Name: histological quantification to determine lung fungal burden in experimental aspergillosis
Uploaded: 2018-03-09T15:00:00.000+00:00
Duration: 9 min 52 s
Description: Watch this Scientific Journal Video about Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis at JoVE.com

这项研究得到了印第安纳大学医学研究增强补助金和 NIH-NIAID 1R03AI122127-01 的部分支持。在这段期间, 位免疫学家美国协会的免疫学研究事业获得了部分支持。

所有动物规程由印第安纳国立大学的动物照料和用途委员会批准, 印第安纳大学医学院的寄宿校园-渔村高级。
1. 用于感染的A. 曲霉孢子的制备
<ol><li>在通风良好的油烟机中工作时, 将125µL曲霉293 库存解决方案添加到900µL 细胞培养级水 (CCGW) 中。然后通过吸管将溶液转移到麦芽萃取琼脂 (多边环境) 板上, 并均匀地与接种循环一起传播。</li>
	<li>在黑暗中储存24摄氏度的真菌片至少14天, 不超过28天。</li>
	<li>使用玻璃珠作为先前描述的9来收获孢子。</li>
	<li>将1.5 克0.5 毫米的玻璃珠倒入板上, 轻轻地来回倾斜, 直到珠子被涂上以提取孢子。在15毫升圆锥管中收集珠/孢子混合物。悬浮在5毫升 Dulbecco 的磷酸盐缓冲盐水 (DPBS) 和漩涡。</li>
	<li>用 hemocytometer 对 DPBS 中上清的50x 稀释中的孢子进行计数。计算图 1所示区域中的生孢子数, 并使用等式 1计算浓度。 #Conidia x 稀释因子 x 104= 孢子/mL (等式 1) 注意: 可以计算额外的 4 x 4 平方角区域, 这些区域的平均值在等式 1中用作 #Conidia。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57155/57155fig1.jpg"> 图 1: 用于分孢子计数的 Hemocytometer 代表性区域 (框、箭头)。
<ol start="6"><li>用无菌生理盐水稀释孢子悬浮液 (DPBS), 以获得所需浓度, 1.0 x 108孢子/毫升, 用于感染 5 x 106 conidia/50 µL. 准备50µL 的孢子溶液每 ' n + 2 ' 小鼠被感染占吹打错误. 注: 标准菌的收获和准备/过滤可以交替使用10。玻璃珠法是最好的方法, 以确保获得最小的小/可溶性菌丝抗原的孢子样品的渴望。</li>
</ol>2. 免疫抑制和小鼠模型感染
<ol><li>使用7-10 周大的老鼠。 注: 本研究采用 BALB、C57BL/6 (B6)、嗜酸细胞缺乏 (ΔdblGATA、BALB/c 背景) 和γδ T 淋巴细胞缺失 (TCRδ/B6 背景) 小鼠。对于每个实验, 年龄和性别匹配 (男性和女性) 小鼠被用于每组。</li>
	<li>耗尽中性粒细胞通过腹腔 (IP) 注射0.1 毫升 (0.5 毫克) anti-mouse-Ly-6G 抗体, 在45°在右下象限的不育盐水中 (为实验计划, 请参阅图 2)。</li>
	<li>24小时后, 通过吸入感染小鼠 5.0 x 106A. 曲霉孢子悬浮在50µL 无菌生理盐水中 (见步骤 2.4-2.10), 如前所述11。继感染后, 在本研究中使用的 DPBS 中注射抗真菌剂 (如5毫克/千克卡泊芬双乙酸) 的动物子集 (每日, 请参阅图 2)。</li>
	<li>用兽医麻醉机麻醉99.9% 异氟醚的小鼠。<ol>
<li>将纸巾放在感应室的地板上, 以捕获粪便和尿液。</li>
		<li>将鼠标放在感应腔内, 确保盖子的安全, 并将蒸发器设置为5% 异氟醚, 2 分钟, 三十年代。 注: 不使用兽医软膏, 因为麻醉总时间少于5分钟。</li>
		<li>将蒸发器减少到三十年代最大设置的一半。</li>
	</ol>
</li>
	<li>通过观察慢呼吸, 缺乏运动, 以及对脚趾捏刺激缺乏反应, 确认老鼠被适当麻醉。</li>
	<li>从感应腔中取出鼠标并放置在斜板上。将鼠标向后放在板上。确保其上切牙被带橡皮筋轻轻地约束, 下切门牙用金属丝轻轻地抑制。</li>
	<li>用小钳和吸管50µL 的孢子/PBS 悬浮在舌头的基础上, 小心地握住舌头。</li>
	<li>保持舌头完全伸展;倾听呼吸急促的声音和渴望的明显的点击声。保持二十年代或直到悬浮完全吸气。</li>
	<li>从斜板上取下鼠标, 轻轻地放在笼子里观察。</li>
	<li>在吸入后, 老鼠应在1分钟内恢复知觉. 监视鼠标直到完全清醒, 并能够绕着笼子走。动物应该被安置在21°c 并且每日被监视两次直到肺被收获。</li>
	<li>重复嗜中性粒细胞 (步骤 2.2) 24 h 感染后的损耗 (图 2)。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57155/57155fig2.jpg"> 图 2: 实验计划.此数字是从以前的出版物8中修改的。
3. 小鼠肺的采集和保存进行组织学分析
<ol><li>深麻醉的动物与致命剂量的0.1 毫升390毫克/毫升戊巴比妥钠溶液 IP。</li>
	<li>通过观察缺乏自发性呼吸和缺乏对肢体刺激的镊子的反应来确认安乐死。一旦安乐死, 用70% 乙醇喷洒尸体以杀菌。</li>
	<li>将屠体放在覆盖有吸收垫的泡沫板上。 注: 该屠体应放在其背上, 用别针将所有四只四肢固定在泡沫板上。</li>
	<li>小心使用剪刀打开上胸腔。为了暴露肺部和心脏, 把肋骨剪掉。切开下腔静脉, 允许灌注。避免切割血管和穿刺器官。</li>
	<li>用25克针慢慢地在心脏右心室灌注5毫升的冷 DPBS。重复灌注5毫升10% 福尔马林。</li>
	<li>小心地暴露气管并分离周围的脂肪垫。慎用, 避免穿刺气管。确保多余的结缔组织被删除, 以充分可视化气管, 然后再继续。一旦气管暴露, 将镊子放在气管下面, 将其抬高。</li>
	<li>用25克针戳上气管上的一个小孔。 注: 孔应定位, 使整个套管适合气管。小心避免气管破裂。</li>
	<li>插入套管通过孔和向肺部。在气管周围松散的外科螺纹固定套管。 注: 如果气管破裂, 套管可能会在气管内尽可能地插入到呼吸道。</li>
	<li>使用注射器, 用1毫升10% 福尔马林缓冲器通过套管充气肺部。确保肺部的膨胀是可见的。如果没有, 调整导管并重复。一旦肺部膨胀, 迅速取出套管小心, 并拧紧线程安全地封闭气管。 注: 如果气管破裂, 肺部不能充气, 肺部可能仍会被收割, 但这可能会阻止正确的固定。</li>
	<li>小心地将气管与镊子的结缔组织连接在一起, 轻轻地拉上螺纹, 轻轻地将气管和肺部从胸腔中取出。将肺部和气管放在50毫升圆锥管中, 15 毫升10% 福尔马林缓冲器。将螺纹的一端置于管外, 并密封盖。倒置管完全淹没在固定的样品。</li>
	<li>将样品在室温下贮存至少24小时, 或直至进一步加工。 警告:福尔马林是危险的。佩戴适当的个人防护设备, 包括护目镜和口罩。处理样品时, 在化学罩下工作。</li>
</ol>4. 小鼠肺部 DNA 真菌负担的收获和保存
<ol><li>按照上面描述的一个单独的鼠标执行步骤 3.1-3.5。</li>
	<li>用25克针慢慢地在心脏右心室灌注10毫升的冷 DPBS。</li>
	<li>用剪刀将肺部和放置在标记的1.5 毫升管上。</li>
	<li>储存肺部在-80 °c, 直到进一步处理。</li>
	<li>使用所描述的标准技术 (嵌入和切片), 并根据制造商的协议 (请参阅材料表)。</li>
</ol>5. 显微和成像
<ol><li>打开图像量化程序 (请参阅材料表)。</li>
	<li>使用光显微镜, 获得至少4个不同领域的 GMS 彩色幻灯片 (10X 目标)。确保图像在肺部内受感染的呼吸道具有类似组织密度的代表性。 注: 如果菌丝病灶的密度和分布明显不同于对照组和实验群之间, 则可采取额外的电场, 或整个肺切片的 GMS 面积可成像 (4X 目标)。如果需要, 获得与苏木精和伊红染色的连续肺切片的肺相同区域 (由等效组织形态学鉴定) 的图像。</li>
	<li>在必要时向图像中添加缩放条, 并保存要量化的图像。选择、编辑 > 添加/编辑校准标记 > 菜单: 选择线条粗细、颜色和缩放栏大小 > 单击要放置缩放条的图像。关闭菜单并选择、编辑 > 合并 > 合并校准标记 > 选择文件保存为 > 命名文件并单击 "保存"。</li>
</ol>6. 使用图像处理程序计算真菌负担 (图 3)
<ol><li>打开图像处理程序。选择带有示例的 "文件" > "文件夹" 以量化 > 图像 (图 3A)。选择 "图像" > "类型" > "转换为" RGB 堆栈 "(图 3B)。</li>
	<li>使用右箭头键选择三给定图像的第二个 (图 3B)。点击 "控制 + shift + T" 以弹出 "阈值" 菜单设置调节器 (图 3C)。将原始. tiff 或. jpg 图像定位为参考, 并使用图像查看程序 (图 3C) 打开。</li>
	<li>将顶部滑块一直向左拖动, 然后调整底部滑块, 直到所选区域 (以红色表示) 是显微图像的代表 (通常从滑块的右侧 (图 3C) 中所示的130-160 不等。</li>
	<li>选中后, 按 "设置" > "确定" (图 3D)。选择 "分析" > "设置测量" > 检查 "区域、区域分数、限制阈值、显示标签", 并保留底部设置 (下拉菜单和小数位) 作为默认 > "确定" (图 3D)。</li>
	<li>空白或背景染色的重要区域可能被排除通过选择适当的组织与区域或多边形工具。</li>
	<li>最后选择 "分析" > "度量" (显示一个带有数据的窗口, 或者 windows 任务栏上的选项卡将出现标记为 "结果") (图 3E)。将数据传输到数据分析程序以进行图形和统计分析。</li>
	<li>输入每个肺样本的四面积百分比的平均值。5-8 样本通常足以检测组之间的显著差异。如果比较两个实验组, 请执行未配对学生的t测试以确定其重要性。</li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57155/57155fig3.jpg"> 图 3: 用于 GMS 组织学场量化的每个步骤的典型截图.(A) 选择带有示例的 "文件" > "文件夹" 以量化 > "选择图像"。(B) 选择图像 > 类型 > 转换为 "RGB 堆栈"。使用右箭头键选择三给定图像的第二个。(C) 命中 "控制 + shift + T" 以提出 "阈值" 菜单设置调节器。将原始. tiff 或. jpg 图像定位为参考, 并使用照片查看器程序打开。向左拉顶部滑块并调整底部滑块, 直到选定区域 (以红色表示) 是显微图像的代表 (通常从滑块右侧显示的130-160 不等)。(D) 一旦选中按下 "设置" > "确定"。选择 "分析" > "设置测量" > 检查 "区域, 区域分数, 限制阈值, 显示标签", 并保留底部设置 (下拉菜单和小数位) 作为默认 > "OK"。(E) 最后选择 "分析" > "度量" (显示一个带有数据的窗口, 或者 windows 任务栏上的选项卡将显示为 "结果")。<a href="//cloudflare.jove.com/files/ftp_upload/57155/57155fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
7. DNA 真菌负担
<ol><li>从-80 摄氏度和 lyophilize 中取出肺部至少4小时 (隔夜是最佳的), 或直到完全干燥的冷冻干燥系统。</li>
	<li>当肺干燥时, 准备 DNA 提取缓冲器 (0.1 米氯化钠, 10 毫米 EDTA ph 值 8.0, 10 毫米三盐酸盐 ph 值 8.0, 和 0.5% SDS)。</li>
	<li>将准备好的 DNA 提取缓冲器放在60摄氏度的水浴中预热。另外, 放置苯酚: 氯仿: 异戊醇 (25:24:1) 在50毫升圆锥管和允许分离。</li>
	<li>将冻干肺放在2毫升螺钉盖管中, 0.2 毫升玻璃珠。使用一个珠搅拌器, 研磨干燥的肺组织为 15-30 s, 直到它形成一个细粉。</li>
	<li>将0.8 毫升的热 DNA 提取缓冲器添加到每个管和涡旋井中。在65摄氏度的珠浴中孵化每管15分钟, 然后在孵化前再进行涡流, 再增加15分钟。孵化后, 涡旋, 然后离心管在 2.5万 x g 10 分钟。</li>
	<li>为每个样品贴上3套1.5 毫升管, 并将上清上一层转移到一组管子中。在吹打水顶层时要小心, 以免干扰中间层。</li>
	<li>加入等量的 phenol:chloroform:isoamyl 醇, 混合均匀。然后离心机在 2.5万 x g 15 分钟之前, 吹打出上层到第二套管。</li>
	<li>增加等量的氯仿: 异戊氯仿, 混合好, 离心机在 2.5万 x 克10分钟. 小心地将上层的吸管移出第三组管子。</li>
	<li>添加500µL 异丙醇和50µL NaoAc (pH 5.2), 混合良好, 并允许样品在室温下孵化10分钟。然后离心机在 2.5万 x g 30 分钟和醒酒上清。</li>
	<li>用750µL 冰冷70% 乙醇和离心机清洗 DNA 颗粒, 在 2.5万 x g 上醒酒乙醇, 并允许颗粒在通风管中空气干燥40分钟。</li>
	<li>并用重悬300µL 的干颗粒。 注: DNA 可以储存在-20 摄氏度, 直到量化。</li>
	<li>使用分光光度计量化 dna, 并准备100µL 0.2 µg/µL 每个 dna 样本进行 qPCR 分析。</li>
	<li>执行标准 qPCR, 如前所述, 1 µg 的 DNA 样本, 真菌 18S rDNA 底漆, 和探针集与改进的探针淬火 (5 '-/56-/AGC CAG CGG/禅宗/CCC),G/3IABkFQ/-3') 12, 13.</li>
	<li>从曲霉基因组 DNA 中准备一个标准曲线, 并使用平均记者染料 Ct 值计算每个样本中 pg 的真菌 DNA 浓度。</li>
	<li>用平均值的标准误差绘制真菌 DNA 的 pg/µg。执行学生的t测试分析以确定其重要性。 注: 作为一种毒性较低的替代苯酚-氯仿 dna 提取在这里使用, 柱基 dna 提取套件可以使用。</li>
</ol>

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作者没有什么可透露的。

本文的目的是通过利用 GMS 染色的肺组织学切片进行图像分析和定量分析, 介绍一种测定 IA 小鼠肺真菌负担的方法。在本研究中, 用β葡聚糖合成靶向抗真菌药物卡泊芬14的治疗并没有改善粒细胞减少野生型小鼠的生存或真菌负担 (IA8)。然而, 在缺乏嗜酸性粒细胞或γδ T 淋巴细胞的情况下, 生存和真菌负担有所改善。我们的研究结果还表明, 与广泛使用的真菌 DNA qPCR 方法6相比, GMS 真菌负担可获得可比结果。
利用 GMS 真菌负担量化有几个好处。首先, 该过程可能利用现有的组织学样本, 从而有可能减少所需的实验数量, 以确定重大差异。第二, 在这项研究中, 比起真菌 DNA 的 qPCR, 对 GMS 的真菌负担量化需要较少的动物 (图 6,图 7)。第三, 在核糖体 DNA 拷贝数15中, 不同分离株的真菌负担的比较可能受 qPCR 的差异的影响。相比之下, GMS 量化不受复制数的影响, 因为真菌负担是由相对水平的肺部真菌生长决定的。因此, 使用 GMS 定量的真菌负担减少了对脊椎动物的使用, 不需要预先确定 rDNA 复制号。最后, 除了改变真菌的形态, 卡泊芬疗法增加真菌碎片, 从而可以人为地增加真菌负担时, 通过孤立的菌落形成单位从肺组织匀浆12, 16 ,17。因此, GMS 定量的真菌负担避免了与其他常用方法固有的一些限制。
然而, GMS 量化和/或这项研究的局限性值得注意。首先, 作者假设在每个实验组的肺部都有可比分布的菌丝生长, 因此使用4个代表性10x 客观领域的量化来作为对整个肺部真菌负担的代表性测量。(图 6B)。在某些情况下, 菌丝灶的相对分布、大小和密度会有很大差异, 从而使真菌的负担与这种方法和 qPCR 方法的等效性不同。然而, 我们的额外结果与整体肺部分量化使用4X 客观的领域显示相似, 虽然统计上不显著, 组之间的差异 (图 6C)。这一策略增加了这个量化指标的标准误差, 可能是由于4X 目标的菌丝分辨率降低和次优场的背景增加。因此, 有代表性的量化更少的领域在更高的放大率是首选。第二, 每个样本只使用一个中央截面。在真菌分离或小鼠菌株的基础上, 一些研究可能导致菌丝生长的不均匀分布。在这些情况下, 每个石蜡区块中的其他部分应量化, 以获得更有代表性的负担。第三, 在诱发粘液大量生产的实验中 (即, 量化过敏性支气管肺曲霉病 (ABPA)18或囊性纤维化 (CF)18) 的气道真菌生长, GMS 反应与富含多糖的粘液19可产生非特异的 GMS + 结果, 从而使一些样品倾斜, 有利于较高的真菌负担。由于仅在本研究中使用了 IA 的粒细胞减少模型, 因此使用其他免疫能力强或抑制模型可能会导致比较少的结果。尽管有这些告诫, 但 GMS 量化提供了一种可比性的方法来确定真菌负担, 其继续在其他研究中的使用可以进一步验证这种方法的实用性, 因为它是一致的、可靠的和具有成本效益的。

IA 是一种机会性感染, 可能会发展在有先天或后天免疫缺陷的敏感个体由于免疫抑制治疗或慢性感染1,2。原发性感染通常发生在肺部, 虽然在某些情况下, 传播曲霉曲霉到肝脏, 肾脏, 心脏和大脑可能发生, 导致广泛组织侵入菌丝伴有严重疾病和高死亡率1,2。此外, 现有 pharmacotherapies 的功效有限, 可能会因环境中抗真菌菌株的出现而进一步减弱3。因此, 重要的是要了解真菌毒力和宿主病理机制, 促进侵袭性真菌疾病的发展或恶化。
小鼠模型仍然是重要的机械 IA 研究, 因为他们允许研究人员评估真菌毒力基因和宿主免疫效应的作用, 以建立和增长的A. 曲霉在体内4,5。因此, 为了有效地量化或比较实验动物组中的真菌负担, 设计了多种策略6,7。这些策略包括基于文化的、生物化学的、免疫分析法或 qPCR 的方法, 每个都有明显的优缺点。此外, 这些方法中的每一个都涉及一个动物子集的奉献, 除了那些为评估免疫效应功能、基因表达分析和比较病理学7而牺牲的那些。因此, 综合 IA 研究往往需要大量的研究动物在一个巨大的成本。因此, 通过利用动物组织进行多种分析, 减少实验时间、动物成本和伦理考虑的有效策略是非常有价值的7。
在本报告中, 介绍了在组织学切片中对 GMS 染色的量化方法, 以比较实验组小鼠与 IA 的相对真菌负担。从真菌培养到感染、组织收获和加工、图像采集和数据分析的每一步都进行了详细描述。粒细胞减少模型中 qPCR 和卡泊芬处理的野生型或嗜酸性粒细胞缺陷小鼠与ia8 进行比较, 得到了 GMS 定量的真菌负担。结果表明, 与 GMS 定量和 qPCR 真菌 DNA 相似。这表明, GMS 量化可能是有用的研究人员进行组织学分析, 作为一种补充或替代方法比较的相对真菌负担的小鼠与 IA, 并可能最终降低成本和使用的研究动物在复杂的, 机械的研究。

<table><tbody><tr><td>&lt;em&gt;Aspergillus fumigatus&lt;/em&gt; 293 Stock Solution</td><td>Fungal Genetics Stock Center</td><td>FGSC #A1100</td><td></td></tr><tr><td>HyPure Cell Culture Grade Water</td><td>Thermo Fisher Scientific</td><td>SH30529.03</td><td></td></tr><tr><td>Malt Extract</td><td>MP Biomedicals</td><td>2155315</td><td>White Powder</td></tr><tr><td>BD&amp;nbsp;BBL Acidicase Dehydrated Culture Media: Peptone</td><td>Fisher Scientific</td><td>L11843</td><td></td></tr><tr><td>Dextrose (D-Glucose) Anhydrous (Granular Powder/Certified ACS)</td><td>Fisher Scientific</td><td>D16-3</td><td></td></tr><tr><td>Fisher BioReagents Agar, Powder / Flakes, Fisher BioReagents</td><td>Fisher Scientific</td><td>BP1423-500</td><td></td></tr><tr><td>Auto Dry Cabinet</td><td>Shanghai Hasuc Instrument Manufacture Co.,LTD</td><td>HSFC160FD</td><td></td></tr><tr><td>1300 Series Class II, Type A2 Biological Safety Cabinet</td><td>Thermo Fisher Scientific</td><td>1375</td><td></td></tr><tr><td>0.5 mm Glass Beads</td><td>BioSpec Products</td><td>11079105</td><td></td></tr><tr><td>15 ml Conical Tubes</td><td>Thermo Fisher Scientific</td><td>339650</td><td></td></tr><tr><td>Hemacytometer</td><td>Fisher Scientific</td><td># 0267110</td><td></td></tr><tr><td>Leica Model DME Microscope</td><td>Leica</td><td>13595XXX</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8662-500 ml</td><td></td></tr><tr><td>1.5 ml tubes</td><td>Fisher Scientific</td><td># 05-408-129</td><td></td></tr><tr><td>BD Precisionglide&amp;nbsp;syringe needles, gauge 27, L 1/2 in.</td><td>Sigma-Aldrich</td><td>Z192384</td><td></td></tr><tr><td>Anti-mouse-Ly-6G antibody</td><td>BioXCell</td><td>BP0075-1</td><td>Clone 1A8</td></tr><tr><td>Caspofungin diacetate</td><td>Sigma-Aldrich</td><td>SML0425</td><td></td></tr><tr><td>Isoflurane</td><td>Henry Schein Animal Health</td><td>1169567762</td><td></td></tr><tr><td>Non-Rebreathing Table Top Veterinary Anesthesia Machine</td><td>Supera Anesthesia Innovations</td><td>M3000</td><td></td></tr><tr><td>Pureline Oxygen Concentrator</td><td>Supera Anesthesia Innovations</td><td>OC8000</td><td></td></tr><tr><td>Slant Board Restraint</td><td>Indiana State University Facilities Management</td><td>Custom made</td><td></td></tr><tr><td>Gilson&amp;nbsp;PIPETMAN Classic 200 ml Pipets</td><td>Fisher Scientific</td><td>F123601G</td><td></td></tr><tr><td>Pentobarbital Sodium (Fatal-Plus)</td><td>Vortech Pharmaceuticals</td><td>0298-9373-68</td><td></td></tr><tr><td>General-Purpose Broad-Tipped Forceps</td><td>Fisher Scientific</td><td>10-300</td><td></td></tr><tr><td>Fisherbrand&amp;nbsp;Standard Dissecting Scissors</td><td>Fisher Scientific</td><td>08-951-20</td><td></td></tr><tr><td>Fisherbrand General-Purpose Curved Forceps</td><td>Fisher Scientific</td><td>10-275</td><td></td></tr><tr><td>Ethyl Alcohol-200 Proof</td><td>PHARMCO-AAPER</td><td>111000200</td><td></td></tr><tr><td>Fisherbrand Absorbent Underpads</td><td>Fisher Scientific</td><td>14-206-63</td><td></td></tr><tr><td>BD Precisionglide&amp;nbsp;syringe needles, gauge 25, L 1 in.</td><td>Fisher Scientific</td><td>Z192406</td><td></td></tr><tr><td>All-Plastic Norm-Ject Syringes</td><td>Fisher Scientific</td><td>14-817-30</td><td></td></tr><tr><td>IV CATH ANGIOCATH 22GX1GIN 50B</td><td>Fisher Scientific</td><td>NC9742754</td><td></td></tr><tr><td>Formalin Solution, neutral buffered, 10%</td><td>Sigma-Aldrich</td><td>HT501128-4L</td><td></td></tr><tr><td>50 ml Conical Tubes</td><td>Thermo Fisher Scientific</td><td>339652</td><td></td></tr><tr><td>Thermo Scientific Shandon Embedding Cassettes II in Tube Packs</td><td>Fisher Scientific</td><td>B1000729</td><td></td></tr><tr><td>Fisherfinest&amp;nbsp;Histoplast Paraffin Wax</td><td>Fisher Scientific</td><td>22-900-700</td><td></td></tr><tr><td>Disposable Base Molds</td><td>Fisher Scientific</td><td>22-363-554</td><td></td></tr><tr><td>Reichert Jung Histocut 820 Microtome</td><td>Labequip.com</td><td>31930</td><td></td></tr><tr><td>Water Bath</td><td>Precision Scientific</td><td>66630-23</td><td></td></tr><tr><td>CO2 Incubator</td><td>Fisher Scientific</td><td>116875H</td><td></td></tr><tr><td>Silver Stain (Modified GMS) Kit</td><td>Sigma-Aldrich</td><td>HT100A-1KT</td><td></td></tr><tr><td>Fast Green FCF</td><td>Fisher Scientific</td><td>AC410530250</td><td></td></tr><tr><td>Frosted Microscope Slides</td><td>Fisher Scientific</td><td>12-550-343</td><td></td></tr><tr><td>Fisherfinest Superslip Cover Glass</td><td>Fisher Scientific</td><td>12-545-89</td><td></td></tr><tr><td>Cytoseal XYL</td><td>Fisher Scientific</td><td>22-050-262</td><td></td></tr><tr><td>Olympus Provis AX70 Microscope</td><td>Olympus</td><td>OLYMPUS-AX70</td><td></td></tr><tr><td>U-PHOTO Universal Photo System</td><td>Olympus</td><td>OLYMPUS-U-PHOTO</td><td></td></tr><tr><td>U-MCB-2 MULTI CONTROL BOX</td><td>Olympus</td><td>OLYMPUS-U-MCB-2</td><td></td></tr><tr><td>U-PS POWER SUPPLY UNIT</td><td>Olympus</td><td>OLYMPUS-U-PS</td><td></td></tr><tr><td>FreeZone 1 Liter Benchtop Freeze Dry System</td><td>LABCONCO</td><td>7740020</td><td></td></tr><tr><td>Maxima C&amp;nbsp;Plus&amp;nbsp;Vacuum Pump</td><td>Fisher Scientific</td><td>01-257-80</td><td>Displacement- 6.1 cfm</td></tr><tr><td>1-Butanol</td><td>Fisher Scientific</td><td>A383-4</td><td></td></tr><tr><td>AMRESCO&amp;nbsp;PHENOL-CHLORFORM-OSOAMYL 100ML&amp;nbsp;DFS</td><td>Fisher Scientific</td><td>NC9573988</td><td></td></tr><tr><td>Free-Standing Microcentrifuge Tubes with Screw Caps</td><td>Fisher Scientific</td><td># 02-682-557</td><td></td></tr><tr><td>Mini-Beadbeater-24</td><td>BioSpec Products</td><td>112011</td><td></td></tr><tr><td>BioSpec ProductsSupplier Diversity Partner 2.3 MM ZIRCONIA BEADS</td><td>Fisher Scientific</td><td>NC0451999</td><td></td></tr><tr><td>Tris Base (White Crystals or Crystalline Powder/Molecular Biology)</td><td>Fisher Scientific</td><td>BP152-1</td><td></td></tr><tr><td>Hydrochloric Acid, Certified ACS Plus</td><td>Fisher Scientific</td><td>A144S</td><td></td></tr><tr><td>Ethylenediaminetetraacetic Acid, Disodium Salt Dihydrate</td><td>Fisher Scientific</td><td>S311</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS), White Powder, Electrophoresis</td><td>Fisher Scientific</td><td>BP166</td><td></td></tr><tr><td>Chloroform/isoamyl alcohol 24:1</td><td>Fisher Scientific</td><td>AC327155000</td><td></td></tr><tr><td>Biotek Epoch Microplate Spectrophotometer</td><td>Fisher Scientific</td><td>11-120-570</td><td></td></tr><tr><td>Thermo Scientific&amp;trade;&amp;nbsp;ABsolute Blue qPCR Mixes</td><td>Fisher Scientific</td><td>AB4137A</td><td></td></tr><tr><td>Hybridization Probe, 5&amp;prime;-FAM-AGCCAGCGGCCCGCAAATG-TAMRA-3&amp;prime;</td><td>Integrated DNA Technologies</td><td>N/A</td><td></td></tr><tr><td>Sense Amplification Primer, 5&amp;prime;-GGCCCTTAAATAGCCCGGT-3&amp;prime;</td><td>Integrated DNA Technologies</td><td>N/A</td><td></td></tr><tr><td>Antisense Amplification Primer, 5&amp;prime;-TGAGCCGATAGTCCCCCTAA-3&amp;prime;</td><td>Integrated DNA Technologies</td><td>N/A</td><td></td></tr><tr><td>Applied Biosystems&amp;trade; MicroAmp&amp;trade; Optical 8-Tube Strip, 0.2mL</td><td>Fisher Scientific</td><td>43-165-67</td><td></td></tr><tr><td>Thermo Scientific&amp;nbsp;Domed and Flat PCR Cap Strips</td><td>Fisher Scientific</td><td>AB-0386</td><td></td></tr><tr><td>Mx3005P QPCR System, 110 Volt</td><td>Agilent</td><td>401443</td><td>Stratagene is now owned by Agilent</td></tr><tr><td>BALB/c mice</td><td>The Jackson Laboratory</td><td>000651</td><td></td></tr><tr><td>C57BL/6 (B6) mice</td><td>The Jackson Laboratory</td><td>000664</td><td></td></tr><tr><td>Eosinophil-deficient (&amp;Delta;dblGATA, BALB/c background) mice</td><td>The Jackson Laboratory</td><td>005653</td><td></td></tr><tr><td>&amp;gamma;&amp;delta; T cell-deficient (TCR&amp;delta;-/-, B6 background) mice</td><td>The Jackson Laboratory</td><td>002120</td><td></td></tr><tr><td>ImageJ Software</td><td>National Institutes of Health</td><td>N/A</td><td>https://imagej.nih.gov/ij/</td></tr><tr><td>Spot Advanced Software</td><td>Spot Imaging</td><td>SPOT53A</td><td>http://www.spotimaging.com/software/spot-advanced/</td></tr><tr><td>GraphPad Prism 6</td><td>GraphPad Software</td><td>N/A</td><td>https://www.graphpad.com/scientific-software/prism/</td></tr><tr><td>Fisherbrand Absorbent Underpads</td><td>Fisher Scientific</td><td>14-206-62</td><td></td></tr><tr><td>Step 4.5</td><td></td><td></td><td>http://www.bdbiosciences.com/sg/resources/protocols/paraffin_sections.jsp ; http://jove.unicartagenaproxy.elogim.com/science-education/5039 ; http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/General_Information/1/ht100.pdf</td></tr></tbody></table>

histological quantification to determine lung fungal burden in experimental aspergillosis

肺真菌负担的量化是确定肺真菌感染小鼠模型中免疫保护和真菌毒力相对水平的关键。虽然用多种方法评估真菌的负担, 但真菌 DNA 的定量聚合酶链反应 (qPCR) 已成为一种技术, 与以往的基于文化的方法相比具有多种优势。目前, 对侵袭性曲霉病 (IA) 小鼠的肺病理学、白细胞招募、真菌负担和基因表达进行综合评估, 必须使用大量的实验和控制动物。这里详细研究了用减少的动物数量来确定真菌负担的肺组织学染色的量化。肺切片染色, 以确定真菌结构与戈木日的改良甲胺银 (GMS) 染色。图像取自 GMS 染色的部分, 从4个离散的领域, 每一个福尔马林固定石蜡嵌入肺。每个图像中的 GMS 染色区域都使用图像分析程序进行量化, 从这个量化方法中, 每个样本都确定了染色面积的平均百分比。采用这一策略, 嗜酸细胞缺乏的小鼠在卡泊芬治疗中表现出真菌负担和疾病的减少, 而与卡泊芬的野生型小鼠没有改善。同样, 卡泊芬, qPCR 和 GMS 的定量测定, 也改善了缺乏γδ T 细胞的小鼠的真菌负担。因此, 将 GMS 量化作为一种测定相对肺部真菌负担的方法, 最终可能减少对侵袭性曲霉病进行综合研究所需的实验动物数量。

图 4包括卡泊芬治疗 IA 的野生型或嗜酸细胞不足小鼠生存图。结果表明, 嗜酸性粒细胞不足的小鼠与野生型小鼠相比 (分别为50% 的死亡率和100% 的死亡率) 表现出更大的生存率。图 5显示了由粒细胞减少野生型和嗜酸性粒细胞不足的小鼠卡泊芬治疗或未治疗的代表性 GMS 染色。卡泊芬治疗导致嗜酸细胞缺乏的相对真菌清除, 但不是野生型小鼠 (右面板), 而两组没有收到卡泊芬是相似的 (左面板)。图 6显示了卡泊芬治疗和控制未经处理的野生型或嗜酸细胞缺乏小鼠的真菌负担的比较, 使用真菌 DNA 的 qPCR (图 6A) 和代表性的 GMS 量化 4 (10X 客观) 领域 (图 6B)。这两种技术产生的结果表明, 卡泊芬治疗导致野生型和嗜酸细胞不足小鼠最显著的真菌负担减少 (图 6A)。然而, 在未经治疗的小鼠中, 只有 GMS 量化导致缺乏嗜酸性粒细胞的小鼠显著减少 (图 6B)。计算全肺 GMS 染色断面平均面积 (4X 目标) 时, 其差异与4个代表性10X 字段的结果相似, 但统计意义较小 (图 6C)。图 7显示了具有代表性的 GMS 染色 (4 个10X 字段) 的生存期、图像和真菌负担的两种方法在γδ T 细胞缺乏 (TCRδKO) 小鼠的治疗与控制, 未经治疗的小鼠的卡泊芬。卡泊芬治疗改善生存 (图 7A) 和真菌负担 (图 7BD) 在 TCRδKO 小鼠。与图 6的结果类似, 由 qPCR (图 7B) 和代表性 GMS 量化 (图 7C) 测量的真菌负担的结果是可比较的。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57155/57155fig4.jpg"> 图 4: 卡泊芬治疗后嗜酸性粒细胞缺乏 (ΔdblGATA) 小鼠的存活率增加.15-30 小鼠/组。3-6 项实验总结。p < 0.0001。此数字是从以前的出版物8中修改的。<a href="//cloudflare.jove.com/files/ftp_upload/57155/57155fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57155/57155fig5.jpg"> 图 5: 由野生型、嗜酸性粒细胞缺乏、卡泊芬治疗或控制未治疗的小鼠与 IA 染色的肺切片的代表性图像.代表3-4 只老鼠/组。刻度条等效于100µm。此数字是从以前的出版物8中修改的。<a href="//cloudflare.jove.com/files/ftp_upload/57155/57155fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57155/57155fig6.jpg"> 图 6: 卡泊芬治疗或控制的野生型嗜酸粒细胞不足小鼠的真菌负担.(A) qPCR 真菌 DNA。15-30 小鼠/组, 总结3-6 项实验。(B) gms 定量的组织学切片, mean% 4 个领域的 gms 染色 (10x 目标)。(C) GMS 量化, mean% 全肺切片 (4X 目标)。B 和 C 是5小鼠/组的总结。* p < 0.05。p < 0.001。p < 0.0001。此数字是使用以前发布的8中报告的数据生成的。<a href="//cloudflare.jove.com/files/ftp_upload/57155/57155fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57155/57155fig7.jpg"> 图 7: 卡泊芬治疗后γδ T 细胞缺陷小鼠 IA 的严重程度下降.(A) 生存。(B) 真菌 DNA 的 qPCR。(C) GMS 对组织学的量化。(D) 由γδ T 细胞与 IA、治疗或未治疗的卡泊芬染色切片的代表性图像。a 和 B 是两个实验的总结, 7-10 只老鼠/组。C 和 D 是4小鼠/组的总结。* p < 0.05。** p < 0.01。p < 0.001。此数字是从以前的出版物8中修改的。<a href="//cloudflare.jove.com/files/ftp_upload/57155/57155fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis at JoVE.com

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

在这里我们描述了一个协议, 以确定侵袭性曲霉病小鼠肺部真菌负担的定量戈木日修饰甲胺银染色的组织学切片。这种方法的应用结果与较少的动物相比, 通过定量 PCR 的肺真菌 DNA 评估的真菌负担比较。

实验性曲霉病肺真菌负担的组织学定量测定

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

histological-quantification-to-determine-lung-fungal-burden

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

10.4K 次观看.  Indiana University School of Medicine - Terre Haute. 在这里我们描述了一个协议, 以确定侵袭性曲霉病小鼠肺部真菌负担的定量戈木日修饰甲胺银染色的组织学切片。这种方法的应用结果与较少的动物相比, 通过定量 PCR 的肺真菌 DNA 评估的真菌负担比较。

视频: Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

使用双光子显微镜在受感染的肺部的嗜中性粒细胞吞噬和NET形成的直接观察

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

科研

免疫与感染

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

使用内核生物测定真菌孢子发生和霉菌毒素生产的定量

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

使用的细胞图像分析仪量化致病真菌与宿主细胞协会

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

利用流细胞测定人类白细胞测定阿斯珀吉鲁斯紫锥体烟曲的噬菌体

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

斑马鱼幼虫感染 阿斯珀吉卢斯 孢子分析宿主-病原体相互作用

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and immunological tolerance, while in immunocompromised patients, conidia can colonize airways and cause severe invasive respiratory disorders. Various cells in different airway compartments are involved in the immune response that prevents fungal invasion; however, the spatio-temporal aspects of pathogen elimination are still not completely understood. Three-dimensional (3D) imaging of optically cleared whole-mount organs, particularly the lungs of experimental mice, permits detection of fluorescently labeled pathogens in the airways at different time points after infection. In the present study, we describe an experimental setup to perform a quantitative analysis of A. fumigatus conidia distribution in the airways. Using fluorescent confocal laser scanning microscopy (CLSM), we traced the location of fluorescently labeled&#160;conidia in the bronchial branches and the alveolar compartment 6 hours after oropharyngeal application to mice. The approach described here was previously used for detection of the precise pathogen location and identification of the pathogen-interacting cells at different phases of the immune response. The experimental setup can be used to estimate the kinetics of the pathogen elimination in different pathological conditions.

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

We describe the method for quantitative analysis of the distribution of Aspergillus fumigatus conidia (3 &#181;m in size) in the airways of mice. The method also can be used for the analysis of microparticles and nanoparticle agglomerate distribution in the airways in various pathological condition models.

聚焦激光扫描显微镜为基础的定量分析 阿斯珀吉卢斯熏蒸 孔尼迪亚分布在全山光学清除鼠肺

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and ...

<meta charset="utf8"></head><body>保留肺部真菌孢子定位以进行荧光显微镜分析

Efficient Method for Imaging Murine Lungs that Preserves Spatial Dynamics of Fungal Spores in the Airways

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and bronchioles, branch until terminating in the alveolar airspace where gas exchange occurs. Infections originating in the bronchioles or alveoli elicit distinct host responses and disease manifestations. Therefore, understanding precisely where spores naturally localize in the lungs, particularly soon after infection, expands opportunities for investigation of host-pathogen interactions. Herein, we detail an in-situ analysis of lungs from mice infected with Coccidioides posadasii cts2/ard1/cts3&#916; arthroconidia. Conventional methods for histological preservation involve liquid inflation of the airways with a fixative solution, which displaces the natural location of aspirated fungal particles, pushing spores from proximal bronchioles to terminal airspaces.
Conversely, this method of air-inflation with blood vasculature perfusion-fixation preserves the physiologic position of fungal spores within the bronchioles. Moreover, we describe a simple approach to cryopreserving, embedding, and imaging lung specimens. We also share high-throughput computational techniques via the open-source QuPath program to analyze the spatial distribution of fungal spores within the lung. The method presented here is simple and quick, requires minimal equipment to perform, and can be easily adapted for use with many respiratory fungal infection models.

<meta charset="utf8"></head><body>保留气道中真菌孢子空间动力学的小鼠肺成像的有效方法

We present a method for a fungal pathogenesis model that preserves the natural positioning of fungal spores in the lung airways for analysis via fluorescent microscopy.

保留气道中真菌孢子空间动力学的小鼠肺成像的有效方法

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and ...

实验性曲霉病肺真菌负担的组织学定量测定

摘要

探索更多视频

此视频中的章节

使用双光子显微镜在受感染的肺部的嗜中性粒细胞吞噬和NET形成的直接观察

使用内核生物测定真菌孢子发生和霉菌毒素生产的定量

使用的细胞图像分析仪量化致病真菌与宿主细胞协会

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

利用流细胞测定人类白细胞测定阿斯珀吉鲁斯紫锥体烟曲的噬菌体

斑马鱼幼虫感染阿斯珀吉卢斯孢子分析宿主-病原体相互作用

聚焦激光扫描显微镜为基础的定量分析阿斯珀吉卢斯熏蒸孔尼迪亚分布在全山光学清除鼠肺

保留气道中真菌孢子空间动力学的小鼠肺成像的有效方法

保留气道中真菌孢子空间动力学的小鼠肺成像的有效方法

保留气道中真菌孢子空间动力学的小鼠肺成像的有效方法

实验性曲霉病肺真菌负担的组织学定量测定

摘要

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此视频中的章节

使用双光子显微镜在受感染的肺部的嗜中性粒细胞吞噬和NET形成的直接观察

使用内核生物测定真菌孢子发生和霉菌毒素生产的定量

使用的细胞图像分析仪量化致病真菌与宿主细胞协会

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

利用流细胞测定人类白细胞测定阿斯珀吉鲁斯紫锥体烟曲的噬菌体

斑马鱼幼虫感染 阿斯珀吉卢斯 孢子分析宿主-病原体相互作用

聚焦激光扫描显微镜为基础的定量分析 阿斯珀吉卢斯熏蒸 孔尼迪亚分布在全山光学清除鼠肺

保留气道中真菌孢子空间动力学的小鼠肺成像的有效方法

保留气道中真菌孢子空间动力学的小鼠肺成像的有效方法

保留气道中真菌孢子空间动力学的小鼠肺成像的有效方法

斑马鱼幼虫感染阿斯珀吉卢斯孢子分析宿主-病原体相互作用

聚焦激光扫描显微镜为基础的定量分析阿斯珀吉卢斯熏蒸孔尼迪亚分布在全山光学清除鼠肺