提交人希望承认由路易斯安那州立的健康科学中心提供的机构支助, 资助了该项目。

所有的任务都是遵守 #8759 和 #9189 的协议, 如 LSUHSC 的 IRB 办公室批准的。所有动物的工作都是遵守协议 #3285 在 LSUHSC IACUC 办事处批准的。
1. 夹细胞片的播种
注: 见图 1。
<ol><li>种子干细胞在大约80% 融合在组织培养板材 (6 cm 或6井板材)。为每一个特警所需, 种子1常规组织培养井和1井相应大小的聚 (n-丙烯酰胺 (pNIPAAm) 涂层组织培养塑料板材。 注: 因此, 一个6井板的斯瓦特将需要种子细胞在一个6良好的标准组织培养板 (基层) 和一个6井 pNIPAAm 涂层组织培养板 (上层)。pNIPAAm 涂层板可以商业或生产在实验室12,13,14。</li>
	<li>保持干细胞在37摄氏度和 5% CO2 Dulbecco 的改良鹰培养基 (DMEM) 补充10% 血清和1x 青霉素/链霉素.每2天更换一次媒体。</li>
	<li>允许细胞联合, 直到它们成为100% 汇合, 并采取一个纹状模式 (大约6–8天)。 注意: 这些细胞将需要作为一个完整的细胞表功能, 以稳定的浮力脂肪组织。没有足够的汇合细胞将片段后, 与笏播种。</li>
</ol>2. 特警装备的准备工作
<ol><li>准备10毫升整除数1x 的平衡盐溶液 (HBSS);准备足够的需要数量的板块与体积备用。</li>
	<li>
准备用于斯瓦特播种的柱塞装置。使用简单的丙烯酸塑料来构造柱塞, 它由连接在圆圆盘上的茎组成。确保它适合组织培养井的周长 (直径: < 6 厘米为 6 cm 菜, < 3.5 cm 为6井板材; 近似质量: 6.7 g 为 6 cm 盘, 5.1 g 为6井板材)。
	<ol>
<li>准备额外的活塞, 以确保该过程将顺利运行, 以防有任何个别的活塞问题。</li>
		<li>将实验室胶带绕在柱塞盘的边缘, 至少 2x, 以防止明胶溶液渗漏。</li>
		<li>用70% 乙醇喷洒生物安全柜 (BSC)。在平衡计分卡内喷洒15毫升锥形管架, 空, 名额15毫升圆锥管;管将有助于确保放置明胶活塞。</li>
		<li>将每个带包的柱塞彻底喷涂70% 乙醇, 放入机架上的15mL 锥形管。喷涂金属垫圈 (近似质量6.3 克), 并将它们放在机架上, 连同一对钩钳;在斯瓦特播种期间, 垫圈会增加活塞的重量。</li>
		<li>关闭平衡计分卡和打开 UV 光, 以方便干燥和灭菌。 注意: 理想情况下, 在斯瓦特播种之前执行此步骤24小时。或者, 在组织收集/斯瓦特播种的当天进行处理;然而, 在这种情况下, 允许15–45最小的 UV 干燥/灭菌。</li>
	</ol>
</li>
</ol>3. 制备明胶活塞-应用于上细胞片
<ol><li>将水浴加热到摄氏75摄氏度。</li>
	<li>
将0.75 克明胶粉加入10毫升的 1x HBSS, 制备明胶溶液。在通风罩下添加100µL 1 米氢氧化钠, 以平衡溶液的 pH 值。
	<ol>
<li>将10毫升的股票添加到水浴中, 每5分钟用力摇动, 直到粉末溶解成溶液。目的在加热水浴后不久将粉末明胶溶解。</li>
		<li>打开 "平衡计分卡", 关闭 UV 光, 并提高窗扇。喷雾平衡计分卡表面和准备过滤器供应 (5 毫升鲁尔锁注射器和0.2 µm 注射器过滤器)。准备多个过滤器, 以有效地应变足够数量的明胶适用于活塞。</li>
	</ol>
</li>
	<li>当明胶溶液达到均匀一致时, 过滤溶液并将其应用于活塞。用明胶装注射器。将明胶应用于塑料活塞, 通过注射器过滤器 (〜2.5 毫升为6井板, 4.5 毫升为 6 cm 盘) 和允许凝固 (~ 20 分钟)。</li>
	<li>一旦凝胶是固体, 从活塞的边缘打开胶带。用钩钳, 从柱塞的外边缘 (即半月板凸起的边缘) 取出明胶。确保柱塞中心的剩余明胶是完全水平的, 以最大限度地与 ADSC 板接触。</li>
	<li>一旦多余的明胶被除去, 轻轻地将明胶活塞到 pNIPAAm 涂层的 ADSC 板上。用金属垫圈称量活塞。应用活塞时不要剪切细胞板。</li>
	<li>
在室温下将活塞放在电池片上1.5 小时。在冰水浴中孵化板/活塞1.5 小时, 以完成从 pNIPAAm 涂层板表面分离的细胞片。
	<ol>
<li>在冰浴中孵化时要小心;不要让冰浴在孵化过程中污染细胞介质。</li>
		<li>完成孵化后, 清洁底部的 pNIPAAm 涂层, 以消除无菌水。</li>
	</ol>
</li>
</ol>4. 白色脂肪组织加工
<ol><li>当从手术室收集人体脂肪组织时, 将所有样品保存在冰上的无菌容器中, 直到斯瓦特被播种。添加不育的维持培养基, 如磷酸盐缓冲盐水 (PBS), 脂肪组织容器的组织稳定性。</li>
	<li>增加冷细胞培养培养基到1.5 毫升离心管为每个井/菜将播种 (100 µL 为6井板, 200 µL 为 6 cm 菜)。</li>
	<li>
切碎脂肪组织。
	<ol>
<li>对于固体脂肪组织段, 请按如下所示进行。<ol>
<li>在无菌 pbs 中清洗大块的脂肪组织 3x, 尽可能多地去除 pbs。</li>
			<li>粗切的组织与镊子和无菌剃刀和删除尽可能多的血管和筋膜 (见讨论更详细)。</li>
			<li>用剃刀切碎的脂肪, 直到切碎的组织采取厚, 液体的一致性。 注: 理想的组织将出现均匀, 没有可见的单独部分的笏, 虽然这并不总是可能的。</li>
		</ol>
</li>
		<li>过程 lipoaspirate 如下所示。<ol>
<li>在平衡计分卡下, 将无菌纱布放在烧杯顶部, 并将烧杯放在较大的烧杯中, 收集多余的液体。</li>
			<li>使用25毫升血清学吸管, 绘制尽可能多的 lipoaspirate, 并将其应用于无菌纱布的表面。将 PBS 直接应用于此表面, 以洗涤 lipoaspirated 脂肪, 清除多余的血液和脂质残留物。</li>
			<li>使用镊子恢复排泄的组织, 转移到一个无菌切碎的表面, 并剁碎 lipoaspirate。</li>
		</ol>
</li>
	</ol>
</li>
	<li>使用无菌剃刀切断 p1000 吸管的远端, 转移切碎的组织;这将减少可能导致脂肪细胞裂解的剪切应力。一旦达到适当的组织一致性, 将所需的碎组织体积转移到每1.5 毫升管 (300–400µL 6 井板, 500–600µL 6 厘米菜)。将碎笏和介质简单地混合在管子里。</li>
	<li>
取基 ADSC 板, 醒酒/吸入介质。更换介质与笏/媒体混合物从每1.5 毫升管。
	<ol>
<li>轻轻地从 pNIPAAm 涂层板上取出明胶活塞, 并将其应用于基 ADSC 板上的笏混合物上。在显微镜下检查 pNIPAAm 涂层板的单层, 以确认细胞脱离。</li>
	</ol>
</li>
	<li>在平衡计分卡下设置一个热块到大约37–40°c。随着活塞仍然到位, 移动板块到热块的表面。加入2–3毫升的温热培养基, 孵化细胞, 促进凝胶融化。</li>
	<li>30分钟后, 轻轻地从板面上取出活塞。更换基部组织培养板的盖子, 移动到细胞培养孵化器。一旦明胶已完全液化37摄氏度, 吸入和取代细胞培养培养基。</li>
	<li>保持斯瓦特在37摄氏度和 5% CO2在苯酚无红 M199 培养基与7µM 胰岛素, 30 µM 地塞米松, 1x 青霉素/链霉素。维护在大约2毫升介质为6井板材和3毫升为 6 cm 菜。每2天更换一次媒体。</li>
</ol>5. 斯瓦特收获
<ol><li>制备胶原酶 (0.5 毫克/毫升胶原酶, 500 nM 腺苷, 在 PBS) 整除数15毫升圆锥管 (大约体积10毫升)。冷冻管, 并储存在-20 摄氏度。</li>
	<li>从细胞中吸取任何培养基, 用 pbs 冲洗 1x, 然后吸入 pbs。在37摄氏度的水浴中解冻胶原酶整除数。 注: 理想情况下, 胶原酶溶液将达到摄氏37摄氏度;紧接着, 添加组织。</li>
	<li>
将斯瓦特板上的所有组织添加到单个整除数。收获斯瓦特使用一个不育细胞刮刀和转移到胶原酶整除数与切断 p1000 吸管提示。将组织直接添加到含有胶原酶溶液的15毫升锥形管中。
	<ol>
<li>或者, 在50毫升圆锥管中孵化组织/胶原酶混合物;增加的表面积将进一步促进酶消化。</li>
	</ol>
</li>
	<li>将取样管放置在一个45°角的孵化轨道振动筛中。孵育在 200 rpm, 在37°c 为30–60分钟。</li>
	<li>将250µm 网格过滤器放入一个新的15毫升锥形管中收集。通过过滤器倒入经消化的脂肪细胞溶液。 注意: 这将允许所有细胞通过过滤纤维组织。</li>
	<li>允许流动通过在室温下坐5分钟, 以允许相分离。 注: 脂肪细胞将漂浮到溶液的顶端, 而脂质基质干细胞 (陶瓷) 将沉淀到较低的阶段。离心5分钟在 500 x g也可以最大化细胞分离或收获周围的干细胞在颗粒。</li>
	<li>
使用切断 p1000 吸管尖端, 转移脂肪细胞 (漂浮层在胶原酶溶液的顶部) 到一个1.5 毫升离心收集管。一次服用250µL, 慢慢地沿着管子的边缘缓慢地收集脂肪细胞。
	<ol>
<li>在收集脂肪细胞的同时, 慢慢地旋转管子, 以最大限度地恢复附着在体内的体细胞。继续绘制更多的细胞, 直到1.5 毫升离心管满。</li>
	</ol>
</li>
	<li>用注射器 (~ 21 克) 从分离的脂肪细胞中取出多余的液体。将针头浸入漂浮的脂肪细胞层下。简单地搅动针, 使任何细胞粘在针轴上, 然后等待脱落的脂肪胞漂浮到顶端。</li>
	<li>慢慢地画出多余的液体, 小心避免意外去除脂肪细胞。使用离心管毕业, 以一贯隔离样本量 (例如,.每个样品0.1 毫升)。</li>
	<li>利用分离细胞进行 DNA/RNA 提取、葡萄糖摄取测定、脂肪测定等.
</li>
</ol>

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作者没有什么可透露的。

本议定书详细介绍了干细胞在三明治人体白色脂肪组织中的应用;人类 ADSC 细胞系可以通过建立完善的协议15进行隔离。然而, 该系统可以适应个性化的研究要求 (如使用 3T3L 1 细胞三明治小鼠笏)。这个过程涉及处理主要的人体组织。应采用标准安全预防措施;作为 BSL-2 病原体 (如HIV、HepC) 处理人体组织。只在平衡计分卡下直接处理组织。根据机构安全标准, 佩戴所有合适的 PP...

脂肪组织是肥胖的主要器官, 其在美国1的直接每年医疗费用为1470亿美元和2100亿美元。脂肪组织的积聚也会导致其他主要死因, 如心脏病、II 型糖尿病和某些类型的癌症2。体外培养模型对代谢研究和药物开发是必不可少的, 但目前脂肪组织的研究模式有很大的不足。脂肪细胞是脆弱的, 浮力的, 和晚期分化, 不会坚持细胞培养塑料, 因此不能使用传统的细胞培养方法培养。自二十世纪七十年代以来, 一些方法被用来克服这些障碍, 包括使用玻璃盖玻片, 天花板文化, 悬浮培养, 细胞外基质3,4,5,6,7. 然而, 这些方法的特点是细胞死亡和分化, 通常只用于为期两周的研究期。此外, 这些模型不尝试重述本机脂肪微环境, 因为它们不维持完整的 ECM, 脂肪细胞和基质支持细胞之间的相互作用, 也不是在体内相互施加的收缩力单元. 笏。
在缺乏金标准的原发脂肪培养方法的情况下, 脂肪的研究主要依赖于分化前脂肪细胞 (diffAds)。DiffAds 是 multilocular, 黏附和新陈代谢活跃。相比之下, 原发性白脂肪细胞单房性, nonadherent, 并表现出相对较低的新陈代谢。目前的脂肪培养模型未能概括健康成熟脂肪组织的生理, 可能是缺乏 FDA 批准的药物直接靶向脂肪细胞的主要因素。事实上, 缺乏生理的体外器官模型是一个主要的问题, 在大多数器官和疾病。
美国国立卫生研究院 (NIH) 在其立场文件中宣布创建其 Microphysiological 系统 (MPS) 计划, 报告说, 在所有人类药物临床试验中, 2013 的成功率仅为第二阶段和50% 阶段的18%。临床试验8。MPS 方案的设计是为了直接解决在体外单一的能力, 以模型人类生理学。NIH 将松龙定义为由人类主要或干细胞组成的多细胞3D 结构中的培养系统, 这些细胞概括了器官功能。与简化模型的同类, 永生化细胞培养, 松龙应准确模型细胞, 药物细胞, 药物药物, 和器官-药物相互作用9。与短期初级文化方法不同的是, NIH 标准规定了4周文化8的 MPS 的可持续性。MPS 计划的进一步细节可以在 NIH 的 RFAs (#RFA TR-18-001)10中找到。
我们开发了一种简单、新颖、适应性强、价格低廉的脂肪 MPS, 称为 "夹心白脂肪组织" (斯瓦特)11。我们克服了脂肪细胞的自然浮力的 "夹" 切碎的原始脂肪组织之间的脂质基质干细胞 (干细胞) (图 1)。由此产生的3D 构造概括细胞接触和当地脂肪微环境的周围成熟脂肪体与自然脂肪细胞支持的人群。斯瓦特已经通过展示8周的生存能力、对外源信号的反应、脂肪分泌物和植入成为动物模型来证实。

<table border="0" cellpadding="0" cellspacing="0" width="817">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:219px;">10x HBSS</td>
			<td style="width:92px;">Thermofisher</td>
			<td style="width:115px;">14185052</td>
			<td style="width:393px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin</td>
			<td>Sigma-aldrich</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase</td>
			<td>Sigma-aldrich</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adenosine</td>
			<td>Sigma-aldrich</td>
			<td>A9251</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Thermofisher</td>
			<td>11995065</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M199 Media</td>
			<td>Thermofisher</td>
			<td>11043023</td>
			<td>Phenol red-free&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">250&#181;m Mesh Filter</td>
			<td>Pierce</td>
			<td>87791</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.2&#181;m Syringe Filter</td>
			<td>Celltreat</td>
			<td>229747</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5mL Luer-Lok syringes&#160;</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;">Metal Washers</td>
			<td></td>
			<td></td>
			<td style="width:393px;">These are simple metal washers and can be bought at any hardware store. They simply add leight weight to the backs of the plugers to ensure even contact between cells and gelatin, while being easy to stock and sterilize. Approximate mass: 6.3g&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heated Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Incubated Orbital Shaker</td>
			<td>VWR</td>
			<td>10020-988</td>
			<td style="width:393px;">Samples should be pitched at 45&#176; angle to facilitate collagenase digestion</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heat Block</td>
			<td></td>
			<td></td>
			<td style="width:393px;">Set to 37-40&#176;C and placed under Biosafety Cabinet</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water Bath</td>
			<td></td>
			<td></td>
			<td style="width:393px;">Set to ~75&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Specialized Plastics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="180">
			<td height="180" style="height:180px;">Upcell Dishes 6cm of 6-multiwell</td>
			<td>Nunc</td>
			<td>174902&#160; or 174901</td>
			<td style="width:393px;">These are commerically available pNIPAAm-coated dishes which can be used to grow the upper sheet of ADSCs. Alternatively, pNIPAAm-coated plates can be produced in-lab. diameter: &#60;6cm for 6cm dish, &#60;3.5cm for 6-well plate; approximate mass: 6.7g for 6cm dish, 5.1g for 6-well plate</td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;">Plastic Plunger Apparatus</td>
			<td></td>
			<td></td>
			<td style="width:393px;">These can be fashioned to fit within desired pNIPAAm-coated plastics (multiwell plates, petri dishes). They are comprised of a simple stem attached to a circular disk. They can be produced in-lab or by any facility that can fashion acrylic plastics</td>
		</tr>
	</tbody>
</table>

a microphysiologic platform for human fat: sandwiched white adipose tissue

白色脂肪组织 (笏) 在调节体重和日常健康方面起着至关重要的作用。 尽管如此, 现有的主要养殖模式仍存在重大限制, 所有这些模型都未能忠实地重述脂肪微环境或延长笏的生存能力超过两周。 缺乏可靠的初级文化模型严重阻碍了笏代谢和药物开发的研究。为此, 我们利用 NIH 的 microphysiologic 系统标准, 为 "斯瓦特" (夹心白脂肪组织) 的主要文化开发了一个新的平台。我们克服脂肪细胞的自然浮力的夹碎笏簇之间的薄层之间的脂质基质干细胞。 在这个构造中, 笏样本在八周的文化中是可行的。 斯瓦特保持完整的 ECM, 细胞对细胞的接触, 和物理压力的体内笏条件;此外, 斯瓦特还保持了强健的转录剖面, 对外源化学信号的敏感性, 以及整个组织功能。斯瓦特是一种简单、可重复、有效的原发性脂肪培养方法。 潜在的, 它是一个广泛适用的平台, 研究在笏生理学, 病理生理学, 新陈代谢和药物发展。

斯瓦特的生存能力最初是由序列 brightfield 成像的单个笏簇 (n = 12) 大约7.6 周。在这段时间内, 簇仍然固定在单层上。微小的形态学变化观察到个别脂肪细胞翘曲轻微或移位的位置。然而, 脂肪细胞并没有成为 multilocular 随着时间的推移, 表明缺乏分化, 也没有表现出任何明显的迹象死亡, 如细胞起泡, 裂解, 或分裂 (图 2)。两周大的特勤组的脂肪细胞特征?...

Watch this Scientific Journal Video about A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue at JoVE.com

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

白色脂肪组织 (笏) 在其目前的主要培养模式中存在严重缺陷, 阻碍了药理学的发展和代谢研究。在这里, 我们提出一个协议, 以产生一个脂肪 microphysiological 系统的夹笏之间的基质细胞。该构造为原笏培养提供了一个稳定、适应性强的平台。

人体脂肪 Microphysiologic 平台: 夹心白色脂肪组织

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health.  Still, there are significant limitations to available primary ...

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Research

JoVE Journal

Behavior

6.2K 次观看.  Louisiana State University Health Sciences Center. 白色脂肪组织 (笏) 在其目前的主要培养模式中存在严重缺陷, 阻碍了药理学的发展和代谢研究。在这里, 我们提出一个协议, 以产生一个脂肪 microphysiological 系统的夹笏之间的基质细胞。该构造为原笏培养提供了一个稳定、适应性强的平台。

视频: A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

脂肪偏好:饮食行为的一种新模型大鼠

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

科研

行为学

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal&#8217;s brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal&#8217;s head.

平地板的空气解禁平台:一种新方法与显微镜或电对清醒结合行为自由移动鼠害

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a ...

Determining Pain Detection and Tolerance Thresholds Using an Integrated, Multi-Modal Pain Task Battery

Human pain models are useful in the assessing the analgesic effect of drugs, providing information about a drug&#39;s pharmacology and identify potentially suitable therapeutic populations. The need to use a comprehensive battery of pain models is highlighted by studies whereby only a single pain model, thought to relate to the clinical situation, demonstrates lack of efficacy. No single experimental model can mimic the complex nature of clinical pain. The integrated, multi-modal pain task&#160;battery presented here encompasses the electrical stimulation task, pressure stimulation task, cold pressor task, the UVB inflammatory model which includes a thermal task and a paradigm for inhibitory conditioned pain modulation. These human pain models have been tested for predicative validity and reliability both in their own right and in combination, and can be used repeatedly, quickly, in short succession, with minimum burden for the subject and with a modest quantity of equipment. This allows a drug to be fully characterized and profiled for analgesic effect which is especially useful for drugs with a novel or untested mechanism of action.

Human pain models are valuable tools used to assess the analgesic potential of novel compounds and predict their clinical efficacy, especially when used in an integrated manner. Although implementation of these models is complex, with proper execution, the pain models described in this protocol can provide predictive and reliable results.

确定疼痛的检测和容忍阈值使用集成的，多模式疼痛任务电池

Human pain models are useful in the assessing the analgesic effect of drugs, providing information about a drug&#39;s pharmacology and identify ...

Substantiating Appropriate Motion Capture Techniques for the Assessment of Nordic Walking Gait and Posture in Older Adults

Nordic walking (NW) has become a safe and simple form of exercise in recent years, and in studying this gait pattern, various data collection techniques have been employed, each with positives and negatives. The aim was to determine the effect of NW on older adult gait and posture and to determine optimal use of different data collection systems in both short and long duration analysis. Gait and posture during NW and normal walking were assessed in 17 healthy older adults (age: 69 &#177; 7.3). Participants performed two trials of 6 Minute Walk Tests (6MWT) (1 with poles (WP) and 1 without poles (NP)) and 6 trials of a 5m walk (3 WP and 3 NP). Motion was recorded using two systems, a 6-sensor accelerometry system and an 8-camera 3-dimensional motion capture system, in order to quantify spatial-temporal, kinematic, and kinetic parameters.
With both systems, participants demonstrated increased stride length and double support and decreased gait speed and cadence WP compared to NP (p &#60;0.05). Also, with motion capture, larger single support time was found WP (p &#60;0.05). With 3-D capture, smaller hip power generation and moments of force were found at heel contact and pre-swing as well as smaller knee power absorption at heel contact, pre-swing, and terminal swing WP compared to NP, when assessed over one cycle (p &#60;0.05). Also, WP yielded smaller moments of force at heel contact and terminal swing along with larger moments at mid-stance of a gait cycle (p &#60;0.05). No changes were found for posture.
NW seems appropriate for promoting a normal gait pattern in older adults. Three-dimensional motion capture should primarily be used during short duration gait analysis (i.e. single gait cycle), while accelerometry systems should be primarily employed in instances requiring longer duration analysis such as during the 6MWT.

The aim was to substantiate optimal use of data collection techniques for Nordic walking gait and posture analysis. Three-dimensional motion capture should be used during short duration analysis (i.e. single gait cycle), while accelerometry should be employed for longer duration analysis (i.e. repeated cycles) like a 6 Minute Walk Test.

充实适当的运动捕捉技术的越野行走步态中老年人的评估和姿势

Nordic walking (NW) has become a safe and simple form of exercise in recent years, and in studying this gait pattern, various data collection techniques ...

Sit-to-stand-and-walk from 120% Knee Height: A Novel Approach to Assess Dynamic Postural Control Independent of Lead-limb

Individuals with sensorimotor pathology e.g., stroke have difficulty executing the common task of rising from sitting and initiating gait (sit-to-walk: STW). Thus, in clinical rehabilitation separation of sit-to-stand and gait initiation - termed sit-to-stand-and-walk (STSW) - is usual. However, a standardized STSW protocol with a clearly defined analytical approach suitable for pathological assessment has yet to be defined.
Hence, a goal-orientated protocol is defined that is suitable for healthy and compromised individuals by requiring the rising phase to be initiated from 120% knee height with a wide base of support independent of lead limb. Optical capture of three-dimensional (3D) segmental movement trajectories, and force platforms to yield two-dimensional (2D) center-of-pressure (COP) trajectories permit tracking of the horizontal distance between COP and whole-body-center-of-mass (BCOM), the decrease of which increases positional stability but is proposed to represent poor dynamic postural control.
BCOM-COP distance is expressed with and without normalization to subjects&#39; leg length. Whilst COP-BCOM distances vary through STSW, normalized data at the key movement events of seat-off and initial toe-off (TO1) during steps 1 and 2 have low intra and inter subject variability in 5 repeated trials performed by 10 young healthy individuals. Thus, comparing COP-BCOM distance at key events during performance of an STSW paradigm between patients with upper motor neuron injury, or other compromised patient groups, and normative data in young healthy individuals is a novel methodology for evaluation of dynamic postural stability.

Here, we present a novel protocol to measure positional stability at key events during the sit-to-stand-to-walk using the center-of-pressure to the whole-body-center-of-mass distance. This was derived from the force platform and three-dimensional motion-capture technology. The paradigm is reliable and can be utilized for the assessment of neurologically compromised individuals.

坐姿到站姿和步行距离120％膝盖高度:新方法来评估铅肢体的动态姿势控制独立

Individuals with sensorimotor pathology e.g., stroke have difficulty executing the common task of rising from sitting and initiating gait (sit-to-walk: ...

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits (Oryctolagus Cuniculus) Via Behavioral Ethogram, Monitoring, and Interventions

New Zealand White (NZW) laboratory rabbits (Oryctolagus cuniculus), as well as their ancestors the European Rabbit, are a social species that exhibit numerous benefits to being housed accordingly. Although these rabbits are innately gregarious, certain behaviors can still arise when kept in captivity, which if left unchecked, can confound research results or lead to wounding, which in extreme cases can be severe. To prevent these issues, there must be a well-structured plan for the monitoring and maintenance of paired laboratory rabbits. The purpose of this protocol is to present effective procedures for establishing newly paired NZW rabbits as well as methods for successful maintenance. Multiple methods have been tested for the creation of newly paired female rabbits from the vendor, but the most efficacious technique emphasizes capitalizing on the stress bonding from transport, urine marking, pairing in a neutral cage with no forced sharing of resources and a system of monitoring and intervention. To determine the best method of housing paired rabbits in a standard caging environment, data were collected to generate a behavioral ethogram. Behaviors were then quantified as positive, neutral or negative and were tracked across the lifespan of the pair to determine which behaviors indicated pair success or failure. With the newfound knowledge of socially housed laboratory NZW rabbit behavior, enrichment intervention was applied to alleviate aggression and prevent wounding, thus resulting in a higher percentage of successful pairs. Through several years of trialing different pairing methods, the development of the ethogram and the resulting enrichment interventions, understanding of the highly complex social constructs that dominate pair housed rabbit behavior has dramatically increased and allowed for the provision of more species-specific care and increased standards of welfare.

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits Oryctolagus Cuniculus Via Behavioral Ethogram, Monitoring, and Interventions

Though European rabbits are a social species, socially housing them can be challenging. Therefore, there must be a thorough understanding of behaviors and social structures of pair-housed laboratory rabbits. Here we present a protocol to identify pairing methods, species-typical hierarchy establishment behaviors and behaviors that warrant appropriate intervention.

新西兰白兔的配对和配对维护方法 (穴兔家兔) 通过行为 Ethogram、监视和干预

New Zealand White (NZW) laboratory rabbits (Oryctolagus cuniculus), as well as their ancestors the European Rabbit, are a social species that exhibit ...

A Novel Single Animal Motor Function Tracking System Using Simple, Readily Available Software

We have recently demonstrated that implanting intracortical microelectrodes in the motor corteces of rats&#160;results in immediate and lasting motor deficits. Motor impairments were manually quantified through an open field grid test to measure the gross motor function and through a ladder test to measure the fine motor function. Here, we discuss a technique for the automated quantification of the video-recorded tests using our custom Capadona Behavioral Video Analysis System: Grid and Ladder Test, or BVAS. Leveraging simple and readily available coding software (see the Table of Materials), this program allows for the tracking of a single animal on both the open field grid and the ladder tests. In open field grid tracking, the code thresholds the video for intensity, tracks the position of the rat over the 3 min duration of the grid test, and analyzes the path. It then computes and returns measurements for the total distance traveled, the maximum velocity achieved, the number of left- and right-handed turns, and the total number of grid lines crossed by the rat. In ladder tracking, the code again thresholds the video for intensity, tracks the movement of the rat across the ladder, and returns calculated measurements including the time it took the rat to cross the ladder, the number of paw slips occurring below the plane of the ladder rungs, and the incidence of failures due to stagnation or reversals. We envision that the BVAS developed here can be employed for the analysis of motor function in a variety of applications, including many injury or disease models.

The current study aimed to automate the quantification of motor deficits in rats. The initial evaluation model assesses motor loss resulting from an intracortical microelectrode implantation in the motor cortex. We report on the development and use of a tracking algorithm using easily adaptable, simple, and readily available coding software.

一种新的单动物运动功能跟踪系统, 使用简单、易于使用的软件

We have recently demonstrated that implanting intracortical microelectrodes in the motor corteces of rats&#160;results in immediate and lasting motor ...

A Novel Digital Platform for a Monitored Home-based Cardiac Rehabilitation Program

Despite the evidence that cardiac rehabilitation (CR) reduces the risk of recurrent cardiac events, only a minority of eligible patients are willing to join existing programs at cardiac rehabilitation centers. The unique remote patient monitoring system presented here enables healthcare providers to monitor CR patients at home in real-time and at low cost. The system combines mobile technology, artificial intelligence, and supportive services, expanding the delivery of medical care to the patient&#39;s home. The primary aim of the study is to increase the long-term adherence to physical activity in patients who participate in CR via the addition of a home-based digitally monitored CR component to the standard CR program in patients with ischemic heart disease (IHD), with the idea of forming new habitual health behaviors and increasing the long-term motivation for physical exercise (PE) habits at home. Secondary aims are to assess the program&#39;s impact on the physical activity level measured by average steps per day, minutes of exercise per week, blood pressure, metabolic parameters, body mass index, and waist-to-hip ratio, as well as a quality-of-life (QoL) questionnaire.The study has two arms: (1) home-based monitored exercise using a smart digital garment and wristband, in addition to motivation and reinforcement via text messages; (2) standard CR facility-based exercise. The study design is a randomized, controlled trial comparing standard CR to a home-based monitoring and reinforcement program. The study program is designed for 12 weeks.Clinical tests and anthropometric measurements are performed before and after the study, measuring height, weight, waist circumference, visceral fat and body mass index (BMI), blood pressure, and HbA1c and lipid profile. Patients have to complete a baseline survey including socio-demographic characteristics and QoL questionnaire SF-36. At the end of the study, patients complete a survey regarding the use of the smart digital garment&#39;s benefits and usability. The study is currently underway.

The aim is to promote a new approach to cardiac rehabilitation (CR), using a unique remote patient monitoring system that will enable healthcare providers to monitor CR patients at home at low cost, with the intention of making CR services more accessible and improving compliance. The study is currently underway.

一种新型的家庭心脏康复监控数字平台

Despite the evidence that cardiac rehabilitation (CR) reduces the risk of recurrent cardiac events, only a minority of eligible patients are willing to ...

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human populations, the Drosophila model allows strict control over genetic background, and virtually unlimited numbers of individuals of the same genotype can be reared rapidly under well-controlled environmental conditions without regulatory restrictions and at relatively low cost. Flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. Here, we describe a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The assay is based on video recording of single flies introduced without anesthesia in 24-well cell culture plates in a set-up that enables synchronous initiation of alcohol exposure. The system enables a single person to collect individual ethanol sedation data on as many as 2,000 flies within an 8 h work period. The assay can, in principle, be extended to assess the effects of exposure to any volatile substance and applied to measure effects of acute toxicity of volatiles on other insects, including other fly species.

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Current methods to measure alcohol sensitivity in Drosophila are designed to test groups of flies. We present a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The method does not require specialized tools and can be performed in any laboratory using common materials.

测量个体果蝇黑色素气器酒精镇分时间的高通量方法

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human ...

Tactile Vibrating Toolkit and Driving Simulation Platform for Driving-Related Research

Collision warning system plays a key role in the prevention of driving distractions and drowsy driving. Previous studies have proven the advantages of tactile warnings in reducing driver&#8217;s brake response time. At the same time, tactile warnings have been proved effective in take-over request (TOR) for partially autonomous vehicles.
How the performance of tactile warnings can be optimized is an ongoing hot research topic in this field. Thus, the presented low-cost driving simulation software and methods are introduced to attract more researchers to take part in the investigation. The presented protocol has been divided into five sections: 1) participants, 2) driving simulation software configuration, 3) driving simulator preparation, 4) vibrating toolkit configuration and preparation, and 5) conducting the experiment.
In the exemplar study, participants wore the tactile vibrating toolkit and performed an established car-following task using the customized driving simulation software. The front vehicle braked intermittently, and vibrating warnings were delivered whenever the front vehicle was braking. Participants were instructed to respond as quickly as possible to the sudden brakes of the front vehicle. Driving dynamics, such as the brake response time and brake response rate, were recorded by the simulation software for data analysis.
The presented protocol offers insight into the exploration of the effectiveness of tactile warnings on different body locations. In addition to the car-following task that is demonstrated in the exemplar experiment, this protocol also provides options&#160;to apply other paradigms to the driving simulation studies by making simple software configuration without any code development. However, it is important to note that due to its affordable price, the driving simulation software and hardware introduced here may not be able to fully compete with other high-fidelity commercial driving simulators. Nevertheless, this protocol can act as an affordable and user-friendly alternative to the general high-fidelity commercial driving simulators.

This protocol describes a driving simulation platform and a tactile vibrating toolkit for the investigation of driving-related research. An exemplar experiment exploring the effectiveness of tactile warnings is also presented.&#160;

触觉振动工具包和驾驶模拟平台，用于驾驶相关研究

Collision warning system plays a key role in the prevention of driving distractions and drowsy driving. Previous studies have proven the advantages of ...