作者希望对 Mr. 波 (上海市公共卫生临床中心) 在本议定书中的µCT 评估提供技术援助表示感谢。

注意: OE 模型已经生成并用于以前报告的研究 30 、 31 、 32 。所有动物实验均经上海交通大学机构动物护理与使用委员会 (IACUC) 批准. 
 1. 鼠标 <ol> <li> 在受控温度 (20 和 #176; c) 和湿度 (40-60%) 的动物设施的单个通风笼中, 无病原体、7至9周老的雌性 balb/c/c 小鼠。提供12和 #160; h 光和 12 #160; 设备中的暗循环。提供食物和水 ad 随意 . </li>
</ol> 2。臭氧或空气暴露 <ol> <li> 在密封丙烯酸 ( 如 有机玻璃) 框中生成带有电发生器的臭氧。用小风机吹出箱内的空气, 通过排气管连接在箱体内外。使用臭氧探针监测盒子中的臭氧浓度。等到臭氧浓度达到2.5 分之百万分之一 (ppm). 
	注: 臭氧探头可自动开启或关闭臭氧发生器, 并可在 2.5 ppm 时保持盒内的臭氧水平. </li>
	<li> 当臭氧水平达到 2.5 ppm 时, 将小鼠放入盒子中。把老鼠放在盒子里, 每次3小时, 让它们暴露在臭氧中. 
	注: 该盒可在3小时内保持臭氧水平 2.5 ppm, 通过自动开关臭氧发生器打开或关闭和吹出的 CO 2 , 这是由小鼠产生的方块. </li>
	<li> 每周 (每3天) 给两次臭氧暴露 (每次接触持续3小时), 为期7周; 同时将控制小鼠暴露在同一时间内, 并同时进行. </li>
</ol> 3。微计算机断层扫描 <ol> <li> 在7周结束时, 麻醉小鼠腹腔注射 pelltobarbitalum 孢 (1%、0.6-0.8 ml/100 g) 和 #160; 根据个别情况调整剂量以查看鼠标不响应脚趾捏. 和 #160; 监测和保持鼠标在一个稳定的呼吸频率;并确保在程序中不存在自愿的动作. </li>
	<li> 将麻醉小鼠放置在微计算机断层扫描室 (#181; CT). </li>
	<li> 校准和 #181; 使用标准协议和制造商和 #39 的说明进行 CT 检查。设置 X 射线管在50伏和电流在450和 #181; A. 
	注: X 射线和探测器都围绕鼠标旋转. </li>
	<li> 执行和 #181; CT 分析通过获得515投影, 有效像素大小为 0.092 mm, 一个切片的曝光时间为300毫秒, 切片厚度为 0.093 mm. </li>
	<li> 使用软件重建肺部与获得的图像。通过分别设置-750 和-550 Hounsfield 单元的灰度最小值和最大值来调整灰度图像亮度. 
	注: 该软件将自动计算肺实质的体积和 low-attenuation 区 (LAA) 34 , 35 。
	<li> 通过将 LAA 体积除以肺总容积来计算 LAA (LAA%) 的百分比. </li> </ol>
 4. 平板电脑测试 <ol> <li> 在跑步机上为小鼠提供10米/分钟的适应性测试. #160; 注意: 当程序进行时, 电总是关闭的. #160; </li> <li> 管理对小鼠进行疲劳试验。
	<ol> <li> 以10米/分钟的速度将鼠标预热到5分钟 </li>
		<li> 将速度提高到15米/分钟, 10 分钟. </li>
		<li> 增加运动强度: 提高速度5米/分钟, 从20米/分钟开始, 每30分钟, 直到鼠标不能继续运行 36 . </li>
	</ol> </li> <li> 将总运行距离和运行时间分别记录为疲劳距离和疲劳时间. </li> </ol>
 5. 肺功能测量 <ol> <li> 麻醉腹腔注射 pelltobarbitalum 孢 (1%、0.6-0.8 ml/100 g) 的小鼠, 根据个别情况调整剂量, 以查看鼠标是否不响应脚趾捏和 #160; 并等待, 直到老鼠保持自发性呼吸. #160; 监测和保持鼠标在一个稳定的呼吸频率;并确保在程序中不存在自愿的动作. </li>
	<li> 小心地 tracheostomize 老鼠, 并将它们放在与计算机控制的呼吸机相连的身体体积中. 
	注: 通风通过靠近气管导管的下部阀门进行控制。该装置提供了不同的半自动机动, 包括准静态压力容积的机动和快速流动量的机动. </li>
	<li> 通过压力控制通气将平均呼吸频率设置为150呼吸/分钟到麻醉小鼠, 直到每一个呼吸循环都得到一个正常的呼吸模式和完全失效. </li>
	<li> 使用体积中产生的负压力对设备执行准静态压力-容积机动. </li>
	<li> 在准静态压力-容积回路内执行快速流动容积机动, 以记录 FVC 和 FEV。将肺部充气到 +30 厘米 h 2 O, 然后立即将其连接至高度负压, 以强制执行有效期, 直到剩余体积在-30 厘米 h 2 o. 记录 FEV 在第一25、50和75毫秒中的呼气 (FEV 25 , 分别 FEV 50 和 FEV 75 。拒绝次优演习。对于每一个测试, 每个单一的鼠标, 进行至少三可接受的演习, 以获得一个可靠的平均所有数值参数. </li>
</ol> 6。BALF 集合 <ol> <li> 使用 pelltobarbitalum 孢 (1%、1.8-2.4 ml/100 g) 和 #160 进行晚期麻醉; 根据个别情况调整剂量, 以观察鼠标不响应脚趾捏和失去呼吸), #160; 灌洗2毫升 PBS 通过一个1毫米口径的气管插管, 然后检索 BALF 10 . </li>
	<li> 将检索到的灌洗等分, 并将其离心4和 #176; C 和 250 x g 为10分钟. </li>
	<li> 收集上清液以便立即使用, 并将剩余物存储在-80 和 #176; C 或液氮. </li>
	<li> 使用例计算单元总数. </li>
	<li> 重在 PBS 中的细胞颗粒, 然后旋转 (1400 x g, 6 分钟) 250 和 #181; L 悬浮细胞在幻灯片上使用滑动微调离心机. </li>
	<li> 根据制造商和 #39 的协议将赖特染色应用于幻灯片上的单元格. </li>
	<li> 每只鼠标计数200单元格; 根据标准形态学, 在400X 放大倍数下, 将细胞识别为巨细胞或嗜中性白血球; 并计算它们的数量. </li>
</ol> 7。心脏血液取样 <ol> <li> 通过心脏穿刺收集血液, 将其加载到1.5 毫升的管中, 并将其放在冰上30分钟. </li>
	<li> 在 2000 x g 和4和 #176 上离心血样5分钟; C. </li>
	<li> 将上清液 (血清) 转移到新管上, 并将其存储在-80 和 #176; C 或液氮. </li>
	<li> 为 IL-1 和 #946、IL-10、TNF 和 #945 制备血清; 使用各自的 ELISA 试剂盒进行检测测试. </li>
</ol> 8。肺形态计量学分析 <ol> <li> 从老鼠身上解剖肺部和气管。
	<ol> <li> 在牺牲后立即将每个被安乐死的鼠标置于手术板上. </li>
		<li> 解剖阔和前气管肌肉, 以可视化和进入气管环. </li>
		<li> 打开胸腔。解剖肺部和气管, 但不要把心脏和肺部分开. </li>
	</ol>
</li> <li> 通过 PE90 聚乙烯管将气管导管连接到含有4% 甲醛的注射器. 
	警告: 甲醛是有毒的。戴上手套和安全眼镜, 并使用通风罩内的解决方案. </li>
	<li> 用4% 甲醛 (10 滴, ~ 200 和 #181; L) 通过气管导管完全充气肺部。在通货膨胀完成后取出心脏. </li>
	<li> 保持15和 #160 的肺部; ml 管包含10毫升的4% 甲醛, 至少4小时. </li>
	<li> 将肺部嵌入石蜡中。通过石蜡切片和旋转切片获得5和 #181; m 段。在切片过程中, 暴露支气管树区内肺组织的最大表面积. </li>
	<li> 用于形态分析, 在切片上进行苏木精和曙红 (H 和 #38; E) 染色. </li>
	<li> 用明亮的场直立显微镜 (物镜, 20X; 曝光时间, 1.667 毫秒) 来图像剖面. </li>
	<li> 有两名调查人员对治疗方案视而不见, 独立计算组织学部分。使用平均线性截距 (L m ) 作为测量 inter-alveolar 间隔壁距离的参数。使用以下步骤确定 L m : <ol> <li> 打开 Photoshop 中各节的图像, 并在图像上绘制一个手提五550和 #181; m 和 #160; 长行. </li>
		<li> 计算网格线上的肺泡数. </li>
		<li> 通过将网格线的长度除以肺泡的数目来计算 L m 。对于量化, 每只鼠标图像五节。获取每个部分的十图像 (每个字段一个图像) 并随机评估。在野外选择的过程中, 通过在前方或另一个方向移动一个磁场来避免气道和容器的电场. 
		注: 数据显示为平均值和 #177; S.E.M。对 air-exposed 小鼠和臭氧暴露小鼠进行了 un-paired t 检验。每组三只动物被用来计算显著的差异。p 值的和 #60; 0.05 被认为是重要的. </li>
	</ol>
	</li>
</ol>

Z.W.S. 和. 是目前的雇员和细胞生物医学组 (纳斯达克: CBMG) 的股票期权持有人。其他作者宣称他们没有竞争利益。

在这项研究中, 我们提出了一个可靠的方法来生成一个新的 COPD 模型。与其他模型 (即LPS 或 PPE 模型) 相比, 此 OE 模型重述 COPD 患者的病理过程。由于香烟烟雾是导致人患 copd 的主要危险物质40, CS 模型仍然是最受欢迎的 copd 模型41,42。然而, CS 模型需要 3-到12月的 r-和 #38;D 期新药。与 CS 模型相比, 目前的 OE 模型将生成周期缩短为6-...

据估计, COPD, 包括肺气肿和慢性支气管炎, 可能是世界上的第三个主要死因在 2020年1,2。在未来40年3中, 在40岁以上的人口中, COPD 的潜在发病率估计为12.7% 男性, 8.3% 为女性。目前尚无药物可逆转 COPD 患者的渐进性恶化4。可靠的 COPD 动物模型不仅要求对疾病的病理过程进行模拟, 而且需要较短的周期。目前的 COPD 模型, 包括 LPS 或 PPE 诱发的模型, 可以诱发肺气肿样症状5,6。一个单一的管理或一个星期的挑战, LPS 或 PPE 的小鼠或大鼠结果在支气管肺泡灌洗液 (BALF) 的显着中性, 增加炎调解人 (例如, TNF-α和 IL-1β) 在 BALF 或血清, 产生肺实质破坏-扩大空气空间, 并限制气流5,6,7,8,9,10。然而, LPS 或 PPE 不是人类慢性阻塞性肺病的原因, 因此不模仿病理过程11。CS 诱导模型产生持续气流限制, 肺实质破坏, 并减少功能锻炼能力。但是, 传统的 CS 协议至少需要3月才能生成 COPD 模型12,13,14,15。因此, 重要的是要产生一个新的, 更有效的动物模型, 满足这两个要求。
最近, 除了吸烟, 空气污染和职业暴露已成为更常见的原因 COPD16,17,18。臭氧作为主要污染物之一 (虽然不是空气污染的主要成分), 可以直接与呼吸道发生反应, 损害儿童和青年成人的肺组织19,20,21 ,22,23,24,25。臭氧, 以及其他刺激, 包括 LPS, PPE, 和 CS, 参与了严重的生化途径的肺氧化应激和 DNA 损伤, 并与启动和促进 COPD26,27。另一个因素是, 一些 COPD 患者的症状在暴露于臭氧后恶化, 表明臭氧会扰乱肺部功能18,28,29。因此, 我们产生了一个新的 COPD 模型, 反复暴露小鼠到高浓度的臭氧7周;这导致气流缺陷和肺部实质损伤类似于以前的调查30,31,32。我们在本研究中推广了对雌性小鼠的 OE 协议, 并成功地再现了我们以前研究的雄性小鼠的肺气肿30,31,32。由于 copd 死亡率在男性减少, 但在许多国家的妇女增加了33, 需要对女性 copd 模型进行研究, 以探讨慢性阻塞性肺病患者的发病机制和治疗方法。OE 模型对所有性别的适用性进一步支持它作为 COPD 模型的使用。

<table width="1024">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">BALB/c mice</td>
			<td style="width:217px;">Slac Laboratory Animal,Shanghai, China</td>
			<td style="width:143px;">N/A</td>
			<td style="width:455px;">7-to-9-week-old female BALB/c mice were used in this study.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Individual ventilated cages</td>
			<td style="width:217px;">Suhang, Shanghai, China</td>
			<td style="width:143px;">Model Number: MU64S7</td>
			<td>The cages were used for housing mice in the animal facility.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Sealing perspex-box</td>
			<td style="width:217px;">Suhang, Shanghai, China</td>
			<td style="width:143px;">N/A</td>
			<td style="width:455px;">The box was used&#160; to contain the ozone generator. Mice were exposed to ozone within the box.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Electric generator</td>
			<td style="width:217px;">Sander Ozoniser, Uetze-Eltze, Germany</td>
			<td style="width:143px;">Model 500&#160;</td>
			<td>The device was used for generating ozone.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Ozone probe</td>
			<td style="width:217px;">ATi Technologies, Ashton-U-Lyne, Greater Manchester, UK</td>
			<td style="width:143px;">Ozone 300</td>
			<td>The device was used for monitoring and controlling the generation of ozone.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Pelltobarbitalum natricum</td>
			<td style="width:217px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:143px;">P3761</td>
			<td style="width:455px;">Mice were anesthetized by intraperitoneal injection of pelltobarbitalum natricum.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Micro-Computed Tomography</td>
			<td style="width:217px;">GE Healthcare, London, ON, Canada</td>
			<td style="width:143px;">RS0800639-0075</td>
			<td>This device was used for acquiring images of the lung.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Micro-view 2.01 ABA software</td>
			<td style="width:217px;">GE Healthcare, London, ON, Canada</td>
			<td style="width:143px;">Micro-view 2.01&#160;</td>
			<td>This device was used for reconstruct the lung and analyze volume, LAA of the lung.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Treadmill machine&#160;</td>
			<td style="width:217px;">Duanshi, Hangzhou, Zhejiang, China</td>
			<td style="width:143px;">DSPT-208</td>
			<td>This machine was usd for fatigue test.</td>
		</tr>
		<tr height="38">
			<td height="38" style="width:211px;height:38px;">Body plethysmograph</td>
			<td style="width:217px;">eSpira&#8482; Forced Manoeuvres System, EMMS, Edinburgh, UK</td>
			<td style="width:143px;">Forced Manoeuvres System</td>
			<td height="80">This device was used to test spirometry pulmonary function.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Ventilator</td>
			<td style="width:217px;">eSpira&#8482; Forced Manoeuvres System, EMMS, Edinburgh, UK</td>
			<td style="width:143px;">Forced Manoeuvres System</td>
			<td height="80">This device was used to test spirometry pulmonary function.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Slide spinner centrifuge</td>
			<td style="width:217px;">Denville Scientific, Holliston, MA, USA</td>
			<td>C1183&#160;</td>
			<td>It was used to spin BALF cells onto slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">Wright Staining</td>
			<td style="width:217px;">Hanhong, Shanghai, China</td>
			<td style="width:143px;">RE04000054</td>
			<td>&#160;It was used to staining macrophages, neutrophils in the suspended BALF.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Hemocytometer</td>
			<td style="width:217px;">Hausser Scientific, Horsham, PA, USA</td>
			<td style="width:143px;">4000</td>
			<td>It was used to count cells.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">IL-1&#946;</td>
			<td style="width:217px;">Abcam, Cambridge, MA, USA</td>
			<td style="width:143px;">ab100704</td>
			<td height="63">They were used to test the respective factors in serum.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">IL-10</td>
			<td style="width:217px;">Abcam, Cambridge, MA, USA</td>
			<td style="width:143px;">ab46103</td>
			<td height="63">They were used to test the respective factors in serum.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">TNF-&#945;</td>
			<td style="width:217px;">Abcam, Cambridge, MA, USA</td>
			<td style="width:143px;">ab100747</td>
			<td height="63">They were used to test the respective factors in serum.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">Paraformaldehyde&#160;</td>
			<td style="width:217px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:143px;">P6148</td>
			<td>The lung was inflated by 4% paraformaldehyde.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">Paraffin</td>
			<td style="width:217px;">Hualing, Shanghai, China</td>
			<td style="width:143px;">56#</td>
			<td>It was used to embed the lung.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">Rotary Microtome</td>
			<td style="width:217px;">Leica, Wetzlar,&#160; Hesse, Germany</td>
			<td style="width:143px;">RM2255</td>
			<td>It was used for sectioning the lung.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Hgaematoxylin and Eosin (H&#38;E) staining solution</td>
			<td style="width:217px;">Solarbio, Beijing, China</td>
			<td style="width:143px;">G1120</td>
			<td>H&#38;E staining was done for morphometric analysis.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Upright bright field microscope</td>
			<td style="width:217px;">Olympus, Center Valley, PA, USA</td>
			<td style="width:143px;">CX41</td>
			<td>It was used to image the H&#38;E staining slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">Adobe Photoshop 12</td>
			<td style="width:217px;">Adobe, San Jose, CA, USA</td>
			<td style="width:143px;">Adobe Photoshop 12</td>
			<td>It was used to count the number of alveoli on the H&#38;E stained images.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">GraphPad prism 5</td>
			<td style="width:217px;">Graphpad Software Inc., San Diego, CA</td>
			<td style="width:143px;">GraphPad prism 5</td>
			<td>It was used for data analysis and production of figures.</td>
		</tr>
	</tbody>
</table>

generation of a chronic obstructive pulmonary disease model in mice by repeated ozone exposure

慢性阻塞性肺疾病 (COPD) 的特点是持续气流限制和肺实质破坏。它在老龄人口中的发病率很高。目前常规的 COPD 治疗主要集中在症状修饰药物;因此, 迫切需要开发新的治疗方法。合格的慢性阻塞性肺病动物模型有助于确定基本的机制, 并可用于新的药物筛选。目前的 copd 模型, 如脂多糖 (LPS) 或猪胰弹性蛋白酶 (PPE) 诱导肺气肿模型, 产生 copd 样病变的肺部和呼吸道, 但没有其他类似的发病机制的人 copd。香烟烟雾 (CS) 诱导模型仍然是最流行的之一, 因为它不仅模拟 copd 样病变的呼吸系统, 但它也基于其中一个主要的危险物质, 导致 copd 的人。然而, CS 诱导模型的耗时和 labor-intensive 的方面极大地限制了它在新药筛选中的应用。在这项研究中, 我们成功地产生了一个新的 COPD 模型, 暴露小鼠到高水平的臭氧。该模型显示如下: 1) 减少了强迫呼气体积 25, 50, 75/强迫肺活量 (FEV25/FVC, FEV50/FVC, 和 FEV75/FVC), 表明肺功能恶化;2) 肺肺泡肿大, 肺实质破坏;3) 减少疲劳时间和距离;和 4) 炎症增加。综合起来, 这些数据表明, 臭氧暴露模型是一种与人类相似的可靠动物模型, 因为臭氧过度曝光是 COPD 的病因之一。此外, 根据我们以前的工作, 它只花了 6-8 周, 创建一个 OE 模型, 而它需要 3-12 月, 以诱导卷烟烟雾模型, 表明 oe 模型可能是一个很好的选择 COPD 的研究。

每个组的3D µCT 图像的示例显示在图 1a中。臭氧暴露的小鼠的总肺容积明显较大 (图 1a 和 b) 和 LAA% (图 1c) 比 air-exposed 控制小鼠。六周的臭氧暴露后, 肺容积和 LAA% 保持升高31,32。肺容积和 LAA% 的增加代表肺气肿的表型。肺肺...

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Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

这项研究描述了一个新的慢性阻塞性肺疾病 (COPD) 动物模型的成功生成, 反复暴露小鼠到高浓度的臭氧。

反复臭氧暴露对小鼠慢性阻塞性肺疾病模型的产生

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

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Research

JoVE Journal

Medicine

10.8K 次观看.  Cellular Biomedicine Group, Shanghai. 这项研究描述了一个新的慢性阻塞性肺疾病 (COPD) 动物模型的成功生成, 反复暴露小鼠到高浓度的臭氧。

视频: Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

科研

医学

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

膀胱胀诱发内脏运动反应为膀胱疼痛小鼠模型

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated measurements over weeks or months of accessory respiratory muscle activity while simultaneously measuring ventilation in a non-anesthetized, freely behaving mouse. The technique includes the surgical implantation of a radio transmitter and the insertion of electrode leads into the scalene and trapezius muscles to measure the electromyogram activity of these inspiratory muscles. Ventilation is measured by whole-body plethysmography, and animal movement is assessed by video and is synchronized with electromyogram activity. Measurements of muscle activity and ventilation in a mouse model of amyotrophic lateral sclerosis are presented to show how this tool can be used to investigate how respiratory muscle activity changes over time and to assess the impact of muscle activity on ventilation. The described methods can easily be adapted to measure the activity of other muscles or to assess accessory respiratory muscle activity in additional mouse models of disease or injury.

This paper introduces a method for repeated measurements of ventilation and respiratory muscle activity in a freely behaving amyotrophic lateral sclerosis (ALS) mouse model throughout disease progression with whole-body plethysmography and electromyography via an implanted telemetry device.

在神经肌肉疾病的模型小鼠呼吸肌肉活动和通风的重复测量

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

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

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

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

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

Repeated Orotracheal Intubation in Mice

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ventral neck for direct confirmation of cannula placement in the trachea. The relative difficulty or the tissue trauma induced to the subject by such procedures can be an impediment to an investigator&#8217;s ability to perform longitudinal studies. This article illustrates a technique in which physical manipulation of the mouse following the use of a depilatory to remove hair from the ventral neck permits transcutaneous visualization of the trachea for orotracheal intubation regardless of degree of skin pigmentation. This method is innocuous to the subject and easily achieved with a limited understanding of murine anatomy. This refined approach facilitates repeated intubation, which may be necessary for monitoring progression of disease or instillation of treatments. Using this method may result in a reduction of the number of animals and technical skill required to measure lung function in mouse models of respiratory disease.

The goal of this article is to describe a refined method of intubation of the laboratory mouse. The method is noninvasive and, therefore, ideal for studies that require serial monitoring of respiratory function and/or instillation of treatments into the lung.

小鼠重复的奥内切管插管

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ...

A Model of Reverse Vascular Remodeling in Pulmonary Hypertension Due to Left Heart Disease by Aortic Debanding in Rats

Pulmonary hypertension due to left heart disease (PH-LHD) is the most common form of PH, yet its pathophysiology is poorly characterized than pulmonary arterial hypertension (PAH). As a result, approved therapeutic interventions for the treatment or prevention of PH-LHD are missing. Medications used to treat PH in PAH patients are not recommended for treatment of PH-LHD, as reduced pulmonary vascular resistance (PVR) and increased pulmonary blood flow in the presence of increased left-sided filling pressures may cause left heart decompensation and pulmonary edema. New strategies need to be developed to reverse PH in LHD patients. In contrast to PAH, PH-LHD develops due to increased mechanical load caused by congestion of blood into the lung circulation during left heart failure. Clinically, mechanical unloading of the left ventricle (LV) by aortic valve replacement in aortic stenosis patients or by implantation of LV assist devices in end-stage heart failure patients normalizes not only pulmonary arterial and right ventricular (RV) pressures but also PVR, thus providing indirect evidence for reverse remodeling in the pulmonary vasculature. Using an established rat model of PH-LHD due to left heart failure triggered by pressure overload with subsequent development of PH, a model is developed to study the molecular and cellular mechanisms of this physiological reverse remodeling process. Specifically, an aortic debanding surgery was performed, which resulted in reverse remodeling of the LV myocardium and its unloading. In parallel, complete normalization of RV systolic pressure and significant but incomplete reversal of RV hypertrophy was detectable. This model may present a valuable tool to study the mechanisms of physiological reverse remodeling in the pulmonary circulation and the RV, aiming to develop therapeutic strategies for treating PH-LHD and other forms of PH.

The present protocol describes a surgical procedure to remove ascending-aortic banding in a rat model of pulmonary hypertension due to left heart disease. This technique studies endogenous mechanisms of reverse remodeling in the pulmonary circulation and the right heart, thus informing strategies to reverse pulmonary hypertension and/or right ventricular dysfunction.

大鼠主动脉切除术治疗左心病肺动脉高压反向血管重塑模型

Pulmonary hypertension due to left heart disease (PH-LHD) is the most common form of PH, yet its pathophysiology is poorly characterized than pulmonary ...

A Silicosis Mouse Model Established by Repeated Inhalation of Crystalline Silica Dust

Silicosis can be caused by exposure to respiratory crystalline silica dust (CSD) in an industrial environment. The pathophysiology, screening, and treatment of silicosis in humans have all been extensively studied using the mouse silicosis model. By repeatedly making mice inhale CSD into their lungs, the mice can mimic the clinical symptoms of human silicosis. This methodology is practical and efficient in terms of time and output and does not cause mechanical injury to the upper respiratory tract due to surgery. Furthermore, this model can successfully mimic acute/chronic transformation process of silicosis. The main procedures were as follows. The sterilized 1-5 &#181;m CSD powder was fully ground, suspended in saline, and dispersed in an ultrasonic water bath for 30 min. Mice under isoflurane-induced anesthesia switched from shallow rapid breathing to deep, slow aspiration for approximately 2 s. The mouse was placed in the palm of a hand, and the thumb tip gently touched the lip edge of the mouse's jaw to straighten the airway. After each exhalation, the mice breathed in the silica suspension drop by drop through one nostril, completing the process within 4-8 s. After the mice's breathing had stabilized, their chest was stroked and caressed to prevent the inhaled CSD from being coughed up. The mice were then returned to the cage. In conclusion, this model can quantify CSD along the typical physiological passage of tiny particles into the lung, from the upper respiratory tract to the terminal bronchioles and alveoli. It can also replicate the recurrent exposure of employees due to work. The model can be performed by one person and does not need expensive equipment. It conveniently and effectively simulates the disease features of human silicosis with high repeatability.

This protocol describes a method for establishing a mouse model of silicosis through repeated exposure to silica suspensions via a nasal drip. This model can efficiently, conveniently, and flexibly mimic the pathological process of human silicosis with high repeatability and economy.

通过反复吸入结晶二氧化硅粉尘建立的矽肺小鼠模型

Silicosis can be caused by exposure to respiratory crystalline silica dust (CSD) in an industrial environment. The pathophysiology, screening, and ...

Acupoint Application Combined with Acupoint Massage for Treating Constipation in a Patient with Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a common and frequent disease in elderly patients, with a tendency for progressive exacerbation. Constipation that reduces the quality of life and triggers the risk of diseases is a common concomitant symptom in patients with COPD. Currently, western medical treatment does not achieve the desired results for patients. A high recurrence rate accompanies it, whereas traditional Chinese medicine (TCM) has a long history and rich experience in treating chronic diseases. Both acupoint application and acupoint massage are characteristic therapies of TCM, with minor side effects, high safety, simple operation, and outstanding advantages. They are effective in treating constipation for patients with COPD and are considered an ideal alternative therapy for patients with chronic constipation. The purpose of this article is to introduce the method of acupoint application combined with acupoint massage for the treatment of constipation in patients with COPD, including the selection of points, items, treatment time, and operation procedure.

Here we demonstrate acupoint application combined with acupoint massage for treating constipation in a patient with chronic obstructive pulmonary disease (COPD).

穴位应用联合穴位按摩治疗慢性阻塞性肺疾病患者便秘

Chronic obstructive pulmonary disease (COPD) is a common and frequent disease in elderly patients, with a tendency for progressive exacerbation. ...

针灸刺激耳朵以缓解 COPD 患者的睡眠障碍

Auricular Acupuncture as a Traditional Chinese Medicine Therapy for Chronic Obstructive Pulmonary Disease Combined with Sleep Disorders

Chronic obstructive pulmonary disease (COPD) is a clinical syndrome characterized by persistent and irreversible airflow limitation and chronic respiratory symptoms. It has a wide spectrum of complications, and sleep disorders, as part of it, are common in severe cases, especially in elderly patients. Long-term lack of sleep may lead to the aggravation of the original disease, reducing patients' quality of life. Benzodiazepines are mainly used for symptomatic treatment of COPD combined with sleep disorders. However, such drugs have the side effect of respiratory central inhibition and could probably aggravate hypoxia symptoms. Auricular acupuncture is a special method of treating physical and psychosomatic dysfunctions by stimulating specific points in the ear. This article explains the specific methods of clinical operation of auricular acupuncture in detail, including assessment of patient eligibility, medical devices used, acupuncture points, course of treatment, post-treatment care, responses to emergencies, etc. The Pittsburgh sleep quality index (PSQI) and chronic obstructive pulmonary disease assessment scale (CAT) were used as the observational index of this method. So far, clinical reports have proved that auricular acupuncture has a definite curative effect in the treatment of COPD combined with sleep disorders, and its advantages of simple operation, few adverse reactions are worthy of further study and promotion, which provide a reference for the clinical treatment of such diseases.

作者聚焦:利用中医耳针疗法改善睡眠障碍 

This protocol introduces an effective method to improve the sleep condition of patients with chronic obstructive pulmonary disease and alleviate cough symptoms by using auricular acupuncture techniques to percutaneously stimulate specific acupoints in the patients' ears.