这项工作得到了SRB慈善公司，美国国立卫生研究院（NIH）P30EY002520的支持，以及预防失明研究（RPB）对贝勒医学院眼科的无限制机构资助。W.L.得到了圣殿骑士团眼科基金会眼科基金会基金会的支持。

所有动物工作，包括实验程序和安乐死，均由贝勒医学院机构动物护理和使用委员会 （IACUC） 批准，协议编号为 AN-8790。
1. 1 N NaOH的制备
<ol><li>将 4 mL 无菌去离子水加入 15 mL 离心管中。称出 400 毫克氢氧化钠 （NaOH），小心地加入试管中。</li>
	<li>通过使用玻璃棒缓慢搅拌溶液来溶解NaOH。通过向试管中加入无菌去离子水，将体积补足至10mL，然后通过轻轻地上下倒置试管再次混合。盖紧盖子，将溶液储存在室温下。</li>
	<li>每月准备新鲜溶液，因为吸收空气中二氧化碳的溶液可能会降低 NaOH 溶液的浓度。</li>
	<li>使用前务必轻轻混合 NaOH 溶液。 注意：在化学防护罩内准备溶液，并穿戴适当的个人防护装备 （PPE）。</li>
</ol>2.4%多聚甲醛（PFA）溶液的制备
<ol><li>向玻璃烧杯中加入 30 mL 1x 磷酸盐缓冲盐水 （PBS）。称出 4 克多聚甲醛 （PFA），并将其加入烧杯中。</li>
	<li>搅拌下将烧杯保持在60°C的热板上。滴加1N NaOH溶液以提高pH值，直到溶液澄清。</li>
	<li>使用 1 N 盐酸 （HCl） 检查并将 pH 值调节至 7.4。用 1x PBS 将最终体积调节至 50 mL。</li>
	<li>冷却并过滤溶液。将溶液储存在4°C。 注意： 在通风橱中准备溶液，同时穿着适当的个人防护装备。</li>
</ol>3. 准备氯胺酮/甲苯噻嗪鸡尾酒
<ol><li>通过将 0.8 mL 氯胺酮（原液浓度：100 mg/mL）和 0.16 mL 甲苯噻嗪（原液浓度：100 mg/mL）加入 9.4 mL 生理盐水中来制备氯胺酮/甲苯噻嗪混合物。</li>
	<li>在室温 （RT） 下将混合物储存在无菌注射瓶中。</li>
</ol>4.碱烧伤小鼠角膜
<ol><li>在手术前 30 分钟皮下注射美洛昔康（4-6 mg/kg 体重）以缓解疼痛。使用腹腔注射氯胺酮/甲苯噻嗪混合物（氯胺酮80mg / kg和甲苯噻嗪16mg / kg体重）麻醉小鼠（C57BL / 6J，6-8周龄，雄性）。</li>
	<li>通过捏住鼠标的脚趾来检查反射反应（踏板退出），并确认没有反射。在一只眼睛的角膜表面滴一滴局部麻醉剂，0.5%的丙美卡因，在另一只眼睛上滴一滴人工泪液。</li>
	<li>使用 2 毫米活检打孔器，打出 Whatman 滤纸盘。</li>
	<li>将 2 μL 1N NaOH 加入干净的培养皿中。将 2 mm 滤纸盘放在 1 N NaOH 液滴上，并使其浸泡 15 秒。</li>
	<li>用镊子拿起滤纸，将滤纸涂在角膜中心的丙美卡因处理的眼睛上30秒。 注意： 滤纸必须仅接触角膜中心，并且必须注意避免滤纸在放置后移动，因为移动滤纸会导致邻近组织灼伤。</li>
	<li>在无菌注射器中用 20 mL 无菌盐水溶液冲洗眼睛。 注意：必须注意确保彻底清洗角膜和结膜囊，以确保不会对角膜或周围组织造成进一步损害。清洗结膜囊将进一步预防眼睑。</li>
	<li>使用一次性软湿巾轻轻擦拭眼睛和周围区域多余的盐水。之后，将小鼠保持在温暖的加热垫上的恢复笼中，直到可以走动。 注：碱烧伤3天后每天监测小鼠。如果观察到疼痛或压力症状，则皮下注射美洛昔康（4-6mg / kg体重）。</li>
</ol>5.新生血管渗入和混浊的检查和评估
<ol><li>在麻醉小鼠中，在烧伤后第10天在解剖显微镜下检查眼睛，并使用连接到解剖镜的相机获得图像以对混浊和新生血管进行评分。 注意：带有摄像头的常规解剖镜就足够了。</li>
	<li>在通过解剖显微镜观察角膜的同时，根据以下等级12 对烧伤后的不透明度进行评分： 0 = 无不透明度;透明角膜 1 = 轻度混浊;虹膜和瞳孔区域有轻微的朦胧感;虹膜和瞳孔清晰可见 2 = 中度不透明度;虹膜和瞳孔几乎看不见 3 = 严重混浊;虹膜或瞳孔不可见 4 = 不透明角膜;虹膜和瞳孔不可见</li>
	<li>在通过解剖显微镜观察角膜时，根据以下等级12 对 CoNV 进行评分： 0 = 无新生血管形成;没有来自角膜缘的新血管 1 = 轻度新生血管形成;源自角膜缘的新血管 2 = 中度新生血管形成;血管起源于角膜缘，向角膜中心生长 3 = 严重新生血管形成;起源于角膜缘并到达和/或穿过角膜中心的血管</li>
	<li>使用学生 t 检验对碱烧伤和健康眼组之间的混浊和新生血管形成评分进行统计比较。</li>
	<li>在第10天通过5%的异氟醚暴露对小鼠实施安乐死，直到呼吸停止后1分钟，然后进行颈椎脱位，并收集角膜进行平坦成像。</li>
</ol>6. 光学相干断层扫描（OCT）成像
<ol><li>在烧伤后第10天拍摄麻醉小鼠眼睛前段的OCT图像。使用 IR + OCT 模式以 30° 视场和 100% IR 强度的 IR + OCT 模式以体积扫描形式执行 OCT 图像采集。</li>
	<li>使用 ImageJ 软件量化角膜的厚度。</li>
	<li>要测量厚度，请使用 ImageJ 软件中的 线选择 工具在中央角膜的前表面和后表面之间创建一条直线。</li>
	<li>单击软件工具中的 “分析>测量 ”，将值传输到数据窗口。</li>
	<li>将这些值复制到电子表格文件中，并使用学生 t 检验对碱烧伤和健康眼组之间的角膜厚度进行统计比较。 注意：角膜的厚度是从角膜前表面的一点到角膜中心角膜后表面最近点的距离。</li>
</ol>7. 扁平角膜上CoNV的免疫染色
<ol><li>在碱烧伤后第10天对小鼠实施安乐死，并通过钝性解剖摘除眼睛。</li>
	<li>用拇指和食指拉开眼睑，将镊子放在眼球下方。关闭镊子，轻轻地将眼球从眼眶上拉开。</li>
	<li>将眼球放入1x PBS中。对于每个眼球，首先在角膜缘区域下方用 30 G 针切开一个切口，从眼球上取下角膜。</li>
	<li>用角膜微剪刀在角膜缘周围剪开，以切口为起点，慢慢将角膜和角膜缘与眼球分开。</li>
	<li>使用细画笔轻轻清洁角膜以去除虹膜。将角膜固定在4%多聚甲醛中1小时。</li>
	<li>在室温（RT）下在1x PBS中洗涤角膜三次，每次20分钟。</li>
	<li>在室温下在封闭缓冲液（补充有0.1%Triton-X 100和5%牛血清白蛋白[BSA]的1x PBS）中孵育1小时。</li>
	<li>将角膜转移到含有一抗的抗体溶液中。在补充有 1% BSA、0.1% Triton-X 100、Dylight550 偶联的抗 CD31 mAb （1：100） 和 Alexa Fluor488 偶联的抗 LYVE-1 mAb （1：100） 的 1x PBS 中制备抗体溶液。</li>
	<li>在4°C孵育3天。 在1x PBS中洗涤角膜3次，每次20分钟。</li>
	<li>在黑暗中使用Hoechst染色溶液（1：1,000）染色细胞核5分钟。</li>
	<li>用径向切口压平角膜，并使用安装介质和盖玻片将它们安装在预先清洁的载玻片上。用透明指甲油密封盖玻片，并在黑暗中干燥载玻片过夜，然后通过共聚焦显微镜进行分析。</li>
	<li>通过拼接单个 Z 堆栈图像，使用共聚焦显微镜对平坦的角膜进行成像;在非共振振流扫描仪上使用 10 倍物镜、488 nm 和 561 nm 激光器，每层分辨率为 512 像素 x 512 像素。</li>
	<li>使用 ImageJ 软件量化 CD31+ 血液和 LYVE-1+ 淋巴管的密度。</li>
	<li>要确定血管密度，请将共聚焦图像转换为 8 位图像。</li>
	<li>从插件中选择血管密度。</li>
	<li>选择图像上的感兴趣区域，然后单击 “确定”。测量值将在新的数据窗口中打开。</li>
	<li>将这些值复制到电子表格文件中，并使用学生 t 检验对碱烧伤和健康眼组之间的血管密度进行统计比较。 注：CD31，也称为血小板内皮细胞粘附分子-1 （PECAM-1），是一种参与血管生成的细胞粘附分子，在早期和成熟血管的内皮细胞中高度表达13。LYVE-1（淋巴管内皮透明质酸受体-1）是淋巴内皮细胞上的细胞表面标志物，可用作淋巴管生成标志物14。</li>
</ol>

碱烧伤诱导的小鼠角膜新生血管形成

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作者声明没有利益冲突。

角膜是研究血管生成和炎症的极好组织，因为它是可触及的和无血管的，这意味着可以方便地检测和记录新生血管形成。兔子、大鼠和小鼠的角膜烧伤已被用于研究角膜血管生成、炎症和混浊、溃疡、角膜穿孔和纤维化15,16,17。此外，角膜烧伤小鼠模型对于测试血管生成和炎症的各种治疗策略很有价值，因为小鼠的免疫系统与人类密切相关18。基因操纵小鼠基因组的技术的可用性也使该物种成为此类研究的绝佳选择19。这项研究的挑战是开发一种角膜烧伤方法，以提供一致、可重复的病理生理学。
碱烧伤模型对于调节血管生成、炎症和纤维化的药物的药理学筛选特别有用。对试剂和资源的最低要求，进行碱烧伤的简单性，以及方案持续时间短和结果的直接观察的好处，使碱烧伤在小鼠角膜上成为药物筛选的首选。但是，在执行此程序时应考虑一些预防措施，以确保一致性和可重复性。首先，滤纸必须放置在角膜中央，以免灼伤眼睛的其他区域，尤其是角膜缘、眼睑和结膜;其次，NaOH的体积和浓度应适当，以便从角膜上的碱烧伤中获得一致的结果。过滤器不得滴湿，而应浸泡在 NaOH 溶液中。该方法中使用的过滤器尺寸和过滤器类型以及溶液的正态性和体积经过优化，以避免 NaOH 溢出。使用不同尺寸的滤纸或更高或更低体积的 NaOH 会导致新生血管形成不一致。第三，重要的是防止 NaOH 溶液吸收室内空气中的 CO2 ，方法是在使用后立即拧紧溶液的管帽并降低空气/溶液比。必须注意使用新鲜的碱溶液，以防止新生血管形成不一致并避免角膜溃疡。最后，必须用生理盐水从眼睛和结膜中广泛清洗所有NaOH溶液，以防止对角膜和眼睛周围组织的进一步损害。彻底清洗角膜和邻近组织也将防止眼睑。
这里描述的方案是研究角膜血管生成的病理生理学的有效和可靠的方法。该方案可进一步用于研究角膜炎症、纤维化和伤口愈合。

角膜是一种无血管组织，通过建立血管生成特权来保持其透明度 1,2。角膜损伤可导致炎症以及血液和淋巴管的发育，以及纤维化3.角膜新生血管形成 （CoNV） 会导致视力障碍，是全球失明的第二大原因4.CoNV 每年影响美国约 140 万人5.CoNV 可由多种因素诱发，包括化学灼伤、感染、炎症和缺氧 3,6。化学烧伤是最常见的眼部急症之一，约占眼外伤的 13.2%，需要立即评估和治疗7.化学烧伤可能是碱烧伤或酸烧伤，但碱烧伤会造成更严重的伤害，因为碱会更深入地渗透到组织中8.
碱烧伤小鼠模型被广泛用于研究CoNV和伤口愈合。与角膜袋血管生成模型 9,10 相比，碱烧伤模型的创建相对简单，也可用于研究角膜炎症、纤维化和上皮增生。这些模型与临床化学烧伤的关系也比血管生成的角膜缝合模型更密切11。碱烧伤时，由于炎症以及抗血管生成和促血管生成因子的不平衡，其他缺血性角膜会形成血管1,2。角膜碱烧伤模型的缺点是难以控制碱烧伤的面积和严重程度、角膜新生血管的变化以及由于碱溶液过量而导致的邻近组织无意烧伤。本研究的目的是描述使用预浸泡在氢氧化钠溶液中的滤纸在小鼠中受控的角膜碱烧伤模型。该模型可用于研究血管生成因子、抗血管生成治疗试剂以及其他可以调节炎症和纤维化的因子和试剂。

<table><tbody><tr><td>0.9% Sodium Chloride Injection</td><td>Hospira</td><td>KL-7302</td><td></td></tr><tr><td>30 G Needle</td><td>McKesson</td><td>16-N3005</td><td></td></tr><tr><td>A1R Confocal</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>Anti-CD31</td><td>Novus Biologicals</td><td>NB100-1642R</td><td></td></tr><tr><td>Anti-LYVE-1</td><td>Life technologies</td><td>53-0443-82</td><td></td></tr><tr><td>ASM Module</td><td>Heidelberg Engineering</td><td></td><td>Anterior segment objective</td></tr><tr><td>Biopsy Punch</td><td>McKesson</td><td>16-1309</td><td></td></tr><tr><td>BSA</td><td>Thermoscientific</td><td>9048-46-8</td><td></td></tr><tr><td>Coverslip</td><td>VWR International</td><td>22X22-1-601640G</td><td></td></tr><tr><td>Dissection Microscope</td><td>AmScope</td><td>SM-4TZ-30WY-10M3</td><td></td></tr><tr><td>Fluoromount-G</td><td>Electron Microscopy Sciences</td><td>17984-25</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>15000-02</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11049-10</td><td></td></tr><tr><td>Forceps</td><td>Fisherbrand</td><td>12-000-157</td><td></td></tr><tr><td>Forceps&amp;nbsp;</td><td>Roboz</td><td>RS-4905</td><td></td></tr><tr><td>Gonak Hypromellose&amp;nbsp;</td><td>Akorn</td><td>17478006412</td><td></td></tr><tr><td>GraphPad Prism 9</td><td>GraphPad Sotware, Inc</td><td></td><td></td></tr><tr><td>Heating pad</td><td>K&amp;amp;H Pet Products</td><td>100213018</td><td></td></tr><tr><td>Hoescht</td><td>Life Technologies</td><td>62249</td><td></td></tr><tr><td>HRA + OCT Spectralis</td><td>Heidelberg Engineering</td><td></td><td></td></tr><tr><td>Insulin Syringe</td><td>Mckesson</td><td>102-SN310C31516P</td><td></td></tr><tr><td>Kimwipe</td><td>Kimberly Clark Professional</td><td>34155</td><td></td></tr><tr><td>Micro Cover Glass</td><td>VWR</td><td>48366-067</td><td></td></tr><tr><td>Microscissors</td><td>Roboz</td><td>RS-5110</td><td></td></tr><tr><td>Microscopic Slide</td><td>Fisherbrand</td><td>12-550-15</td><td></td></tr><tr><td>NaOH</td><td>Sigma Aldrich</td><td>55881-500G</td><td></td></tr><tr><td>Neomycin and Polymyxin B Sulfates and Dexamethasone&amp;nbsp;</td><td>Bausch &amp;amp; Lomb</td><td>24208-0795-35</td><td></td></tr><tr><td>Normal Serum</td><td>Jackson Immuno</td><td>008-000-121</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>158127-500G</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>20012-027</td><td></td></tr><tr><td>Proparacaine HCl</td><td>Bausch &amp;amp; Lomb</td><td>24208073006</td><td></td></tr><tr><td>Saline</td><td>Henry Schein</td><td>1531042</td><td></td></tr><tr><td>SMZ125</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>Syringe 10 mL</td><td>McKesson</td><td>16-S10C</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>TX1568-1</td><td></td></tr><tr><td>Whatmann Filter Paper</td><td>Cytiva</td><td>WHA1003323</td><td></td></tr></tbody></table>

a mouse model for corneal neovascularization by alkali burn

角膜新生血管形成 （CoNV） 是血管生成的一种病理形式，涉及血液和淋巴管从角膜缘生长到缺血性角膜，并对透明度和视力产生不利影响。碱烧伤是导致CoNV的最常见眼外伤形式之一。在该协议中，使用氢氧化钠溶液以受控方式实验诱导CoNV，以确保可重复性。碱烧伤模型有助于了解 CoNV 的病理学，并且可以扩展到研究一般的血管生成，因为角膜的无血管性、透明度和可及性。在这项工作中，通过在解剖显微镜下直接检查并使用抗 CD31 mAb 对扁平角膜进行免疫染色来分析 CoNV。使用抗 LYVE-1 mAb 进行免疫染色，在扁平角膜上检测到淋巴管生成。角膜水肿使用光学相干断层扫描 （OCT） 进行可视化和量化。总之，该模型将有助于推进现有的新生血管化检测，并发现病理性眼部和眼外血管生成的新治疗策略。

本研究描述了一种通过碱烧诱导小鼠眼角膜血管生成的方法。解剖显微镜（图1A，B）显示碱烧伤组角膜新生血管形成和混浊评分显著升高（P < 0.05;图1C，D）。在第 10 天收集的角膜分别用血管抗 CD31 mAb 和淋巴管抗 LYVE-1 mAb 进一步免疫染色（图 2A-I）。碱烧伤组10 d后血液和淋巴管密度显著升高（P < 0.001和P < 0.05;图2J，K）。使用OCT成像和量化的角膜厚度（图3A，B）观察到碱烧伤组的角膜厚度显着更高（P < 0.01;图3C）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65289/65289fig01.jpg"> 图 1：碱烧伤引起的角膜新生血管形成和混浊。 （甲，乙）角膜新生血管从角膜缘血管向角膜中心萌发，在受伤后 10 天，碱烧伤的小鼠眼 （A） 但不是健康的眼睛。（C，D）图 A 和 B 中 （C） 角膜新生血管和 （D） 混浊的量化（± SEM;t 检验;*P < 0.05;n = 3 只眼睛，1 只眼睛/小鼠）。红色箭头表示角膜缘，黄色箭头表示萌发的新血管。<a href="https://www.jove.com/files/ftp_upload/65289/65289fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65289/65289fig02.jpg"> 图2：碱烧伤引起的角膜新生血管形成和淋巴管生成。 免疫组化分别使用抗 CD31 和抗 LYVE-1 mAb 显示 （A、D、G） 血液和 （B、E、H） 淋巴管。（空调）健康的小鼠角膜。（D-I）碱烧伤角膜 受伤后 10 天。（C，F，I）CD31 和 LYVE-1 信号的叠加图像。（G-I）面板 D-F 的放大图像。比例尺 = （A-F） 200 μm 和 （G-I） 500 μm。 （J，K） 如图所示，图 A-F 中血液和淋巴管密度的量化（± SEM;t 检验;*P < 0.05;***P < 0.001;n = 3 只眼睛，1 只眼睛/只小鼠）。<a href="https://www.jove.com/files/ftp_upload/65289/65289fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65289/65289fig03.jpg"> 图3：碱烧伤引起的角膜厚度增加 。 （A） 健康小鼠眼睛的 OCT 图像。（B） 碱烧伤后 10 天小鼠角膜的 OCT 图像。（C） 在角膜中心测量的图 A 和 B 中角膜厚度的量化（± SEM;t 检验;**P < 0.01;n = 3 只眼睛，1 只眼睛/只小鼠）。 <a href="https://www.jove.com/files/ftp_upload/65289/65289fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Decoding Corneal Neovascularization with Alkali Burn Model for Future Therapeutic Strategies at JoVE.com

作者聚焦：用碱烧伤模型解码角膜新生血管化，用于未来的治疗策略 

该方案侧重于碱烧伤诱导的小鼠角膜新生血管形成。该方法生成了可重复和可控的角膜疾病模型，以研究病理性血管生成和相关分子机制，并测试新的药物以预防角膜新生血管形成。

碱烧伤角膜新生血管化的小鼠模型

Corneal neovascularization (CoNV), a pathological form of angiogenesis, involves the growth of blood and lymph vessels into the avascular cornea from the ...

我们正试图开发一种高度可重复的诱导角膜新生血管形成的模型，并确定最相关的组织学和免疫染色方法来监测预防和减少新生血管形成的疗法。角膜碱烧伤模型的缺点是难以控制碱烧伤的面积和严重程度、角膜新生血管的变化以及由于碱溶液过量导致的边缘和组织意外烧伤。角膜新生血管形成是一种威胁部位的疾病。这种疾病的发病机制知之甚少。该模型将用于获得对病情的新见解，并确定相关的治疗靶点 与角膜袋血管生成模型相比，碱烧伤模型的创建相对简单，也可用于研究角膜炎症、纤维化和上皮增生。与血管生成的角膜缝合模型相比，这些模型与临床化学烧伤的关系也更密切。我们将使用碱烧伤角膜作为疾病模型，研究一种新的抗血管生成疗法来治疗角膜新生血管形成和混浊。

a-mouse-model-for-corneal-neovascularization-by-alkali-burn

Research

JoVE Journal

Biology

A Mouse Model for Corneal Neovascularization by Alkali Burn

2.2K 次观看.  Baylor College of Medicine. 我们正试图开发一种高度可重复的诱导角膜新生血管形成的模型，并确定最相关的组织学和免疫染色方法来监测预防和减少新生血管形成的疗法。角膜碱烧伤模型的缺点是难以控制碱烧伤的面积和严重程度、角膜新生血管的变化以及由于碱溶液过量导致的边缘和组织意外烧伤。角膜新生血管形成是一种威胁部位的疾病。这种疾病的发病机制知之甚少。该模型将用于获得?...

视频: 作者聚焦：用碱烧伤模型解码角膜新生血管化，用于未来的治疗策略 

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

角膜新生血管在小鼠碱烧伤模型

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

科研

医学

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

生物学

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

眼毛刺角膜上皮磨损与角膜创面愈合的模型研究

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析:在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

角膜上皮机械和化学损伤的离体和体内动物模型

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

在使用肌球蛋白重链免疫鼠肌肉横断面的快速自动化方案为肌纤维人口分析

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

A Mouse 5/6th Nephrectomy Model That Induces Experimental Uremic Cardiomyopathy

Chronic kidney disease (CKD) is a great risk factor for cardiovascular disease events and mortality, and progressively develops to the clinical phenotype called "uremic cardiomyopathy". We describe here an experimental CKD mouse model, named 5/6th partial nephrectomy (PNx) with pole ligation, which developed uremic cardiomyopathy at four weeks post-surgery. This PNx model was performed by a two-step surgery. In step-one surgery, both poles of the left kidney were ligated. In step-two surgery, which was performed 7 days after the step-one surgery, the right kidney was removed. For the sham surgery, the same surgery procedures were performed but without pole ligation of the left kidney or removal of the right kidney. The surgical procedures are easier and less time-consuming, compared to other methods. However, the remnant functional renal mass is not as easily controlled as the renal artery ligation. Four weeks after surgery, in comparison with the sham-operated mice, the PNx mice developed impaired renal function, anemia, cardiac hypertrophy, cardiac fibrosis, and decreased heart systolic and diastolic function.

A Mouse 5/6th Nephrectomy Model That Induces Experimental Uremic Cardiomyopathy

This manuscript provides a detailed two-step surgical procedure to perform mouse 5/6th partial nephrectomy (PNx) with pole ligation. Four weeks after surgery, in comparison with sham-operated mice, the PNx mice developed impaired renal function, anemia, cardiac hypertrophy, cardiac fibrosis, and decreased heart systolic and diastolic function.

一种诱导实验性尿毒症心肌病的小鼠 5/6th肾切除模型

Chronic kidney disease (CKD) is a great risk factor for cardiovascular disease events and mortality, and progressively develops to the clinical phenotype ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

一种用于小鼠精确碱烧伤的改良活检穿刺法

Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of corneal injury models, researchers aim to enhance their understanding of how the cornea heals and develop strategies to prevent and manage corneal opacities. Chemical injury is one of the most popular injury models that has extensively been studied on mice. Most previous investigators have used a flat paper soaked in sodium hydroxide to induce corneal injury. However, inducing corneal and limbal injury using flat filter paper is unreliable, since the mouse cornea is highly curved. Here, we present a new instrument, a modified biopsy punch, that enables the researchers to create a well-circumscribed, localized, and evenly distributed alkali injury to the murine cornea and limbus. This punch-trephine method enables researchers to induce an accurate and reproducible chemical burn to the entire murine cornea and limbus while leaving other structures, such as the eyelids, unaffected by the chemical. Moreover, this study introduces an enucleation technique that preserves the medial caruncle as a landmark for identifying the nasal side of the globe. The bulbar and palpebral conjunctiva, and lacrimal gland are also kept intact using this technique. Ophthalmologic examinations were performed via slit lamp biomicroscope and fluorescein staining on days 0, 1, 2, 6, 8, and 14 post-injury. Clinical, histological, and immunohistochemical findings confirmed limbal stem cell deficiency and ocular surface regeneration failure in all experimental mice. The presented alkali corneal injury model is ideal for studying limbal stem cell deficiency, corneal inflammation, and fibrosis. This method is also suitable for investigating pre-clinical and clinical efficacies of topical ophthalmologic medications on the murine corneal surface.

作者聚焦:推进角膜混浊治疗和再生的研究 

This protocol describes a method to induce an accurate and reproducible corneal and limbal alkali injury in a mouse model. The protocol is advantageous as it allows for an evenly distributed injury to the highly curved mouse cornea and limbus.

在小鼠模型中使用 Punch-Trephine 技术诱导角膜和角膜缘碱损伤

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of ...