We thank Ellen Ytebrouck for her valuable information regarding method application in rat. This work was supported by the Research Council of the KU Leuven (OT09/22) and the Fund for Scientific Research-Flanders (FWO Vlaanderen). Jeroen Aerts is supported by a Ph.D. fellowship of the Agency for Innovation through Science and Technology Flanders (IWT Vlaanderen) and Julie Nys by a Ph.D. grant from FWO Flanders.

所有实验均批准了鲁汶大学的研究伦理委员会，均严格按照欧共体理事会指令2010年9月22日（2010/63 /欧盟），并与比利时的法律（KB 29日2013年5月）。尽一切可能的努力，以尽量减少动物的痛苦，并减少动物的数量。 1，动物诊疗和麻醉剂<ol><li>麻醉小鼠腹膜内注射盐酸氯胺酮（75毫克/毫升）和盐酸美托咪定（1毫克/千克）在盐水的混合物。 </li><li>按捏脚趾用钳，以保证鼠标完全镇静检查反射。 </li><li>应用70％乙醇消毒眼睑，并用棉签眼睛周围的区域。检查眼睑反射到另外评估镇静程度。 </li></ol> 2，取出眼<ol><li>确保动物驻留在平整，干燥和光滑的表面。 </li><li>消毒镊子用弯曲的，锯齿状的尖端（首选头尺寸:0.5×0.4毫米）。 </li><li>轻轻按到眼角（眼角），与之前的眼球从插槽中流离失所，视神经可达镊子。 </li><li>引导视线背后的镊子。按住视神经牢固，优选与弯道的开始和钳子的不是很尖。这将有助于从插座全球，用镊子将完整的视神经。 </li><li>作圆周运动的手拿着镊子与阻力最小的方向，而鼠标仍然在平坦的表面上。鼠标将根据手移动的方向沿着表面摆动。 </li><li>与逐渐增加的速度执行这个动作，直到视神经收缩的两个（通常在7至15圆周运动，约半至每秒一整圈）。因此，分离的眼球删除。 </li></ol> 3，术后护理<ol><li>如果出血（罕见）的，充满粘性凝结和止血剂的轨道。 </li><li>通过注射1毫克/千克盐酸atipamezol在盐水腹膜内扭转麻醉。 </li><li>辖1毫克/千克美洛昔康的腹腔内，每24小时，以减轻疼痛。 </li><li>应用眼膏剩余的眼睛，以防止角膜脱水。 </li><li>让动物恢复在加热板上或包裹在一个单独的笼子，以控制体温绝缘材料的动物。 </li><li>每天测量小鼠的体重，至少2天。重量的损失可以表示痛苦和在这种情况下，继续美洛昔康治疗，直到动物被完全恢复。 </li></ol>

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The authors have nothing to disclose.

根据我们的方法执行成功摘除术，要考虑的最重要的步骤是:1）使用镊子与适当大小的弯曲和锯齿尖; 2）表演光滑，干燥的表面去核;和3）逐渐加快了圆周运动中，用最少的摩擦方向。 对于一个有效的结果，就必须使用特点是弯曲的，锯齿状的尖端合适的镊子（最好头尺寸:鼠标:0.5×0.4毫米;鼠:2.15×1.3毫米）。曲率使得眼球移位后容易进入视神经和进行圆周运动时，有必要正确的手的位置。流畅的提示不推荐，因为他们持有的视神经时缺乏必要的抓地力。故障期间的圆运动适当保持视神经导致眼动脉，眼的剥离差和再现性，因此较差的断裂。因此，建议首先实践本技术的实施安乐死的动物进行处理，以保证最大的动物福利一旦施加的方法在体内钳子的最优化。成功的眼眼球摘除最近也已在大鼠中使用相同的技术，除了手工转动动物体和保持镊子固定的执行在我们的实验室中。 该技术的一个限制是，它可能损害视网膜。因此，该方法是不太适合用于收集视网膜进行组织学5。此外，我们的方法是有限的张贴睁眼青睐，因为眼球需要从插座位移不除去或切断眼睑。 在不同的物种，包括啮齿类动物的眼睛剜除术，是经常进行使用替代方法，这往往带来切除眼睑和切断视神经18,23-25。这些实现方法具DS往往更侵入，并有比这里所描述的技术，较高的学习曲线。无需去除或缝合眼睑，在手术后的恢复时间被最小化，从而获得更高的动物福利和更可重复的结果。

取出眼，从而不可逆转地自毁感觉受体表面（视网膜），规定了相当的损失感觉输入沿视觉通路。在幼年和成年视觉系统中的去核的模型已经被证明是在理解的不同的视觉中心1-4的发展，可塑性和功能有价值。这种感觉丧失的分子，细胞和生理后果可以提供深入了解皮质电路发展如何正常的调节，以及如何既定处理，并响应改变其结构和功能，以经验这样一个广泛的改变。 视觉剥夺的方法不同，存在的，他们都有自己的视力相关的研究特定的优势。例如暗饲养具体地说消除视觉驱动活动但它不影响自发视网膜活性。同样地，盖缝线或眼罩去除图案visuaL输入，而不会干扰自发活动，但他们可以通过闭上眼睛分散的光线穿透。这些方法都是可逆的，并已被证明是在理解的双眼输入图案的视觉和低级别的相关性在发展中6-8塑身皮质回路的作用价值。在青光眼的研究，在成年动物的视神经损伤模型已被广泛使用，因为它确立了构成视神经9,10视网膜神经节细胞的输入累进损失。另一方面，去核，其中所述眼，从而在视网膜完全和立即除去，是剥夺的适当的选择，当目标是在一次不可逆地除去是自发性的，图案化的视野。它也导致一个强大的眼压活性不平衡，可提高信号噪声比在活性作图研究11,12。比较功能和结构的变化响应眼球摘除与日OSE剥夺通过温和的方法，例如盖缝合例如之后，也可能会暴露出新的见解自发视网膜活性的两种稳态和突触可塑性类型的角色。 眼球摘除触发营养的影响，在视网膜的直接目标的损失。例如，BDNF的水平显著在外侧膝状体（LGN）和成年大鼠去核13的上丘下调。活性氧，它充当信使分子介导的结构重塑，也分别在成年大鼠视觉系统14的皮层下结构检测。此外，小胶质细胞和星形胶质细胞的活化在不同的皮层下的视觉目标结构中的鼠标发生在一个15星期的特定后眼球摘除的时间框架。一起，视神经传入神经阻滞导致的神经胶质，结构和分子水平不同的皮层下的反应。尽管有这些皮层下的影响，并不一定牵涉在皮层水平16特效。这两个单眼后值得注意的是，跨通道皮质可塑性，包括修改在其他感官方面旁边的非视觉输入到被剥夺视觉皮层的加强会发生（ME）的3,4,17,18和双眼（BE）1,17眼球摘除。 除了 ​​有助于视觉神经，去核的类型传入神经 ​​阻滞可被用来研究对中枢神经系统的神经保护19和神经变性20-22性能之间的平衡。 不同的程序进行眼球摘除文献中已经有说明。用于体内 ME在大鼠和小鼠中的某些方法是较复杂的，由于轨道的肌肉和组织23-25 ​​不必要的切片。其他出版物如马哈詹等人 （2011）5使用提供一份详细的协议<html>钝性分离对眼睛的一种高通量的收集，研究基因型 - 表型的相关性，可能验尸。对于他们的目的，该方法简便，快速。然而，该方法是不太适合体内摘除术时人们选择学习的传入视觉通路下列摘除术（在活的动物），而不是眼睛本身。在这样的设置，后期摘除术的生存是非常重要。此外，最小的体内损伤和保存视神经和眼眶组织是有利的。这里，我们提出了一种替代眼球摘除方法，更类似于由Faguet 等人所描述的（2008）26，它提供某些有利的特性:它具有快速的术后恢复相关联，其特征是一个非常低的学习阈研究人员。一般情况下，不同的方法是互补取决于后续研究的重点:眼部形态或视觉通路的研究。 ve_content"&gt;总之，剜除术可以从视觉研究应用朝着稳态和跨模态脑的可塑性，胶质反应性，以及轴突稳定性调查，在这个可视化的文章中，我们证明在体内摘除眼球的一种可行和可靠的方法鼠标。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ketamine hydrochloride (Anesketin)</td>
			<td>Dechra Veterinary Products (Eurovet)</td>
			<td>BE-V136516</td>
			<td></td>
		</tr>
		<tr>
			<td>Medetomidine hydrochloride (Domitor)</td>
			<td>Orion Corporation (Janssen Animal Health)</td>
			<td>BE-V151742</td>
			<td></td>
		</tr>
		<tr>
			<td>Atipamezol hydrochloride (Antisedan)</td>
			<td>Orion Corporation (Elanco Animal Health)</td>
			<td>BE-V153352</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam (Metacam)</td>
			<td>Boehringer Ingelheim Vetmedica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye ointment (Fucithalmic)</td>
			<td>Leo Pharma nv-sa</td>
			<td>BE 144654</td>
			<td></td>
		</tr>
		<tr>
			<td>Moria MC31 Forceps - Serrated Curved</td>
			<td>Fine Science Tools</td>
			<td>11370-31</td>
			<td>For application in the mouse. Any forceps with similar dimensions can be used as long as the tip is curved and serrated.</td>
		</tr>
		<tr>
			<td>Narrow Pattern Forceps - Curved</td>
			<td>Fine Science Tools</td>
			<td>11003-13</td>
			<td>For application in the rat. Any forceps with similar dimensions can be used as long as the tip is curved and serrated.</td>
		</tr>
		<tr>
			<td>Hemostatic cotton wool</td>
			<td>Qualiphar</td>
			<td>N/A</td>
			<td>Other hemostatic agents are equally suitable (e.g. Viscostat, #649, Ultradent Products)</td>
		</tr>
	</tbody>
</table>

a highly reproducible and straightforward method to perform in vivo ocular enucleation in the mouse after eye opening

剜除术或手术切除的眼睛一般可以视为典范神经传入神经阻滞。它提供了可视化研究，跨模态的发育可塑性的不同方面以及哺乳动物的视觉系统1-4的宝贵工具。 在这里，我们证明了优雅和简单的技术，用于除去的一个或两个眼睛的小鼠，其在20天龄达小鼠对成年人进行验证。简言之，将消毒的弯钳是用来夹持眼后面的视神经。随后，圆周运动中进行，以收缩视神经和取出眼球。这种技术的优点是高的再现性，最小或没有出血，快速术后恢复和对实验者非常低的学习阈值。因此，大量的动物可以被操纵和精力最小量的处理。该技术的性质可引起轻微损坏在手术过程中的视网膜。这种副作用使得这种方法不适合作为比较马哈詹等人（2011）5，如果目标是要收集和分析视网膜组织。此外，我们的方法仅限于后睁眼年龄（鼠标:P10 - 13日起），因为眼球需要从插座移位不去除眼睑。在这个手稿中描述的体内摘除术的技术最近已成功地与小的修改适用于大鼠和研究啮齿类动物的传入视觉通路一般会出现非常有用。

图1示出了使用所描述的协议的成功摘除眼球，并且特征在于没有出血或眶组织或眼圈（ 图1A，1B）的任何明显的物理破坏。此外，除去眼具有平滑的角膜，脉络膜和光盘中，表示为一个完全不变地球（ 图1C）。因为我们的协议包括眼球和机械车削后视神经的夹紧，将除去的眼的视神经是缩在视网膜上（ 图1D）的基极。在执行上述过程的结果在一个干净的切割视神经而不向周围脑区域（ 图1E）的任何损坏。 单眼摘除术，与活动的映射（ 图2）的组合，使人们得以大幅描绘功能或眼睛特定输入区域在对侧visua鼠标12,27甚至眼优势列在高阶哺乳动物象猴子28的升皮质。 在小鼠实验中，除去一个的（ME）或双眼（BE）结合有针对性的视觉刺激和zif268基因或c-Fos蛋白的表达水平，检测施加到露出的区域的神经元激活在视觉皮层12,27 。相反，在视觉刺激的控制（ 图2A），BE小鼠显示出在由于完全缺乏视觉输入（ 图2B）的视觉皮质基底活动。正因为如此，使用非视觉皮层（ 即躯体感觉皮层在更前的部分和在更后段听觉皮层）视觉之间的边界被揭露。结果从ME的小鼠进行为期一周的存活时间可视眼睛特定的输入地区，在对侧视觉皮层。这两个单眼带动地区是机能减退和位于MEDI人与横向的中央双目区（ 图2C）的。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51936/51936fig1highres.jpg" width="500" /> 眼窝中，取出眼球和视神经的后眼球摘除状态图1定性评价。摘除眼球用弯钳（ 一）无出血或损伤眼窝（ 二）观察后。被删除的眼睛是完全不变所反映的角膜和脉络膜（C，D）的正常外观。视神经被缢缩在视盘，其中它离开眼睛（D）。考试大脑的腹侧部分揭示了一个干净的切割视神经（星号）和其他结构（E）没有明显的损伤。比例尺在C，D:1毫米。在电子商务比例尺:5毫米。答:前; L:左;病人:后路; R:对。 图中的鼠标视觉皮层所揭示的眼球摘除2眼功能输入特定的细分 。连接眼睛和皮质黑色和灰色线代表了跨越传入视网膜和眼睛特定的输入区域。神经活动可视化的控制（ 一）冠状切片，BE（B）和ME（C）小鼠放射性原位杂交（ISH）的zif268（灰度）前囟门左右的水平-3.40毫米。在对照组动物（一），在两个半球的视觉皮层表达了以下的视觉刺激活性高。当一个或两个眼睛（s）值摘除，在活动信号清晰的降低是在对应剥夺皮层区域可见。单眼去核（C）的小鼠表现出较高的ACTi的区域VITY在视觉皮层由下降信号在单眼区对侧取出的眼睛所包围的双目区。比例尺:2毫米。转载自范布鲁塞尔等[12]的权限。

Watch this Scientific Journal Video about A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening at JoVE.com

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

去除眼，也叫剜除，为研究沿哺乳动物视觉系统的可视化，跨模态和发育可塑性方面，因为它导致不可逆的部分（单眼）或完全（双眼）视力减退有用的策略。在这里，我们描述了一个高度可重复的和直接的方法， 在体内摘除术表演。

一个高度可重复的和直接的方法来执行<em&gt;体内</em&gt;眼剜除术中的鼠标眼睛开放后

Enucleation or the surgical removal of an eye can generally be considered as a model for nerve deafferentation. It provides a valuable tool to study the ...

a-highly-reproducible-straightforward-method-to-perform-vivo-ocular

Research

JoVE Journal

Biology

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

23.2K 次观看.  KU Leuven - University of Leuven. 去除眼，也叫剜除，为研究沿哺乳动物视觉系统的可视化，跨模态和发育可塑性方面，因为它导致不可逆的部分（单眼）或完全（双眼）视力减退有用的策略。在这里，我们描述了一个高度可重复的和直接的方法， 在体内摘除术表演。 

视频: A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

焦磷酸测序：一个准确的基因分型的简单方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

科研

生物学

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 ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

小鼠模型的验证扰乱LINC配合物在细胞特异性的方式

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

A Method for Islet Transplantation to the Omentum in Mouse

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

小鼠骨髓胰岛移植的一种方法

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

原发性小鼠视网膜色素上皮细胞的有效解剖与培养

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) human corneal epithelial cell cultures. Cells were exposed to UV radiation and toxic doses of benzalkonium chloride (BAK), hydrogen peroxide (H2O2), and sodium dodecyl sulfate (SDS). Metabolic activity was measured using a metabolic assay. The release of inflammatory cytokines was measured using a multi-plex interleukin (IL)-1&#946;, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-&#945;) assay, and cells were evaluated for viability using fluorescent dyes. 
The damaging effects of UV on cell metabolic activity and cytokine release occurred at 5 min of UV exposure for iHCEC and 20 min for pHCEC. Similar percent drops in metabolic activity of the iHCEC and pHCEC occurred after exposure to BAK, H2O2, or SDS, and the most significant changes in cytokine release occurred for IL-6 and IL-8. Microscopy of fluorescently stained iHCEC and pHCEC BAK-exposed cells showed cell death at 0.005% BAK exposure, although the degree of ethidium staining was greater in the iHCECs than pHCECs. Utilizing multiple methods of assessing toxic effects using microscopy, assessments of metabolic activity, and cytokine production, the toxicity of UV radiation and chemical toxins could be determined for both primary and immortalized cell lines.

This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line.

确定紫外线辐射和化学物质对原代和永生化的人角膜上皮细胞的毒性

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) ...

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative sparsity of information about C. elegans in its natural context comes from the challenges involved in the identification of the small nematode in nature. Despite these challenges, an increasing focus on the ecology of C. elegans has caused a wealth of new information regarding its life outside of the laboratory. The intensified search for C. elegans in nature has contributed to the discovery of many new Caenorhabditis species and revealed that congeneric nematodes frequently cohabitate in the wild, where they feed on microbial blooms associated with rotting plant material. The identification of new species has also revealed that the androdioecious mating system of males and self-fertilizing hermaphrodites has evolved three times independently within Caenorhabditis. The other two selfing species, C. briggsae and C. tropicalis, share the experimental advantages of C. elegans and have enabled comparative studies into the mechanistic basis of important traits, including self-fertilization. Despite these advances, much remains to be learned about the ecology and natural diversity of these important species. For example, we still lack functional information for many of their genes, which might only be attained through an understanding of their natural ecology. To facilitate ecological research of selfing Caenorhabditis nematodes, we developed a highly scalable method to collect nematodes from the wild. Our method makes use of mobile data collection platforms, cloud-based databases, and the R software environment to enhance researchers&#39; ability to collect nematodes from the wild, record associated ecological data, and identify wild nematodes using molecular barcodes.

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

This protocol can be used to perform large-scale ecological surveys of selfing Caenorhabditis nematodes. The primary advantage of this method is the efficient organization and analysis of ecological and molecular data associated with the nematodes collected from nature.

一种高度可扩展的方法，用于对自发 性Caenorhabditis 线虫进行生态调查

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

从人眼高效、一致地生成视网膜色素上皮/脉络膜平面安装，用于组织学分析

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...