特拉华谷狮子会眼库的支持是非常宝贵的，这项工作。鼠标主，单克隆抗体ICP8的是一种礼物博士大卫Knipe（哈佛医学院）。我们也感谢斯蒂芬·詹宁斯（Drexel大学医学院）和彼得·莱布森（Wills眼科研究所）博士的有益讨论和专业知识。

所有的试剂和设备购买无菌或灭菌前的程序。本文介绍的步骤建立体外模型与一个去核的眼睛开始。在我们的实验中，我们使用新鲜全眼球从年轻的白兔（8-12周）（PEL-FREEZ生物制品，罗杰斯，AR）。此外，我们使用人获得当地眼库的角膜。如果执行剜除自己，照顾，以避免损坏角膜或角膜缘，在操作过程中。兔剜除术协议可以在别处找到2。 第1部分：切除角巩膜按钮<ol><li>与PBS浸湿一个狭长的纱布，并用它周围的眼球，以方便在操作过程中。 </li><li>使用锋利的手术刀，使巩膜切口，切向的角膜缘约0.5厘米的距离。 </li><li>使用锋利的剪刀延长切口，完全切除的corneoscleral按钮。 </li><li>后巩膜按钮是孤立的，逗了虹膜，巩膜缘用钳子。避免损坏对角膜内皮细胞或施加过大的压力。 </li></ol>第2部分：介绍器官培养角膜进入<ol><li>取出的角膜在最小必需培养基（MEM），补充有非必需氨基酸（1X），（2毫摩尔），L-谷氨酰胺，青霉素（200单位/毫升）和链霉素（200微克/毫升）培养。如有必要，也可加入抗真菌剂，例如两性霉素，可能。 </li><li>制备1％的琼脂糖溶液培养基中，在55℃下，并保持准备</li><li>彻底冲洗角膜在无菌PBS中含有青霉素（200单位/毫升）和链霉素（200微克/毫升）。 </li><li>角膜上皮侧放置下来到井的无菌点板。 </li><li>添加含琼脂糖介质角膜内皮凹部刚够填写完全。允许1分钟的琼脂糖巩固。 </li><li>将凝胶支架上皮面朝上插入一个35毫米组织培养皿，并添加培养基表面覆盖上皮与角膜。 </li><li>可以以这种方式培养的角膜超过一个星期，与介质发生变化，每48小时。 </li></ol>第3部分：感染了HSV-1和实验性药物治疗<ol><li>添加1毫升到35 mm的组织培养皿中含有所需量的病毒的培养基中。我们通常为1×10 4 PFU应变KOS每角膜的HSV-1感染。我们的股票效价是由斑块检测CV-1细胞单层。 </li><li>一起放置在角膜与琼脂糖脚手架上皮侧下降到含有病毒的培养基。 </li><li>将角膜组织培养孵化器，感染1小时间歇性摇摆每10-15分钟。 </li><li> 1小时后，吸出病毒含有多种宁培养基并用PBS冲洗2-3次，以除去任何残留的病毒颗粒。 </li><li>将角膜回到它以前的位置，加入新鲜的培养基覆盖的上皮细胞表面。 </li><li>实验性治疗前或感染后，可以开始，根据实验的性质。 </li><li>改变培养基每48小时。 </li></ol>第4部分：样品采集<ol class="lalpha"><li> 中斑块的检测分析。混合介质，并立即使用的空斑法或存储在-80°C以备后用，如果在不同时间点采集样本。 </li><li> DNA，RNA，蛋白质，或从角膜上皮细胞 <ol><li>删除的琼脂糖凝胶支架，并用PBS冲洗角膜。 </li><li>海关巩膜组织从角膜环，以确保仅角膜上皮材料被收集。 </li><li> ü唱一把手术刀，刮去角膜上皮细胞进入适当的缓冲液中，根据以进行分离的类型的材料。 DNA和RNA的隔离，我们使用的DNeasy血液及组织试剂盒和RNeasy Mini试剂盒（QIAGEN，Hilden，德国）。对于蛋白质的裂解物的集合，我们使用Laemmli缓冲液。对于角膜上皮细胞的流式细胞仪分析，我们预孵化角膜在少量的胰蛋白酶松开细胞，然后赶走他们到胰蛋白酶用解剖刀。由于刮角膜上皮不产生等量的组织，它是重要的，包括分析样品时，适当的控制。我们使用的18S rRNA，GAPDH定量RT-PCR的成绩单参照基因在qPCRs，和核仁素的免疫印迹加载控制作为参考。 </li></ol></li><li> 组织样本进行免疫组化或免疫荧光 <ol><li>删除琼脂糖凝胶支架和冲洗玉米EA与PBS。 </li><li>根据实验性的目标上，使用角膜使石蜡组织块或冰冻组织块，在视频中所示。准备角膜组织切片时，我们遵循的标准程序。 </li></ol></li></ol>代表性的成果大多数的方法可用于研究细胞在组织培养中，可以很容易地适应用于角膜感染HSV-1 体外 。这里展示的是代表的一些研究HSV-1感染的最常见的技术-定量PCR（ 图1D），定量RT-PCR（ 图1B），Western blot检测（ 图1F），空斑法（ 图1C），组织的免疫荧光（ 图1E）。 <img src="/files/ftp_upload/3631/3631fig1.jpg" alt="图1" /> 图1。分析角膜感染HSV-1 EX v龙腾。 （A）急性角膜上皮HSV-1感染的体外模型示意图。（BF）分析使用视频文章中描述的方法角膜感染。取出的兔角膜与1×10 4 PFU /角膜应变KOS HSV-1感染和培养（PAA）的存在或不存在（模拟）膦酰乙酸（400微克/毫升），一种已知的单纯疱疹病毒复制抑制剂（B）在指定的时间点收集的总RNA，和病毒的转录与HSV-1聚合酶的引物，通过定量RT-PCR进行评估。 18S rRNA基因的引物对被用来作为参考。 n = 3时。误差条表示±扫描电镜（SEM）（C）的培养基，收集在48 HPI，传染性颗粒生产的CV-1单层上的噬斑分析评估，（D）的总DNA，收集在48 HPI，病毒基因组的复制，评估一个定量PCR检测的引物为HSV-1基因组。镨imers对GAPDH用作参考。 n = 3时。误差条表示±扫描电镜（SEM）（E）的模拟处理的角膜闪存冻结在48 HPI，切片，并通过间接免疫荧光法分析的存在下，受感染的上皮细胞内的病毒的早期蛋白质（ICP8）。核复染与Höchst的33258。（F）蛋白裂解液收集在48 HPI，并通过Western blot检测病毒蛋白的表达（ICP0）进行了评估。 BCA法，以确保样品相等的加载。 HPI小时后感染。

<ol>
	<li>Kaye, S., Choudhary, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+keratitis.">Herpes simplex keratitis.</a> Prog. Retin. Eye Res. 25, 355-380 (2006).</li><li>Holmberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enucleation+of+exotic+pets.">Enucleation of exotic pets.</a> Journal of Exotic Pet Medicine. 16, 88-94 (2007).</li><li>Remeijer, L., Osterhaus, A., Verjans, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+herpes+simplex+virus+keratitis:+the+pathogenesis+revisited.">Human herpes simplex virus keratitis: the pathogenesis revisited.</a> Ocul. Immunol. Inflamm. 12, 255-285 (2004).</li><li>Foreman, D. M., Pancholi, S., Jarvis-Evans, J., McLeod, D., Boulton, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+organ+culture+model+for+assessing+the+effects+of+growth+factors+on+corneal+re-epithelialization.">A simple organ culture model for assessing the effects of growth factors on corneal re-epithelialization.</a> Exp. Eye Res. 62, 555-564 (1996).</li><li>Xu, K. P., Li, X. F., Yu, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+organ+culture+model+for+assessing+epithelial+responses+to+surfactants.">Corneal organ culture model for assessing epithelial responses to surfactants.</a> Toxicol. Sci. 58, 306-314 (2000).</li><li>Klausner, E. A., Peer, D., Chapman, R. L., Multack, R. F., Andurkar, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+gene+therapy.">Corneal gene therapy.</a> J. Control Release. 124, 107-133 (2007).</li><li>Halford, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ICP0+antagonizes+Stat+1-dependent+repression+of+herpes+simplex+virus:+implications+for+the+regulation+of+viral+latency.">ICP0 antagonizes Stat 1-dependent repression of herpes simplex virus: implications for the regulation of viral latency.</a> Virol. J. 3, 44 (2006).</li><li>Bhattacharjee, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+treatment+of+ocular+HSK+with+a+human+apolipoprotein+E+mimetic+peptide+in+a+mouse+eye+model.">Effective treatment of ocular HSK with a human apolipoprotein E mimetic peptide in a mouse eye model.</a> Invest Ophthalmol Vis. Sci. 49, 4263-4268 (2008).</li><li>Novitskaya, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difficulties+imaging+herpes+simplex+keratitis+with+fluorescein+isothiocynate-labeled+anti-HSV-1+antibodies+in+an+ex+vivo+model.">Difficulties imaging herpes simplex keratitis with fluorescein isothiocynate-labeled anti-HSV-1 antibodies in an ex vivo model.</a> Cornea. 28, 421-425 (2009).</li><li>Sharma, A., Shimeld, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+immunofluorescence+to+diagnose+herpes+simplex+virus+keratitis+in+mice.">In vivo immunofluorescence to diagnose herpes simplex virus keratitis in mice.</a> Br. J. Ophthalmol. 81, 785-788 (1997).</li></ol>

没有利益冲突的声明。

在这个视频文章中，我们描述了急性角膜上皮细胞在体外模型中的HSV-1感染的研究方法。之间的在体外和体内模型中，该模型是一个有用的踏脚石，因为它允许对生理精确验证细胞培养的结果，并且由此限制动物实验的量必须执行的。此外，体外模型提供了一种独特而宝贵的机会，学习完整的人角膜上皮细胞单纯疱疹病毒感染。下面列出的是采用这种模式?...

<table border="1"><tbody><tr><td> 试剂/耗材 </td><td> 公司 </td><td> 目录＃ </td></tr><tr><td>最小必需培养基（MEM） </td><td>敏达</td><td> 10-010-CV </td></tr><tr><td>非必需氨基酸（100倍的溶液） </td><td>敏达</td><td> 25-025-CI </td></tr><tr><td> L-谷氨酰胺</td><td>敏达</td><td> 25-005-CI </td></tr><tr><td>青霉素G钠盐</td><td>西格玛</td><td> P3032 </td></tr><tr><td>链霉素硫酸盐</td><td>西格玛</td><td> S9137 </td></tr><tr><td>琼脂糖</td><td> Invitrogen公司</td><td> 15510-027 </td></tr><tr><td>磷酸盐缓冲液（PBS） </td><td>在实验室制备</td><td> N / A </td></tr><tr><td>胰蛋白酶EDTA（1X）&lt;/ TD&gt; <td>敏达</td><td> 25-053-CI </td></tr><tr><td>的DNeasy血液及组织试剂盒</td><td> QIAGEN </td><td> 69504 </td></tr><tr><td> RNeasy Mini试剂盒</td><td> QIAGEN </td><td> 74104 </td></tr><tr><td> Laemmli缓冲液</td><td>在实验室制备</td><td> N / A </td></tr><tr><td>最佳切削温度（OCT）化合物</td><td>组织康</td><td> 4583 </td></tr><tr><td>全兔眼（年轻） </td><td> PEL-FREEZ </td><td> 41211-2 </td></tr><tr><td>人类角膜和全眼</td><td>当地眼库</td><td> N / A </td></tr><tr><td> 12井瓷现货板</td><td>阿伏加德罗常数的实验室供应</td><td> 1877年</td></tr><tr><td> 35毫米组织培养皿</td><td>鹰</td><td> 353001 </td></tr><tr><td> 6井细胞培养板</td><td>格雷纳生物之一</td><td> 657160 </td></tr><tr><td> Cryomold中级</td><td>组织康</td><td> 4566 </td></tr></tbody></table>

ex vivo organotypic corneal model of acute epithelial herpes simplex virus type i infection

疱疹性角膜炎是一种与单纯疱疹病毒1型（HSV-1）相关联的最严重的病症。疱疹性角膜炎是目前在发达国家都角膜和来自感染相关性失明的首要原因。典型的表现包括感染疱疹性角膜炎的角膜上皮，有时更深的角膜基质和内皮细胞，导致永久性角膜病理疤痕，变薄，不透明度1。 角膜HSV-1感染的传统研究了两种类型的实验模型。在被感染的培养皿中培养的单层角膜上皮细胞的体外模型，提供简单，高层次的可复制性，快速实验，和相对低廉的成本。在另一方面， 在体内模型，在其中直接进行接种的动物（如兔或小鼠）在角膜上，提供了一个高度复杂的生理系统，但具有较高的成本，更长的实验中，必要的动物护理，和更大程度的可变性。 在这个视频文章中，我们提供了详细演示了一个新的离体角膜上皮HSV-1感染模型，它结合的优势，无论是在体外和体内模型。 体外模型采用完整的角膜organotypically维持在文化和HSV-1感染。使用的体外模型允许高度的生理基础的结论，但它是相当便宜，需要投入的时间可比的体外模型。

Watch this Scientific Journal Video about Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection at JoVE.com

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

在这个视频文章中，我们描述了使用一个新的<em&gt;体外</em&gt;模型急性疱疹单纯病毒I型角膜上皮感染。

体外器官角膜模型急性上皮疱疹，单纯疱疹病毒I型感染

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1).  Herpes keratitis is currently the leading ...

ex-vivo-organotypic-corneal-model-acute-epithelial-herpes-simplex

Research

JoVE Journal

Neuroscience

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

12.6K 次观看.  Drexel University College of Medicine. 在这个视频文章中，我们描述了使用一个新的体外模型急性疱疹单纯病毒I型角膜上皮感染。

视频: Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

前全体体内培养，发展的果蝇大脑

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

科研

神经科学

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

完整的犁鼻器和副嗅球的体外制剂

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

脉络丛上皮细胞的纤毛运动的观察<em&gt;离体</em

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

在脑片恐惧电路体外光遗传学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

溶剂清除脑组织狂犬病病毒感染的高分辨率3D成像

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

原位 急性 离体 胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

成人皮层的器官型培养作为人类干细胞移植和验证的 离体 模型

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

用于 AMD 研究的基于 OCT 的人类供体眼多模态成像

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging could add prognostic value to laboratory findings. In this work, ex vivo OCT and MMI were applied to human donor eyes prior to retinal tissue sectioning. The eyes were recovered from non-diabetic white donors aged &#8805;80 years old, with a death-to-preservation time (DtoP) of &#8804;6 h. The globes were recovered on-site, scored with an 18 mm trephine to facilitate cornea removal, and immersed in buffered 4% paraformaldehyde. Color fundus images were acquired after anterior segment removal with a dissecting scope and an SLR camera using trans-, epi-, and flash illumination at three magnifications. The globes were placed in a buffer within a custom-designed chamber with a 60 diopter lens. They were imaged with spectral domain OCT (30&#176; macula cube, 30 &#181;m spacing, averaging = 25), near-infrared reflectance, 488 nm autofluorescence, and 787 nm autofluorescence. The AMD eyes showed a change in the retinal pigment epithelium (RPE), with drusen or subretinal drusenoid deposits (SDDs), with or without neovascularization, and without evidence of other causes. Between June 2016 and September 2017, 94 right eyes and 90 left eyes were recovered (DtoP: 3.9 &plusmn; 1.0 h). Of the 184 eyes, 40.2% had AMD, including early intermediate (22.8%), atrophic (7.6%), and neovascular (9.8%) AMD, and 39.7% had unremarkable maculas. Drusen, SDDs, hyper-reflective foci, atrophy, and fibrovascular scars were identified using OCT. Artifacts included tissue opacification, detachments (bacillary, retinal, RPE, choroidal), foveal cystic change, an undulating RPE, and mechanical damage. To guide the cryo-sectioning, OCT volumes were used to find the fovea and optic nerve head landmarks and specific pathologies. The ex vivo volumes were registered with the in vivo volumes by selecting the reference function for eye tracking. The ex vivo visibility of the pathology seen in vivo depends on the preservation quality. Within 16 months, 75 rapid DtoP donor eyes at all stages of AMD were recovered and staged using clinical MMI methods.

作者聚焦：基于离体 OCT 的人类供体眼多模态成像，用于年龄相关性黄斑变性的研究  

Laboratory assays can leverage prognostic value from the longitudinal optical coherence tomography (OCT)-based multimodal imaging of age-related macular degeneration (AMD). Human donor eyes with and without AMD are imaged using OCT, color, near-infrared reflectance scanning laser ophthalmoscopy, and autofluorescence at two excitation wavelengths prior to tissue sectioning.

离体 基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging ...