缚, 并适当地把头向后倾斜。还要确保鼠标能正常呼吸。 注: 一旦葡萄糖管理开始, 良好的时间管理是非常重要的。</li>
		<li>小心地管理葡萄糖溶液 (基于步骤 2.5) 直接进入胃使用喂养针。小心地将喂食针通过口朝食道。让老鼠吞下针头: 针头完全下沉到小鼠的下食管/胃里。然后注入葡萄糖溶液 (图 3a)。<ol>
<li>如果遇到任何阻力, 或如果动物立即挣扎, 撤回针, 并重新定位它。在第一次胃后立即启动计时器, 并在1分钟间隔内将葡萄糖管理给所有其他小鼠。 注意: 将一滴葡萄糖溶液直接从喂食针放到老鼠嘴里可能会有帮助, 这会刺激舔舐和吞咽, 从而方便插入针头。插入喂食针时不要施加压力, 因为这可能会严重伤害动物。</li>
		</ol>
</li>
	</ol></li>
	<li>15分钟后, 测量血糖水平与血糖, 并另外采取血液样本 (〜30µL) (详见详细说明在步骤 2.4) 的每一个鼠标在相同的顺序, 因为他们被注射。 注意: 时间管理是非常重要的;尽可能使用与胃相同的时间间隔。让小鼠尽可能自由地移动, 并在整个过程中限制最小化以减少压力, 这可能会改变结果。用一只手的牛奶尾巴, 并收集与其他血液。</li>
	<li>根据预期结果重复步骤 2.7 (例如, 在30、45、60、90、120、150和葡萄糖管理后180分钟)。如果选定的时间点超过120分钟, 确保小鼠能够获得饮用水。确保老鼠总是能接触到饮用水。当实验完成后, 将小鼠返回到装有食物和水的笼子里。 注意: OGTT 对老鼠来说很累人。因此, 在进行下一次代谢测试之前至少要等待1周, 例如 ITT。</li>
	<li>实验后, 离心机血液样本在 2500 x g, 30 分钟, 4 ° c。将上清液 (等离子) 转移到板的空井, 并将其贮存在-20 ° c 直到分析。<ol>
<li>如果存在, 则记录样本的溶血 (见3节)。</li>
	</ol></li>
	<li>使用商业上可用的 ELISA 试剂盒确定血浆胰岛素水平 (请参见材料表), 按照制造商的说明进行操作。 注: 根据禁食状态以及对被调查小鼠的新陈代谢情况, 在这种化验过程中可能出现困难: 隔夜空腹胰岛素水平 (时间点 0) 非常低, 因此接近检测限度。为了避免这个问题, 建议的血浆体积的数量加倍, 相应地将 ELISA 法的结果减半。另一方面, 如果小鼠在 OGTT 时达到胰岛素峰值, 特别是在风喂养的小鼠, 胰岛素水平可能超过检测限度: 稀释样本 (例如, 10 倍与 0.9% NaCl) 和重复 ELISA 检测。血浆样品中的溶血可能导致胰岛素的降解, 导致读数值的减少。降解取决于样品中的时间、温度和血红蛋白浓度。始终保持溶血样品冷或冰, 以减少胰岛素的退化。</li>
</ol>3. ITT
注意: 在执行 ITT 时, 同样的预防措施也必须适用于 OGTT (处理老鼠、血液、血糖和凡士林等)。例如, 所有的注射应在15分钟内进行1分钟的间隔, 如果15只老鼠进行并行测试。对于 ITT, 随后收集的血液样本毛细管管是可选的。
<ol><li>实验前的准备<ol>
<li>在注射胰岛素前至少2小时的快速小鼠, 同时确保小鼠能够饮用饮用水 (例如, 在上午8:00 时取出食物, 在2-5 小时后测试小鼠)。</li>
		<li>稀释胰岛素 1:1, 000 在0.9% 氯化钠 (股票: 100 u/毫升胰岛素; 工作浓度 0.1 u/毫升), 并准备20% 葡萄糖 (d-(+) 葡萄糖溶解在蒸馏水中), 以管理, 如果老鼠变得低血糖。 注意: ITT 通常是在短时间内进行, 以避免低血糖, 否则可能发生在夜间禁食动物。所有用于动物的试剂都必须是药理级和无菌的。</li>
	</ol>
</li>
	<li>测量小鼠的体重, 标记尾巴, 用锋利的剪刀剪尾尖, 并测量2.4 步中 OGTT 的基本血糖水平。</li>
	<li>胰岛素注射液<ol>
<li>注射胰岛素腹腔 (0.75 U 胰岛素/千克体重, 预先计算), 用颈背法抑制小鼠。</li>
		<li>使用新鲜的, 无菌27或30针为每种动物, 以避免不适和任何注射现场感染的风险。 注意: 皮肤的消毒可以延长胰岛素的持续时间, 因此会对动物造成额外的干扰。因此, 不建议这样做。</li>
		<li>将小鼠头部向下倾斜, 露出动物腹侧。将无菌针放在动物腹部右下象限30°角的斜面上 (图 3b)。在第一个鼠标插入后立即启动计时器。 注: 低剂量 ITTs (0.1 U/千克) 可进行特别评估肝胰岛素敏感性。至于 OGTT, 根据体重计算注射量是标准的程序, 而根据瘦体质量的剂量是首选, 如果身体组成的数据是可用的。</li>
	</ol>
</li>
	<li>测量所选时间点的血糖水平 (例如, 在15、30、45、60和90分钟之后)。 注: 由于胰岛素在小鼠13中有短时间的10分钟, 胰岛素治疗后的晚期差异 (如如, 2 小时后) 可能不反映胰岛素作用的直接作用。管理20% 葡萄糖溶液, 以防老鼠变成低血糖 (血糖水平低于35毫克/dL), 并有死亡的危险。</li>
	<li>在最后的时间点后, 将小鼠放回他们准备好大量食物和水的笼子里。</li>
</ol>

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+phenotyping+guidelines:+assessing+glucose+homeostasis+in+rodent+models.">Metabolic phenotyping guidelines: assessing glucose homeostasis in rodent models.</a> J Endocrinol. 222 (3), G13-G25 (2014).</li><li>Arioli, V., Rossi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Errors+related+to+different+techniques+of+intraperitoneal+injection+in+mice.">Errors related to different techniques of intraperitoneal injection in mice.</a> Appl Microbiol. 19 (4), 704-705 (1970).</li><li>Miner, N. 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作者没有什么可透露的。

随着糖尿病和相关疾病在世界人口中的高患病率, 对疾病的分子机制、预防和治疗的研究有很强的要求19。该协议描述了建立风小鼠的成熟方法, 一种用于代谢研究的健壮动物模型, 以及 OGTT 和 ITT 的传导, 它们是评估全身代谢改变的有力工具。如胰岛素抵抗。本文所提出的方法可能有助于研究疑似基因、环境因素以及药理、膳食、物理或基因治疗对全身葡萄糖代谢的作用<sup class="x...

在发达世界, 由于缺乏体力活动和加工食品的过量消费, 肥胖和糖尿病已经达到了流行病的程度, 这是由于迅速的城市化、工业化和全球化所造成的影响。虽然研究胰岛素抵抗和它的疾病, 如高脂血症和动脉粥样硬化, 已经在过去的几十年中获得突出, 复杂的生物机制, 调节代谢在健康和疾病仍然不完全了解, 仍然迫切需要新的治疗方法来预防和治疗这些疾病1。
胰岛素, 它的 counter-regulatory 激素胰高血糖素作为主要调节细胞能量供应和营养素平衡, 从而也保持适当的系统性血糖浓度2。葡萄糖本身是胰腺β细胞分泌胰岛素的主要刺激之一, 而其他营养素、体液因子以及神经输入则进一步改变了这种反应。胰岛素通过促进过剩的血糖扩散到肌肉和脂肪细胞, 并进一步激活糖酵解以及蛋白质或脂肪酸的合成, 从而触发了联邦政府的合成代谢过程。此外, 胰岛素抑制糖, 抑制肝脏葡萄糖的输出。慢性过量能量消耗和 meta-inflammation 导致高和周围胰岛素抵抗由于胰岛素受体表达的下调以及下游信号通路的改变, 从而导致受损对胰岛素介导的葡萄糖处理的敏感性以及对肝脏葡萄糖分泌不足的抑制作用3,4,5,6。
广泛的动物模型与遗传, 营养, 或实验性诱导疾病已被证明是优秀的工具, 研究胰岛素抵抗的分子机制和各种形式的糖尿病以及其伴随的疾病7.一个典型的例子是广泛使用, 并建立了良好的风诱导小鼠模型, 这是特点是快速体重增加, 由于饮食摄入与降低代谢效率相结合, 导致胰岛素抵抗8,9. 在动物模型和人类中, 空腹血糖和胰岛素水平的升高, 以及对葡萄糖管理的耐受性受损, 常被用于胰岛素抵抗和其他葡萄糖系统改变的指标代谢.因此, 监测血糖和胰岛素水平的基础状态或刺激后, 容易接近读数。
本议定书概述了风喂养小鼠的产生, 以及两种常用的方法, 口服葡萄糖耐受试验 (OGTT) 和胰岛素抵抗试验 (ITT), 这是有用的表征代谢表型和调查葡萄糖代谢的改变。我们详细地描述了 OGTT, 它评估了口服葡萄糖负荷和胰岛素分泌的处理时间。此外, 我们还提供了关于如何通过监测血糖浓度来对胰岛素的反应来检查全身胰岛素的行为的指导。本文中描述的协议已被广泛采用, 并已在多个研究中使用10、11、12。除了轻微的修改, 这可能有助于增加成功, 我们提供了实验设计和数据分析的指导方针, 以及有用的提示, 以避免潜在的陷阱。本文所描述的协议可以是非常强大的工具, 以研究遗传, 药理, 饮食, 和其他环境因素对全身葡萄糖代谢的影响, 并对其相关的疾病, 如胰岛素抵抗。除了刺激葡萄糖或胰岛素, 其他各种化合物可用于刺激, 根据个人研究的目的。虽然在这份手稿的范围之外, 许多其他下游应用可以在抽取的血样上进行, 例如对血糖和胰岛素以外的血液值进行分析 (例如、血脂和脂蛋白图谱) 以及详细代谢标志物的分析 (例如, 通过定量实时聚合酶链式反应 (PCR), 免疫印迹分析, 酶联免疫吸附试验 (ELISA))。进一步的流式细胞仪和荧光活化细胞分类 (组) 可用于研究不同单细胞种群的影响, 同时也可用于不分析。
总的来说, 我们提供了一个简单的协议来生成一个风诱导的小鼠模型, 同时进一步描述了研究全身代谢变化的两个强有力的方法, OGTT 和 ITT, 这可以成为有用的工具, 研究疾病的发病机制和开发新的治疗方法, 特别是在代谢相关疾病领域, 如胰岛素抵抗 & 糖尿病。

<table>
	<tbody>
		<tr>
			<td>Mouse strain: C57BL/6J</td>
			<td>The Jackson Laboratory</td>
			<td>664</td>
			<td>LFD/HFD</td>
		</tr>
		<tr>
			<td>Accu Chek Performa - Glucometer</td>
			<td>Roche</td>
			<td>6870228</td>
			<td>OGTT/ITT</td>
		</tr>
		<tr>
			<td>Accu Chek Performa - Strips</td>
			<td>Roche</td>
			<td>6454038</td>
			<td>OGTT/ITT</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose solution</td>
			<td>Sigma-Aldrich</td>
			<td>G8769</td>
			<td>OGTT</td>
		</tr>
		<tr>
			<td>Actrapid - Insulin</td>
			<td>Novo Nordisk</td>
			<td>417642</td>
			<td>ITT</td>
		</tr>
		<tr>
			<td>Reusable Feeding Needles</td>
			<td>Fine Science Tools</td>
			<td>#18061-22</td>
			<td>OGTT; 22 gauge (-24 gauge for young mice)</td>
		</tr>
		<tr>
			<td>Omnifix-Fine dosing syringes</td>
			<td>Braun</td>
			<td>9161406V</td>
			<td>OGTT/ITT</td>
		</tr>
		<tr>
			<td>Sterican Insulin needle (30G x 1/3&#34;; &#248; 0.30 x 13 mm)</td>
			<td>Braun</td>
			<td>304000</td>
			<td>ITT; lean mice</td>
		</tr>
		<tr>
			<td>Sterican (G 27 x 3/4&#34;; &#248; 0.40 x 20 mm)&#160;&#160;</td>
			<td>Braun</td>
			<td>4657705</td>
			<td>ITT; mice on HFD</td>
		</tr>
		<tr>
			<td>96 Well PCR Plates, non-skirted, flexible</td>
			<td>Braintree Scientific, Inc.</td>
			<td>SP0016</td>
			<td>OGTT</td>
		</tr>
		<tr>
			<td>Ultrasensitive Mouse Insulin ELISA kit</td>
			<td>Crystam Chem</td>
			<td>90080</td>
			<td>OGTT</td>
		</tr>
		<tr>
			<td>Rodent Diet with 60% kcal% fat</td>
			<td>Research Diets Inc</td>
			<td>D12492</td>
			<td>mice on HFD</td>
		</tr>
		<tr>
			<td>Rodent Diet with 10% kcal% fat.</td>
			<td>Research Diets Inc</td>
			<td>D12450B</td>
			<td>mice on LFD</td>
		</tr>
		<tr>
			<td>BRAND micro haematocrit capillary</td>
			<td>Sigma-Aldrich</td>
			<td>BR749321</td>
			<td>OGTT/ITT</td>
		</tr>
		<tr>
			<td>Vaseline - creme</td>
			<td>Riviera</td>
			<td>P1768677</td>
			<td>OGTT/ITT</td>
		</tr>
	</tbody>
</table>

study of in vivo glucose metabolism in high-fat diet-fed mice using oral glucose tolerance test (ogtt) and insulin tolerance test (itt)

肥胖是2型糖尿病发病的最重要的单一危险因素, 这种疾病的特点是抗胰岛素刺激的葡萄糖摄取和全身糖代谢的严重失代偿。尽管在了解葡萄糖代谢方面取得了相当大的进展, 但它在健康和疾病方面的调控的分子机制仍然 under-investigated, 而新的预防和治疗糖尿病的方法是迫切需要的。饮食衍生葡萄糖刺激胰腺分泌胰岛素, 这是主要调节器的细胞合成过程中的美联储状态, 从而平衡血糖水平保持系统的能量状态。慢性过度触发 meta-inflammation, 导致周围胰岛素受体相关信号的改变, 从而降低对胰岛素介导的葡萄糖处理的敏感性。这些事件最终导致空腹血糖和胰岛素水平升高以及葡萄糖耐受减少, 这反过来又成为胰岛素抵抗的重要指标。在这里, 我们提出了一个协议的产生和代谢特征的高脂饮食 (风) 喂养小鼠作为一个常用的模型, 饮食诱导胰岛素抵抗。我们详细说明了口服葡萄糖耐受试验 (OGTT), 它监测的周围处理的口服葡萄糖负荷和胰岛素分泌随着时间的推移。此外, 我们提出了一个协议的胰岛素耐受性测试 (ITT), 以监测全身胰岛素行为。这些方法和它们的下游应用代表了强有力的工具来表征小鼠的一般代谢表型, 以及专门评估葡萄糖代谢的变化。它们可能在胰岛素抵抗、糖尿病和肥胖的广泛研究领域中特别有用, 以便更好地了解发病机制, 并测试治疗干预的效果。

图 1说明了在饮食上对小鼠进行代谢分的示意图时间表。在大约6周的年龄, 老鼠应该放在一个风, 而 LFD 组可以作为对照组。重要的是, 体重应该每周确定, 以观察是否有预期的体重增加。任何形式的压力 (例如、噪音或攻击性的男性行为) 都会影响体重增加, 应立即消除。每组小鼠的饮食实验应包括至少10小鼠, 因为这些饮食-实验是耗时的, 和异?...

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利用口服葡萄糖耐量试验OGTT和胰岛素耐量试验ITT研究高脂饮食喂养小鼠的体内葡萄糖代谢- 博士论文医学|视频

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

口服葡萄糖耐受试验 (OGTT) 和胰岛素耐受试验 (ITT) 对高脂饮食喂养小鼠体内葡萄糖代谢的影响研究 (英文)

本文描述了高脂喂养小鼠的生成和代谢特性, 作为饮食诱导的胰岛素抵抗和肥胖的模型。它还有详细的协议来执行口服葡萄糖耐受试验和胰岛素耐受试验, 监测葡萄糖代谢的全身变化在体内。

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Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

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67.3K 次观看.  Medical University of Vienna. 本文描述了高脂喂养小鼠的生成和代谢特性, 作为饮食诱导的胰岛素抵抗和肥胖的模型。它还有详细的协议来执行口服葡萄糖耐受试验和胰岛素耐受试验, 监测葡萄糖代谢的全身变化在体内。

视频: Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

听力:IMAP测试电池的决策意识

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

科研

医学

The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides1. Thus, identification of cell-free correlates that directly regulate the number of CD4+ T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates.
The number of stem cells that enter blood and are destined to become circulating CD4+ T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion2. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLECS) and the HLECS-reactive active &alpha;1proteinase inhibitor (&alpha;1PI, &alpha;1antitrypsin, SerpinA1)3. In HIV-1 disease, &alpha;1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active &alpha;1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 &mu;M, and to achieve normal levels during the symptomatic categories4, 5. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with &gt;220 CD4 cells/&mu;l, CD4 counts were correlated with serum levels of active &alpha;1PI (r2=0.93, p&lt;0.0001, n=26) and inactive &alpha;1PI (r2=0.91, p&lt;0.0001, n=26) 5. Administration of &alpha;1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting &alpha;1PI participates in regulating the number of CD4+ T cells in blood 3.
With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active &alpha;1PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum &alpha;1PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to &alpha;1PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active &alpha;1PI in saliva. The resulting inhibition of PPE by active &alpha;1PI can be measured by adding the PPE substrate SA3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/&mu;l), the concentration of &alpha;1PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person6. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable7. Thus, active &alpha;1PI in saliva is calculated as a ratio to saliva protein content and is termed the &alpha;1PI Index. Results presented herein demonstrate that the &alpha;1PI Index provides an accurate and precise physiologic method for calculating CD4 counts.

The &#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

A CD4 enumeration method, the &#x03B1;-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The &alpha;-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.

测试:α-细胞快速免费的CD4计数，使用全唾液

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the arteries (&beta;2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. 
Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine.
As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.

We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.

多巴酚丁胺诱导的心脏压力测试和监控通过在小鼠心脏导管插入术

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

一个<em在体内</em分析测试血管通透性

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used techniques require killing of the animals and postmortem recovery of the meal1,2. This approach prevents longitudinal studies to determine changes in gastric emptying with age and progression of disease. The commonly used [13C]-octanoic acid breath test for humans3 has been modified for use in mice4-6 and rats7 and we previously showed that this test is reliable and responsive to changes in gastric emptying in response to drugs and during diabetic disease progression8. In this video presentation the principle and practical implementation of this modified test is explained. As in the previous study, NOD LtJ mice are used, a model of type 1 diabetes9. A proportion of these mice develop the symptoms of gastroparesis, a complication of diabetes characterized by delayed gastric emptying without mechanical obstruction of the stomach10.
This paper demonstrates how to train the mice for testing, how to prepare the test meal and obtain 4 hr gastric emptying data and how to analyze the obtained data. The carbon isotope analyzer used in the present study is suitable for the automatic sampling of the air samples from up to 12 mice at the same time. This technique allows the longitudinal follow-up of gastric emptying from larger groups of mice with diabetes or other long-standing diseases.

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Determination of gastric emptying with a non-invasive [13C]-octanoic acid breath test for tracking gastroparesis in female NOD LtJ mice.

使用的非肥胖型糖尿病小鼠胃排空的评估[<sup&gt; 13</sup&gt; C-辛酸呼气试验

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used ...

Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical profiles, inclusive of blood glucose levels in both the fed and fasted state, HbA1c, insulin levels, clinical history of diet, body composition, and body weight status, an assessment of normal and abnormal glycemic control can be made. Interpretation of an IVGTT is done through measurement of changes in glucose and insulin levels over time in relation to the dextrose challenge. Critical components to be considered are: peak glucose and insulin levels reached in relation to T0 (end of glucose infusion), the glucose clearance rate K derived from the slope of rapid glucose clearance in the first 20 min (T1 to T20), the time to return to glucose baseline, and the area under the curve (AUC). These IVGTT measures will show characteristic changes as glucose homeostasis moves from a healthy to a diseased metabolic state5. Herein we will describe the characterization of nonhuman primates (Rhesus and Cynomolgus macaques), which are the most relevant animal model of Type II diabetes (T2D) in humans and the IVGTT and clinical profiles of these animals from a lean healthy, to obese dysmetabolic, and T2D state 8, 10, 11.

The goal of this protocol is to present a standard method to perform intravenous glucose tolerance tests (IVGTTs) to assess glycemic control in nonhuman primates and assess their metabolic status from healthy to dysmetabolic.

代谢状态的表征非人灵长类动物与静脉葡萄糖耐量试验

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). Impaired uptake and metabolism of glucose in multiple organ systems due to insulin resistance is a hallmark of type 2 diabetes. The ability of this technique to extract an image-derived input function from the vena cava using an iterative deconvolution method eliminates the requirement of the collection of arterial blood samples. Fitting of tissue and vena cava time activity curves to a two-tissue, three compartment model permits the estimation of kinetic micro-parameters related to the 18F-FDG uptake from the plasma to the intracellular space, the rate of transport from intracellular space to plasma and the rate of 18F-FDG phosphorylation. This methodology allows for multiple measures of glucose uptake and metabolism kinetics in the context of longitudinal studies and also provides insights into the efficacy of therapeutic interventions.

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This study describes a protocol that uses 18F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of 18F-FDG into tissues.

确定葡萄糖代谢动力学研究<sup&gt; 18</sup&gt; F-FDG微PET / CT

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). ...

Improving Strength, Power, Muscle Aerobic Capacity, and Glucose Tolerance through Short-term Progressive Strength Training Among Elderly People

This protocol describes the simultaneous use of a broad span of methods to examine muscle aerobic capacity, glucose tolerance, strength, and power in elderly people performing short-term resistance training (RET). Supervised progressive resistance training for 1 h three times a week over 8 weeks was performed by RET participants (71&#177;1 years, range 65-80). Compared to a control group without training, the RET showed improvements on the measures used to indicate strength, power, glucose tolerance, and several parameters of muscle aerobic capacity. Strength training was performed in a gym with only robust fitness equipment. An isokinetic dynamometer for knee extensor strength permitted the measurement of concentric, eccentric, and static strength, which increased for the RET group (8-12% post- versus pre-test). The power (rate of force development, RFD) at the initial 0-30 ms also showed an increase for the RET group (52%). A glucose tolerance test with frequent blood glucose measurements showed improvements only for the RET group in terms of blood glucose values after 2 h (14%) and the area under the curve (21%). The blood lipid profile also improved (8%). From muscle biopsy samples prepared using histochemistry, the amount of fiber type IIa increased, and a trend towards a decrease in IIx in the RET group reflected a change to a more oxidative profile in terms of fiber composition. Western blot (to determine the protein content related to the signaling for muscle protein synthesis) showed a rise of 69% in both Akt and mTOR in the RET group; this also showed an increase in mitochondrial proteins for OXPHOS complex II and citrate synthase (both ~30%) and for complex IV (90%), in only the RET group. We demonstrate that this type of progressive resistance training offers various improvements (e.g., strength, power, aerobic capacity, glucose tolerance, and plasma lipid profile).

The effect of short-term resistance training on elderly people was investigated through the simultaneous use of several methods. Compared to a control group, many improvements were seen, including on muscle aerobic capacity, glucose tolerance, strength, power, and muscle quality (i.e., protein involved in cell signaling and muscle fiber type composition).

通过长期进步力量训练提高力量，力量，肌肉有氧能力和葡萄糖耐量

This protocol describes the simultaneous use of a broad span of methods to examine muscle aerobic capacity, glucose tolerance, strength, and power in ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

持续药物输注系统对小鼠脑内下丘脑胰岛素信号的研究

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

口服葡萄糖耐受试验 (OGTT) 和胰岛素耐受试验 (ITT) 对高脂饮食喂养小鼠体内葡萄糖代谢的影响研究 (英文)

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