作者感谢 j. Lebacq 教授对手稿的严谨编辑。Cftrtm1Eur (纯合 F508del-CFTR (FVB/129) 小鼠是由荷兰鹿特丹的伊拉斯 MC 开发的, 支持欧洲经济共同体欧洲协调行动, 为囊性纤维化的研究欧盟 FP6LHHM-CT-2005-018932。

研究和规程由学院的动物研究的道德委员会 (2017/伦敦大学/马里兰州/015) 批准, 并与欧洲共同体关于研究动物使用的条例 (中欧 n° 86/609) 达成一致。调查人员在欧洲议会 2010/63/欧盟和2010年9月22日理事会关于保护用于科学目的的动物的指导下, 有资格进行动物试验。
1. 试验前评估和管理
<ol><li>准备双腔鼻导管。 注: 鼻导管为双腔毛细管, 一腔用于连续灌注溶液, 另一种为测量通道。<ol>
<li>加热一件的中心部分, 约20厘米长, 聚乙烯管 (2.0 毫米内径; 3.0 毫米外径;图 1a) 在气体燃烧器的火焰, 直到它是足够软拉 (10-十五年代)。</li>
		<li>拉两个两端, 以获得一个非常薄的毛细管适当长度 (〜15厘米) 和外径 (~ 0.1 毫米) 的鼻腔探头 (图 1b)。</li>
		<li>清洁和脱脂两个这样的毛细血管与纯乙醇, 加入他们与磁带 (图 1c), 并粘合在一起使用氰胶水。</li>
		<li>用剃刀刀片或手术刀截去探头的多余长度, 以获得8厘米左右的双腔导管的最佳长度 (图 1d)。</li>
		<li>通过两个流明注入水, 以验证它们是否具有渗透性。</li>
		<li>在距尖端5毫米的距离处应用一个标记。 注: 探头插入到鼻孔中, 以便所有测量都在同一部位的鼻腔内进行。</li>
		<li>将双腔导管存放在干燥箱中。 注: 如果在测试后立即进行适当清洗, 可以使用 6-10 次。</li>
	</ol>
</li>
	<li>准备奶油混合。<ol>
<li>在1:1 的比例 (体积/体积) 中轻轻地混合电解质霜和饱和3米氯化钾溶液, 避免形成微小的气泡。 注: 奶油混合用于建立测量和参考 Ag/AgCl 电极的电极桥, 如图 1所示。</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57934/57934fig1.jpg"> 图 1: 探头与电极和桥梁.该图说明了聚乙烯管 (a) 和加热和拉 (b) 后, 连接双腔导管 (d) 的两个毛细管部分 (c)、硅胶管接头 (e) 和电极 (f) 的胶带。对于图片清晰度, 连接器尚未填充奶油混合。<a href="https://www.jove.com/files/ftp_upload/57934/57934fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>解决方案标签</td>
			<td>解决方案说明</td>
		</tr>
<tr>
<td>一个</td>
			<td>基底缓冲盐溶液</td>
		</tr>
<tr>
<td>B</td>
			<td>无氯缓冲盐溶液</td>
		</tr>
<tr>
<td>C</td>
			<td>10-2 M 阿米洛利库存解决方案</td>
		</tr>
<tr>
<td>D</td>
			<td>10-3 M 佛司可林库存解决方案</td>
		</tr>
</tbody></table>表 1: 小鼠鼻腔 PD 试验的库存解决方案。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>盐</td>
			<td>毫米</td>
			<td>分子量</td>
			<td>g/升</td>
		</tr>
<tr>
<td>氯化钠 (氯化钠)</td>
			<td>135</td>
			<td>58.44</td>
			<td>7889</td>
		</tr>
<tr>
<td>氯化钙二水合物 (CaCl22H2O)</td>
			<td>2.25</td>
			<td>147</td>
			<td>0.331</td>
		</tr>
<tr>
<td>六水合氯化镁 (氯化镁26H2O)</td>
			<td>1。2</td>
			<td>203。3</td>
			<td>0.244</td>
		</tr>
<tr>
<td>磷酸二钾 (K2HPO4)</td>
			<td>2。4</td>
			<td>174。2</td>
			<td>0.418</td>
		</tr>
<tr>
<td>磷酸钾盐 (2PO4)</td>
			<td>0。4</td>
			<td>136。1</td>
			<td>0.054</td>
		</tr>
</tbody></table>表 2: 基底缓冲盐溶液的组成 (库存溶液 A)。
<ol start="3"><li>准备解决方案。<ol>
<li>准备实验协议所需的四个库存解决方案 (表 1)。<ol>
<li>准备基础缓冲液 (股票溶液 A;表 2)和无氯缓冲溶液 (库存溶液 B;表 3)在室温下将盐与纯净水混合。将 ph 值调整为 7.4 (范围 7.0-7.6), 使用 1 N 氢氧化钠来提高 ph 值, 1 n HCl 以降低 ph 值。检查解决方案的渗透 (275 mOsm/升)。</li>
			<li>在4摄氏度的标签玻璃瓶中贮存长达3月或在塑料瓶中冷冻6月。</li>
			<li>10-2米阿米洛利溶液 (股票溶液 C) 加入26.6 毫克盐酸阿米洛利到10毫升纯净水中。将混合物加热5到10分钟, 在70摄氏度。由于阿米洛利对光线敏感, 所以将溶液 C 保存在一个深色容器中。标签的容器和存储在4°c 长达3月。</li>
			<li>通过加入10毫克佛司可林到24.36 毫升纯净水中, 准备 10-3米佛司可林溶液 (股票溶液 D)。标签0.1 毫升整除数的佛司可林库存解决方案和存储在-20 摄氏度, 长达6月。</li>
		</ol>
</li>
	</ol>
</li>
	<li>准备新的解决方案 A1, B1 和 B2 (表 4)。<ol>
<li>通过获得10毫升的基盐溶液 (股票溶液 A) 和添加0.1 毫升 10-2 M 阿米洛利溶液 (A1 溶液 C), 制备新鲜溶液 (基底缓冲盐溶液加10-4米阿米洛利)。标签的容器和使用24小时内的准备。</li>
		<li>通过获得10毫升的无氯缓冲盐溶液 (库存溶液 B) 并加入0.1 毫升 10-2 M 阿米洛利溶液 (B1 溶液 C), 制备新溶液 (氯化物缓冲溶液加10-4米阿米洛利)。标签的容器和使用24小时内准备。</li>
		<li>制备新鲜溶液 B2 (无氯缓冲盐溶液加10-4米阿米洛利和 10-5米佛司可林) 通过获得10毫升无氯缓冲盐溶液 (股票溶液 B) 和增加0.1 毫升 10-2 M阿米洛利溶液 (股票溶液 C) 和0.1 毫升 10-3 M 佛司可林溶液 (股票溶液 D)。标签的容器和使用2小时内准备。</li>
	</ol>
</li>
</ol><table fo:keep-together.within-page="1"><tbody>
<tr>
<td>盐</td>
			<td>毫米</td>
			<td>分子量</td>
			<td>g/升</td>
		</tr>
<tr>
<td>葡萄糖酸钠 (味精)</td>
			<td>135</td>
			<td>218。1</td>
			<td>29444</td>
		</tr>
<tr>
<td>葡萄糖酸钙 (无水粉)</td>
			<td>2。2</td>
			<td>430。4</td>
			<td>0.947</td>
		</tr>
<tr>
<td>硫酸镁水合物 (氯化镁26H2O)</td>
			<td>1。2</td>
			<td>246。5</td>
			<td>0.296</td>
		</tr>
<tr>
<td>磷酸二钾 (K2HPO4)</td>
			<td>2。4</td>
			<td>174。2</td>
			<td>0.418</td>
		</tr>
<tr>
<td>磷酸钾盐 (2PO4)</td>
			<td>0。4</td>
			<td>136。1</td>
			<td>0.054</td>
		</tr>
</tbody></table>表 3: 无氯缓冲溶液的组成 (库存溶液 B)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57934/57934fig2.jpg"> 图 2: 小鼠在鼻腔粘膜灌注过程中的位置.该图说明了加热垫 (a), 所述的电压表 (b)、在后肢 (c)、近端 (d) 和蠕动泵的远端 (e) 插座、枕头 (f) 和床单 (g) 中插入的参考电极。<a href="https://www.jove.com/files/ftp_upload/57934/57934fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
2. 测试期间的评估和管理
<ol><li>准备实验装置。<ol>
<li>为了防止在测试期间和之后体温过低, 使用两个加热垫保持在生理温度, 一个为测量设置 (18.8 x 37.5 cm;图 2a), 另一条 (15.5 x 15.5 厘米) 用于在测试结束时将动物放置在其上进行恢复的盒子。</li>
		<li>使用软件加载的计算机上的数据捕获连接到数据存储器高输入阻抗 (> 1012Ω) 和高分辨率 (0.1 mV) 电压表 (图 2b)。</li>
		<li>将电极连接到电压表。将正测量电极与鼻腔导管和负参考电极连接到后部皮下腔内插入的导管, 如图 2c所示。</li>
		<li>将电极的尖端蘸在奶油混合物中。检查初始电极偏移值。如果值大于 2 mv (理想的 0 mv), 则拒绝电极。</li>
		<li>打开蠕动泵并将泵管插入位置, 如图 2所示。</li>
		<li>将泵管的近端出口连接到含有所选溶液进行鼻腔灌注的瓶子 (图 2d)。将泵管的远端出口连接至鼻腔导管的灌注腔 (图 2e)。</li>
		<li>用电解质奶油混合通过10µL 吸管尖来填充鼻腔导管的一腔。将导管调整成硅胶管接头 (图 1e), 用奶油混合填充接头, 并将正极插入接头 (图 1f)。检查测量电极桥距值。如果值大于 2 mv (理想的 0 mv), 则拒绝桥接。</li>
		<li>用新鲜溶液 A1 填充鼻腔导管的第二腔。将鼻腔导管的尖端浸入含有溶液的瓶子中. 反向蠕动泵的方向, 以填补与十年代的灌注相应的鼻腔导管长度。 注意: 这允许在应用阿米洛利之前记录十年代的基本 PD 值。将鼻腔导管的尖端浸入参考电极连接器内的奶油混合物中 (图 1)。</li>
	</ol></li>
	<li>开始处理鼠标。<ol>
<li>记录鼠标重量以计算腹腔 (ip)17号干线注射的麻醉剂的确切剂量。</li>
		<li>使用 26G (0.45 x 10 毫米) 针, 注射术前用药腹腔促进麻醉的诱导: 固定剂量50µL 5 毫克/毫升咪达唑仑。等5分钟。</li>
		<li>准备麻醉混合物 (芬太尼, medetomidine, 氟哌利多在最后浓度 0.05, 0.40 和20毫克/千克体重分别和可乐定剂量0.375 µg), 允许最佳和稳定的麻醉深度 (第三阶段, 平面 2)17.注射麻醉药混合腹腔, 再注射可乐定量。 注:15 分钟后, 鼠标应睡着, 麻醉可以持续至少30分钟。</li>
		<li>折叠一张吸收的组织, 使一个3厘米宽的 ' 枕头 ' (图 2f), 将覆盖在加热垫的上部 (图 2a) 作为对鼠标头的支持。</li>
		<li>用一张吸收纸巾盖住暖气垫 ("床单";图 2g). 把鼠标放在暖气垫上。将四肢和尾部胶带 (图 2)。</li>
		<li>将参考静脉导管 (图 1g) 插入后肢皮下空间。取下针并调整硅胶管接头 (图 1e, 2c)。</li>
		<li>用奶油混合填充导管和接头, 并将负极插入接头 (图 1f)。</li>
		<li>用胶带固定导管和电极以防止任何移位。在参考电极和桥梁放置后, 通过将鼻腔导管的尖端浸入 IV 导管连接器内的电极膏内, 再次验证测量电极桥距值。记录稳定的最终偏移值 (理想小于 2 mV)。</li>
		<li>在两块吸收组织 ("枕头" (图 2f) 和 "床单" 之间的自由空间, 用 "柔和" 胶带固定在加热垫上的鼠标耳朵;(图 2g))。固定触须 (在鼻孔附近生长的僵硬毛发) 而不接触眼睛。</li>
		<li>要从口腔中吸收液体, 请将舌头侧移 (图 3a) 并插入一根尖芯的滤纸 ("管子";图 3b) 到口中大约1厘米。要吸收流出鼻孔的液体, 请放置第二片滤纸 ("手帕";图 3c) 在鼻尖处举行。</li>
		<li>通过放置鼻腔导管的尖端与口中的内侧接触, 检查上皮电位的正控制值。 注意: 读数应稳定, 范围介于-10 mv 和-20 mv 之间。</li>
		<li>把它与优良的钳和良好的直接照明, 微妙地介绍鼻腔导管到一个鼻孔 4-6 毫米从鼻子尖端。 注: 鼻腔 pd 导管位于中鼻甲的位置, 使最大稳定基线 PD 值17,20。</li>
		<li>麻醉药物注射5分钟后, 用动物头部向下轻轻倾斜加热垫。监测最大基底 PD 值。</li>
		<li>当其稳定在三十年代左右, 开始灌注鼻腔粘膜, 以恒定率为10µL/分钟, 与4缓冲溶液 (a, A1, B1, B2) 相继。灌注的第一个解决方案 (步骤 1.3.1.1) 为十年代和每个后续解决方案 (步骤 1.4) 为5分钟或直到达到稳定的值。当瞬态佛司可林响应开始下降时, 停止灌注佛司可林 (B2)。</li>
		<li>选择1秒的数据记录间隔。在开始灌注时开始记录数据。将数据显示为计算机屏幕上时间的函数。</li>
		<li>在解决方案 B2 的灌注结束时停止数据捕获。将数据另存为电子表格文件。</li>
		<li>通过减去最终偏移值来更正数据。</li>
	</ol></li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57934/57934fig3.jpg"> 图 3: 鼠标放置在加热垫上, 用鼻腔导管和滤纸放到位.该图说明了侧 (a)、管子 (b) 和手帕 (c) 的舌。<a href="https://www.jove.com/files/ftp_upload/57934/57934fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
3. 测试后评估和管理
<ol><li>从加热垫上松开鼠标。</li>
	<li>用 "床单" 擦拭鼠标鼻子 (图 2g)。</li>
	<li>注射腹腔麻醉拮抗剂混合组成的固定剂量 (4 µg) 纳洛酮, 一个竞争性的吗啡拮抗剂, 和 atipamezole, medetomidine 特定的解毒剂 (2 毫克/千克体重)。</li>
	<li>将鼠标放在恢复箱中较小的加热垫上, 直到完全恢复, 这通常在1到2小时后被观察到。 注: 在伦敦大学学院实验室, 无论性别或基因型, 都观察到大约10% 的死亡率, 主要与 bronchoaspiration 在鼻腔内灌注的溶液有关。</li>
</ol>

作者没有什么可透露的。

本文的目的是描述一个适当的协议, 以测量连续灌注的解决方案, 在自发呼吸小鼠的时间长度, 以测试的完整性, 离子转运体, 主要是 CFTR 和 ENaC。该议定书的所有步骤都经过仔细的优化, 以确保完整的动物恢复和良好的质量和可重现的数据。特别是, 关键步骤是麻醉评估和管理, 并在测试期间和之后适当的动物位置和护理。
先前的研究表明, 2 级的麻醉暴露, 可以通过应用这里使用的鸡尾酒混合物达到17, 是与定期呼吸和没有负强心剂效应和眨眼, 瞳孔和踏板撤退反射。在麻醉深度的这一水平, 鼻腔导管可以保持原位, 以连续灌注鼻腔, 并用作测量电极的桥梁, 具有良好的耐受性。此外, 插入导管在皮下的空间, 作为参考电极的桥梁, 并没有随后任何痛苦的反应或迹象的有害影响。在啮齿类动物中, nasofacial 区域的感官输入在控制外界情况 (包括威胁) 方面的重要作用, 使得在鼻腔手术时有足够的麻醉深度尤其具有挑战性。在一些小鼠研究33,34, 采用雾化代替连续鼻腔灌注进行鼻腔 PD 试验。然而, 由于鼻腔探针的反复清除和 reinsertions, 这种方法导致了不可靠的结果。事实上, 由于在小鼠鼻腔黏膜20中细胞类型的非均匀分布, 在鼻孔的同一位置重新定位探针的尖端是至关重要的。此外, 对解决方案的变化, 特别是无氯溶液的反应, 在灌注中断时迅速消失。
支气管--吸入导致呼吸骤停的方法是手术过程中的一个重要限制, 也是检验死亡率的主要原因。一些基本措施的目的是防止它, 包括动物的背部压疮位置, 轻轻地倾斜它与它的头部向下, 并吸收过剩的液体从口腔和鼻腔17。在试验结束时应用麻醉药品的解毒剂, 确保了快速、可逆的麻醉和完全恢复动物的水平。在这里提出的协议允许可靠的测量在自发呼吸的老鼠和重复测试在同一动物。它影响到统计意义35和遵守 3R (替换, 提炼和减少) 的动物使用规则在实验程序36所需的老鼠数量。在人类和小鼠中, 总氯的反应之间存在着最低的试验变异性, 这表明它是检测新 CF 治疗策略有效性的最可靠的鼻腔 PD 参数。在 CF 小鼠中, 总氯化物反应的测量误差显示为小于±1.7 mV35。换言之, 当评估 CFTR 药物在 F508del-CF 小鼠体内的生物活性时, 在缺席和超过 2 mV 的治疗中, 总氯化物反应的差异表明与药物相关的改善效果的几率为95%。
对典型追查的数据解释表明, 鼻腔 PD 试验对 CF 和野鼠18、19、20、21、35的鉴别能力, 表明F508del-CF 的鼠标模型30模仿人鼻粘膜的典型临床离子运输异常。然而, 在动物模型中, 残留的氯化物电导率是可检测的, 产生于残余 F508del-CFTR 函数或由替代非 CFTR 依赖的氯化物通道的贡献。从临床前期的 CF 研究中将结果转化为临床医学, 意味着要处理这两种设置之间的几个主要差异。小鼠的表型表现为减毒呼吸综合征。缺乏多疗法与事实老鼠模型被安置在特权条件以卫生障碍也贡献到区别37。这里描述的协议显示一个非常低的变异性35 , 它已经适应了猪38,39和鼬模型40。在前一项研究中, 通过将小鼠鼻腔粘膜的灌注与替代氯转运体的抑制剂结合, 对实验性协议进行了修改, 探讨了非 CFTR 依赖钙活性氯的可能贡献频道18。该试验还用于研究β-ENaC overexpressing 小鼠模型41中的钠转运, 其设计目的是模拟 CF-肺部疾病42。今后, 可以考虑进一步应用该试验, 以研究其他转运体, 如 ATP12A, CFTR 独立的 H+泵蛋白表达在人和猪, 但没有在小鼠气道43。总之, 这里描述的协议允许可靠的测量 transepithelial 氯和钠转运体在自发性呼吸小鼠的功能状态, 减少测试相关的死亡率和多次试验在同一动物。

近三年来, 电电位差 (PD) 测量已被用来评估跨膜离子转运蛋白在鼻腔粘膜表达的功能状态, 作为远端气管1的代表。作为一个多步法的动态测试2,3, 鼻腔 PD 允许功能解剖的囊性纤维化跨膜电导调节剂 (CFTR) 和上皮钠通道 (ENaC) 活动, 均在顶端膜局部上皮细胞及在气道表面水化中发挥关键作用。鼻腔 PD 试验的主要临床应用是协助诊断 CF, 这是高加索人群中最常见的致命遗传紊乱, 平均发病率为 1, 在欧洲国家2500活产。这项试验长期证明有助于诊断的减毒, 寡聚或单症状的 cf 通常诊断的形式, 在以后的生活, 和 cf 相关的障碍, 如先天性双侧缺输精管, 特发性慢性胰腺炎, 过敏支气管肺曲霉病, 支气管扩张症4。最近, clinometric 评估的治疗调制基本 CFTR 缺损5,6,7,8,9,10,11 ,12,13,14,15,16已利用鼻腔 PD 在临床试验的新 CF 疗法。在临床前的设置, 测试已经适应了鼠标17 , 以允许调查的生物活性的新 CF 靶治疗18,19,20,21。在小鼠, 该技术是微妙的, 基于物种相关的解剖差异的鼻子区域之间的啮齿动物和人类, 主要是对感官输入的重要作用, 从 nasofacial 地区的啮齿动物。它需要训练有素和熟练的操作员、专用设备和用品。
CF 是一种多系统的外分泌腺体紊乱, 其中慢性呼吸道疾病占主导地位的临床图片。该病是由基因编码的突变所造成的循环腺苷磷酸 (阵营) 调节 CFTR 氯通道22。到目前为止, 已经发现了2000多 CFTR 突变23。最常见的突变24,25, 发现在约90% 的 CF 等位基因, 对应于删除的苯丙氨酸在位置508的多肽链的蛋白质 (F508del-CFTR)。CFTR 蛋白是纯欧姆的小电导氯通道。还有大量证据表明, CFTR 管制其他运输机制, 特别是 ENaC26、27。有缺陷的电解质输送, 包括降低 CFTR 依赖氯电导率和增加 ENaC 依赖性钠电导率, 是 CF 上皮的一个标志。前者的缺陷是由减少或废除的复极化反应的电化学梯度有利于氯外流和添加肾上腺素 (β肾上腺素激动剂, 增加细胞内的阵营) 或佛司可林 (腺苷酸酶激动剂, 不批准用于临床使用)。后者的缺陷是由鼻粘膜基底极化 (一个更负的 PD) 反映, 并增加对阿米洛利的反应, 一种利尿药物, 阻止 ENaC28。
cf 小鼠模型在 cf 研究中得到了广泛的应用, 对 cf 病理学的解剖具有重要的价值。目前, 至少有十五种模型被描述为29, 其中三是纯合的, 最临床相关的 F508del 变异30,31,32。其中一个三菌株30, 在鹿特丹的伊拉斯大学开发, 已经使用了近20年在大学天主教 de 鲁汶 (伦敦学院) 实验室。Cftrtm1Eur模型30已证明是非常有用的研究的多器官病理生理学的 CF 疾病和测试疗效的新的治疗策略18,19,20, 21。小鼠鼻腔 PD 试验 (< 24 小时) 后或术后早期可能出现许多问题。本文叙述了保持鼻腔导管原位连续灌注所需的足够的麻醉深度, 以及避免鼻内灌注的支气管的方法。在测试结束时的动物护理也被描述, 包括管理麻醉药物解毒剂的组合, 导致迅速逆转麻醉与完全恢复的动物。总之, 这些程序允许可靠的测量在自发呼吸的老鼠, 减少测试相关的死亡率和重复测试在同一动物。显示并讨论了在 CF 和野生型小鼠鼻腔 PD 试验中获得的代表性数据。
小鼠鼻腔 PD 测试协议报告在三次会议: 评估和管理之前, 期间和之后的测试。在试验前评估和管理中, 详细介绍了双腔鼻腔导管的制备和连续鼻腔灌注的解决方案。在测试的评估和管理部分, 对鼠标的实验设置和处理进行了详细的分析。最后, 对动物在试验结束时的管理进行描述, 以改善动物的完整恢复。

<table><tbody><tr><td>Portex polyethylene tube&amp;nbsp;</td><td>Smiths Medical, Hythe, Kent, England CT21 6JL</td><td>Portex 800/100/500;2.0mm ID, 3.0 mmOD</td><td>to prepare capillary tubes for nasal probe</td></tr><tr><td>Electrode cream</td><td>Parker, Fairfield, NJ, USA</td><td>Redux cream</td><td>to build electrode bridges</td></tr><tr><td>Ag/AgCl electrodes</td><td>Biomedical, Clinton Township, MI, USA</td><td>JNS BNT131-1,0</td><td>measuring and reference electrodes</td></tr><tr><td>amiloride hydrochloride</td><td>Sigma, St Louis, MI, USA</td><td>A7410</td><td>to prepare perfusion solutions</td></tr><tr><td>forskolin</td><td>Sigma, St Louis, MI, USA</td><td>F6886</td><td>to prepare perfusion solutions</td></tr><tr><td>Knick Portamess voltmeter</td><td>Elektronisch Me&amp;beta;ger&amp;auml;te, Berlin, Germany</td><td>Portavo 904 pH</td><td>to measure potential difference</td></tr><tr><td>Paraly SW 112 Software&amp;nbsp;</td><td>Elektronisch Me&amp;beta;ger&amp;auml;te, Berlin, Germany</td><td>Paraly SW112 software</td><td>to capture potential difference data</td></tr><tr><td>midazolam&amp;nbsp;</td><td>Mylan, Hoeilaart, Belgium</td><td>Dormicum 15mg/3ml</td><td>to serve as anaesthetic premedication</td></tr><tr><td>fentanyl</td><td>Janssen Cilag, Berchem, Belgium</td><td>Fentanyl-Janssen 0.05 mg/ml</td><td>to serve as anaesthetic medication</td></tr><tr><td>medetomidine</td><td>Orion Pharma, Espoo, Finland</td><td>Domitor 1 mg/ml</td><td>to serve as anaesthetic medication</td></tr><tr><td>droperidol&amp;nbsp;</td><td>Janssen&amp;nbsp; Cilag, Berchem, Belgium</td><td>Dehydrobenzperidol 2.5 mg/ml</td><td>to serve as anaesthetic medication</td></tr><tr><td>clonidine&amp;nbsp;</td><td>Boehringer Ingelheim Pharma KG, Ingelheim am Rhein, Germany</td><td>Catapressan 0.15 mg/ml,</td><td>to serve as anaesthetic medication</td></tr><tr><td>refernce IV catheter</td><td>Becton Dickinson, Sandy, UT, USA</td><td>24 GA x 0.75 IN, BD Insyte-W</td><td>to build electrode bridges</td></tr><tr><td>forceps&amp;nbsp;</td><td>Fine science Tools, Heidelberg, Germany</td><td>Dumont #5, Fine science Tools</td><td>to place the nasal catheter</td></tr><tr><td>naloxone&amp;nbsp;</td><td>Braun Medical, Brussels, Belgium</td><td>Narcan, 0.4 mg/ml</td><td>to serve as anaesthetic antagonist</td></tr><tr><td>atipamezole&amp;nbsp;</td><td>Zoetis, Bloomberg, Belgium</td><td>Antisedan, 5 mg/ml</td><td>to serve as a medetomedine specific antidote&amp;nbsp;</td></tr><tr><td>Heating pads&amp;nbsp;</td><td>Harvard Apparatus, Holliston, MA, USA</td><td>18,8x37,5 cm; 15,5x15,5 cm</td><td>to avoid hypothermia during and after the test</td></tr><tr><td>Peristaltic pump P1</td><td>GE Life Sciences, Uppsala, Sweden</td><td>18111091</td><td>to perfuse solutions in the mouse nose</td></tr><tr><td>cyanoacrylate glue</td><td>Loctite, Henkel, D&amp;uuml;sseldorf, Germany&amp;nbsp;</td><td>super glue 3</td><td>to glue together two capillary tubes&amp;nbsp; for nasal probe</td></tr><tr><td>NaCl</td><td>Sigma, St Louis, MI, USA</td><td>RES0926S-A7</td><td>Pharma-Grade, USP</td></tr><tr><td>CaCl2.2H2O</td><td>Sigma, St Louis, MI, USA</td><td>M7304</td><td>Pharma-Grade, USP</td></tr><tr><td>MgCl2.6H2O</td><td>Sigma, St Louis, MI, USA</td><td>1551128</td><td>Pharma-Grade, USP</td></tr><tr><td>K2HPO4</td><td>Sigma, St Louis, MI, USA</td><td>1551139</td><td>Pharma-Grade, USP</td></tr><tr><td>Na gluconate</td><td>Sigma, St Louis, MI, USA</td><td>S2054</td><td>Pharma-Grade, USP</td></tr><tr><td>Ca gluconate</td><td>Sigma, St Louis, MI, USA</td><td>C8231</td><td>Pharma-Grade, USP</td></tr><tr><td>MgSO4.7H2O</td><td>Sigma, St Louis, MI, USA</td><td>RES0089M-A7</td><td>Pharma-Grade, USP</td></tr><tr><td>BD needle&amp;nbsp;</td><td>Becton Dickinson, Franklin Lakes, USA</td><td>BD 26G (0.45x10 mm)</td><td>intraperitoneal injection</td></tr></tbody></table>

nasal potential difference to quantify trans-epithelial ion transport in mice

鼻电位差试验已用于近三年来协助诊断囊性纤维化 (CF)。它已证明是有益的, 在减毒, 寡聚或单症状形式的 cf 通常诊断的生活后期, 和 CF 相关的障碍, 如先天性双侧缺失输精管, 特发性慢性胰腺炎, 过敏支气管肺曲霉病和支气管扩张症。在临床和前期的设置, 该测试已被用作一个生物标志, 以量化反应的靶向治疗策略的 CF. 将测试调整为鼠标是有挑战性的, 可能会导致相关的死亡率。本文介绍了保持鼻腔导管原位连续灌注所需的足够的麻醉深度。它列出了避免支气管在鼻腔内灌注溶液的方法。它还描述了动物护理在测试结束时, 包括管理麻醉药物解毒剂的组合, 导致迅速逆转麻醉与完全恢复的动物。从 cf 和野生型鼠标获得的代表性数据表明, 该测试在 cf 和非 cf 之间有区别。总之, 这里描述的协议允许可靠地测量在自发性呼吸小鼠中的反式上皮氯和钠转运体的功能状态, 以及在同一动物中进行多次试验, 同时减少与试验有关的死亡率。

为了说明 CF 中的特征离子传输异常, 在 F508del-CF 小鼠的上述协议和 FVB/129 遗传背景的野生型控制下, 进行了鼻 PD 测量, 从布鲁塞尔殖民地Cftrtm1Eur小鼠30。这个临床相关的模型, 窝藏最常见的和最严重的 F508del-CFTR 突变23,24,25, 是最好的目前可用 CF 鼠标模型30,31, 32。
代表鼻腔 PD 追查, 获得在一个4月大的雌性小鼠纯合为 F508del-CF 突变和在年龄和性别匹配野生型 littermate, 如图 4所示。在试验的前两个阶段, 灌注溶液 A 和 A1 (后者含有阿米洛利) 研究了 ENaC 的功能状态。在试验的最后两个阶段, 评估了 CFTR (以及替代氯转运体在没有佛司可林的情况下) 的功能状况, 当时 ENaC 的贡献仍然被阿米洛利所阻止。
在 F508del-CF 鼠标中, 超极化搏基线值 (相对于野类型的鼠标值比较负 PDmax), 并观察到阿米洛利响应的增加;这两项发现都反映了 CFTR 相关的 ENaC overactivity。更一贯地说, 在反应的电化学梯度对氯化物流出有利和加法的佛司可林的一个极大地减少的复极化, 这里命名作为总氯化物反应, 被观察了。尽管野生型小鼠的佛司可林反应量小 (-3 mV), 但在 CF 中, 反应通常是钝的, 与 CFTR 丧失功能一致。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57934/57934fig4.jpg"> 图 4-有代表性的鼻腔 PD 追查.有代表性的鼻 pd 追查从纯合正常小鼠 (a) 和小鼠纯合为 F508del-CFTR 突变 (B), 连同为鼻腔 pd 参数 (C 和 D) 获得的单个值。PDmax: 最大基线稳定值。阿米洛利反应: 鼻粘膜的价值与鼻腔黏膜灌注时的不同值之间的差异, 基底缓冲盐溶液含有阿米洛利 (溶液 A1)。无氯反应: 在鼻粘膜的端部和在鼻腔黏膜灌注开始时, 与无氯缓冲盐溶液加阿米洛利 (溶液 B1) 的值之间的差异。佛司可林反应: 鼻粘膜的值在端部和鼻腔黏膜灌注开始时的差异与无氯缓冲盐溶液加佛司可林和阿米洛利 (溶液 B2)。总氯化物反应: 在零氯灌注下获得的最后两个参数的总和。箭头表示在鼻孔中灌注的溶液的变化。<a href="https://www.jove.com/files/ftp_upload/57934/57934fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
麻醉药的解毒剂在测试结束时应用, 从而减少了麻醉的持续时间, 可达45分钟以上的测试完成。由于动物的恢复没有事后效应, 他们在七天的时间间隔后再次测试, 当应用相同的协议。在两项试验中都探讨了同一鼻孔。在这两项试验中, 第二次试验和配对不同的鼻腔 PD 参数的例子如图 5所示。正如以前报告的35, 之间的测试差异接近零, 特别是对总氯化物反应, 反映了功能状态的 CFTR 依赖氯化物运输, 有缺陷的 CF。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57934/57934fig5.jpg"> 图 5-鼻腔 PD 参数的个体值.在二次测试 (t2) 中获得的值是在纯合正常的小鼠 (a) 和 F508del-CFTR 突变 (B) 的小鼠纯合中进行的, 以及每个对应参数的第二个和第一个测试 (t1) 之间的配对差异。PDmax: 最大基线稳定值。阿米洛利反应: 鼻粘膜的价值与鼻腔黏膜灌注时的值之间的差异, 其基底缓冲盐溶液含有阿米洛利 (溶液 A1)。无氯反应: 鼻粘膜的值在端部和鼻腔黏膜灌注开始时的差异与无氯缓冲盐溶液加阿米洛利 (溶液 B1)。佛司可林反应: 鼻腔 PD 的价值在末端和在鼻腔黏膜灌注开始时的差异与无氯缓冲盐溶液加佛司可林和阿米洛利 (溶液 B2)。总氯化物反应: 在零氯灌注下获得的最后两个参数的总和。<a href="https://www.jove.com/files/ftp_upload/57934/57934fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>解决方案标签</td>
			<td>解决方案说明</td>
		</tr>
<tr>
<td>A1</td>
			<td>基底缓冲盐溶液 (A) 加 10-4米阿米洛利</td>
		</tr>
<tr>
<td>B1</td>
			<td>无氯缓冲盐溶液 (B) 加 10-4米阿米洛利</td>
		</tr>
<tr>
<td>B2</td>
			<td>无氯缓冲盐溶液 (B) 加 10-4米阿米洛利加 10-5米佛司可林</td>
		</tr>
</tbody></table>表 4: 小鼠鼻腔 PD 试验新鲜溶液的清单。

Watch this Scientific Journal Video about Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice at JoVE.com

Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice

在这里, 我们提出了一个测量小鼠鼻电位差异的协议。该试验量化了囊性纤维化跨膜电导调节剂和上皮钠通道等跨膜离子转运蛋白的功能。评价新疗法对囊性纤维化的疗效有重要意义。

鼻电位差量化小鼠跨上皮离子转运

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful ...

nasal-potential-difference-to-quantify-trans-epithelial-ion-transport

Universidad San Sebastian

Research

JoVE Journal

Biology

7.7K 次观看.  Université Catholique de Louvain. 在这里, 我们提出了一个测量小鼠鼻电位差异的协议。该试验量化了囊性纤维化跨膜电导调节剂和上皮钠通道等跨膜离子转运蛋白的功能。评价新疗法对囊性纤维化的疗效有重要意义。

视频: Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

模型和方法来评价药物输送系统在整个细胞屏障的运输

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

科研

生物工程

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

鼠标嗅觉感官神经元的完整神经上皮穿孔膜片钳记录:神经元表达鉴定的气味受体功能分析

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

神经科学

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

在小鼠犁鼻急性组织切片定义细胞群的深入生理学分析

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

用四甲基铵进行实时离子电渗疗法，以量化体细胞分数和脑细胞外空间的变性

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD)

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) function are monitored by the change in voltage across the nasal epithelium after the superfusion of solutions that modify ion channel activity. This is enabled by the measurement of the potential difference between the subcutaneous compartment and the airway epithelium in the nostril, utilizing a catheter in contact with the inferior nasal turbinate.
The test allows the measurement of the stable baseline voltage and the successive net voltage changes after perfusion of 100 &#181;M amiloride, an inhibitor of Na+ reabsorption in Ringer&#39;s solution; a chloride-free solution containing amiloride to drive chloride secretion and 10 &#181;M isoproterenol in a chloride-free solution with amiloride to stimulate the cyclic adenosine monophosphate (cAMP)-dependent chloride conductance related to CFTR.
This technique has the advantage of demonstrating the electrophysiological properties of two key components establishing the hydration of the airway surface liquid of the respiratory epithelium, ENaC, and CFTR. Therefore, it is a useful research tool for phase 2 and proof of concept trials of agents that target CFTR and ENaC activity for the treatment of cystic fibrosis (CF) lung disease. It is also a key follow-up procedure to establish CFTR dysfunction when genetic testing and sweat testing are equivocal. Unlike sweat chloride, the test is relatively more time consuming and costly. It also requires operator training and expertise to conduct the test effectively. Inter- and intra-subject variability has been reported in this technique especially in young or uncooperative subjects. To assist with this concern, interpretation has been improved through a recently validated algorithm.

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference NPD

Here, we present a standardized protocol to measure the nasal potential difference (NPD). Cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) function are evaluated by the change in the voltage across the nasal epithelium after superfusion of solutions that modify ion channel activity, providing an outcome measure.

鼻膜 Transepithelial 电位差的标准化测量 (NPD)

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) ...

医学

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

使用标准化肠道循环模型对小鼠肠道渗透性和中性粒细胞转导的功能评估

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

免疫与感染

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

电生理测量在中性转运体活性研究中的应用

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...

生物学

Functional Assessment of Intestinal Tight Junction Barrier and Ion Permeability in Native Tissue by Ussing Chamber Technique

The Ussing chamber technique was first invented by the Danish scientist Hans Ussing in 1951 to study the transcellular transport of sodium across frog skin. Since then, this technique has been applied to many different tissues to study the physiological parameters of transport across membranes. The Ussing chamber method is preferable to other methods because native tissue can be used, making it more applicable to what is happening in vivo. However, because native tissue is used, throughput is low, time is limited, and tissue preparation requires skill and training. These chambers have been used to study specific transporter proteins in various tissues, understand disease pathophysiology such as in Cystic Fibrosis, study drug transport and uptake, and especially contributed to the understanding of nutrient transport in the intestine. Given the whole epithelial transport process of a tissue, not only transepithelial pathways, but also paracellular pathways are important. Tight junctions are a key determinant of tissue specific paracellular permeability across the intestine. In this article, the Ussing chamber technique will be used to assess paracellular permselectivity of ions by measuring transepithelial conductance and dilution potentials.

Intestinal epithelium confers not only nutrient absorption but protection against noxious substances. The apical-most epithelial intercellular junction, i.e., the tight junction, regulates paracellular solute and ion permeability. Here, a protocol for the preparation of mucosal sheets and assessment of the ion selectivity of tight junctions using Ussing chamber technique is described.

通过Ussing Chamber技术评估天然组织中肠道紧密连接屏障和离子渗透性的功能评估

The Ussing chamber technique was first invented by the Danish scientist Hans Ussing in 1951 to study the transcellular transport of sodium across frog ...

Primary Human Nasal Epithelial Cells: Biobanking in the Context of Precision Medicine

Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing. Patient-derived primary HNE cells can be amplified and differentiated into a pseudo-stratified epithelium in air-liquid interface conditions to quantify cyclic AMP-mediated Chloride (Cl-) transport as an index of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function. If critical steps such as quality of nasal brushing and cell density upon cryopreservation are performed efficiently, HNE cells can be successfully biobanked. Moreover, short-circuit current studies demonstrate that freeze-thawing does not significantly modify HNE cells&#39; electrophysiological properties and response to CFTR modulators. In the culture conditions used in this study, when less than 2 x 106 cells are frozen per cryovial, the failure rate is very high. We recommend freezing at least 3 x 106 cells per cryovial. We show that dual therapies combining a CFTR corrector with a CFTR potentiator have a comparable correction efficacy for CFTR activity in F508del-homozygous HNE cells. Triple therapy VX-445 + VX-661 + VX-770 significantly increased correction of CFTR activity compared to dual therapy VX-809 + VX-770. The measure of CFTR activity in HNE cells is a promising pre-clinical biomarker useful to guide CFTR modulator therapy.

Here we describe the isolation, amplification, and differentiation of primary human nasal epithelial (HNE) cells at the air-liquid interface and a biobanking protocol allowing to successfully freeze and then thaw amplified HNE. The protocol analyzes electrophysiological properties of differentiated HNE cells and CFTR-related chloride secretion correction upon different modulator treatments.

原代人鼻上皮细胞:精准医学背景下的生物样本库

Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing. Patient-derived primary HNE cells can be amplified and ...

A Fluorescence-Based Assay of Membrane Potential for High-Throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines

Fluorescence-based studies are suitable for high-throughput plate reader assays of cells in culture. They have been commonly employed for drug discovery campaigns targeting recombinant ion channel proteins overexpressed in cells such as HEK-293 cells. However, there is increasing emphasis on the use of tissue-relevant cell lines for studying the effects of small molecule interventions. The following protocol describes the adaptation of a fluorescence-based membrane potential assay for the study of ion channels endogenously expressed in epithelial cell lines. The membrane potential assay details a high-throughput assay for chloride channel activity of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in two commonly studied epithelial cell lines, Caco-2 and Calu-3. In addition, this paper describes a novel application of this system to measure the activity of the Epithelial Sodium Channel (ENaC) in a high-throughput format in the same epithelial cell lines. Together, these fluorescence-based assays provide a robust and flexible platform for studying small molecule modulators, targeting two epithelial channels in a relevant cellular context.

This protocol describes a method for the study of electrogenic membrane proteins by measuring changes in membrane potential. This assay provides a platform for the functional readout of multiple ion channels endogenously expressed in epithelial cell lines.

基于荧光的膜电位测定，用于两个上皮细胞系中两个内源性离子通道的高通量功能研究

Fluorescence-based studies are suitable for high-throughput plate reader assays of cells in culture. They have been commonly employed for drug discovery ...