我们的研究是由美国国立卫生研究院，美国国家眼科研究所院内研究计划的支持。

所有的设备和试剂使用无菌。所有动物实验获得批准的动物护理和使用的聂/美国国立卫生研究院委员会（ACUC）（动物研究协议聂- 570），并根据美国国立卫生研究院的准则和法规执行。 1。基线逆行标记视网膜神经节细胞（RGC的）第1天这一程序的目的是标签的视网膜神经节细胞的神经示踪染料注射入小鼠视神经挤压伤的前三天，上丘逆行。染料将逆行采取由视网膜神经节细胞，并提供生活RGC的一个标记（图1）。这种方法产生的变化不大可行的视网膜神经节细胞1-5重现标签。 <ol><li>深麻醉鼠标。清洁的头顶上的头发。放置在一个小的立体定位仪鼠标。保护oinment是适用于两只眼睛。三次使用碘伏和70％的酒精擦洗消毒头部皮肤。 </li><li>做一个切口，在皮肤暴露的头骨，并保持干燥和清洁用3％双氧水。 </li><li>识别和标记的前囟门。钻前囟门后面，和2.9毫米，0.5毫米，横向到每个半球7中线以上的孔上丘。在钻井过程中，应用生理盐水孔钻，以防止二次热损伤的网站。 </li><li>使用立体测量设备和汉密尔顿注射器，注入每个半球上丘荧光金神经示踪染料（1微升3％生理盐水中荧光色素）非常缓慢的脑骨表面1.6毫米的深度。 </li><li>缝合切口部位。为镇痛丁丙诺啡皮下注射。 </li><li>将鼠标移动到一个温暖而干燥的地区和监测，直到它能够保持直立的姿势，并返回到其家笼。首3天的标签程序后，全身性镇痛药（例如，丁丙诺啡）和外用抗生素软膏，每天两次给予密切监测和鼠标。 </li></ol> 2。第4天的视神经挤压伤在此过程中，我们将应用挤压伤到视神经，导致主损伤的轴突（图2），这将导致的视网膜神经节细胞逐渐死亡。 <ol><li>三天后示踪染料应用，鼠标深感麻醉。使用解剖显微镜，一只眼结膜切开约4点钟位置，用一双弹簧剪刀。 </li><li>轻轻地转移轨道的肌肉，并把它们放在一边。退出眼球暴露在白色的视神经。非常小心，不要破坏任何血管。 </li><li>随着跨动作钳对援助，适用于约2毫米的眼球一个挤压伤到视神经，约3秒钟。非常谨慎，不损伤眼动脉，引起出血。 </li><li>挤压伤完成后，切口缝合。 </li><li>在小鼠从麻醉中复苏，给一个皮下注射丁丙诺啡镇痛。 </li><li>移动鼠标到一个温暖，干燥的地区，监视它，直至它能够保持直立的姿势，并返回到其家乡笼。前三后挤压伤过程，全身镇痛药（例如，丁丙诺啡）和外用抗生素软膏，每日两次，鼠标密切监察。 </li></ol> 3。收获的视网膜和分析研资局生存11天这一程序的目的是收获retinaeto分析视网膜神经节细胞的存活。 <ol><li>在第11天（ONC后7天），鼠标是由CO 2输液和颈椎脱位安乐死。 </li><li>眼睛被摘除的一双轨道施加压力钳的帮助。 </li><li>两个小时的眼睛是在4％多聚甲醛溶液固定，PBS洗3次。剖析视网膜。视网膜清扫的过程已经被发表在2010年通过的Jove Gustmann，S. 等 。6，因此不会在这里描述。 </li><li>尚存的视网膜神经节细胞的图像在视网膜定义地区使用荧光显微镜，可以算作视网膜神经节细胞密度。带或不带荧光标记的视神经挤压伤视网膜神经节细胞的一个代表性的结果如图3所示。 </li></ol> 4。代表性的成果: 分析视网膜神经节细胞的生存，视神经挤压伤后7天，收获的视网膜，固定，夷为平地，安装。视网膜图像，分别用荧光显微镜。与健侧正常řetina（左，正常），可行的视网膜神经节细胞的数量（绿色，逆行到上丘注射荧光金神经示踪染料标记）是显着的视网膜与视神经挤压伤（右，ONC）（图3） 。 <img src="/files/ftp_upload/2685/2685fig1.jpg" alt="图1" /> 图1。基线逆行标记视网膜神经节细胞 为了算可行的视网膜神经节细胞，视神经挤压伤后不同时间点逆行标记视网膜神经节细胞注射到大脑中的三天丘的视神经前一个神经示踪染料（绿色）（红色），挤压伤。由于视网膜神经节细胞的轴突位于上丘，将采取示踪染料由视网膜神经节细胞逆行，并提供生活RGC的一个标志。 <img src="/files/ftp_upload/2685/2685fig2.jpg" alt="图2" /> 图2。视神经挤压伤 为了分析视网膜神经节细胞存活的状态，挤压伤是适用于视神经（红色），导致主轴突的损害。这将导致的视网膜神经节细胞逐渐死亡。三天示踪染料（绿色）注射后，老鼠被麻醉。一只眼结膜切开。偏离轨道的肌肉，并把它们放在一边。暴露在退出其眼球的视神经。应用挤压伤到视神经约2毫米的眼球约3秒钟，用一双跨动作钳。 <img src="/files/ftp_upload/2685/2685fig3.jpg" alt="图3" /> 图3。荧光标记的视网膜神经节细胞，视神经挤压伤或不 分析视网膜神经节细胞的生存，视神经挤压伤后7天，收获的视网膜，固定，夷为平地，安装。视网膜图像，分别用荧光显微镜。可行的视网膜神经节细胞（绿色荧光金神经示踪染料注射到上丘逆行标记）与未受伤的正常的视网膜（左，普通）相比，明显的视网膜，视神经挤压伤（右，ONC ）（图3）。比例尺:50微米

<ol>
	<li>Yoles, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM1+reduces+injury-induced+metabolic+deficits+and+degeneration+in+the+rat+optic+nerve.">GM1 reduces injury-induced metabolic deficits and degeneration in the rat optic nerve.</a> Investigative ophthalmology &#38; visual science. 33, 3586-3591 (1992).</li><li>Fisher, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccination+for+neuroprotection+in+the+mouse+optic+nerve:+implications+for+optic+neuropathies.">Vaccination for neuroprotection in the mouse optic nerve: implications for optic neuropathies.</a> J Neurosci. 21, 136-142 (2001).</li><li>Levkovitch-Verbin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RGC+death+in+mice+after+optic+nerve+crush+injury:+oxidative+stress+and+neuroprotection.">RGC death in mice after optic nerve crush injury: oxidative stress and neuroprotection.</a> Investigative ophthalmology &#38; visual science. 41, 4169-4174 (2000).</li><li>Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-B+inhibits+apoptosis+via+VEGFR-1-mediated+suppression+of+the+expression+of+BH3-only+protein+genes+in+mice+and+rats.">VEGF-B inhibits apoptosis via VEGFR-1-mediated suppression of the expression of BH3-only protein genes in mice and rats.</a> The Journal of clinical investigation. 118, 913-923 (2008).</li><li>Tang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+effect+of+PDGF-CC+rescues+neurons+from+apoptosis+in+both+brain+and+retina+by+regulating+GSK3beta+phosphorylation.">Survival effect of PDGF-CC rescues neurons from apoptosis in both brain and retina by regulating GSK3beta phosphorylation.</a> The Journal of experimental medicine. 207, 867-880 (2010).</li><li>Gustmann, S., Dunker, N. <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-like+organotypic+murine+retinal+wholemount+culture.">In vivo-like organotypic murine retinal wholemount culture.</a> J Vis Exp. , (2010).</li><li>Franklin, K., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (1997).</li></ol>

视神经挤压伤的小鼠模型研究RGC的死亡和生存的过程中是非常有用。这种模式也常常用于研究不同的试剂和RGC的凋亡和生存的基因的影响。这个模型的一个优点是它具有高度的重复性，最小的变化。 然而，需要特别照顾，在此模型中的几个步骤。首先，使视神经挤压伤时，它是必不可少的，不要用力过猛，而不是粉碎太久，因为它们可能导致眼动脉，因此随后的视网膜缺血?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Stereotaxic apparatus</td>
<td>&nbsp;</td>
<td>ASI Instruments</td>
<td>SAS-4100</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>PZMIV-BS </td>
<td>&nbsp;</td>
<tr>
<td>Scalpel handle</td>
<td>&nbsp;</td>
<td>Fine Science Tools </td>
<td>10003-12 </td>
<td>&nbsp;</td>
<tr>
<td>Scalpel Blades #15</td>
<td>&nbsp;</td>
<td>Fine Science Tools </td>
<td>10015-00</td>
<td>&nbsp;</td>
<tr>
<td>Microdrill</td>
<td>&nbsp;</td>
<td>Fine Science Tools </td>
<td>18000-17</td>
<td>&nbsp;</td>
<tr>
<td>Suture needle</td>
<td>&nbsp;</td>
<td>Fine Science Tools </td>
<td>12050-03</td>
<td>&nbsp;</td>
<tr>
<td>Spring scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools </td>
<td>16144-13</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools </td>
<td>11252-50</td>
<td>&nbsp;</td>
<tr>
<td>Hamilton syringe</td>
<td>&nbsp;</td>
<td>Hamilton Company </td>
<td>88400</td>
<td>&nbsp;</td>
<tr>
<td>Gauze sponges</td>
<td>&nbsp;</td>
<td>Office Depot</td>
<td>674889</td>
<td>&nbsp;</td>
<tr>
<td>Fluorochrome</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>39286</td>
<td>&nbsp;</td>
<tr>
<td>Alcohol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>459844</td>
<td>70%</td>
</tr>
<tr>
<td>H2O2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3410</td>
<td>3%</td>
</tr>		</tbody>
		</table>
		</div>

an optic nerve crush injury murine model to study retinal ganglion cell survival

视神经损伤可导致轴索变性，逐渐死亡的视网膜神经节细胞（RGC的），在不可逆的视力丧失结果。在人类疾病的例子包括:外伤性视神经病变和青光眼视神经病变。它的特点是典型的视神经头的变化，逐步视神经病变，和视网膜神经节细胞的损失，如果不加控制，导致视力下降和失明。 视神经粉碎（ONC）损伤的小鼠模型，是一个重要的外伤性视神经病变的实验疾病模型，青光眼等，在此模型中，挤压伤到视神经，导致逐步视网膜神经节细胞的凋亡。这种疾病模型可以用来研究神经元死亡和生存，这是必要的治疗措施的发展的一般过程和机制。此外，药理学和分子的方法，可以在此模型中，识别和测试潜在的治疗试剂，治疗视神经病变的不同类型。 在这里，我们提供了一步一步演示（一）基线逆行标记视网膜神经节细胞（RGC的）第1天，（二）在4天的视神经挤压伤，（三）收获的视网膜和分析研资局在一天的生存11，（四）代表结果。

Watch this Scientific Journal Video about An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival at JoVE.com

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

该协议显示了如何逆行标记视网膜神经节细胞，以及如何随后视神经挤压伤，以分析视网膜神经节细胞的存活和凋亡。这是一个为不同类型的视神经病变，包括青光眼的实验性疾病模型。

一个视神经挤压伤的小鼠模型，研究视网膜神经节细胞生存

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

an-optic-nerve-crush-injury-murine-model-to-study-retinal-ganglion

Research

JoVE Journal

Neuroscience

30.4K 次观看.  NIH. 该协议显示了如何逆行标记视网膜神经节细胞，以及如何随后视神经挤压伤，以分析视网膜神经节细胞的存活和凋亡。这是一个为不同类型的视神经病变，包括青光眼的实验性疾病模型。

视频: An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

视神经横断:一个成人在中枢神经系统的神经元凋亡模型

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

科研

神经科学

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

微创技术注入到老鼠视神经

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

在实验视神经脱髓鞘模型大鼠视觉诱发电位记录

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

视网膜神经节细胞定义子集的单细胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

背根神经节内注射和背根挤压伤为感官轴突再生模型

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

大鼠部分视神经切断术: 建立一种新的手术方法评价视网膜神经节细胞二次变性的模型

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Ethanol-Induced Cervical Sympathetic Ganglion Block Applications for Promoting Canine Inferior Alveolar Nerve Regeneration Using an Artificial Nerve

Polyglycolic acid collagen (PGA-C) tubes are bio-absorbable nerve tubes filled with collagen of multi-chamber structure, which consist of thin collagen films. Favorable clinical outcomes have been achieved when using these tubes for the treatment of damaged inferior alveolar nerve (IAN). A critical factor for the successful nerve regeneration using PGA-C tubes is blood supply to the surrounding tissue. Cervical sympathetic ganglion block (CSGB) creates a sympathetic blockade in the head and neck region thus increasing blood flow in the area. To ensure an adequate effect, the blockade must be administered with local anesthetics one to two times a day for several consecutive weeks; this poses a challenge when creating animal models for investigating this technique. To address this limitation, we developed an ethanol-induced CSGB in a canine model of long-term increase in blood flow in the orofacial region. We examined whether IAN regeneration via PGA-C tube implantation can be enhanced by this model. Fourteen Beagles were each implanted with a PGA-C tube across a 10-mm gap in the left IAN. The IAN is located within the mandibular canal surrounded by bone, therefore we chose piezoelectric surgery, consisting of ultrasonic waves, for bone processing, in order to minimize the risk of nerve and vessel injury. A good surgical outcome was obtained with this approach. A week after surgery, seven of these dogs were subjected to left CSGB by injection of ethanol. Ethanol-induced CSGB resulted in improved nerve regeneration, suggesting that the increased blood flow effectively promotes nerve regeneration in IAN defects. This canine model can contribute to further research on the long-term effects of CSGB.

We evaluated the effect of cervical sympathetic ganglion block on nerve repair using artificial nerve conduits. Male beagle dogs were each implanted with an artificial nerve across a 10-mm gap in the left inferior alveolar nerve; left cervical sympathetic ganglion was blocked by injecting 99.5% ethanol via lateral thoracotomy.

乙醇诱导的颈椎交交型神经节阻滞在促进犬下肺泡神经再生中的应用

Polyglycolic acid collagen (PGA-C) tubes are bio-absorbable nerve tubes filled with collagen of multi-chamber structure, which consist of thin collagen ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

大鼠模型中的面部神经外科研究同氧抑制与再生

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...