我们感谢 Paul 奥布莱恩, Marizel 米娜和伊莎贝拉弗里德曼的动物实验, Jacquie 冈萨雷斯和丹妮尔从 NIH/NIAID 帮助与伽玛辐照兔组织之前, 激光捕获显微切割和 Jansy Sarathy 的手稿想法和建议。这项工作得到了比尔和梅琳达盖茨基金会 (OPP1174780) 和 NIH 共享仪器赠款1S10OD018072 的资助。我们感谢 Eliseo a Eugenin 提供了对莱卡 LMD 6500 显微镜的访问, 并分享了专门知识和建议。LMD 6500 的采购和持续支持由国家心理健康补助金研究所、MH096625、国家神经疾病和中风研究所、NS105584、PHRI 资助 (e.a. e) 和葛兰素 (e.a. e) 资助。

所有动物研究都是按照《国家卫生研究院动物护理和使用指南》的规定进行的, 并得到了 NIAID (NIH)、马里兰州的兽医学会和使用委员会的批准。
1. 动物实验和组织收集
《议定书》这一部分描述了生物安全等级 3 (BSL3) 条件下的动物程序和样本收集。在兔子的结核分枝杆菌气溶胶感染程序和药物管理协议的详细协议已经描述了以前的11,12。
<ol><li>使用仅鼻气溶胶系统, 如前所述的11, 感染新西兰白兔 (男性和女性在 4-5 月大) 与m. 结核HN878。</li>
	<li>管理所选择的药物 (乙胺在这里提出的例子) 通过首选的路线和弄死动物在 2, 6 和 24 h 以下管理。首先, 用35毫克/千克和甲苯噻嗪的肌肉注射氯胺酮麻醉兔, 5 毫克/千克。等待十分钟, 并确认正确的麻醉通过捏尾巴和轻轻地触摸眼睛。如果没有反应, 弄死静脉注射戊巴比妥和苯妥英 (参见材料表), 在2毫升不育生理盐水1毫升/4.5 公斤。 注意: 这些 timepoints 是最佳的涵盖乙胺的药代动力学的轮廓, 可能需要调整/优化其他研究药物。</li>
	<li>使用镊子、剪刀和/或手术刀, 从胸腔中取出肺部, 切除包含在周围无关肺组织内的巨大坏死肉芽肿的肺活检 (如前所述3)。坏死性肉芽肿的颜色呈米色, 通常从周围的红色/粉红色的肺部微微凸出。为了方便 cryosectioning, 确保活检不大于 2 x 1.5 x 1.5 厘米。</li>
	<li>使用镊子, 将活检放置在预先标记的 cryomold 托盘上, 其所需的切割面与托盘底座直接接触。冷冻后, 这将提供一个平坦的表面, cryosections 将被削减。</li>
	<li>在液氮蒸气中冷冻活检。将发泡胶容器装入2英寸深的液态氮气, 然后放置金属线管架。该机架应凸出在液氮表面上, 提供了放置组织托盘的平坦表面。将盖子放回发泡胶容器上, 并将纸巾留10分钟, 以完全冻结。</li>
	<li>取出组织托盘, 快速包装铝膜, 并放置在单独标记密封塑料袋和印章。转移到-80 °c 冷藏库存放。 注意: 步骤 1.1-1.6 是在 BSL3 条件 (包括所有动物工作和处理感染的器官和组织) 执行。伽玛照射肺活检在 3 Megarads, 以使处理以外的 BSL3 遏制。如果已批准的安全协议到位, 可在 BLS3 设施内进行激光捕获显微切割生料组织。但是, 本协议的其余部分描述了 BSL-2 设施中的下游处理。</li>
</ol>2. 组织切片
<ol><li>将恒温器设置为所需的切削温度。将伽玛辐照肺活检从-80 摄氏度贮存到恒温器, 离开30分钟平衡组织温度。注:-20 至-22 °c 是结核病病灶活检的最佳选择。</li>
	<li>使用镊子, 修复活检恒温器恰克使用少量的最佳切削温度胶粘剂 (OCT) 坚持基地的组织到恰克。将组织定向, 使平面 (与 cryomold 的底部接触) 为切割的外露表面。确保 OCT 不会污染组织表面, 因为这可能会干扰随后的质谱分析。</li>
	<li>切割三个组织切片在25µm 厚度和安装到 PET 膜幻灯片。轻轻地将膜接触到组织切片并取出。如果施加太多的压力, 薄膜可能会撕裂。<ol>
<li>避免在安装前过度处理幻灯片, 因为这会导致 PET 膜被充电, 组织切片的粘附性较差。确保膜的滑动保持在室温下, 使组织的解冻和成功地附着在膜上。</li>
	</ol>
</li>
	<li>从恒温器中取出幻灯片, 并允许风干3分钟。如果 LCM-LC/ms/ms 将不会立即执行, 密封在一个小密闭密封袋的幻灯片, 并转移到-80 °c 存储, 直到需要进行解剖。</li>
	<li>在 10-12 µm 上切下相邻的部分, 在标准玻片上进行解冻, 用于苏木精和伊红 (H & E) 染色和参考。另外的部分可以被切开在这个时间为其他想要的组织化学污渍 (例如萋 Niellsen 为可视化结核分枝杆菌)。</li>
</ol>3. 显微切割
<ol><li>从-80 °c 存储中取出包含该幻灯片的密封袋, 并允许达到室温5分钟。 注: 如果冷滑片立即暴露在实验室的气氛中, 组织将会被涂上凝结, 并且药物的空间完整性可能会受到损害。</li>
	<li>打开显微镜和激光 (激光需要 5-10 分钟预热, 才能开始切割)。将扁帽0.20 毫升 PCR 管装入支架。</li>
	<li>从袋子中取出幻灯片, 并使用平板扫描仪在 PET 滑块上的组织切片上取一个光学图像。</li>
	<li>将幻灯片放到幻灯片持有者 (组织侧朝下), 并将不同的收集管分配给感兴趣的特定肉芽肿区域使用显微镜软件。通常, 这些将是 ' 无关肺, ' 细胞肉芽肿, ' 和 ' caseum ' (坏死中心), 但可能会因肉芽肿/活检的具体病理而异。</li>
	<li>用5X 显微镜的目的聚焦于组织。这个放大倍数应该提供一个很好的概述, 包括细胞和坏死性肉芽肿地区的组织。在软件中选择管指定的 "caseum" 将其移动到组织下的位置。</li>
	<li>输入所需的解剖参数。典型的设置为25µm 厚的肺部分是激光功率 30, 速度15和光圈 35 (任意单位)。然而, 这些将不同的视镜使用和潜在的下降功率由于激光的年龄。</li>
	<li>选择 "自由绘制" 工具, 并使用鼠标或触摸屏笔, 勾勒出所需的区域进行解剖。该区域的表面积将显示在软件中。将选定区域保留在50万µm2 (0.5 毫米2) 之下, 以方便解剖。重复解剖直到300万µm2 (3 毫米2) 在管帽中总共收集到。<ol>
<li>有时, 解剖区可能仍然粘在周围的膜 (例如由于静态吸引力), 而不是落入集合帽。通过在软件中手动选择和删除, 从累计曲面区域总计中移除这些区域。</li>
	</ol>
</li>
	<li>使用步骤3.7 中概述的相同过程, 选择 "细胞病变" 的 cap 并收集300万µm2的组织。</li>
	<li>选择 "无关肺" 的 cap, 并使用步骤3.7 中概述的相同过程收集300万µm2的组织。注意, 无关肺组织包含许多性细支气管和肺泡空间。仔细注意排除这些从定义的组织区域进行解剖。</li>
	<li>取下瓶盖, 仔细松开, 密封并贴上每个管子的标签。保护被解剖的组织免受周围的空气干扰 (例如从打开的门的气流中断)。立即分析解剖组织, 或贮存在-80 摄氏度和解冻之前的加工和 LCMS 分析。</li>
</ol>4. 提取和 LCMS 分析
<ol><li>制备含有乙胺 d-10 内部标准的1:1 乙腈/甲醇的萃取液。当选择一个内部标准, 使用一个稳定的标记形式的分析药物 (如在这个示范使用的氘标记的教统局) 与足够的质量转移, 以避免同位素交叉交谈的分析药物和标准 (通常至少4道尔顿). 注: 在每个组织类型的匀浆中创建标准是困难的, 因为有非常有限的控制组织, 以创建匀浆标准。作为一个替代的标准从一个尖锐的匀浆样品, 一个标准可以创造通过添加空白组织和测试化合物一起和提取。匀浆中的控制组织体积与研究样本组织切片的靶体积相匹配, 直接与一定浓度下存在的测试化合物的数量相结合。</li>
	<li>根据组织切片的表面积和厚度计算靶组织体积, 并利用将添加到标准和 QC 样品中的匀浆量来确定匀浆的必要稀释因子。下面说明了300万µm2 (3 毫米2) 目标解剖区域的计算结果, 其中有25µm 厚度和2µL 匀浆量。 <img alt="Equation 1" src="/files/ftp_upload/57402/57402eq1.jpg"> <img alt="Equation 2" src="/files/ftp_upload/57402/57402eq2.jpg"> <img alt="Equation 3" src="/files/ftp_upload/57402/57402eq3.jpg"> <img alt="Equation 4" src="/files/ftp_upload/57402/57402eq4.jpg">
</li>
	<li>假设组织密度为1克/毫升, 通过称量50毫克的控制组织和添加 PBS 缓冲稀释 (使用步骤4.2 中计算的26.67 匀浆稀释因子, 稀释剂为1.283 毫升), 制备匀浆料。融汇由珠殴打肺组织和 PBS 缓冲5分钟在 1750 rpm 上的珠均质体。 <img alt="Equation 5" src="/files/ftp_upload/57402/57402eq5.jpg"> <img alt="Equation 6" src="/files/ftp_upload/57402/57402eq6.jpg">
</li>
	<li>稀释1毫克/毫升药物储存浓度在1:1 乙腈/水, 以创建标准的曲线峰值解决方案。根据穗体积和靶组织体积确定峰值标准浓度。插图的例子是一个 100 ng/毫升标准使用10µL 穗量。 <img alt="Equation 7" src="/files/ftp_upload/57402/57402eq7.jpg"> <img alt="Equation 8" src="/files/ftp_upload/57402/57402eq8.jpg">
</li>
	<li>从-80 摄氏度的储藏中取出含有 microdissected 组织的管子, 使室温达到5分钟。</li>
	<li>添加10µL 1:1 乙腈/水溶液和2µL 的 PBS 缓冲器的管含有 microdissected 组织。</li>
	<li>对于标准曲线和质量控制管, 添加10µL 的峰值溶液到2µL 的控制肺匀浆。</li>
	<li>在每管中添加50µL 的萃取液。</li>
	<li>涡旋每管5分钟, 油脂实验5分钟, 离心机在 5000 RPM 5 分钟内形成一粒膜和组织在每管。</li>
	<li>将50µL 的上清液转移到96井深井板上, 并在每个井中稀释外加50µL 的去离子水。</li>
	<li>使用优化的乙胺和 Ethambutol-d10 内部标准的仪器参数执行 LC/ms/毫秒分析 (如前面详细介绍的12)。</li>
	<li>使用稀释因子纠正每个样本的组织解剖的确切数量。 <img alt="Equation 9" src="/files/ftp_upload/57402/57402eq9.jpg">
</li>
</ol>5. 方法验证
<ol><li>结合1份肺、2部 PBS 和 3-4 钢珠, 在控制肺组织中建立匀浆。用珠均质器在1750转每分钟打肺组织和 PBS 缓冲器5分。</li>
	<li>将匀浆通过加入10µL 1 毫克/毫升乙胺亚砜股票成990µL 匀浆, 以创造最终浓度 1万 ng/毫升 (10 毫克/毫升) 和漩涡1分钟。</li>
	<li>通过将匀浆倒入 cryomold, 并在干冰上快速冷冻5分钟, 创建一个冷冻匀浆块。</li>
	<li>按照步骤 2.1-2.5 中的描述, 从匀浆块中准备25µm 厚的部分。</li>
	<li>解剖在步骤 3.2-3.10 中指定的目标组织区域。</li>
	<li>将10µL 1:1 乙腈/水和2µL 的 PBS 缓冲器添加到含有 microdissected 组织的管中。</li>
	<li>在每管中添加50µL 的萃取液。按照步骤 4.9-4.12 创建一个标准曲线, 并确定在组织匀浆块中的药物浓度。</li>
	<li>使用下面的公式计算提取效率: <img alt="Equation 10" src="/files/ftp_upload/57402/57402eq10.jpg">
</li>
</ol>

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作者没有什么可透露的。

需要在空间上解决肺结核病变中的药物量化, 以确定药物暴露是否能达到对居住在不同病变隔间的细菌种群的杀菌浓度。这里所描述的 LCM-LC/MS 方法可以在所有病变舱室内绝对量化抗结核药物, 包括富含细菌的 caseum, 总共只使用 1-3 组织切片。传统的组织均匀化和 LC/MS 在组织中药物定量的方法往往缺乏空间特异性, 以解决特定的病变舱室, 甚至当它是可能的, 有很大的潜力交叉污染细胞和人工提取过?...

空间分辨和量化药物水平的能力是确定抗结核药物是否在灭菌浓度的肺部病变中达到细菌亚群的关键要求1。特别重要的是确定药物渗入病变坏死的核心 (称为 caseum), 它通常含有最高的杆菌数量, 由于缺乏血管化, 药物可能无法进入。
传统的评估病灶穿透的方法, 包括肺切除病灶的均匀化, 其次是溶剂萃取和液相色谱质谱 (LC/MS) 分析, 是高度敏感和选择性的药物兴趣。然而, 这些方法提供的空间信息较差, 仅限于原始匀质组织的大小。基于质谱的成像方法, 如基质辅助激光解吸电离 (MALDI)2,3, 解吸电喷雾电离 (方案)4或液体增强表面萃取5, 6提供高度空间解析的成像能力, 但由于不同的细胞的异质离子抑制效应和分析方法的不同提取效率, 直接量化可能是极具挑战性或不可能的。或组织类型7。此外, 大多数直接组织 MS 成像方法本身的敏感性比 LC/MS, 由于缺乏色谱分离的内源物种竞争电离和较低的溶剂萃取效率的药物从组织。
激光捕获显微切割 (LCM) 结合 LC/MS 分析已被常规应用于分离和表征不同的组织区域的蛋白质组学研究8,9和最近用于药物量化的剂量动物组织10。在这里, 我们提出了一个优化的协议, 应用 lcm 结合 lc/ms (LCM-lc/ms) 分析, 以量化抗结核药物在不同的肉芽肿隔间。在激光捕获显微切割过程中, 紫外线激光通过显微镜目标聚焦到组织切片上, 通过遵循用户定义的路径切割和分离所需的组织区域。对于重力辅助 LCM (用于这项研究的技术), 组织部分安装在薄聚合物膜 (PET 或钢笔) 上, 组织被捕获在幻灯片下方的收集管帽中。这些药物从切除的组织中提取出来, 并使用标准的 LC/MS 方法进行量化。所需收集的组织数量最终取决于组织中药物的预期浓度和 LC/MS 方法的敏感性。对于大多数药物治疗水平的分析和分析使用常规三重四极质谱仪, 300万µm2 (3 毫米2) 的组织表面面积是足够的。
该协议描述了由 LCM-LC/MS 提供的空间轮廓和完全量化的强大组合, 在结核肉芽肿的所有隔间提供绝对药物浓度。该技术还可用于确定许多不同疾病组织的药物浓度, 提供重要的药物发现和发展信息。

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			<td height="21" style="height:21px;width:273px;">New Zealand White rabbits</td>
			<td style="width:159px;">Covance</td>
			<td style="width:155px;">N/A</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:273px;">HN878 Mycobacterium tuberculosis</td>
			<td style="width:159px;">BEI Resources</td>
			<td style="width:155px;">NR-13647</td>
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			<td height="42" style="height:42px;width:273px;">Anased (Xylazine) 100 mg/mL</td>
			<td style="width:159px;">Henry Schein Animal Health</td>
			<td style="width:155px;">33198</td>
			<td></td>
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			<td height="42" style="height:42px;width:273px;">Euthasol (pentobarbital sodium and phenytoin sodium) Solution&#160;</td>
			<td style="width:159px;">Virbac</td>
			<td style="width:155px;">710101</td>
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			<td height="21" style="height:21px;width:273px;">Acetonitrile (LC-MS grade)</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:155px;">A955-212</td>
			<td></td>
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			<td height="21" style="height:21px;width:273px;">Methanol (LC-MS grade)</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:155px;">A456-212</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Formic Acid (LC-MS grade)</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:155px;">A117-50</td>
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			<td height="21" style="height:21px;width:273px;">Water (LC-MS grade)&#160;</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:155px;">W6212</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:273px;">0.2 mL flat-cap PCR tubes</td>
			<td style="width:159px;">Corning</td>
			<td style="width:155px;">07-200-392</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Steel frames, PET-membrane</td>
			<td style="width:159px;">Leica</td>
			<td style="width:155px;">11505151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Premium Frosted Microscope Slides</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:155px;">12-544-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">&#160;96 Deep well plate 2.0ML PP RB&#160;</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:155px;">NC0363259</td>
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			<td height="42" style="height:42px;width:273px;">Zorbax SB-C8&#160;column (4.6 by 50 mm; particle size, 3.5 &#956;m)</td>
			<td style="width:159px;">Agilent</td>
			<td style="width:155px;">820631-001D</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:273px;">&#34;Zipper&#8221; Seal Sample Bags</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:155px;">01-816-1B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Name</td>
			<td style="width:159px;">Company</td>
			<td style="width:155px;">Catalog Number</td>
			<td>Comments</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Equipment</td>
			<td style="width:159px;"></td>
			<td style="width:155px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:273px;">CM1850&#160;cryostat</td>
			<td style="width:159px;">Leica</td>
			<td style="width:155px;">Discontinued</td>
			<td>Leica CM1860 is the current model</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Laser Microdissection System 6500</td>
			<td style="width:159px;">Leica</td>
			<td style="width:155px;">Discontinued</td>
			<td>Leica LMD 6 is the current model</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Agilent 1260 Infinity II HPLC&#160;</td>
			<td style="width:159px;">Agilent</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">API 4000 QTRAP Mass Spectrometer</td>
			<td style="width:159px;">Sciex</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

spatial quantification of drugs in pulmonary tuberculosis lesions by laser capture microdissection liquid chromatography mass spectrometry (lcm-lc/ms)

结核病仍然是全球发病率和死亡率的主要原因。迫切需要改进现有药物方案和开发新型疗法。在肺肉芽肿的低血管坏死区 (caseum) 中, 服用结核药物来达到和杀菌细菌的能力是治疗成功的关键。因此, 有效的治疗方案必须含有有利的 caseum 渗透特性的药物。目前用于量化生物组织中药物水平的 LC/MS 方法的空间分辨能力有限, 难以准确确定小组织内的绝对药物浓度, 如坏死性肉芽肿。在这里, 我们提出了一个协议结合激光捕获显微解剖 (LCM) 的病理不同的组织区域与 LC/MS 量化。这种技术提供绝对量化的药物在肉芽肿 caseum, 周围细胞病变和无关肺组织, 因此, 准确地确定是否有杀菌浓度正在实现。除了结核病的研究, 该技术还有许多潜在的应用, 在空间上解决的药物定量的疾病组织。

LCM-LC/MS 方法的概述显示在图 1中。通过伽玛照射对组织进行消毒后, 所有后续步骤 (从组织切片开始) 都发生在 BSL3 条件之外。图 2显示了 LCM 组织隔离前后的病灶活检切片。坏死和细胞区结核病变可以很容易地识别和孤立的光学图像的视觉检查单独 (不需要提到组织学染色相邻的组织切片)。解剖过程产生一个干净的切口, 对周围?...

Watch this Scientific Journal Video about Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS at JoVE.c

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

在这里, 我们描述了一个协议使用激光捕获显微切割结合 LC/MS 分析, 以空间量化的药物分布在肺结核肉芽肿。该方法具有广泛的适用性, 以量化在高空间细节组织内的药物浓度。

激光捕获显微切割液相色谱质谱 (LCM-LC/MS) 对肺结核患者药物的空间量化

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

spatial-quantification-drugs-pulmonary-tuberculosis-lesions-laser

Research

Education

JoVE Core

JoVE Journal

Medical-Surgical Nursing

Medicine

Unit 1

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Lower Respiratory Tract Infections

SCIENTISTS IN ACTION

9.0K 次观看.  Rutgers, The State University of New Jersey. 在这里, 我们描述了一个协议使用激光捕获显微切割结合 LC/MS 分析, 以空间量化的药物分布在肺结核肉芽肿。该方法具有广泛的适用性, 以量化在高空间细节组织内的药物浓度。

视频: Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Magnetic Resonance Imaging Quantification of Pulmonary Perfusion using Calibrated Arterial Spin Labeling

This demonstrates a MR imaging method to measure the spatial distribution of pulmonary blood flow in healthy subjects 
during normoxia (inspired O2, fraction (FIO2) = 0.21) hypoxia (FIO2 = 0.125), and hyperoxia 
(FIO2 = 1.00). In addition, the physiological responses of the subject are monitored in the MR scan environment. MR images 
were obtained on a 1.5 T GE MRI scanner during a breath hold from a sagittal slice in the right lung at functional residual capacity. An arterial 
spin labeling sequence (ASL-FAIRER) was used to measure the spatial distribution of pulmonary blood flow 1,2 and a multi-echo fast 
gradient echo (mGRE) sequence 3 was used to quantify the regional proton (i.e. H2O) density, allowing the quantification 
of density-normalized perfusion for each voxel (milliliters blood per minute per gram lung tissue). 
With a pneumatic switching valve and facemask equipped with a 2-way non-rebreathing valve, different oxygen concentrations 
were introduced to the subject in the MR scanner through the inspired gas tubing. A metabolic cart collected expiratory gas via expiratory tubing. Mixed expiratory O2 and CO2 concentrations, oxygen consumption, carbon dioxide production, respiratory exchange ratio, 
respiratory frequency and tidal volume were measured. Heart rate and oxygen saturation were monitored using pulse-oximetry. 
Data obtained from a normal subject showed that, as expected, heart rate was higher in hypoxia (60 bpm) than during normoxia (51) or hyperoxia (50) and the arterial oxygen saturation (SpO2) was reduced during hypoxia to 86%. Mean ventilation was 8.31 L/min BTPS during hypoxia, 7.04 L/min during normoxia, and 6.64 L/min during hyperoxia. Tidal volume was 0.76 L during hypoxia, 0.69 L during normoxia, and 0.67 L during hyperoxia. 
Representative quantified ASL data showed that the mean density normalized perfusion was 8.86 ml/min/g during hypoxia, 8.26 ml/min/g during normoxia and 8.46 ml/min/g during hyperoxia, respectively. In this subject, the relative dispersion4, an index of global heterogeneity, was increased in hypoxia (1.07 during hypoxia, 0.85 during normoxia, and 0.87 during hyperoxia) while the fractal dimension (Ds), another index of heterogeneity reflecting vascular branching structure, was unchanged (1.24 during hypoxia, 1.26 during normoxia, and 1.26 during hyperoxia). 
Overview. This protocol will demonstrate the acquisition of data to measure the distribution of pulmonary perfusion noninvasively under conditions of normoxia, hypoxia, and hyperoxia using a magnetic resonance imaging technique known as arterial spin labeling (ASL). 
Rationale: Measurement of pulmonary blood flow and lung proton density using MR technique offers high spatial resolution images which can be quantified and the ability to perform repeated measurements under several different physiological conditions. In human studies, PET, SPECT, and CT are commonly used as the alternative techniques. However, these techniques involve exposure to ionizing radiation, and thus are not suitable for repeated measurements in human subjects.

A MR imaging method to study the distribution of pulmonary blood flow under a variety of physiological conditions, in this case exposure to three different inspired oxygen concentrations: hypoxia, normoxia, and hyperoxia, is described. This technique utilizes human pulmonary physiology research techniques in an MR scanning environment.

使用校准动脉自旋标记磁共振肺灌注显像定量

This demonstrates a MR imaging method to measure the spatial distribution of pulmonary blood flow in healthy subjects 
during normoxia (inspired O2, ...

科研

医学

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson&#039;s Disease

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

MALDI成像帕金森病的神经肽质谱

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

从手术标本的人类胰岛细胞激光显微切割的改进方法

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

对源自液体和固体肿瘤癌症细胞定量蛋白质组质谱剖析

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

人类前列腺上皮的激光捕获显微切割的RNA分析

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a specific anatomical region. Within the brain, metals have a highly compartmentalized distribution, depending on the primary function they play within the central nervous system. Imaging the spatial distribution of metals has provided unique insight into the biochemical architecture of the brain, allowing direct correlation between neuroanatomical regions and their known function with regard to metal-dependent processes. In addition, several age-related neurological disorders feature disrupted metal homeostasis, which is often confined to small regions of the brain that are otherwise difficult to analyze. Here, we describe a comprehensive method for quantitatively imaging metals in the mouse brain, using laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) and specially designed image processing software. Focusing on iron, copper and zinc, which are three of the most abundant and disease-relevant metals within the brain, we describe the essential steps in sample preparation, analysis, quantitative measurements and image processing to produce maps of metal distribution within the low micrometer resolution range. This technique, applicable to any cut tissue section, is capable of demonstrating the highly variable distribution of metals within an organ or system, and can be used to identify changes in metal homeostasis and absolute levels within fine anatomical structures.

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry LA-ICP-MS

Quantitatively mapping metals in tissue by laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) is a sensitive analytical technique that can provide new insight into how metals participate in normal function and disease processes. Here, we describe a protocol for quantitatively imaging metals in thin sections of mouse neurological tissue.

在脑组织成像金属激光剥蚀 - 电感耦合等离子体 - 质谱法（LA-ICP-MS）

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

微生物肠道血屏障及肝脏代谢的体内评价方法

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography

Glycomics is a new subspecialty in omics system research that offers significant potential in discovering next-generation biomarkers for disease susceptibility, drug target discovery, and precision medicine. Alternative IgG N-glycans have been reported in several common chronic diseases and suggested to have great potential in clinical applications (i.e., biomarkers for diagnosis and prediction of diseases). IgG N-glycans are widely characterized using the method of hydrophilic interaction chromatography (HILIC) ultra-performance liquid chromatography (UPLC). UPLC is a stable detection technology with good reproducibility and high relative quantitative accuracy. In addition, the structure of IgG N-glycan is clearly separated, and glycan composition and relative abundance in plasma are characterized.

Immunoglobulin G (IgG) N-glycan is characterized using hydrophilic interaction chromatography UPLC. In addition, the structure of IgG N-glycan is clearly separated. Presented here is an introduction to this experimental method so that it can be widely used in research settings.

免疫球蛋白 G N-甘油分析通过超高性能液色谱分析

Glycomics is a new subspecialty in omics system research that offers significant potential in discovering next-generation biomarkers for disease ...

Analysis of Raw and Processed Cyperi Rhizoma Samples Using Liquid Chromatography-Tandem Mass Spectrometry in Rats with Primary Dysmenorrhea

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is enhanced after processing with vinegar, CR processed with vinegar (CRV) is generally used clinically. However, the mechanism by which the analgesic effect is enhanced by vinegar processing is unclear. In this study, the ultra-high pressure liquid chromatographytandem mass spectrometry (UPLC-MS/MS) technique was used to examine changes in the blood levels of the exogenous constituents and metabolites between CR-treated and CRV-treated rats with dysmenorrhea. The results revealed differing levels of 15 constituents and two metabolites in the blood of these rats. Among them, the levels of (-)-myrtenol and [(1R,2S,3R,4R)-3-hydroxy-1,4,7,7-tetramethylbicyclo[2.2.1]hept-2-yl]acetic acid in the CRV group were considerably higher than in the CR group. CRV reduced the level of 2-series prostanoids and 4-series leukotrienes with proinflammatory, platelet aggregation, and vasoconstriction activities and provided analgesic effects by modulating arachidonic acid and linoleic acid metabolism and the biosynthesis of unsaturated fatty acids. This study revealed that vinegar processing enhances the analgesic effect of CR and contributes to our understanding of the mechanism of action of CRV.

Here, a comparative analysis of raw and processed Cyperi rhizoma (CR) samples is presented using ultra-high performance liquid chromatography-high-resolution tandem mass spectrometry (UPLC-MS/MS) in rats with primary dysmenorrhea. The changes in blood levels of the metabolites and the sample constituents were examined between rats treated with CR and CR processed with vinegar (CRV).

使用液相色谱-串联质谱分析原发性痛经大鼠的原始和加工的Cyperi Rhizoma样品

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is ...

捕获口腔粘膜下纤维化组织样本用于空间组学分析

Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina propria and the submucosa are often found on histopathological slides. Epithelial dysplasia, epithelial atrophy, and senescent fibroblasts have been proposed to be associated with the malignant transformation of OSF. However, because of the heterogeneity of potentially malignant oral disorders and oral squamous cell carcinoma, it is difficult to identify the specific molecular mechanisms of malignant transformation in OSF. Here, we present a method to obtain a small number of epithelial or mesenchymal cells carrying morphological data and spatial information by laser capture microdissection on formalin-fixed paraffin-embedded tissue slides. Using a microscope, we can precisely capture microscale (~500 cells) dysplastic or atrophic epithelial tissue and fibrotic subepithelial tissue. The extracted cells can be evaluated by genome or transcriptome sequencing to acquire genomic and transcriptomic data with morphological and spatial information. This approach removes the heterogeneity of bulk OSF tissue sequencing and the interference caused by cells in non-lesioned areas, allowing for precise spatial-omics analysis of OSF tissue.

作者聚焦：揭开口腔潜在恶性肿瘤的神秘面纱 

Laser capture microdissection of oral submucous fibrosis tissues allows for precise extraction of cells from histological regions of interest for the analysis of multi-omics data with morphological and spatial information.

激光捕获显微切割从口腔粘膜下纤维化样本中分离具有形态学和空间信息的细胞

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina ...