Name: deep dermal injection as a model of candida albicans skin infection for histological analyses
Uploaded: 2018-06-13T13:00:00.000+00:00
Duration: 10 min 45 s
Description: Watch this Scientific Journal Video about Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses at JoVE.com

Associazione 意大利每 la Ricerca Cancro (2016 号 18842), Cariplo 基金会 (赠款 2014-0655), 乔瓦尼博物馆每 la Ricerca 牛津 (FRRB) 支持。 伊兹获得 NIH 赠款 1R01AI121066-01A1, 1R01DK115217, HDDC P30 DK034854 补助金, 哈佛医学院弥尔顿发现, CCFA 高级研究奖 (412708), 埃莉诺和迈尔斯海岸第五十周年奖学金计划, Cariplo 基金会 (2014-0859)。

所有程序均经机构动物护理和使用委员会 (IACUC) 批准, 并在波士顿儿童医院儿童医院动物资源部的监督下运作, 或经意大利卫生部并在米兰大学 Bicocca 的机构动物护理委员会下进行。
1.白念珠菌制剂
<ol><li>文化C. 白念珠菌, 应变 CAF3-113, 在含有丰富培养基的管 (酵母萃取蛋白胨葡萄糖 (YEPD), 1% (w/v) 酵母提取物, 2% (w/v) Bacto 蛋白胨, 2% (瓦特/v) 葡萄糖) 补充苷 (50 毫克/升) 在25摄氏度。在这些条件下,白色念珠菌生长为酵母。 注: 其他c. 白念珠菌可使用, 如临床分离c. 白色念珠菌 SC531413。因为我们最近证明了溃疡过程是由先天免疫系统的活化引起的14, 理论上每一个C. 白念珠菌保持其细胞壁成分和结构, 以及有能力发起菌丝预测, 可以使用。</li>
	<li>使用细胞计数器分析仪对细胞浓度进行计数, 以此来监测文化。</li>
	<li>当它达到大约 8 x 106细胞/毫升的浓度时, 收获它的文化。或者, 使用分光光度计测量 OD600。 注: 通常, OD600 = 1 的值对应于 3 x 107细胞/毫升的酵母浓度, 但建议滴定。</li>
	<li>并用重悬 YEPD-苷培养基中的培养, 以 HEPES (50 毫米, pH 7.5) 为缓冲剂, 孵育37°c 的培养, 诱导菌丝形成。用100X 放大显微镜检查菌丝的形成。在37摄氏度培养后, 菌丝形成可见5小时;然而, 当菌丝百分比达到总培养的近95% 时, 在孵化后的37摄氏度使用细胞16小时。</li>
	<li>为了进一步丰富制剂的菌丝浓度, 离心机整除数的培养在 3300 x g 5 分钟. 弃上清, 并并用重悬在无菌 PBS 中的颗粒浓度 1 x 108菌丝/毫升。</li>
</ol>2. 深部真皮注射
<ol><li>24 h 在感染程序之前, 麻醉小鼠腹腔注射氯胺酮 (80-100 毫克/千克) 和甲苯噻嗪 (10 毫克/千克), 然后用电动剪刀剃掉老鼠的侧面。 注: 在任何麻醉过程中, 将老鼠放在加热灯下, 直到有效地恢复。</li>
	<li>休息的小鼠24小时, 以避免任何炎症由于剃须过程。</li>
	<li>24小时后剃须程序, 麻醉小鼠与腹腔注射氯胺酮 (80-100 毫克/千克) 和甲苯噻嗪 (10 毫克/千克)。</li>
	<li>检查爪子反射, 以确保小鼠被深深麻醉。通过牢牢捏爪来评估爪子反射。如果麻醉达到了, 动物不会感到疼痛, 也不会移动。</li>
	<li>用0.3 毫升胰岛素注射器在深真皮中注射白色念珠菌菌丝, 其最终容积为50µL/注射液, 对应 5 x 106菌丝/注射液。<ol>
<li>用两个手指拉紧剃的侧面的皮肤, 以便直接在深真皮中注入体积。用针朝上的斜角插入 10-半角的针。</li>
		<li>以这个角度, 病原体将被注入大约 300-500 µm 的深度。为了确保注射良好, 检查注射的体积是否形成一个肿块, 在注射后吸收几分钟。 注: 注射后形成的肿块约为1厘米2。这是将被删除的区域, 在指定的时间点, 无论是分子或组织学分析。</li>
		<li>为少于 24 h 的时间点 (推荐) 标记传染区域与标记笔, 因为与射入形成的凹凸持续几分钟, 在之后它被吸收。对于24小时或以上的时间点, 这一步骤是不需要的, 因为溃疡或囊肿将明显可见。</li>
	</ol>
</li>
</ol>3. 组织学染色标本的收集和准备
<ol><li>根据制度程序弄死老鼠。</li>
	<li>使用手术钳, 拉在注射部位的边界, 以前标记与标记笔的皮肤。使用手术剪刀, 通过切割的皮肤后, 标记线的感染部位。 注意: 小心用圆尖剪刀将皮肤与腹膜分开。</li>
	<li>将皮肤浸泡在一次性的基模中, 填充最佳切削温度 (OCT) 化合物, 与皮肤的内部面朝上。在液氮中冷冻样品, 直到化合物变成白色。不要让液氮覆盖样品之前, 它完全冻结, 因为这将损害样品。 注意: 小心!在步骤3.3 中, 戴上防护罩, 防止液态氮。</li>
	<li>如果样品不立即用于幻灯片准备, 迅速储存在-80 摄氏度。 注: 样品可以无限期储存在-80 摄氏度。</li>
</ol>4. 幻灯片的制作
<ol><li>将样品切成两半, 从样品的中心开始为组织学准备切片。</li>
	<li>用恒温器切开5µm 厚片。 注: 对于皮肤样品, 恒温器的温度不应高于摄氏-25 摄氏度。较高的温度会导致 H OCT 部分熔化, 因此样品在切割过程中不会保持在正确的位置。</li>
	<li>使用带正电荷的显微镜幻灯片收集切片。</li>
	<li>如果在准备后不久不使用收集的切片, 请将其存储在摄氏-20 摄氏度。此时, 切片可以无限期地存储在-20 摄氏度。</li>
</ol>5. 苏木精 & 伊红染色
<ol><li>浸泡在鳃的苏木素4分钟, 使幻灯片着色. 在自来水中冲洗幻灯片5分钟。</li>
	<li>浸泡在1分钟内, 用浸没在红 Y 溶液中的幻灯片来着色. 用蒸馏水冲洗幻灯片, 然后继续执行该协议。</li>
	<li>浸泡在连续增加的乙醇溶液浓度 (50%, 70%, 80%, 95%, 100%) 脱水。在每个乙醇溶液中浸泡十五年代的幻灯片。</li>
	<li>通过浸泡在组织学清除剂中, 清除幻灯片至少2分钟。</li>
	<li>使用安装介质和盖子滑块装入幻灯片。</li>
	<li>以40X 倍的比例获取带有显微镜幻灯片扫描仪的幻灯片图像。</li>
</ol>6. 周期性酸希夫 (PAS) 染色
<ol><li>用≥99.5% 丙酮固定在室温下的幻灯片1分钟. 在自来水中冲洗幻灯片1分钟。</li>
	<li>浸泡在周期性酸溶液中 (1 克/dL), 以5分钟的方式对幻灯片进行着色. 在3的蒸馏水中冲洗幻灯片。</li>
	<li>浸泡在席夫的试剂15分钟, 使幻灯片着色. 在自来水中冲洗幻灯片5分钟。</li>
	<li>浸泡在鳃的苏木素5分钟, 使幻灯片着色. 在三十年代的自来水中冲洗滑梯。</li>
	<li>浸泡3的100% 乙醇的变化, 脱水的幻灯片。每一次将幻灯片浸入十五年代。</li>
	<li>通过浸泡在组织学清除剂中, 清除幻灯片至少2分钟。</li>
	<li>使用安装介质和盖子滑块装入幻灯片。</li>
	<li>使用显微镜幻灯片扫描仪获取幻灯片的图像。</li>
</ol>7. RNA 提取的样品收集和准备
<ol><li>根据制度程序弄死老鼠。删除步骤3.2 中的外观示例。</li>
	<li>将样品浸入2毫升的盖管中, 用1毫升的试剂胍硫氰酸盐-苯酚-氯仿萃取液。 注意: 协议可以在这里暂停, 样品可以存储在-80 摄氏度。</li>
	<li>用手术钳将样品切成大约0.2 厘米2块。 注意: 小心!大多数 RNA 分离试剂是有毒和诱变的。步骤7.3 和 7.4必须在通风罩下完成。</li>
	<li>以20振荡/秒的速度在20分钟内粉碎样品与珠磨房. 或者, 可以使用机械波特。</li>
	<li>检查样品的有效均匀性, 寻找是否有完整的皮肤片。如果样品不是完全均匀的, 重复此步骤。</li>
	<li>离心样品在 1.6万 x g 1 分钟保持上清液 RNA 提取和丢弃颗粒。执行此步骤可消除可能阻止抽取列的毛发和碎片。</li>
	<li>使用 rna 纯化柱14进行 rna 提取。</li>
	<li>使用分光光度计评估 RNA 的纯化和浓度。如果 RNA 提取物不立即用于 cDNA 准备, 保存样品在-80 °c。</li>
</ol>

<ol>
	<li>Belkaid, Y., Segre, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dialogue+between+skin+microbiota+and+immunity.">Dialogue between skin microbiota and immunity.</a> Science. 346 (6212), 954-959 (2014).</li><li>Kashem, S. W., Kaplan, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+Immunity+to+Candida+albicans.">Skin Immunity to Candida albicans.</a> Trends Immunol. 37 (7), 440-450 (2016).</li><li>Havlickova, B., Czaika, V. A., Friedrich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiological+trends+in+skin+mycoses+worldwide.">Epidemiological trends in skin mycoses worldwide.</a> Mycoses. 51, 2-15 (2008).</li><li>Wisplinghoff, H., Bischoff, T., Tallent, S. M., Seifert, H., Wenzel, R. P., Edmond, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nosocomial+Bloodstream+Infections+in+US+Hospitals:+Analysis+of+24,179+Cases+from+a+Prospective+Nationwide+Surveillance+Study.">Nosocomial Bloodstream Infections in US Hospitals: Analysis of 24,179 Cases from a Prospective Nationwide Surveillance Study.</a> Clin Infect Dis. 39 (3), 309-317 (2004).</li><li>Romani, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity+to+fungal+infections.">Immunity to fungal infections.</a> Nat Rev Immunol. 4 (1), 11-24 (2004).</li><li>Odds, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Candida+infections.">Pathogenesis of Candida infections.</a> J Am Acad Dermatol. 31 (3), S2-S5 (1994).</li><li>MacCallum, D. M., Odds, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+events+in+the+intravenous+challenge+model+for+experimental+Candida+albicans+infections+in+female+mice.">Temporal events in the intravenous challenge model for experimental Candida albicans infections in female mice.</a> Mycoses. 48 (3), 151-161 (2005).</li><li>Dai, T., Kharkwal, G. B., Tanaka, M., Huang, Y. -. Y., Bil de Arce, V. J., Hamblin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+external+traumatic+wound+infections.">Animal models of external traumatic wound infections.</a> Virulence. 2 (4), 296-315 (2018).</li><li>Gaspari, A. A., Burns, R., Nasir, A., Ramirez, D., Barth, R. K., Haidaris, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD86+(B7-2),+but+not+CD80+(B7-1),+expression+in+the+epidermis+of+transgenic+mice+enhances+the+immunogenicity+of+primary+cutaneous+Candida+albicans+infections.">CD86 (B7-2), but not CD80 (B7-1), expression in the epidermis of transgenic mice enhances the immunogenicity of primary cutaneous Candida albicans infections.</a> Infect Immun. 66 (9), 4440-4449 (1998).</li><li>Koh, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+models+of+Candida+gastrointestinal+colonization+and+dissemination.">Murine models of Candida gastrointestinal colonization and dissemination.</a> Eukaryot Cell. 12 (11), 1416-1422 (2013).</li><li>Mear, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+Airway+Exposure+Primes+the+Lung+Innate+Immune+Response+against+Pseudomonas+aeruginosa+Infection+through+Innate+Lymphoid+Cell+Recruitment+and+Interleukin-22-Associated+Mucosal+Response.">Candida albicans Airway Exposure Primes the Lung Innate Immune Response against Pseudomonas aeruginosa Infection through Innate Lymphoid Cell Recruitment and Interleukin-22-Associated Mucosal Response.</a> Infect Immun. 82 (1), 306-315 (2014).</li><li>Leoni, G., Neumann, P. -. A., Sumagin, R., Denning, T. L., Nusrat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair:+role+of+immune-epithelial+interactions.">Wound repair: role of immune-epithelial interactions.</a> Mucosal Immunol. 8 (5), 959-968 (2015).</li><li>Fonzi, W. A., Irwin, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isogenic+strain+construction+and+gene+mapping+in+Candida+albicans.">Isogenic strain construction and gene mapping in Candida albicans.</a> Genetics. 134 (3), 717-728 (1993).</li><li>Santus, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+infections+are+eliminated+by+cooperation+of+the+fibrinolytic+and+innate+immune+systems.">Skin infections are eliminated by cooperation of the fibrinolytic and innate immune systems.</a> Sci Immunol. 2 (15), (2017).</li><li>Florescu, D. F., Brostrom, S. E., Dumitru, I., Kalil, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+Skin+Abscess+in+a+Heart+Transplant+Recipient.">Candida albicans Skin Abscess in a Heart Transplant Recipient.</a> Infect Dis Clin Pract. 18 (4), 243-246 (2010).</li><li>Pradeu, T., Cooper, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+danger+theory:+20+years+later.">The danger theory: 20 years later.</a> Front Immunol. 3, 287 (2012).</li><li>Cheng, A. G., DeDent, A. C., Schneewind, O., Missiakas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+play+in+four+acts:+Staphylococcus+aureus+abscess+formation.">A play in four acts: Staphylococcus aureus abscess formation.</a> Trends Microbiol. 19 (5), 225-232 (2011).</li></ol>

作者没有什么可透露的。

在这里, 我们描述了一种C. 白念珠菌感染的方法, 以研究在深层真皮真菌入口引发的炎症过程。
虽然皮肤脓肿的形成是一个相对罕见的事件后, C. 白色念珠菌感染15, 直接在深真皮中注射真菌不仅可以研究真菌驱动脓肿的形成, 而且还分析特异免疫参与控制真菌扩散的细胞。在这里描述的方法, 可以研究免疫细胞之间的相互作用, 以及免疫细胞的招募是重要的控制真菌传播, 同时避免任何炎症反应机械损伤或伤口形成。事实上, 保持上皮结构完好的可能性对于避免由于机械刺激 (如磨损) 引起的炎症环境的发展非常重要。正如波莉 Matzinger 在1994年首次提出的理论, 损伤信号实际上可以激活免疫应答, 因此完全影响病原体引起的免疫应答。
损伤相关的分子模式 (抑) 可以起源于死细胞, 但也可以释放在压力条件下。由于这些信号可以在组织损伤的情况下产生, 伤口的形成即使在感染的背景下也会引起超炎症的环境。我们所描述的方法限制了抑的释放, 从而减少了混杂元素的存在。
此外, 直接在深层真皮中注射病原体是一种方法, 不仅可以用于真菌感染, 而且还可用于细菌挑战。另一种病原体是皮肤和血流感染病例增加的原因是金黄色葡萄球菌4。我们最近展示了该协议也是研究金黄色葡萄球菌感染14的重要工具。特别是, S. 金黄色葡萄球菌是已知的在临床上, 它的能力形成脓肿在身体的几个地区17。在深真皮中注射金黄色葡萄球菌导致脓肿的形成, 从而使研究早期发生的事件引起的金黄色葡萄球菌接触以及免疫应答的参与, 导致愈合相。
最后, 这里描述的方法的另一个重要特点是, 保持一个完整的上皮结构提供了组织学分析免疫细胞的定位和激活不同途径的可能性, 可以空间分割。在这些实验条件下, 事实上, 皮肤的不同解剖结构没有改变, 免疫细胞和炎症介质可以在一个精确的位置被可视化。
因此, 使用这种方法保留免疫细胞的空间分布是理想的, 以更好地了解每个细胞类型的作用, 在真菌和细菌性皮肤感染。

人体上覆盖着极高数量的微生物。皮肤表面是大约100万细菌的栖所每平方厘米1。然而, 这个数字并没有反映出殖民皮肤的各种微生物。除了细菌外, 人体还被许多真菌物种所殖民, 包括白色念珠菌, 它能够在粘膜和皮肤2级存活。
近年来, 被诊断为真菌感染的人的百分比大大增加。这主要是由于免疫机能丧失者的数量增加,即HIV 阳性患者和3移植后经过化疗或免疫抑制剂的患者。在美国进行的一项监测研究中, Wisplinghoff等人发现, 9.5% 的医院血流感染是由念珠菌4种引起的。由于真菌感染的增加, 特别是由于血液感染中发现的念珠菌的比例升高, 了解这种病原体如何逃脱免疫系统的控制是非常重要。
白色念珠菌是一种二形真菌, 生长在不同形态形态, 如酵母, blastospores, 假菌丝和菌丝, 取决于环境条件5。在其菌丝的形式,白色念珠菌显示其最高的侵袭能力, 并有能力穿透上皮6。
用几种实验方法对白念珠菌感染进行了研究。最常见的感染模型是静脉注射白色念珠菌酵母7。然而, 这个模型没有考虑到所有的过程中发生的真菌传播到血液中。另一种模型利用C. 白念珠菌侵入上皮细胞的能力。这种方法, 也称为砂纸模型8, 是由 Gaspari等在1998年开发的9, 并包括使用沙纸擦伤皮肤, 从而消除角质层之前, 应用C. 白念珠菌。这个过程允许真菌穿透上皮, 从而能够分析这种病原体的侵袭能力。最后, 其他模型的胃肠道10和呼吸道感染11已被用于不同的研究。
伤口的形成 (如在沙纸模型中) 导致了几个途径的活化, 包括免疫细胞的招募和活化, 以促进愈合过程12。这可以改变或掩盖对病原体特别诱发的免疫反应, 从而导致混淆结果。
在这里, 我们描述一种皮肤感染的方法, 避免最初的伤口形成和诱导的基底炎症环境。为了保持完整的上皮结构, 我们直接在深真皮中注射菌丝形式的白色念珠菌。尽管单个注射可以引起轻度炎症, 但与在沙纸模型中的开放性伤口的形成相比, 炎症的数量受到限制和限制。我们在这里描述的方法允许研究真菌感染和传播的免疫反应, 同时避免机械损伤造成的过度和预先存在的炎症环境。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PBS</td><td>Euroclone</td><td>ECB4053L</td><td>warm in 37 &amp;deg;C bath before use</td></tr><tr><td>H-OCT compound</td><td>histo-line laboratories</td><td>R0030</td><td></td></tr><tr><td>Gill&#039;s Hematoxilyn</td><td>histo-line laboratories</td><td>09-178-2</td><td></td></tr><tr><td>Eosin Y solution, alcoholic</td><td>histo-line laboratories</td><td>09-209-05</td><td></td></tr><tr><td>Ethanol absolute</td><td>scharlau</td><td>ET00232500</td><td></td></tr><tr><td>Citro-HISTOCLEAR</td><td>histo-line laboratories</td><td>R0050</td><td></td></tr><tr><td>Eukitt, mounting medium</td><td>bio-optica</td><td></td><td></td></tr><tr><td>Acetone</td><td>sigma-aldrich</td><td>179124</td><td></td></tr><tr><td>PAS staining system</td><td>sigma-aldrich</td><td>395B-1KT</td><td></td></tr><tr><td>Bacto Peptone</td><td>BD</td><td>211677</td><td></td></tr><tr><td>Bacto Yeast Extract</td><td>BD</td><td>212750</td><td></td></tr><tr><td>D(+)-Glucose anhydrous for molecular biology</td><td>Applichem PanReac</td><td>50-99-7</td><td></td></tr><tr><td>Uridine</td><td>Merck Millipore</td><td>8451</td><td></td></tr><tr><td>HEPES</td><td>Applichem PanReac</td><td>A1070,0500</td><td></td></tr><tr><td>Safe-Lock tubes 2 mL</td><td>eppendorf</td><td>30121597</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Life Technologies</td><td>15596018</td><td>Toxic, corrosive and mutagen. Use all precaution needed</td></tr><tr><td>Rneasy Mini Kit</td><td>QIAGEN</td><td>74104</td><td></td></tr><tr><td>liquid nitrogen</td><td></td><td></td><td>Wear eye protection</td></tr><tr><td>&lt;strong&gt;Instrument&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Coulter Counter-Particle Count</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Centrifuge 5415 R</td><td>eppendorf</td><td></td><td></td></tr><tr><td>MC 3000 Microtome Cryostat</td><td>histo-line laboratories</td><td></td><td></td></tr><tr><td>TissueLiser</td><td>QIAGEN</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.3 ml Insulin Syringe with a 30G x 8mm needle&amp;nbsp;</td><td>BD</td><td>324826</td><td></td></tr><tr><td>Surgical forceps</td><td></td><td></td><td></td></tr><tr><td>Surgical scissors</td><td></td><td></td><td></td></tr><tr><td>Base mould disposable</td><td>histo-line laboratories</td><td>2781</td><td></td></tr><tr><td>Positively charged bio microscope slides</td><td>bio-optica</td><td>09-2000</td><td></td></tr><tr><td>Cover slips 24 x 50 mm</td><td>thermo scientific</td><td>11911998</td><td></td></tr></tbody></table>

deep dermal injection as a model of candida albicans skin infection for histological analyses

皮肤是身体的一个非常扩展的器官, 并且, 由于这个大表面, 它持续暴露于微生物。皮肤损伤很容易导致真皮的感染, 进而导致病原体向血液中传播。了解免疫系统在早期如何对抗感染, 以及宿主如何消除病原体是确定今后治疗干预的基础的重要步骤。在这里, 我们描述了一种白色念珠菌感染模型, 可以可视化的过程中发生的早期感染, 包括当病原体已经通过了上皮屏障, 以及由C. 白念珠菌诱发的免疫反应入侵。我们利用这种感染模型进行组织学分析, 显示了渗透皮肤的免疫细胞以及病原体的存在和定位。在感染后收集的样本可以处理 RNA 提取。

通过直接在深部真皮中注射病原体, 组织结构形态保持不变 (图 1A)。
皮肤结构完整性的维护允许检测免疫细胞及其在感染部位的定位。图 1B显示的放大倍数显示, 脓肿主要由中性粒细胞组成, 其中含有病原体的传播, 其受感染部位的限制。如中性粒细胞的招聘, 可以形成脓肿, 从而避免在组织14中传播真菌。更高的放大 (图 1C, D) 显示了周围地区的脓肿也丰富了在市政局。
通过保持结构完整性, 可以确定病原体在感染过程中的定位。利用白念珠菌细胞壁中多糖含量升高的特点, 采用 PAS 染色法对病原体进行鉴定, 给出了一种紫红色的真菌。如图 2A所示, PAS 染色清楚地表明, 病原体仅限于脓肿内, 由14粒细胞的招募形成。白色念珠菌在更高的放大倍数下明显可见 (图 2B), 它似乎被坏死和免疫细胞包围。
使用上述方法, 感染过程和随后的炎症可以随着时间的推移。起初, 免疫细胞的招募导致脓肿的形成 (图 1A和图 3A)。当皮肤组织的分析延长到 48-72 小时, 皮肤结构的破坏和疤痕的形成被可视化 (图 3B, C)。这种结构的形成是由于在病原体14被驱逐之后的愈合阶段。
导致溃疡形成的过程可以在感染后的几天内被遵循。如图 4A所示, C57BL/6 野生型小鼠显示了随着时间推移而增加的溃疡过程。大约 70-80% 的老鼠在感染后显示溃疡72小时的形成。由于实验的变异性, 每项实验中至少应使用8只小鼠。在统计分析中, 如果比较不同的基因型或治疗方法, 建议对每个溃疡评分使用最少 8-10 小鼠。在这种情况下, 即使是细微的差别也应该是可见的。图 4B显示了溃疡过程的说明性图片。在特定的治疗或遗传缺陷, 溃疡的形成可以减少和/或废除14。在某些情况下, 而不是溃疡, 囊肿形成, 如图 4C所示。
除了苏木精 & 染色和 PAS 染色之外, 这些幻灯片可以被染色为特定的细胞标记或与其他免疫组化更好地可视化: 胶原纤维;细胞因子/趋化因子在组织内的位置;和免疫细胞的身份和空间分布。
除了组织学准备, 样品也可以用于分子分析。整个注射部位可以切除, 如组织学准备, 并处理 RNA 提取。RNA 提取从组织允许研究和辨认那些基因上调在传染期间与病原体。对样品进行分子分析处理的可能性是一个重要的工具, 连同组织学分析, 以便更好地表征对病原体的免疫应答类型及其在早期和晚期的调控在挑战14以后。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57574/57574fig1.jpg"> 图 1: 保持皮肤形态学和细胞定位.(A) 所有层均保留。上皮, 脂肪细胞和肌肉单元很容易辨认。(B) 可以在脓肿内确定中性粒细胞 (用箭头突出显示)。(C、 D)更高的放大显示在脓肿周围区域存在中性粒细胞 (箭头)。<a href="/files/ftp_upload/57574/57574fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57574/57574fig2.jpg"> 图 2:白念珠菌的鉴定.(A)白念珠菌可以在组织内检测到 PAS 染色。(B) 更高的放大倍数, 以更好地可视化的真菌, 染色的紫色, 局限在脓肿内。<a href="/files/ftp_upload/57574/57574fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57574/57574fig3.jpg"> 图 3: 炎症过程的随访.(A) 在由C. 白念珠菌注射液引起的炎症过程开始的时候, 为了形成脓肿来限制病原体, 我们招募了粒细胞。(B、 C)溃疡的形成是排出真菌所必需的。愈合组织的特点是改变的皮肤结构完整性, 并可能导致形成疤痕。<a href="/files/ftp_upload/57574/57574fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57574/57574fig4.jpg"> 图 4: 溃疡的动力学.(A) 溃疡过程在感染后的头几天内呈上升趋势。感染后72小时, 70-80% 的小鼠在感染部位显示溃疡。使用了 16 C57BL/6 野生型动物。(B) 感染后形成48小时溃疡的说明性图片。(C) 感染后囊肿形成的图解图。<a href="/files/ftp_upload/57574/57574fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses at JoVE.com

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

深部真皮注射液作为白色念珠菌皮肤感染模型的组织学分析

在这里, 我们描述了一个协议, 允许皮肤标本的组织学和分子分析后,白色念珠菌皮注射液。该协议维护皮肤的结构完整性, 并允许组织居民或新招募的免疫细胞的定位以及病原体的分布。

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

deep-dermal-injection-as-model-candida-albicans-skin-infection-for

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

10.7K 次观看.  University of Milano-Bicocca. 在这里, 我们描述了一个协议, 允许皮肤标本的组织学和分子分析后,白色念珠菌皮注射液。该协议维护皮肤的结构完整性, 并允许组织居民或新招募的免疫细胞的定位以及病原体的分布。

视频: Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

科研

免疫与感染

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

白色念珠菌生物膜发展在医学上，国外相关机构在小鼠皮下型号其次是生物发光成像

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

医学

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

使用荧光原位杂交对毛尿胃肠道中的念珠菌的可视化

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

澄清和成像念珠菌生物膜

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

前体内分析研究胃 肠道的 念珠菌血脂磷

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

In vivo Function of Differential Subsets of Cutaneous Dendritic Cells to Induce Th17 Immunity in Intradermal Candida albicans Infection

The skin is the outermost barrier organ in the body, which contains several types of dendritic cells (DCs), a group of professional antigen-presenting cells. When the skin encounters invading pathogens, different cutaneous DCs initiate a distinct T cell immune response to protect the body. Among the invading pathogens, fungal infection specifically drives a protective interleukin-17-producing Th17 immune response. A protocol was developed to efficiently differentiate Th17 cells by intradermal Candida albicans infection to investigate a subset of cutaneous DCs responsible for inducing Th17 immunity. Flow cytometry and gene expression analyses revealed a prominent induction of Th17 immune response in skin-draining lymph nodes and infected skin. Using diphtheria toxin-induced DC subset-depleting mouse strains, CD301b+ dermal DCs were found to be responsible for mounting optimal Th17 differentiation in this model. Thus, this protocol provides a valuable method to study in vivo function of differential subsets of cutaneous DCs to determine Th17 immunity against deep skin fungal infection.

In vivo Function of Differential Subsets of Cutaneous Dendritic Cells to Induce Th17 Immunity in Intradermal Candida albicans Infection

Here, we demonstrate the in vivo function of cutaneous dendritic cell subsets in Th17 immunity of deep dermal Candida albicans infection.

The skin is the outermost barrier organ in the body, which contains several types of dendritic cells (DCs), a group of professional antigen-presenting ...

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic changes are regulated by a complex signaling network controlled in response to environmental stimuli. These regulatory components have been highly studied, but almost all studies use a variety of in vitro stimuli to trigger filamentation. To determine how morphogenesis is regulated during the pathogenesis process, we developed an in vivo microscopy system to obtain high spatial resolution images of organisms undergoing hyphal formation within the mammalian host. The protocol presented here describes the use of this system to screen small collections of C. albicans mutant strains, allowing us to identify key regulators of morphogenesis as it occurs at the site of infection. Representative results are presented, demonstrating that some regulators of morphogenesis, such as the transcriptional regulator Efg1, have consistent phenotypes in vitro and in vivo, whereas other regulators, such as adenyl cyclase (Cyr1), have significantly different phenotypes in vivo compared to in vitro.

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

This manuscript describes a method for screening moderately sized Candida albicans mutant libraries for morphogenesis phenotypes during active infection in a mammalian host using non-invasive confocal microscopy.

使用 体内 成像筛选哺乳动物宿主活动性感染期间 白色念珠 菌突变菌株的形态发生表型

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic ...

使用姜黄素类化合物治疗小鼠口腔念珠菌病的有效 aPDT

Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for evaluating alternative treatments prior to clinical trials. This study describes the efficacy of aPDT in a murine model of oral candidiasis. Forty mice were immunosuppressed with subcutaneous injections of prednisolone, and their tongues were inoculated using an oral swab previously soaked in a C. albicans cell suspension. Tetracycline was administered via drinking water during the course of the experiment. Five days after fungal inoculation, mice were randomly distributed into eight groups; a ninth group of untreated uninfected mice was included as a negative control (n = 5). Three concentrations (20 &#181;M, 40 &#181;M, and 80 &#181;M) of a mixture of curcuminoids were tested with a blue LED light (89.2 mW/cm2; ~455 nm) and without light (C+L+ and C+L- groups, respectively). Light alone (C-L+), no treatment (C-L-), and animals without infection were evaluated as controls. Data were analyzed using Welch&#39;s ANOVA and Games-Howell tests (&#945; = 0.05). Oral candidiasis was established in all infected animals and visualized macroscopically through the presence of characteristic white patches or pseudomembranes on the dorsum of the tongues. Histopathological sections confirmed a large presence of yeast and filaments limited to the keratinized layer of the epithelium in the C-L- group, and the presence of fungal cells was visually decreased in the images obtained from mice subjected to aPDT with either 40 &#181;M or 80 &#181;M curcuminoids. aPDT mediated by 80 &#181;M curcuminoids promoted a 2.47 log10 reduction in colony counts in comparison to those in the C-L- group (p = 0.008). All other groups showed no statistically significant reduction in the number of colonies, including photosensitizer (C+L-) or light alone (C-L+) groups. Curcuminoid-mediated aPDT reduced the fungal load from the tongues of mice.

作者聚焦:探索姜黄素在口腔念珠菌病小鼠模型中的光动力疗法

This protocol describes the application of antimicrobial Photodynamic Therapy (aPDT) in a murine model of oral candidiasis. aPDT was performed using a water-soluble mixture of curcuminoids and blue LED light.

深部真皮注射液作为白色念珠菌皮肤感染模型的组织学分析

摘要

探索更多视频

此视频中的章节

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

白色念珠菌生物膜发展在医学上，国外相关机构在小鼠皮下型号其次是生物发光成像

使用荧光原位杂交对毛尿胃肠道中的念珠菌的可视化

澄清和成像念珠菌生物膜

前体内分析研究胃肠道的念珠菌血脂磷

In vivo Function of Differential Subsets of Cutaneous Dendritic Cells to Induce Th17 Immunity in Intradermal Candida albicans Infection

使用体内成像筛选哺乳动物宿主活动性感染期间白色念珠菌突变菌株的形态发生表型

姜黄素介导的抗菌光动力疗法在口腔念珠菌病小鼠模型上的应用

姜黄素介导的抗菌光动力疗法在口腔念珠菌病小鼠模型上的应用

姜黄素介导的抗菌光动力疗法在口腔念珠菌病小鼠模型上的应用

深部真皮注射液作为白色念珠菌皮肤感染模型的组织学分析

摘要

探索更多视频

此视频中的章节

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

白色念珠菌生物膜发展在医学上，国外相关机构在小鼠皮下型号其次是生物发光成像

使用荧光原位杂交对毛尿胃肠道中的念珠菌的可视化

澄清和成像念珠菌生物膜

前体内分析研究胃 肠道的 念珠菌血脂磷

In vivo Function of Differential Subsets of Cutaneous Dendritic Cells to Induce Th17 Immunity in Intradermal Candida albicans Infection

使用 体内 成像筛选哺乳动物宿主活动性感染期间 白色念珠 菌突变菌株的形态发生表型

姜黄素介导的抗菌光动力疗法在口腔念珠菌病小鼠模型上的应用

姜黄素介导的抗菌光动力疗法在口腔念珠菌病小鼠模型上的应用

姜黄素介导的抗菌光动力疗法在口腔念珠菌病小鼠模型上的应用

前体内分析研究胃肠道的念珠菌血脂磷

使用体内成像筛选哺乳动物宿主活动性感染期间白色念珠菌突变菌株的形态发生表型