这项研究得到了标准化委员会 (临床试验网络、欧洲囊性纤维化社会) 和国家资源中心工作组 (治疗发展网络、囊性纤维化) CFTR 功能工作组的支持。基础)。CF 基金会 (FY09 对 GMS) 和 NIH (DK072482 SMR 和 GMS) 提供了额外支助。

涉及人类主体的议定书得到所有参与机构研究委员会的批准。实施该技术的每个中心负责向其机构人类研究伦理委员会申请批准。
1. 解决方案准备
<ol><li>
准备解决方案 #1, #2 和 #3, 这是基础解决方案, 在 1 L 批次之前的过程和存储在现场 (表 1)。 注: 阿米洛利是轻敏感的, 必须存储在黑暗中 (见表 1为解决方案组成) (见 SOP 为详细的解决方案准备11)。<ol>
<li>在 pH 值为7.4 的情况下缓冲所有解决方案, 并使用0.22 µm 瓶顶过滤器进行过滤。</li>
		<li>对于溶液 #3, 首先加入含磷酸盐的盐, 允许它们电离以防止结晶 (见表 2为溶液成分)。 注: 混合序列对解决方案 #3 至关重要。</li>
		<li>存储这些解决方案在4°c (稳定3月) 或-20 摄氏度 (稳定6月)。</li>
		<li>通过在 NPD 测试当天添加代理来准备解决方案 #4 和 #5。异丙肾上腺素是轻和氧化敏感性和它丢失它的活动在室温 (证明4% 朽烂 4 h 在4–8°c)。存储在4摄氏度。 注: ATP 是光和氧化敏感 (见表 3)。</li>
	</ol>
</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>复合</td>
			<td>分子量</td>
			<td>浓度 (毫米)</td>
			<td>组成 (g/升)</td>
		</tr>
<tr>
<td>Nacl</td>
			<td>58</td>
			<td>148</td>
			<td>8.58</td>
		</tr>
<tr>
<td>CaCl2 2H2O</td>
			<td>147</td>
			<td>2.25</td>
			<td>0.33</td>
		</tr>
<tr>
<td>氯化钾</td>
			<td>75</td>
			<td>4.05</td>
			<td>0。3</td>
		</tr>
<tr>
<td>K2HPO4
</td>
			<td>174</td>
			<td>2。4</td>
			<td>0.42</td>
		</tr>
<tr>
<td>2 PO4
</td>
			<td>136</td>
			<td>0。4</td>
			<td>0.05</td>
		</tr>
<tr>
<td>氯化镁2 6H2O</td>
			<td>203</td>
			<td>1。2</td>
			<td>0.24</td>
		</tr>
</tbody></table>表 1: 解决方案组成。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>复合</td>
			<td>分子量</td>
			<td>浓度 (毫米)</td>
			<td>组成 (g/升)</td>
		</tr>
<tr>
<td>葡萄糖酸钠</td>
			<td>218</td>
			<td>148</td>
			<td>33.26</td>
		</tr>
<tr>
<td>葡萄糖酸钙</td>
			<td>430</td>
			<td>2.25</td>
			<td>0.97</td>
		</tr>
<tr>
<td>葡萄糖酸钾</td>
			<td>234</td>
			<td>4.05</td>
			<td>0.95</td>
		</tr>
<tr>
<td>K2 HPO4
</td>
			<td>174</td>
			<td>2。4</td>
			<td>0.42</td>
		</tr>
<tr>
<td>2 PO4
</td>
			<td>136</td>
			<td>0。4</td>
			<td>0.05</td>
		</tr>
<tr>
<td>MgSO4 7H2O</td>
			<td>246</td>
			<td>1。2</td>
			<td>0.24</td>
		</tr>
</tbody></table>表 2: 解决方案组成。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>解决 方案</td>
			<td>解决方案编号</td>
			<td>内容</td>
			<td>标记</td>
		</tr>
<tr>
<td>枪手注射液</td>
			<td>解决方案 #1/A</td>
			<td>注射用缓冲枪手</td>
			<td>枪手</td>
		</tr>
<tr>
<td>枪手 + 阿米洛利</td>
			<td>解决方案 #2/B</td>
			<td>缓冲枪手 + 100 微米阿米洛利</td>
			<td>AMIL</td>
		</tr>
<tr>
<td>零 Cl + 阿米洛利</td>
			<td>解决方案 #3/C</td>
			<td>缓冲零氯 + 100 微米阿米洛利</td>
			<td>Ocl</td>
		</tr>
<tr>
<td>零氯阿米洛利 + 异丙肾上腺素</td>
			<td>解决方案 #4/D</td>
			<td>缓冲零氯 + 100 微米阿米洛利 + 10 微米异丙肾上腺素</td>
			<td>Iso</td>
		</tr>
<tr>
<td>零氯阿米洛利 + 异丙肾上腺素 + ATP</td>
			<td>解决方案 #5/E</td>
			<td>缓冲零氯 + 100 微米阿米洛利 + 10 微米异丙肾上腺素 + 100 微米 ATP</td>
			<td>Atp</td>
		</tr>
</tbody></table>表 3: 解决方案列表。
2. 导管
<ol><li>
使用 PVC, 无菌, 单一用途, 2 流明 (直径0.7 毫米内径) 导管与圆形和平滑的四肢 (直径2.5 毫米), 这是专门为新产品开发。
	<ol>
<li>通过一个侧面孔, 与粘膜接触, 2 毫米远的尖端与尖孔在灌注 (见步骤 10.1)。</li>
		<li>将导管的两个鲁尔锁连接其中一个连接到测量电极, 另一条与灌注泵相连。使用蓝色染色的通道作为测量腔。在每0.5 厘米间隔10厘米处标记导管。 注: 死亡空间为0.3 毫升。如果在接下来的两个步骤中无法做到这一点, 最好使用上述程序来制备导管。</li>
		<li>PE50 和 PE90 导管管的长度相等 (~ 76 厘米)。</li>
		<li>把这些贴在一块1厘米的硅橡胶油管上。将25克钝尖针 snuggly 插入 PE-90 油管的另一端。插入一个25克的蝴蝶针 snuggly 到对面的 PE50 油管, 确保不刺穿油管, 因为它被放置。</li>
	</ol>
</li>
</ol><img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57006/57006fig4.jpg"> 图 4: 用于 NPD 测量的导管.插入框说明导管尖端与测量孔。
3. 琼脂皮桥 (蝶形针) 和导管的制备
注: 对熔化的琼脂的操作可能导致烧伤, 并且这应该谨慎地做。
<ol><li>
将3克琼脂与100毫升溶液 #1 在宽口瓶中混合, 制备3% 琼脂。将琼脂在微波炉中熔化直至可溶性 (透明)。
	<ol>
<li>用温琼脂填充10毫升注射器。</li>
		<li>随后将注射器连接到蝶形针 (23 克) 和导管的显腔。注射琼脂直到它出现在尖端。</li>
		<li>让它冷却至少10分钟。</li>
		<li>确保皮肤桥和导管充分填充, 并可视化无气泡。</li>
		<li>将散装皮桥存放在溶液中 #1 4 摄氏度, 1 周后不要使用。</li>
	</ol>
</li>
</ol>4. 如果使用心电霜
<ol><li>
用溶液 #1 (1:1, v/v 铃声) 稀释心电霜。让它休息, 直到没有气泡。
	<ol>
<li>用稀释的心电图膏填充10毫升注射器。</li>
		<li>将注射器连接到导管的标记腔内, 慢慢注入心电图膏, 直到出现在下侧孔。</li>
		<li>确保导管完全填充并无气泡。</li>
	</ol>
</li>
</ol>5. 数据采集系统
注: 数据采集系统的一般设置如图 5所示。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57006/57006fig5.jpg"> 图 5: 建立数据采集系统.展示 bioamplifier 和 headstage 的连接到计算机接口并且电极连接到 headstage11。<a href="https://www.jove.com/files/ftp_upload/57006/57006fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol><li>
连接
	<ol>
<li>用 USB 电缆将计算机连接到数据采集系统 (材料表)。</li>
		<li>要将数据采集系统连接到 bioamplifier, 请将 BNC 电缆从数据采集系统 (前端) 上的通道1输入连接到 bioamplifier (后端) 上的输出。</li>
		<li>将 bioamplifier 连接到 headstage, 并将自定义电缆预连接到 headstage 螺钉插入 bioamplifier 前面的输入部分。</li>
		<li>将 headstage 连接到电极和接地电极上。使用标准的 2 mm 女-女性连接器连接 headstage 的前端与电极。 注: 端口为凹槽, 仅适合2毫米电缆的一个方向: 红色测量电极至患者鼻 (经鼻导管);黑参考电极对患者皮肤桥梁;白色至心电电极接地于主体的皮肤。</li>
		<li>设置 bioamplifier 如所述: 偏移: 拉动, 转向调节, 在调整后留在拉位置;电压: 直流;1 mV 卡尔: 中性位置;电源: 在收集数据, 关闭充电电池;增益: 设置为 10;带通: LoFreq (外旋钮): DC;带通: HighFreq (内旋钮): 1kHz;音量: 关闭。</li>
	</ol>
</li>
</ol>6. 调整头级偏移量
<ol><li>
按照 SOP11中所示的顺序将笔记本电脑和放大器连接起来。在数据采集系统上进行切换, 然后使用笔记本电脑 (该序列对于软件识别用于数据采集的设备来说是很重要的)。
	<ol>
<li>根据 SOP 顺序调整头级偏移量。</li>
	</ol>
</li>
</ol>7. 抵消
注: 为了保证电气测量系统的稳定性, 需要测试多个偏移量。(见图 6)
<ol><li>
对于电极偏移, 将参考 (负极) 电极和测量 (正极) 电极一起放置在稀释的心电霜或3米氯化钾中. 确保电极之间的电位差读数接近零 headstage。
	<ol>
<li>为设置导管和/或皮桥偏移, 将鼻腔导管的鲁尔锁端或皮桥的鲁尔锁端置于与测量 (正) 电极的浴缸中。将导管的另一端置于心电霜浴或3米氯化钾中, 其中含有参考 (负极) 电极, 确保 headstage 上的电位差接近零。</li>
		<li>要设置闭环偏移量, 请确保在电极的浴缸中更换鼻腔导管时关闭电路。检查闭环偏移读数接近 0 mv (= ' 偏移量 '; ±2.5 mv)。调整 headstage 偏移旋钮, 将偏移量调至 0 mV。 注意: 这确认电路内的所有连接都完好无损。如果情况并非如此, 鼻腔导管可能不完整 (琼脂中的气泡或心电霜)。改变琼脂桥或推心电图霜在设置。电极偏移必须首先执行, 其次是闭合系统 (闭合回路偏移) 与电极和桥梁 (图 6)。</li>
	</ol>
</li>
</ol><img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57006/57006fig6.jpg"> 图 6:设置 (A) 电极偏移, (B) 导管 (或桥) 偏移, (C) 闭环偏移。
8. 注射器安装
注意: 建议的设置如下。
<ol><li>
解冻解答 #1, #2 和 #3 大约1小时在测量之前。
	<ol>
<li>将延伸线连接到离导管最近的旋塞阀。</li>
		<li>打开所有泵, 用溶液冲洗导管 #1 彻底冲洗导管, 直到 stopcocks 没有气泡。</li>
	</ol>
</li>
</ol>9. 参考和测量电极的放置
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57006/57006fig7.jpg"> 图 7: 用测量电极和皮下桥准备测量的主题。
<ol><li>
让学习主体坐在面向 NPD 操作员的位置上。为了更舒适, 请把脚放在可选的防静电垫上, 头放在直视的下巴上休息。
	<ol>
<li>将接地引线连接到放置在主体臂上的心电垫 (图 7)。</li>
		<li>将皮下针插入前臂背部 (琼脂系统), 或将参考电极应用于前臂先前微擦伤部位的心电霜上 (见下文7至10点)。</li>
		<li>检查与皮下空间的连接, 通过测量与皮肤的潜在差异 (指 PD), 并要求主题关闭 "测量孔" 通过捏在他/她的拇指和食指尖端之间的导管。</li>
		<li>如果手指 PD 不是-30 mV 或更多阴性, 检查插入蝶针。重复磨损 (为心电霜设置) 和检查桥梁。</li>
		<li>启动溶液 #1 注射器泵在80毫升/小时. 从右鼻孔开始。</li>
		<li>测量手指 PD 作为一个稳定的负电压 (典型范围-40 到-80 mV)。</li>
		<li>如果使用心电霜系统: 稀释心电霜 1:1, 并填充导管后, 完全冲洗出探头孔, 如前所见的琼脂。</li>
		<li>连接导管到一个50毫升注射器半填充与心电图膏, 以沐浴电极, 允许检查电极偏移和桥关闭设置。</li>
		<li>将参考 Ag/Cl 电极连接到皮下空间, 经过轻微的皮肤磨损, 当达到真皮水平时, 皮肤会出现 ' 粉红色和发亮 '。</li>
		<li>将测量电极, 用心电霜覆盖在擦伤的皮肤上。检查手指 PD, 如前所示的琼脂系统。</li>
	</ol>
</li>
</ol>10. 基础 PD 的测量
<ol><li>
将鼻腔导管插入右鼻孔, 使用照明鼻内窥镜 (或等同) 可视化下鼻甲。以前尖端为标志, 推进导管靶向下鼻甲下壁部位的呼吸粘膜。或者, 如果放置困难, 探头孔可以放置在与鼻孔的地板接触。 注: 导管刚性足够, 由操作者引导进入鼻孔。为便于放置, 导管的一个通道呈蓝色, 在与下鼻甲接触时含有探针侧孔。这可防止导管旋转。在导管上标明的标记从1到10厘米提供容易的参考点。<ol>
<li>测量下鼻甲的 PD。为此, 应确保导管的测量孔因其放置于下鼻甲粘膜 (标记右基底) 而关闭。</li>
		<li>测量 PD 在 3.0, 2.0, 1.5, 1.0 和 0.5 cm (距离在下鼻道从下鼻甲): 标记正确的基底 PDs。</li>
		<li>保持每一个测量在指定的距离约5秒, 以确保一个稳定的读数 (1 mV), 并有助于准确解释基本 PD 值。</li>
		<li>在左鼻孔重复上述步骤, 使用功能键标记左基底 PDs (3 厘米, 2 厘米等) 和左基部。</li>
		<li>以基底局部放电测量为指导, 将鼻腔导管探头插入到最负信号的部位 (从下鼻甲前尖端到3厘米), 并在鼻尖 (或等效) 上用一小块胶带固定。</li>
	</ol>
</li>
</ol>11. 新产品跟踪序列显影
<ol><li>
右鼻孔
	<ol>
<li>验证解决方案是否从病人的鼻子滴水。让主题假设一个舒适的位置, 他们的头向下 (经常帮助的主题休息他们的头上他们的手或使用 chinrest 或其他固定装置)。提醒主体尽量减少运动, 避免接触鼻子或油管, 避免说话。</li>
		<li>转动溶液 #1 (枪手) 泵 (5 毫升/分, 或300毫升/小时)。记录直到获得稳定的值 (< 1 mV change/30 s)。 注意: 这需要大约3分钟来实现稳定性。</li>
		<li>用溶液 #1 关闭灌注。</li>
		<li>用溶液 #2 (阿米洛利) 开始灌注。记录 NPD 至少3分钟 (如果高原电压有疑问, 请继续录制多达5分钟)。</li>
		<li>用溶液 #3 (零氯) 开始灌注。记录 NPD 至少3分钟 (如果高原电压不稳定, 则继续录制最多5分钟)。</li>
		<li>用溶液 #4 (异丙肾上腺素) 开始灌注。记录 NPD 至少3分钟 [如果高原电压不稳定 (至少在三十年代 < 1 mV 漂移的稳态电压跟踪), 继续录音最多5分钟)。</li>
		<li>用溶液 #5 (ATP) 开始灌注。记录 NPD 至少1分钟, 直到获得峰值极化响应。</li>
		<li>用溶液 #1 (枪手) 打开灌注, 并允许三十年代冲洗导管。</li>
		<li>关闭 #1 灌注的溶液。</li>
		<li>对左鼻孔重复操作。</li>
	</ol>
</li>
</ol>12. 测试结束
<ol><li>
复查并记录稳定的手指 PD ("后指") 5 秒。
	<ol>
<li>从皮肤上的插入部位取出主体的皮桥和绷带。对于 AgCl/心电霜系统, 从手臂上取出电极。</li>
		<li>记录用于测量初始闭环偏移量的 "最终闭环偏移" 电压 (参见步骤 7.1.3)。</li>
		<li>用函数键标记最终偏移量。</li>
		<li>停止数据采集 (按 "开始")。 注: 目前的 SOP 建议使用100µM ATP 激活嘌呤钙依赖性氯分泌物, 作为一个积极的控制测试;但是, 这是一个可选测试。</li>
	</ol>
</li>
</ol>

<ol>
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作者声明他们没有竞争的财政利益。

在体内, NPD 提供了一个独特的测量, 可重复执行的纵向基础上, 并表明, 通过反复测量, 类似的纵向结果, 观察到在小组和个人基础上14, 15。有确凿的证据表明, NPD 具有很好的判别有效性, 区分 cf 从非 cf。25项研究一致表明, CF 与健康对照组的氯-钠+电导率有统计学意义的差异10。虽然以前制定的一些指数证明了这一能力, 但鉴于最近标准化方法7、8, 我们预计新的更新是必要的。
修改和疑难解答
此测试需要几个关键步骤, 以确保准确的测量。这包括电极和导管闭环偏移, 以确保系统执行推荐标准。病人必须保持静止, 避免说话, 因为这减少了工件和导管变位。这使得非合作性患者的测试难度很大, 而这种技术仅在6岁以下儿童的一项研究中被报道过7岁。
对鼻腔上皮进行预先检查是必要的, 以确保上皮没有结壳或粘液, 这可能影响测量。
非常重要的是, 必须指出, 放置导管的位置是辩论的主题。这里提出的 SOP 利用下鼻甲 (它) 下的测量。在多中心试验中, 导管的放置是标准化和进行的, 因此, 这是推荐的技术。测量下, 它是执行与侧孔导管, 这可能是很难保持与鼻腔粘膜的牢固接触, 而与解决方案接触。其他组可以测量鼻腔底部的 PD, 这在技术上比较容易。重要的是, 韦尔默朗 (2011) 证明2方法是可比较的16。
解决方案变暖仍然是欧洲和美国--中心17,18之间的争论问题。有人主张, 使用溶液在37°c 而不是22°c 增加观察到的总氯化物反应约25% 和异丙肾上腺素依赖氯化物反应约 95%18。然而, 变暖增加了可变性, 这是由总氯化物反应17的更大标准偏差评估的。因此, 随着升温的解决方案是一个额外的因素的可变性, 建议不要温暖的解决方案, 除非需要在研究的基础上。
我们以前比较了这两种电极技术, 发现 AgCl 和甘汞电极系统在正常受试者的基础和受激电流上同样操作13。
技术的局限性
此测试受主体内变异的影响。评分的变异性在不确定追查患者中尤为普遍, 应在诊断应用19中加以说明。变异因素包括急性上呼吸道感染、广泛的鼻息肉、既往鼻窦手术和 CF 相关炎症, 降低其特异性和敏感性20,10。此外, 读者对追查的解释可能有所不同, 尽管专家读者在 cf 和非 cf 追查中表现出了优异的定量评分和解读的一致性, 但与追踪19的信心。
内在变异性与显著阈值
非常重要的是, 测量的生理变异性是相当可观的, 如不同的研究10所示, 如 CFTR 基因治疗试验, 表明氯总运输的变化有相当大的变异性和阿米洛利范围21,22。横断面评价表明, 在-5 至 -7 mV 阈值以上的零 Cl 加异丙肾上腺素反应是 cf 和非 cf 科目10之间的切断。
然而, 我们对这一参数的变化程度缺乏明确的认识, 这代表了在疾病改良疗法的二期试验中有效的 CFTR 矫正。为了评估个体的反应, 可能需要反复的测试来监测对干预的反应, 以区分重要的变化和内在的变异性。非常重要的是, 未来长期的疾病改良药物研究需要证明 CFTR 功能的改善与临床相关结局或替代结果 (如 FEV1的改善) 有关。疾病。事实上, 最近的第二阶段 Ivacaftor 研究表明, 尽管氯分泌物的小改善23, 但临床上的益处显著。
这类研究将有助于确定在反式上皮内电导率的改善是否可以作为临床疗效的替代参数。这将是指导 CFTR 改良疗法发展的一个重要参数。
对现有方法的意义: 汗液测试和肠道电流测量 (ICM)
在有 "可疑" 囊性纤维化的患者中, 由30至 60 毫米的中间汗氯浓度评定, NPD 综合评分提供了一种高度敏感的工具来诊断患者为 "cf" 和 "cf-不太可能"10.肠道电流测量 (ICM) 提供了一个在直肠上皮内的净氯通量的前体测量, 也允许测定残留 CFTR 功能高灵敏度, 因为 CFTR 是高度表达在这个上皮细胞。
考虑到 CFTR 调制器对 CFTR 功能的修正, 这些不同 CFTR 生物标志物变化之间的关系目前尚不清楚。虽然最近基于 Ivacaftor 的工作确定了 NPD 和汗水测试是相关的4, 但它尚未建立, 如果测量在呼吸道是一个更好的预测呼吸结果比, 例如, 汗水测试24,25或在 ICM 中更改。此外, 修饰药物也可能在其器官特异性疗效上有所不同。对于 NPD, 重要的是要注意到, 基础 PD 和阿米洛利反应的变化表达 Na+运输, 而改变0氯和异丙肾上腺素反应表达氯转运 。还有待确定哪些对改善疾病更重要。
该技术的未来应用
这项技术的使用预计在 CF 领域之外。由于该技术特别适合于显示 Na+和 Cl 离子通道, 它可用于显示呼吸道疾病的功能障碍, 包括哮喘26, 慢性支气管炎27, 非 CF 支气管扩张症28复发性胰腺炎29。此外, 在下呼吸道 (洛城警署) 对该技术的修改已被用于证明慢性阻塞性肺病 (COPD) 患者慢性支气管炎30的低气道聚焦 CFTR 功能障碍。
NPD 提供了一个敏感的体内生物标志物的 CFTR 功能, 这既可用于诊断, 也可以用于概念验证研究, 旨在纠正 CFTR 和 ENaC 通道活动的翻译研究。这允许对跨上皮功能进行纵向评估, 并将承诺作为个性化医学的策略, 为每个患者量身定做最有效的校正器。

该方法的总体目标是测量鼻电位差 (NPD), 目的是研究1体内的跨上皮离子转运。这种技术允许测量钠 (钠+) 和氯化物 (Cl) 运输。NPD 已被用作研究工具, 自二十世纪八十年代代末, 并被接受1998年作为诊断程序的囊性纤维化基金会 (CFF) 共识声明2和2017年在囊性纤维化基金会 (CFF) 共识诊断指南3. 事实上, 生物 CFTR 功能障碍, 这是 CF 的原因, 是证明了钠+吸收在顶端膜和氯分泌的缺陷.这种功能测试提供了一个额外的诊断工具的优势, 当遗传学是非决定性的患者的不确定的中间汗水测试结果3。虽然这些信息也可以通过肠道电流测量活检 (icm) 获得, 但是, icm 仅在全球少数几个中心提供, 需要进一步标准化。NPD 在大约60个全球性中心更是可利用的, 并且, 目标呼吸道上皮是疾病的主要位置。
考虑到它提供的关于 CFTR 活动的信息, 它也用于概念验证研究, 目的是通过调制疗法4、5、6、7来评估 CFTR 蛋白的功能恢复, 8。事实上, 来自研究与 CFTR mRNA/基因编辑, CFTR 增强剂和校正疗法的数据, 突出了 Cl 和 Na +运输与治疗6,9的显著变化, 并确认 NPD 可以是一个临床试验中的应答终点。由于我们缺乏敏感的临床端点, 能够在短期内检测到患者临床状况的细微变化, 所以这个临床前生物标志物可能具有很高的信息量。CFTR 调制疗法的领域迅速扩大, 我们迫切需要在体内的测试, 能够快速破译活性化合物, 然后再进行大3期试验10。
该技术的生理原理是以测量鼻孔和皮下腔内气道上皮的电位差为基础的。通过测量稳定最大基线电位差 (PD), 通过不同顶端氯转运体阻断 ENaC 相关 Na+吸收和驱动 Cl 分泌物, 探讨离子通道活动.包括 CFTR。CFTR 功能障碍显示的潜在差异的最小变化后, 通过一个阵营依赖途径刺激 Cl 分泌和增加的 ENaC 介导的钠+吸收被发现的更负基线电位差以及对阿米洛利的增强反应。图 1总结了 CF 与正常 PD 的机械基础。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57006/57006fig1.jpg"> 图 1: 离子通道活动的摘要图.(A) 呼吸道上皮中的离子活性表明正常受试者的 ENaC 和 CFTR 平衡活动, (B) CFTR 活动的丧失导致 ENaC 介导的钠转运量增加, CFTR 依赖氯化物减少运输。ENaC: 上皮钠通道, Na+: 钠, CFTR: 囊性纤维化跨膜调节剂, 氯-: 氯化, mV: 毫伏, PD: 电位差, 最小: 分钟/秒<a href="https://www.jove.com/files/ftp_upload/57006/57006fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
然而, 这项试验表明, 在同一病人的重复测量和同一基因型患者中有一定程度的变异性。这是非常重要的, 以促进解释的变化后, 调制治疗。此外, 我们仍然缺乏验证的阈值辨别 CF 和健康的对象。这可能部分是由于临床设施的可用性和所使用的技术之间的差异。因此, 正在进行一项旨在标准化测试的重大国际努力。美国 CFF-TDN (囊性纤维化基金会-治疗发展网络) 和从此之后 (欧洲囊性纤维化社会-临床试验网络) 创建了一个 NPD 标准操作程序 (SOP) 用于多中心和研究试验。最近由各箱和 TDN 合作开展的工作, 促成了一套联合的国际 SOP, 汇集了各箱和 TDN (2014)11的专门知识。本文介绍了使用 NPD 进行 CF 诊断或调查人员发起的概念验证试验的协议和测试技术。实施该技术的每个中心负责向其机构人类研究伦理委员会申请批准。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57006/57006fig2.jpg"> 图 2: 整个推荐的 NPD 安装示意图.请注意, 建议的安装程序显示, 包括顺序灌注泵和4停止公鸡系列设置。具体的连接和组件的例子显示在 SOP。(图由所罗门的许可修改, 通用胸, 201013)<a href="https://www.jove.com/files/ftp_upload/57006/57006fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
在图 2中概述了一般的实验流程, 即在位于上皮表面的探索桥与在皮下空间中放置的参考桥之间测量 NPD, 两者都连接到电极和高阻抗伏特计。
这是由2不同的系统确保: 有2可接受的参考电极设置: (i) 平衡 Ag/AgCl 电极和心电图 (心电) 奶油填充桥连接到皮下空间由轻微磨损或 (ii) 饱和甘汞半细胞和琼脂填充22到24口径针被引入皮下。鼻粘膜的接触是由双腔导管引起的。一个腔内充满琼脂或心电霜, 并连接到测量电极, 另一个允许灌注到鼻腔粘膜的不同的解决方案。
探查导管的尖端放置在下鼻甲下的呼吸粘膜上 (图 3)。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57006/57006fig3.jpg"> 图 3: 探索导管在呼吸道粘膜上的放置.(A) 显示位置的外部视图。(B) Rhinoscopic 的观点展示位置。(C) 显示导管放置的解剖位置的图示。潜在差异
为了研究 PD 对几种药物的反应, 通过导管的第二腔 superfusion 溶液。从最初的准备到数据分析, 在编写和执行 NPD 测量方面有几个关键步骤。
在制备溶液和电极后, 对电极和导管进行足够的质量测试, 使测试的基本进行。基础测量是沿下鼻甲进行的, 它允许选择最佳的测量地方, 通常是以最负的测量。然后顺序显影通过鼻上皮的电压变化来确定 Na+ (ENaC) 和 Cl (CFTR 依赖) 离子通量。

<table>
	<tbody>
		<tr>
			<td>KD Scientific infusion pump (or equivalent &#8211; such as programmable infusion pumps provided by the institution/hospital)</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Powerlab 4/30</td>
			<td>AD Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BMA-200 AC/DC portable bioamplifier</td>
			<td>AD Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IS0-Z isolation headstage for BMA-200</td>
			<td>AD Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Windows compatible PC - Minimum requirements of Windows XP or higher</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AD Instruments software: GLP Client V6 (Windows) or higher</td>
			<td>AD Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ECG electrode (ground for study subject)</td>
			<td>Hospital standard</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2 mini calomel reference electrodes</td>
			<td>Fisher Scientific</td>
			<td>13-620-79</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride KCl, Granular &#8211; USP, formula weight 76, qty: 500 gm</td>
			<td>Spectrum</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile container (such as specimen collection container , or similar) to be used for KCl calomel bath, with holes cut in lid to hold electrodes in place. (If not provided by electrode manufacturer.)</td>
			<td>Hospital standard</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2 electrodes: Ag/AgCl 8 mm TP electrode</td>
			<td>BIOPAC Systems</td>
			<td>UNSHLD-EL258</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Ag/AgCl electrodes, B0194, plug 4 mm</td>
			<td>SLE Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Signacreme&#174; Conductive Electrode Cream</td>
			<td>Fisher Scientific</td>
			<td>Parker Labs ref # 17-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Skin abrasion device</td>
			<td>PROMED Feeling</td>
			<td>Ref 374901</td>
			<td></td>
		</tr>
		<tr>
			<td>Hi Di 541 M, Diamond tipped dental burrs</td>
			<td>Ash Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Becton Dickinson PE 50 tubing</td>
			<td>Fisher Scientific</td>
			<td>427411</td>
			<td></td>
		</tr>
		<tr>
			<td>Becton Dickinson PE 90 tubing</td>
			<td>Fisher Scientific</td>
			<td>427421</td>
			<td></td>
		</tr>
		<tr>
			<td>Silastic tubing, 0.062&#8221; ID, 0.095&#8221; OD</td>
			<td>Fisher Scientific</td>
			<td>508-007</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropore Surgical Tape Paper (25 mm x 9.1 m)</td>
			<td>3M</td>
			<td>1530-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Marquat double lumen catheter Length: 80 cm; Outer diameter: 2.5 mm; Internal diameter of the channels: 0.8 mm; Distance of the side-holes to the tip: 2 mm. EU label Agreement for NPD: I0202US</td>
			<td>Marquat</td>
			<td>I0202US</td>
			<td></td>
		</tr>
		<tr>
			<td>1&#34; X 10 yards silk tape</td>
			<td>3M Durapore</td>
			<td>1538-1</td>
			<td></td>
		</tr>
		<tr>
			<td>IV extension tubing (30&#34;, 50/box)</td>
			<td>International Limited</td>
			<td>IMN30</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-way stopcock (50/box)</td>
			<td>Medex</td>
			<td>MX5311L</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringe filters (ANOTOP 25 sterile 50pk; 0.22-micron or smaller filters; or equivalent)</td>
			<td>Fisher Scientific</td>
			<td>09-926-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Becton Dickinson Intramedic Luer stub adapter (20G, for connection to PE90 if using nasal catheter produced at study site)</td>
			<td>Fisher Scientific</td>
			<td>427564</td>
			<td></td>
		</tr>
		<tr>
			<td>Becton Dickinson 23G, 0.75&#8221; Vacutainer (&#8220;butterfly&#8221;) needles (0.6 x 19 mm; 50U/box) (for connection to PE50) if using nasal catheter produced at study site)</td>
			<td>Fisher Scientific</td>
			<td>367283</td>
			<td></td>
		</tr>
		<tr>
			<td>Becton Dickinson Syringe 60 ml without needle Luer-Lok tip (40/Box)</td>
			<td>Fisher Scientific</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>Becton Dickinson Syringe 10 ml without needle Luer-Lok tip (100/Box</td>
			<td>Fisher Scientific</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>Single use sterile wipes (per institutional availability)</td>
			<td>Hospital standard</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% EtOH (1 pint), Aaper Alcohol and Chemical Co. catalog number NC9274019 (or equivalent)</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning single use sterile bottle-top filters, 0.22 &#956;m pore size (0.15 &#8211; 1.0 litre volumes acceptable)</td>
			<td>Fisher Scientific</td>
			<td>430624</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer Cert Ph 10.00 (1L Sn04332) &#8211; for pH meter calibration</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer Cert Ph 4.00 (1L Sn04327) &#8211; for pH meter calibration</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer Cert Ph 7.00 (500 ml Sn04328) &#8211; for pH meter calibration</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable underpads (Blue Pads; 23&#34;X36&#34; 150/Box; or equivalent per hospital standard)</td>
			<td>SureCare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>23G, 0.75&#8221; Vacutainer &#8220;butterfly&#8221; needles (0.6x19 mm; 50U/box)</td>
			<td>Becton Dickinson</td>
			<td>367283</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Laboratories Agar (Noble 100g 0142-15-2; or equivalent)</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Welch Allyn Rhinoscope 71000-C (or equivalent)</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Welch Allyn Convertible Handle Battery 72300 (or equivalent) OR Otoscope with battery</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Head and chin rest (or equivalent; optional)</td>
			<td>Richmond Products, Inc</td>
			<td>629R</td>
			<td></td>
		</tr>
		<tr>
			<td>Static Dissipative Anti-Fatigue Matting&#160; (or equivalent)</td>
			<td>Fisher Scientific</td>
			<td>No. 791</td>
			<td></td>
		</tr>
		<tr>
			<td>REAGENTS FOR SOLUTIONS MIXED ON SITE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride, Granular &#8211; USP NaCl</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 58; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Calcium Chloride CaCl2&#8226;2H2O &#8211; USP</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 147; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Magnesium Chloride Hexahydrate Crystal, MgCl2&#8226;6H2O &#8211; USP</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 203; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic, Anhydrous, Granular, K2HPO4 &#8211; USP</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 174; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic Crystals &#8211; NF (KH2PO4)</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 136; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Sodium Gluconate- USP (monosodium salt)</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 218; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Calcium Gluconate &#8211; USP (Anhydrous Powder)</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 430; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Potassium Gluconate- USP (Anhydrous)</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 234; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Heptahydrate &#8211; USP MgSO4&#8226;7H2O</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 246; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Amiloride HCl &#8211; USP</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 302; Size: 5gm</td>
		</tr>
		<tr>
			<td>Adenosine 5&#8217;-Triphosphate (ATP) (Disodium salt)</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 551; Size: 5gm</td>
		</tr>
		<tr>
			<td>Magnesium Chloride, Hexahydrate, Crystal &#8211; USP MgCl2&#8226;6H2O</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 203; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Double-distilled water (ddH2O)</td>
			<td>Hospital Pharmacy</td>
			<td></td>
			<td>Formula Weight: NA; Size: 1 L</td>
		</tr>
		<tr>
			<td>Isoproterenol HCL Injection - USP 1 mg/5 ml ampule</td>
			<td>Hospital Pharmacy</td>
			<td></td>
			<td>Formula Weight: 248; Size: single use</td>
		</tr>
		<tr>
			<td>Ringers Injection, USP or Ringers Irrigation</td>
			<td>Hospital Pharmacy</td>
			<td></td>
			<td>Formula Weight: NA; Size: 5 L</td>
		</tr>
	</tbody>
</table>

standardized measurement of nasal membrane transepithelial potential difference (npd)

我们描述一个标准的测量鼻电位差 (NPD)。在这项技术中, 囊性纤维化跨膜电导调节剂 (CFTR) 和上皮钠通道 (ENaC) 功能是监测的变化, 在 superfusion 的解决方案, 改变离子通道的鼻腔上皮细胞活动。这是由测量的潜在差异的皮下室和呼吸道上皮的鼻孔, 利用一个导管与下鼻甲。
该测试允许测量的稳定基线电压和后续的净电压变化后, 100 µM 阿米洛利, 一抑制剂的钠+吸收在响铃的解决方案;一种无氯溶液, 含有阿米洛利, 以促进氯分泌和10µM 异丙肾上腺素在无氯溶液中与阿米洛利, 以刺激与 CFTR 有关的循环腺苷 (阵营) 依赖性氯电导。
该技术具有显示两个关键成分的电生理特性, 建立呼吸上皮、ENaC 和 CFTR 气道表面液体的水化。因此, 它是一个有用的研究工具的2阶段, 并证明了药物的概念试验的目标 CFTR 和 ENaC 活动的治疗囊性纤维化 (CF) 肺部疾病。这也是建立 CFTR 功能障碍的关键后续程序, 当基因检测和汗水测试是模棱两可的。与汗液不同的是, 测试的耗时和成本较高。它还需要操作员培训和专门知识来有效地进行测试。这项技术中, 特别是在年轻或不合作的科目中, 报告了学科间和内部的变异。为了帮助这一关注, 通过最近验证的算法改进了解释。

在正常气道上皮中, Na+吸收是主要的离子转运活动。这导致间质的气道表面电位差。ENaC 通道阻滞剂阿米洛利的灌注导致了较低的负电位差。然后, superfusion-自由溶液产生了一个化学梯度的 cl-, 这产生了一个更负的潜在差异, 并激活所有的氯转运体, 包括 CFTR。异丙肾上腺素, 增加细胞内的阵营, 进一步增加氯分泌通过具体激活 CFTR, 并增加潜在的差异。
相比之下, 在 CF 科目, 缺席或功能失调的 CFTR 导致增加 ENaC 介导的 Na+吸收12。因此, 基线电位差更负。应用阿米洛利观察到的退极化量较大, 而在 CFTR 依赖通路刺激 Cl 分泌物时, 电位差有极小或无变化。这可以在图 8的代表追查中看到, 显示 "健康" vs "CF" 追查。
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57006/57006fig8.jpg"> 图 8: "健康" 主题的代表追查与 CF 主题.pd: 潜在的差异, ΔAmiloride: 三角洲阿米洛利, 0氯/Iso: 低氯: 在 PD 之间的变化 #2 和解决方案 #4 灌注, S1-S4: 阶段 1-4, 绿线上图 A 和 B 表明 NPD 跟踪和黑色箭头表示潜在差异的差异

Watch this Scientific Journal Video about Standardized Measurement of Nasal Membrane Transepithelial Potential Difference NPD at JoVE.com

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference NPD

在这里, 我们提出了一个标准化的协议, 以测量鼻电位差 (NPD)。囊性纤维化跨膜电导调节剂 (CFTR) 和上皮性钠通道 (ENaC) 的功能是评估的变化, 在 superfusion 的解决方案, 改变离子通道活动后的鼻上皮的电压, 提供了一个结果测量。

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) ...

This method can help answer key questions in the CF and ion transport fields about whether CF patients can mount a meaningful response to activation or restoration of CFTR function.The main advantage of this technique is that it measures the coordinated activity of the CFTR and epithelial sodium channel in the airway epithelium.Although this method can provide insight into cystic fibrosis, it can also be applied to other disease systems, such as genetic disorders of hyperactive epithelial sodium channel function.Demonstrating the procedure will be Thao Nguyen, biologist, and Aur

standardized-measurement-nasal-membrane-transepithelial-potential

Research

JoVE Journal

Medicine

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD)

15.9K 次观看.  University of Alabama at Birmingham. This method can help answer key questions in the CF and ion transport fields about whether CF patients can mount a meaningful response to activation or restoration of CFTR function.The main advantage of this technique is that it measures the coordinated activity of the CFTR and epithelial sodium channel in the airway epithelium.Although this method can provide insight into cystic fibrosis, it can also be applied to other disease systems, such as genetic disorders of hyperactive epithelial sod...

视频: Standardized Measurement of Nasal Membrane Transepithelial Potential Difference NPD

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

鼻腔和支气管液的吸收: 人体呼吸道黏膜的精确取样和样品的实验室处理

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

科研

医学

Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the genotype-directed therapy model currently in use has several limitations. First, rare or understudied mutation groups are excluded from definitive clinical trials. Moreover, as additional modulator drugs enter the market, it will become difficult to optimize the modulator choices for an individual subject. Both of these issues are addressed with the use of patient-derived, individualized preclinical model systems of CFTR function and modulation. Human nasal epithelial cells (HNEs) are an easily accessible source of respiratory tissue for such a model. Herein, we describe the generation of a three-dimensional spheroid model of CFTR function and modulation using primary HNEs. HNEs are isolated from subjects in a minimally invasive fashion, expanded in conditional reprogramming conditions, and seeded into the spheroid culture. Within 2 weeks of seeding, spheroid cultures generate HNE spheroids that can be stimulated with 3',5'-cyclic adenosine monophosphate (cAMP)-generating agonists to activate CFTR function. Spheroid swelling is then quantified as a proxy of CFTR activity. HNE spheroids capitalize on the minimally invasive, yet respiratory origin of nasal cells to generate an accessible, personalized model relevant to an epithelium reflecting disease morbidity and mortality. Compared to the air-liquid interface HNE cultures, spheroids are relatively quick to mature, which reduces the overall contamination rate. In its current form, the model is limited by low throughput, though this is offset by the relative ease of tissue acquisition. HNE spheroids can be used to reliably quantify and characterize CFTR activity at the individual level. An ongoing study to tie this quantification to in vivo drug response will determine if HNE spheroids are a true preclinical predictor of patient response to CFTR modulation.

Here we describe a method to generate three-dimensional spheroid cultures of human nasal epithelial cells. Spheroids are then stimulated to drive Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-dependent ion and fluid secretion, quantifying the change in the spheroid luminal size as a proxy for CFTR function.

人鼻上皮细胞球体的生成与个体化囊性纤维膜电导调节的研究

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the ...

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

流式细胞仪测定缺氧/复氧后心肌细胞线粒体膜电位的实验研究

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

Microelectrode Impalement Method to Record Membrane Potential from a Cannulated Middle Cerebral Artery

Membrane potential (Vm) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ion channels and electrogenic pumps that regulate Vm in disease conditions could potentially alter Vm, vascular tone, and blood flow. Thus, a basic understanding of electrophysiology and the methods necessary to accurately record Vm in healthy and diseased states are essential. This method will allow modulating Vm using different pharmacological agents to restore Vm. Although there are several methods, each with its advantages and disadvantages, this article provides protocols to record Vm from cannulated resistance vessels such as the middle cerebral artery using the microelectrode impalement method. Middle cerebral arteries are allowed to gain myogenic tone in a myograph chamber, and the vessel wall is impaled using high resistance microelectrodes. The Vm signal is collected through an electrometer, digitized, and analyzed. This method provides an accurate reading of the Vm of a vessel wall without damaging the cells and without changing the membrane resistance.

The primary goal of this article is to provide details of how to record membrane potential (Vm) from the middle cerebral artery using the microelectrode impalement method. The cannulated middle cerebral artery is equilibrated to gain myogenic tone, and the vessel wall is impaled using high resistance microelectrodes.

微电极镀膜法记录从可吸入性中脑动脉的膜电位

Membrane potential (Vm) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ...

DIPLOMA Approach for Standardized Pathology Assessment of Distal Pancreatectomy Specimens

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant cancers. A minority (20%) of PDACs are found in the pancreatic body and tail. Accurate pathology assessment of the pancreatic specimen is essential for providing prognostic information and it may guide further treatment strategies. The recent 8th edition of the American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC) staging system for pancreatic tumors has incorporated significant changes to tumor (pT) stage, which is predominantly based on tumor size. This change emphasizes the importance of careful block selection. Owing to the greater prevalence of tumors in the head of the pancreas, efforts are made to standardize the assessment of pancreatoduodenectomy specimens. However, consensus regarding the macroscopic assessment of distal (i.e., left) pancreatectomy specimens is lacking. The DIPLOMA approach includes the standardized measurement of pancreas and other resected organs, inking of relevant surgical margins and anatomical surfaces without removing covering layers of fat, measurement of tumor size (for T-stage), together with assessment of splenic vessel involvement (and other organs if present). All relevant margins are assessed, and relevant blocks are selected to confirm these parameters microscopically. The current protocol describes a standardized approach to the macroscopic assessment of distal pancreatectomy specimens. This approach was developed during several meetings with pathologists and surgeons during the preparation phase for an international multicenter trial (DIPLOMA, ISRCTN44897265), which focuses on radicality of distal pancreatectomy for pancreatic ductal adenocarcinoma. This standardized approach can be instrumental in the design of studies and will uniform reporting on the outcomes of distal pancreatectomy. The described technique is used in the DIPLOMA trial for pancreatic ductal adenocarcinoma but may also be useful for other indications.

The current study highlights a standardized approach to the macroscopic assessment of distal pancreatectomy specimens for pancreatic ductal adenocarcinoma, with special emphasis on the measurement of pancreatic dimensions and those of other organs, inking of margins, measurement of tumor size and proximity to margins, lymph node sampling and block selection.

分离性造形切除术标本标准化病理学评估的DIPLOMA方法

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant cancers. A minority (20%) of PDACs are found in the pancreatic body and tail. ...

Standardized Model of Ventricular Fibrillation and Advanced Cardiac Life Support in Swine

Cardiopulmonary resuscitation after cardiac arrest, independent of its origin, is a regularly encountered medical emergency in hospitals as well as preclinical settings. Prospective randomized trials in human subjects are difficult to design and ethically ambiguous, which results in a lack of evidence-based therapies. The model presented in this report represents one of the most common causes of cardiac arrests, ventricular fibrillation, in a standardized setting in a large animal model. This allows for reproducible observations and various therapeutic interventions under clinically accurate conditions, hence facilitating the generation of better evidence and eventually the potential for improved medical treatment.

Cardiopulmonary resuscitation and defibrillation are the only effective therapeutic options during cardiac arrest caused by ventricular fibrillation. This model presents a standardized regimen to induce, assess, and treat this physiological state in a porcine model, thus providing a clinical approach with various opportunities for data collection and analysis.

猪心室颤动和高级心脏生命支持标准化模型

Cardiopulmonary resuscitation after cardiac arrest, independent of its origin, is a regularly encountered medical emergency in hospitals as well as ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

手术小鼠模型中骨关节炎的标准化形态学评价

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

人鼻上皮类器官的培养和成像

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary dyskinesia. However, the wider application of these techniques is limited by the extreme susceptibility of ciliary function to changes in environmental factors e.g., temperature, humidity, and pH. In the airway of patients with Cystic Fibrosis (CF), mucus accumulation impedes cilia beating. Cilia function has been investigated in primary airway cell models as an indicator of CF Transmembrane conductance Regulator (CFTR) channel activity. However, considerable patient-to-patient variability in cilia beating frequency has been found in response to CFTR-modulating drugs, even for patients with the same CFTR mutations. Furthermore, the impact of dysfunctional CFTR-regulated chloride secretion on ciliary function is poorly understood. There is currently no comprehensive protocol demonstrating sample preparation of in vitro airway models, image acquisition, and analysis of Cilia Beat Frequency (CBF). Standardized culture conditions and image acquisition performed in an environmentally controlled condition would enable consistent, reproducible quantification of CBF between individuals and in response to CFTR-modulating drugs. This protocol describes the quantification of CBF in three different airway epithelial cell model systems: 1) native epithelial sheets, 2) air-liquid interface models imaged on permeable support inserts, and 3) extracellular matrix-embedded three-dimensional organoids. The latter two replicate in vivo lung physiology, with beating cilia and production of mucus. The ciliary function is captured using a high-speed video camera in an environment-controlled chamber. Custom-built scripts are used for the analysis of CBF. Translation of CBF measurements to the clinic is envisioned to be an important clinical tool for predicting response to CFTR-modulating drugs on a per-patient basis.

This protocol describes nasal epithelial cell collection, expansion, and differentiation to organotypic airway epithelial cell models and quantification of cilia beat frequency via live-cell imaging and custom-built scripts.

原代人鼻上皮细胞模型的收集、扩增和分化，用于定量纤毛搏动频率

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary ...

A Standardized Approach to Extra-Oral and Intra-Oral Digital Photography

Contemporary dentistry mandates a more comprehensive and personalized analysis of each patient. Technological advances in digital photography have played vital roles in diagnostic accuracy, treatment planning, execution of therapies, and outcome evaluations, including esthetic enhancement. Digital photography also provides an excellent platform for patient education, communication, and co-management of cases with other healthcare providers. However, intra-oral photography often faces challenges such as inaccessibility of areas to be captured, different moveable vs. fixed tissues involved, contamination with saliva or blood, and differing illumination needs on various locations. Thus, a more standardized and systematic approach is proposed for intra- and extra-oral documentation via digital photography to overcome the existing technical challenges. The current work will outline the appropriate equipment specifications (camera bodies, macro lens, and flashes), positions and postures of the operator and patients, proper techniques of tissue retraction, the use of appropriate intra-oral mirrors, and the essential elements such as aperture settings (F-stop), ISO, shutter speed, and white balance. This article aims to provide all dental professionals with an approachable linear array of guidelines to produce simplified and standardized visual tools for more efficient and effective documentation.

The present protocol describes a method to capture intra- and extra-oral photographs for digital documentation in the clinical practice of dentistry. The protocol highlights all the critical components involved and the commonly utilized settings to capture dental photographs for clinical documentation.

口腔外和口腔内数码摄影的标准化方法

Contemporary dentistry mandates a more comprehensive and personalized analysis of each patient. Technological advances in digital photography have played ...