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><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>Antibiotics (cefazolin, Kefzol)</td><td>Eurocept Pharmaceuticals</td><td>BE 106267</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 (&lt;em&gt;e.g.&lt;/em&gt;, 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

Universidad de Cartagena UDEC

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

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

老鼠的眼睛剜除术的远程高通量的表型

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

科研

医学

Video-oculography in Mice

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).
Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system5. Second, the oculomotor system is relative simple6; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles7. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis8. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes9.
Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels10. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice8.

Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.

视频oculography小鼠

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the ...

神经科学

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

在体内的器官型小鼠视网膜Wholemount文化

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

生物学

Monocular Visual Deprivation and Ocular Dominance Plasticity Measurement in the Mouse Primary Visual Cortex

Monocular visual deprivation is an excellent experimental paradigm to induce primary visual cortical response plasticity. In general, the response of the cortex to the contralateral eye to a stimulus is much stronger than the response of the ipsilateral eye in the binocular segment of the mouse primary visual cortex (V1). During the mammalian critical period, suturing the contralateral eye will result in a rapid loss of responsiveness of V1 cells to contralateral eye stimulation. With the continuing development of transgenic technologies, more and more studies are using transgenic mice as experimental models to examine the effects of specific genes on ocular dominance (OD) plasticity. In this study, we introduce detailed protocols for monocular visual deprivation and calculate the change in OD plasticity in mouse V1. After monocular deprivation (MD) for 4 days during the critical period, the orientation tuning curves of each neuron are measured, and the tuning curves of layer four neurons in V1 are compared between stimulation of the ipsilateral and contralateral eyes. The contralateral bias index (CBI) can be calculated using each cell&#39;s ocular OD score to indicate the degree of OD plasticity. This experimental technique is important for studying the neural mechanisms of OD plasticity during the critical period and for surveying the roles of specific genes in neural development. The major limitation is that the acute study cannot investigate the change in neural plasticity of the same mouse at a different time.

Here, we present detailed protocols for monocular visual deprivation and ocular dominance plasticity analysis, which are important methods for studying the neural mechanisms of visual plasticity during the critical period and the effects of specific genes on visual development.

小鼠主视觉皮质中的单眼视觉剥夺和眼部优势可塑性测量

Monocular visual deprivation is an excellent experimental paradigm to induce primary visual cortical response plasticity. In general, the response of the ...

发育生物学

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

穿刺诱导的虹膜新生血管虹膜虹膜小鼠模型的建立

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

植入颅窗以在清醒小鼠 中重复体内 成像

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

用于轴突再生研究的角膜感觉纤维损伤方案

Three Strategies to Induce Neurotrophic Keratitis and Nerve Regeneration in Murine Cornea

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation provides sensation and trophic function to the eye and contributes to preserving corneal integrity. The pathological disruption of this innervation is termed neurotrophic keratitis. This can be triggered by injury to the eye, surgery, or disease. In this study, we propose three different protocols for inflicting damage on the innervation in ways that recapitulate the three types of cases generally encountered in the clinic.
The first method consists in making an abrasion of the epithelium with an ophthalmic burr. This involves the removal of the epithelial layer, the free nerve endings, and the subbasal plexus in a manner similar to the photorefractive keratectomy surgery performed in the clinic. The second method&#160;only targets the innervation by sectioning it at the periphery with a biopsy punch, maintaining the integrity of the epithelium. This method is similar to the first steps of lamellar keratoplasty and leads to a degeneration of the innervation followed by regrowth of the axons in the central cornea. The last method damages the innervation of a transgenic mouse model using a multiphoton microscope, which specifically localizes the site of cauterization of the fluorescent nerve fibers. This method inflicts the same damage as photokeratitis, an overexposure to UV light.
This study describes different options for investigating the physiopathology of corneal innervation, particularly the degeneration and regeneration of the axons. Promoting regeneration is crucial for avoiding such complications as epithelium defects or even perforation of the cornea. The proposed models can help test new pharmacological molecules or gene therapy that enhance nerve regeneration and limit disease progression.

作者聚焦：通过创新模型推进角膜神经支配研究 

Here, we propose three different methods of damaging the sensory fibres innervating the cornea. These methods facilitate the study of axon regeneration in mice. These three methods, which are adaptable to other animal models, are ideal for the study of corneal innervation physiology and regeneration.

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation ...

Microdissection of the Rodent Eye

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, to obtain the anterior and posterior eyecups. With this technique, the sub-parts of the eye, including the corneal tissue, neural tissue, retinal pigment epithelial (RPE) tissue, and lens, can be obtained for wholemounts, cryo-sectioning, and/or single-cell suspensions of a specific ocular tissue. The presence of the third eyelid presents unique and significant advantages, as it benefits the maintenance of the orientation of the eye, which is important for understanding eye physiology following any localized intervention or in studies involving ocular analysis relating to the eye&#39;s spatial topography.
In this method, we enucleated the eyeball at the socket along with the third eyelid by carefully and slowly cutting through the extraocular muscles and severing the optic nerve. The eyeball was pierced through the corneal limbus using a microblade.&#160;The incision was used as the point of entry, allowing for cutting along the corneal-scleral junction by inserting micro-scissors through the incision point. Small and continuous cuts along the circumference were made until the cups separated. These could be further dissected by gently peeling the translucent layer of the neural retina using Colibri suturing forceps to obtain the neural retina and RPE layers. Further, three/four equidistant cuts were made from the periphery perpendicularly to the optic center until the optic nerve was reached. This opened the hemispherical cups into a floret shape so that they fell flat and could be easily mounted. This technique has been used in our lab for corneal wholemounts and retinal sections. The presence of the third eyelid delineates the nasal-temporal orientation, which allows for the study of various cell therapy interventions post-transplantation and, thus, the targeted physiological validation vital for visualization and accurate representation in such studies.

This paper presents a protocol for ocular micro-dissection in rodents. The process involves the enucleation of the eyeball along with the nictitating membrane (i.e., the third eyelid). This is then followed by the separation of the posterior and anterior eye cups.

啮齿动物眼睛的显微解剖

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, ...

一个高度可重复的和直接的方法来执行

摘要

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此视频中的章节

老鼠的眼睛剜除术的远程高通量的表型

视频oculography小鼠

在体内的器官型小鼠视网膜Wholemount文化

小鼠主视觉皮质中的单眼视觉剥夺和眼部优势可塑性测量

穿刺诱导的虹膜新生血管虹膜虹膜小鼠模型的建立

植入颅窗以在清醒小鼠中重复体内成像

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略

啮齿动物眼睛的显微解剖

一个高度可重复的和直接的方法来执行

摘要

探索更多视频

此视频中的章节

老鼠的眼睛剜除术的远程高通量的表型

视频oculography小鼠

在体内的器官型小鼠视网膜Wholemount文化

小鼠主视觉皮质中的单眼视觉剥夺和眼部优势可塑性测量

穿刺诱导的虹膜新生血管虹膜虹膜小鼠模型的建立

植入颅窗以在清醒小鼠 中重复体内 成像

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略

啮齿动物眼睛的显微解剖

植入颅窗以在清醒小鼠中重复体内成像