作者感谢沙龙Spana夫人的鼠害监测，处理和试验的帮助。博士的资金支持已经由悉尼大学和澳大利亚研究中心卓越视觉提供的。

该协议遵循悉尼大学的动物护理准则。 1.制作电极<ol><li>构造正极（一个其将接触角膜）从铂丝直径1-2毫米的短（5厘米）的长度。时尚它变成一个循环的几个毫米的直径。这个环路连接到一个常规的引线，足够长的时间，以达到你放大器的输入级（参见图4）。 </li><li>构造负极使用银/氯化银沉淀1-2毫米的直径，也连接到一个会议引线（这将在动物的嘴去）（ 见图4）。 </li><li>作为参比电极（将进入动物的臀部），用干净的皮下注射针头（23 G）的，也连接到一个适当长度的导线（ 见图4）。 </li><li>理想情况下，使用由仪器制造商提供的三引脚电缆，连接三个电极（正极û94;角膜，负→口，参考→臀部）的放大器。 </li></ol> 2.连接光刺激和ERG建立和校准<ol><li>创建（或定位）的小记录实验室，可制成暗。装备有一个以上的非长凳光发红色或红色头灯的任一个或两个。 </li><li>用照度计，以确认红色光的照度在安装过程中到达老鼠的眼睛不会超过1勒克斯。 注意：中性密度滤镜可用于降低灯的亮度和灯光的来源必须明确发出红光。如果光源发出低（可见光）波长暗适应将受到损害。 </li><li>封锁进入录音实验室所有杂散光（这往往需要恒心与不透明的胶带），并准备一个中性密度滤镜（可在张购买）足够大，以适应过来，等昏暗，任何计算机屏幕，您将在实验室。 注：杂散光和屏幕的光足以妨害暗适应鼠眼。 </li><li>连接放大器，以数据采集硬件。连接阳性，阴性和参考引线到放大器。确保计算机和LED Ganzfeld电源单元牢固地连接到一地源。 注：一些实验室有专门的接地点，连接到建筑物地面;水管是一种有效的替代。 </li><li>校准与科研质量的辐射计的LED光源。修复仪的传感器在该动物的眼睛将一个实验过程中所处位置。 </li><li>方案Ganzfeld LED的运行与逐步增加闪光能量，闪光持续时间，闪光灯重复和时间闪烁之间的全场ERG协议，称为interstimuls间隔（ISI），设置。举一个例子全场协议见表1。 注：全视野ERG从闪烁朦胧重复闪烁提高到b右闪在步骤明智的方式。双闪存程序遵循从满场的协议，使杆状和锥状响应隔离。 </li></ol> 3.前一天ERG实验<ol><li>录制黑暗中前适应SD大鼠12小时。是很方便的做到这一点，在记录实验室，一旦杂散光已被淘汰。 </li></ol> 4日ERG实验<ol><li>安排动物轻轻加热的同时录制。我们使用内置使动物的头可以放心在正确的点在入口到Ganzfeld轻金属平台。该平台具有内置管，通过它我们泵送水预热到40℃的水浴中。 注意：经验表明，这保持了动物的核心温度在37℃。 </li><li>权衡黑暗条件下的老鼠。记录体重和化妆正确的氯胺酮（60毫克/公斤）和甲苯噻嗪（5毫克/千克）剂量。抑制大鼠根TLY和腹腔注射麻醉剂。 </li><li>注意注射时间。一旦动物是无意识的（通常在5分钟），轻轻捏一只脚垫，看是否有反射反应存在检查麻醉深度。最好是等到这种反射不存在或弱，继续前。 </li><li>应用阿托品一滴proxmethacaine的，另一个角膜。 </li><li>切黑线的有10厘米长。使用一个简单的结的环路和滑环在眼睛的赤道。稍微拧紧;效果是吸引眼球微微前倾，以最小的压力。这样可以使角膜从眼皮清楚。 </li><li>应用卡波姆滴眼液在角膜表面。确保卡波姆保持在角膜表面，不会波及到眼皮或脸部。 </li><li>放置在加热平台的顶吸收床上用品。 </li><li>位置在老鼠的床上用品，用头在Ganzfeld开幕推荐的地方。 </li><li>插入INTernal温度探头插入直肠。用胶带探头线到尾位置的安全温度探头。 </li><li>插入基准电极（23政针）皮下后支腿，并连接到放大器。 </li><li>放置负电极（银/氯化银沉淀）牢固地固定在口中。为了防止这种情况滑出嘴角，加盖连接导线，以稳定的表面。 </li><li>定位正极在角膜的中心。采用显微，确保电极接触角膜平缓。 </li><li>查体温在37.0 - 37.5℃。 </li><li>一旦动物被适当地定位和电极的地方，悬垂整个安装（Ganzfeld和动物）用不透明材料（以保留暗适应）。我们用黑色的软布。 </li><li>在采集软件设置为2 kHz的采样速率为100-1000毫秒，5毫秒预收款采样的集合时间。设置带通滤波器，以1-1,000赫兹并确保采样触发品尝〜250毫秒以下闪光的时期。 </li><li>检查记录基线。它应该是免费的外来噪音，但表现出一定的放大器的噪声和振动的呼吸。 </li><li>如果基线显示多余的噪音，开始排除故障。大多数问题都与在电极位置，或接地滑脱。使用一个法拉第笼，以确保录音都是免费的外来噪音。 </li><li>运行测试闪存，0.4日志税赋cd.sm -2。一个ERG波形类似于图2A应该出现。在我们的0.4日志苏格兰人cd.sm -2闪光灯实验室典型的回答是：（a波：-474±39μV和b波：1,512±160μV，N = 11）。 </li><li>允许动物到暗重新适应10分钟。方便的是使用这些10分钟，以重新检查基线。 </li><li>稳定的信号的确认之后开始录制。 </li><li>在记录会话结束时，检查身体Temperat .. [温度茜保持。取出电极。重新卡波姆聚合物眼角膜。让动物恢复在散热垫，直到其完全的移动和积极的，返回到动物的住房之前。 </li></ol> 5.远程缺血<ol><li>在任何醒着还是anesthetised啮齿类动物进行远程缺血。 </li><li>如果将动物麻醉，把它在加热平台（上）和滑血压计袖带在后肢，明确膝关节的上部。 </li><li>如果动物被用于被处理，就可以不用麻醉执行此过程;这需要两个人。白岩松抑制动物轻轻第二应用血压计袖带和操作血压计。 </li><li>对于清醒的动物，用一块毛巾约15厘米×30〜50厘米轻轻包裹的动物，一个后肢免费。躺在动物在其上（说）左前臂背面，与持有人的手臂和躯干，以及地方之间的藏头袖带刚刚描述。 </li><li>袖带放气，并保证空气压力阀关闭。泵的袖口到160毫米汞柱的anesthetised动物，以及180毫米汞柱清醒的动物。这超出了收缩压（通常140毫米汞柱和160毫米汞柱分别）。 </li><li>保持这些压力的要求，使用手持式泵。 </li><li>之后在计划时间为缺血（我们使用的5分钟再灌注分离5分钟2期），松开气压阀放气袖带压力。 </li><li>确认远程缺血与附连到足垫皮肤温度探测器的作用。皮肤温度通常瀑布从32-30℃，超过5分钟，恢复再灌注。 </li></ol> 6.光损伤<ol><li>确保大鼠在暗适应过夜，之前的光损伤的过程。 </li><li>在肢体缺血（在我们的实验中无延迟）在适当的时候，每个动物被置于单独成有机玻璃盒，无线日水和食物地板为主的容器。 注意：光诱导损伤，只能在白化动物进行。 </li><li>开关上的预校准1000勒克斯的白光以标准时间（通常上午09点），并保持该条件24小时。 </li></ol> 7. ERG数据提取和分析<ol><li>获得ERG的平均波形。如果需要的话，校正非零基线，由减法。 </li><li>测量一个波（在中间呈现给高刺激强度）的振幅，作为基准，并在第一间的电压差（&lt;30毫秒延迟）槽（ 图1）。 </li><li>测量的b波振幅为a波的峰值和正下面波之间的电压差，通常是在80-100毫秒（ 图1）一个延迟发生。 </li><li>用傅立叶隔离振荡电位变换从60-235赫兹筛选数据，以90赫兹的过渡带<sup&gt; 7。如果需要分离的振荡电位的信号然后可从未经滤波波形中减去，以确认一个波槽的身份。 </li><li> a-和b-波峰的隐式时间（等待时间）也可以是一个有用的度量（ 图1）。使用双闪烁以分离杆响应。减去从混合反应（闪光1）隔离杆响应（ 图2）的视锥细胞反应（闪光2）。 </li><li>正常化个别光强度的波和b波振幅（后处理/后处理基线）或平均为治疗组。强度响应曲线绘制组幅度和对错误闪光能量。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+relationship+of+the+scotopic+and+photopic+ERG+to+photoreceptor+cell+loss+in+light+damaged+rats.">Quantitative relationship of the scotopic and photopic ERG to photoreceptor cell loss in light damaged rats.</a> Experimental Eye Research. 70 (5), 693-705 (2000).</li><li>Machida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P23H+rhodopsin+transgenic+rat:+Correlation+of+retinal+function+with+histopathology.">P23H rhodopsin transgenic rat: Correlation of retinal function with histopathology.</a> Investigative Ophthalmology &amp; Visual Science. 41 (10), 3200-3209 (2000).</li><li>Brandli, A., Stone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+Ischemia+Influences+the+Responsiveness+of+the+Retina.">Remote Ischemia Influences the Responsiveness of the Retina.</a> Observations in the Rat. Investigative Ophthalmology &amp; Visual Science. 55 (4), 2088-2096 (2014).</li><li>Maccarone, R., Di Marco, S., Bisti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saffron+supplement+maintains+morphology+and+function+after+exposure+to+damaging+light+in+mammalian+retina.">Saffron supplement maintains morphology and function after exposure to damaging light in mammalian retina.</a> Investigative Ophthalmology &amp; Visual Science. 49 (3), 1254-1261 (2008).</li><li>Hood, D. C., Birch, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+abnormal+rod+photoreceptor+activity+with+the+a-wave+of+the+electroretinogram:+Applications+and+methods.">Assessing abnormal rod photoreceptor activity with the a-wave of the electroretinogram: Applications and methods.</a> Documenta Ophthalmologica. 92 (4), 253-267 (1996).</li><li>Robson, J. G., Frishman, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rod-driven+a-wave+of+the+dark-adapted+mammalian+electroretinogram.">The rod-driven a-wave of the dark-adapted mammalian electroretinogram.</a> Progress in Retinal and Eye Research. 39, 1-22 (2014).</li><li>Hood, D. C., Birch, D. 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+COMPUTATIONAL+MODEL+OF+THE+AMPLITUDE+AND+IMPLICIT+TIME+OF+THE+B-WAVE+OF+THE+HUMAN+ERG.">A COMPUTATIONAL MODEL OF THE AMPLITUDE AND IMPLICIT TIME OF THE B-WAVE OF THE HUMAN ERG.</a> Visual Neuroscience. 8 (2), 107-126 (1992).</li><li>Wachtmeister, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillatory+potentials+in+the+retina:+what+do+they+reveal.">Oscillatory potentials in the retina: what do they reveal.</a> Progress in Retinal and Eye Research. 17 (4), 485-521 (1998).</li></ol>

乔纳森·斯通是CSCM私人有限公司的董事

上述的暗适应闪光ERG方法是评估在大鼠视网膜功能的可靠方法。两者的a波和b波被光伤害减少。远程缺血预处理减轻光损伤诱导的减少在一个波和B波。这种防护视网膜功能的建议，远程缺血性预处理已引起神经保护作用，类似其他形式的防护预处理，如缺氧，缺血和运动8-10。记录设置中，光刺激的参数，和动物的状态 - 记录ERG的信号是由三组因素来决定。 录像设置<...

ERG的是由视网膜响应于光产生的，并且从眼睛的角膜表面记录的电势。当记录条件精心管理，ERG的可以以各种不同的方式来评估视网膜功能被使用。在这里，我们介绍了如何记录“闪光ERG”，时产生的视网膜暴露在一个简短的，明亮的闪光在Ganzfeld背景呈现的潜力。该Ganzfeld分散的均匀光与光的闪光灯到达视网膜整体大致均匀。如果视网膜记录前暗适应和暗适应被保持为动物准备用于记录，ERG的获得是通过两个杆和视锥感光细胞生成的。 暗适应闪光ERG的具有特征的波形，这已在两个方面进行了分析。首先，ERG的波形的早期和晚期成分被区分开来，并与神经元的序人活化在视网膜上。最早的组件是一个短潜伏期负向电位的一个波（ 图1）。这之后是一个正向电位，称为b波。 b波的上升阶段表示振荡，这被认为是一个单独的部件（振荡电位或有机磷）。在一个波被认为是由光感受器产生，b波由内核层细胞和有机磷由无长突细胞1。 根据刺激强度，反应非常昏暗闪烁称为暗门槛响应是可能的。暗视阈响应被理解为从视网膜神经节细胞2-4生成的。第二，闪光ERG可以通过光适应来分离，或通过两个闪光协议如下所述，成棒状和锥驱动的组件。明视下的条件下，一个波是在大鼠未检测到，因为锥体人口低，但有机磷和b波是明确5。在灵长类动物，其视网膜具有较高的人口锥，既杆状和锥途径产生可探测的一个波6。 常从闪速ERG提取两个有用措施是a-和b-波的振幅，测量为在图1中，与图2中所示的典型闪光的反应。当感光体人口被减少，例如通过暴露于破坏力亮光，ERG的所有成分减少。神经保护措施，如远程缺血预处理（RIP），可以由a-和b-波（ 图3）的振幅的保存进行验证。综上所述，ERG的分析使得健康，光损伤和neuroprotected视网膜之间的比较。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>PC computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Powerlab, 4 channel acquistion hardware</td>
			<td>AD Instruments</td>
			<td>PL 35044</td>
			<td>Acquistion of ERG</td>
		</tr>
		<tr>
			<td>Animal Bio Amp</td>
			<td>AD Instruments</td>
			<td>FE 136</td>
			<td>Amplifier for ERG</td>
		</tr>
		<tr>
			<td>Lab chart</td>
			<td>AD Instruments</td>
			<td></td>
			<td>Signal collection software</td>
		</tr>
		<tr>
			<td>Ganzfield</td>
			<td>Photometric solutions</td>
			<td>FS-250A</td>
			<td>Light stimulus</td>
		</tr>
		<tr>
			<td>Ganzfield operating system</td>
			<td>Photometric solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Research Radiometer</td>
			<td>International light technologies</td>
			<td>ILT-1700</td>
			<td>calibrate light series</td>
		</tr>
		<tr>
			<td>Lux meter</td>
			<td></td>
			<td>LX-1010B&#160;</td>
			<td>check red light illumanation</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lead wires</td>
			<td>AD Instruments</td>
			<td></td>
			<td>Connect postive, negative ground electrodes to amplifier</td>
		</tr>
		<tr>
			<td>Lead wires -aligator</td>
			<td>AD Instruments</td>
			<td></td>
			<td>ground ganzfield and acquistion hardware to computer</td>
		</tr>
		<tr>
			<td>Platinum wire 95%</td>
			<td>A&#38;E metals</td>
			<td></td>
			<td>postive electrode</td>
		</tr>
		<tr>
			<td>Mouth electrode Ag/AgCl Pellet</td>
			<td>SDR</td>
			<td>E205</td>
			<td>negative electode</td>
		</tr>
		<tr>
			<td>26 gauge needle</td>
			<td>BD</td>
			<td></td>
			<td>ground electode</td>
		</tr>
		<tr>
			<td>Water pump</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Homeothermic blanket system with flexible probe</td>
			<td>Harvard Appartus</td>
			<td>507222F</td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine 1% w/v</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>topical mydriasis</td>
		</tr>
		<tr>
			<td>Proxmethycaine 0.5% w/v</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>topical anaesthetic</td>
		</tr>
		<tr>
			<td>Visco tears eye drops</td>
			<td>Novartis</td>
			<td></td>
			<td>carbomer polymer</td>
		</tr>
		<tr>
			<td>Thread</td>
			<td></td>
			<td></td>
			<td>retract eye lid</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reusable adhesive</td>
			<td>Blu tac</td>
			<td></td>
			<td>Dim red headlamp. Affix electrodes</td>
		</tr>
		<tr>
			<td>Absorbent bedding</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamil - ketamine 100 mg/ml - 50 ml</td>
			<td>Troy Laboratories Pty Ltd</td>
			<td></td>
			<td>dissociative</td>
		</tr>
		<tr>
			<td>Xylium - Xylazine 100 mg/ml - 50 ml</td>
			<td>Troy Laboratories Pty Ltd</td>
			<td></td>
			<td>muscle relaxant</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

using the electroretinogram to assess function in the rodent retina and the protective effects of remote limb ischemic preconditioning

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the &#8216;flash&#8217; ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.

该协议可以用于测量啮齿动物视网膜的体内视觉功能。在一个波，光感受器功能的量度，和b波，内视网膜功能的量度，被注释在图1中。 随光刺激的棒为主的ERG的信号增加时， 如图2A所示 。在〜0.4日志苏格兰人cd.sm -2和的一个波增加，直到饱和为2.5日志苏格兰人cd.sm振幅-2（未示出）的一个波变得很明显。双子闪光范式已经用于...

Watch this Scientific Journal Video about Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning at JoVE.com

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

使用电生理评估功能，在啮齿动物的视网膜和远程肢体缺血预适应的保护作用

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

using-electroretinogram-to-assess-function-rodent-retina-protective

Research

JoVE Journal

Neuroscience

16.0K 次观看.  University of Sydney. The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

视频: Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

Strategies for Study of Neuroprotection from Cold-preconditioning

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro model that closely reflects their in vivo counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-&alpha; to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.

We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. We present strategies for such work that require biological systems, experimental manipulations plus technical capacities that are highly reproducible and sensitive.

冷预处理神经保护作用的研究策略

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

科研

神经科学

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

鼠标感觉神经支配的皮肤和四肢骨骼纤维组织的制备及免疫染色

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

血管再生在中枢神经系统中使用的小鼠视网膜评估

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

在斑马鱼幼虫的视觉反应的视网膜电图分析

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

影像学检查HT能纤维在小鼠脊髓使用CLARITY / CUBIC技术

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

爆炸性神经损伤对压生啮鼠中脑动脉的影响

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

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 ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

结合行为和脑电图研究正念冥想对情景记忆的影响

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...