Name: visual evoked potential recording in a rat model of experimental optic nerve demyelination
Uploaded: 2015-07-29T14:00:00.000+00:00
Duration: 6 min 49 s
Description: Watch this Scientific Journal Video about Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination at JoVE.com

这项研究是由眼科研究所澳大利亚（ORIA）的支持。我们感谢教授阿尔吉斯Vingrys和Bang裴博士，墨尔本大学，最初用于帮助我们开发VEP记录技术。

伦理声明:所有涉及动物的程序是按照实践的护理和使用动物用于科学目的和ARVO声明的动物在眼科和视觉研究中使用的指导方针的澳大利亚准则进行的，并经麦考瑞大​​学动物伦理委员会。 1. VEP植入电极<ol><li>麻醉动物通过腹膜内注射氯胺酮（75毫克/千克）和美托咪定（0.5毫克/千克）。 注意:按照麻醉诱导，停药观察反射（捏测试，角膜和眼睑反射等）和它们的不存在作为指示开始手术。持续监测动物在整个手术和管理附加麻醉剂药物（每补足初始氯胺酮剂量的10％），如果反射都存在。成年大鼠（&gt; 12周）中使用的实验。 </li><li>剃手术区的皮肤上。放置在一个加热垫（37℃）的动物的手术过程中保持体温。通过聚维酮碘局部应用准备的皮肤。应用手术铺巾。应用外用眼药膏，以防止在全身麻醉下角膜干燥。通过使用无菌器械无菌维持。 </li><li>做一个纵向切开皮肤上的头部皮肤的中线。清除结缔组织达到颅骨良好的曝光。 </li><li>仔细，钻小毛刺的孔手动用微型手钻在前囱后方7毫米和3mm横向于中线。 </li><li>通过头骨向皮质（区域17）的植入物螺旋电极，穿透皮质到大约0.5mm的深度。植入螺钉参考电极上的中线3毫米延髓前囟门。应用牙科水泥包住并固定螺丝（并不总是需要）。 </li><li>缝合头部的皮肤上，给予抗生素软膏在皮肤上，并允许动物RECO从版本上麻醉变暖垫。 注意:可替换地，电极可被暴露在外面，以便皮肤不需要在每个记录被重新打开。时立即停止手术，但前麻醉恢复，管理一个非甾体抗炎药（NSAID）（如果不施用预可操作）或阿片类镇痛剂。监测动物不断，直到从麻醉剂，并充分卧床完全恢复。 </li><li>允许至少1周为动物从手术VEP记录之前恢复。 </li></ol> 2.视神经注射<ol><li>麻醉动物，备皮和应用悬垂如上（1.1和1.2）。 </li><li>作为1〜1.5厘米的切口在一个随机选择的眼睛的轨道上方的皮肤上。打开皮下组织，达到用细虹膜剪刀眶腔。打开结膜和前在手术显微镜下Tenon囊。 </li><li>收回眼肌肉和眶内泪腺以露出视神经大约3毫米的长度。纵向使用眼用刀片打开视神经周围硬脑膜和蛛网膜物质层。 </li><li>将玻璃吸管进入视神经处到地球的距离为2毫米后。在玻璃微附着于Hamilton注射器。 </li><li>注射1％溶血卵磷脂（0.4 - 1.0微升，0.02％Evan的蓝不具有对髓鞘形成的影响）慢慢进入神经大约过了一段30秒。 </li><li>缝合皮肤切口。应用抗生素软膏以防止感染。对侧眼可作为电生理学记录的内部控制。 </li><li>上放置一个变暖垫动物从麻醉中恢复。 </li></ol> 3. VEP记录<ol><li>麻醉动物，并准备皮肤1.1和1.2。 注意:麻醉药较低剂量可用于电RECO录制（氯胺酮40毫克/公斤和美托咪定0.25毫克/千克）。 </li><li>将大鼠在暗室内，并允许它适应黑暗为5 - 30分钟。在某些情况下，老鼠可能分别为暗适应O / N为暗光或适合适光VEP录音8。 </li><li>保持体温在37±0.5℃，由homoeothermic毯系统与直肠温度计探针。 </li><li>扩张1.0％托眼药水的学生。打开上方的皮肤头骨来访问预放置在原位螺钉电极。 </li><li>连接螺钉在刺激眼睛的对侧视觉皮层和参考螺钉到放大器。插入针电极插入尾部作为接地。测量和保持低于5kΩ的电极的阻抗。 </li><li>直接放置在眼睑周围皮肤迷你Ganzfeld刺激提供卓越的眼睛隔离7。需要的刺激器的照明将被事先标定用光度计。 </li><li>通过闪烁递送光刺激的100倍在1Hz的频率，具有1到100赫兹的低和高带通滤波器的设置，分别。信号采样率是在5千赫。 注意:信号应取样至少在约250 - 300赫兹，确保更多的两个样本在每个周期期间被收集。 </li><li>缝合皮肤背部和保持动物在变暖垫从麻醉中恢复。记录可以重复地记录在个别动物以监测在一段时间的功能变化。 </li><li>在终点，施用过量注射戊巴比妥钠（100毫克/千克，IP）的安乐死的动物。确认安乐死心脏骤停，呼吸停止和降低体温。 </li></ol> 4，组织准备和组织学<ol><li>从实施安乐死的动物中取出视神经显微镜下，修复1％多聚甲醛O / N的组织。 </li> &lt;LI&gt;用生理盐水彻底冲洗组织。对待组织在自动组织处理器和嵌入石蜡。使用旋转切片机 - （10微米5）的切断面。 注:对于免疫组织化学研究，修复组织中1％多聚甲醛，洗净用盐水和孵化与30％蔗糖O / N。在华侨城中嵌入组织包埋剂，用低温恒温器进行冷冻切片。 </li><li>在孵育0.1％快蓝溶液部分，如Luxol（95％乙醇）O / N为56℃。区分在0.05％碳酸锂的部分30秒，然后70％乙醇中再持续30秒。最后，安装部分之前染液以0.1％甲酚紫溶液30秒。使用快速蓝染色，以确定视神经髓鞘5。 </li></ol>

<ol>
	<li>Balcer, 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=Clinical+practice.+Optic+neuritis.">Clinical practice. Optic neuritis.</a> N Engl J Med. 354 (12), 1273-1280 (2006).</li><li>Lassmann, H., Waxman, S. 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> Multiple sclerosis as a neuronal disease. , 153-164 (2005).</li><li>Fahle, M., Bach, M., Heckenlively, J., Arden, 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> Principles and practice of clinical electrophysiology of vision. , 207-234 (2006).</li><li>Halliday, A. M., McDonald, W. I., Mushin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+visual+evoked+response+in+optic+neuritis.">Delayed visual evoked response in optic neuritis.</a> Lancet. 1, 982-985 (1972).</li><li>You, Y., Klistorner, A., Thie, J., Graham, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Latency+delay+of+visual+evoked+potential+is+a+real+measurement+of+demyelination+in+a+rat+model+of+optic+neuritis.">Latency delay of visual evoked potential is a real measurement of demyelination in a rat model of optic neuritis.</a> Invest Ophthalmol Vis Sci. 52 (9), 6911-6918 (2011).</li><li>You, Y., Klistorner, A., Thie, J., Gupta, V. K., Graham, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+loss+in+a+rat+model+of+optic+neuritis+is+closely+correlated+with+visual+evoked+potential+amplitudes+using+electroencephalogram+based+scaling.">Axonal loss in a rat model of optic neuritis is closely correlated with visual evoked potential amplitudes using electroencephalogram based scaling.</a> Invest Ophthalmol Vis Sci. 53, 3662 (2012).</li><li>You, Y., Klistorner, A., Thie, J., Graham, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+reproducibility+of+VEP+recording+in+rats:+electrodes,+stimulus+source+and+peak+analysis.">Improving reproducibility of VEP recording in rats: electrodes, stimulus source and peak analysis.</a> Doc Ophthalmol. 123 (2), 109-119 (2011).</li><li>Heiduschka, P., Schraermeyer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+visual+function+in+pigmented+and+albino+rats+by+electroretinography+and+visual+evoked+potentials.">Comparison of visual function in pigmented and albino rats by electroretinography and visual evoked potentials.</a> Graefes Arch Clin Exp Ophthalmol. 246 (11), 1559-1573 (2008).</li><li>You, Y., Thie, J., Klistorner, A., Gupta, V. K., Graham, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+visual+evoked+potentials+using+underlying+electroencephalogram+levels+improves+amplitude+reproducibility+in+rats.">Normalization of visual evoked potentials using underlying electroencephalogram levels improves amplitude reproducibility in rats.</a> Invest Ophthalmol Vis Sci. 53 (3), 1473-1478 (2012).</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=Animal+models+of+optic+nerve+diseases.">Animal models of optic nerve diseases.</a> Eye (Lond). 18 (11), 1066-1074 (2004).</li><li>Henry, K. R., Rhoades, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+of+albinism+and+drugs+to+the+visual+evoked+potential+of+the+mouse).">Relation of albinism and drugs to the visual evoked potential of the mouse).</a> J Comp Physiol Psychol. 92 (2), 271-279 (1978).</li><li>Murrell, J. C., Waters, D., Johnson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+effects+of+halothane,+isoflurane,+sevoflurane+and+desflurane+on+the+electroencephalogram+of+the+rat.">Comparative effects of halothane, isoflurane, sevoflurane and desflurane on the electroencephalogram of the rat.</a> Lab Anim. 42 (2), 161-170 (2008).</li><li>Makela, K., Hartikainen, K., Rorarius, M., Jantti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+F-VEP+during+isoflurane-induced+EEG+suppression.">Suppression of F-VEP during isoflurane-induced EEG suppression.</a> Electroencephalogr Clin Neurophysiol. 100 (3), 269-272 (1996).</li><li>Boyes, W. K., Padilla, S., Dyer, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+temperature-dependent+and+independent+actions+of+chlordimeform+on+visual+evoked+potentials+and+axonal+transport+in+optic+system+of+rat.">Body temperature-dependent and independent actions of chlordimeform on visual evoked potentials and axonal transport in optic system of rat.</a> Neuropharmacology. 24 (8), 743-749 (1985).</li><li>Hetzler, B. E., Boyes, W. K., Creason, J. P., Dyer, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-dependent+changes+in+visual+evoked+potentials+of+rats.">Temperature-dependent changes in visual evoked potentials of rats.</a> Electroencephalogr Clin Neurophysiol. 70 (2), 137-154 (1988).</li><li>Mitchell, 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+effects+of+lysolecithin+on+non-myelinated+axons+in+vitro.">The effects of lysolecithin on non-myelinated axons in vitro.</a> Acta Neuropathol. 58 (4), 243-248 (1982).</li><li>Meyer, R., 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=Acute+neuronal+apoptosis+in+a+rat+model+of+multiple+sclerosis.">Acute neuronal apoptosis in a rat model of multiple sclerosis.</a> J Neurosci. 21 (16), 6214-6220 (2001).</li><li>Lachapelle, F., 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=Failure+of+remyelination+in+the+nonhuman+primate+optic+nerve.">Failure of remyelination in the nonhuman primate optic nerve.</a> Brain Pathol. 15 (3), 198-207 (2005).</li></ol>

None of the authors have competing interests or conflicting interests.

The optic nerve is very susceptible to mechanical damage. Optic nerve crush injury over a duration of 1 s can lead to about 75% loss of RGC over a period of 2 weeks10. Therefore, extreme care is required while performing the surgical procedures. According to the authors&#8217; experience, it is much better to adapt a blunt dissection approach to expose and make way through the tissues around the optic nerve along the orientation of the nerve, rather than penetrating in a perpendicular orientation to the optic nerve. Also, the dura and arachnoid mater should be unwraped longitudinally to avoid any axotomy-like damage to the RGC axons. Evan&#8217;s blue is usually used to facilitate the microinjection, as presence of the dye makes the nerve light blue in colour at the injection site.
The reproducibility of VEP recording is critical for this experiment. Different types of anaesthetics and the depth of anaesthesia can affect the VEP waveforms11. We do not recommend using inhaled isoflurane anaesthesia for the VEP study, as isoflurane can lead to burst suppress on the electroencephalogram response12, probably due to GABAergic activity, resulting in more variable VEP traces13. Combination of ketamine/medetomidine anaesthesia provides stable anaesthesia that can quickly be reversed by administration of an alpha receptor antagonist such as atipamazole (2.5 mg/kg i.p). Body temperature can also have a significant impact on the speed of nerve conduction, especially in demyelinated nerves. Temperature-dependant changes in visual evoked potentials have been observed in rat VEPs and latencies were observed to be significantly longer as body temperature was lowered14,15. Hence, it is important to maintain a uniform body temperature using a homoeothermic blanket system during the VEP recordings.
Also, it is important to understand that the pathogenesis of lysolecithin-induced demyelination differs from that in optic neuritis or multiple sclerosis. Lysolecithin-induced demyelination results from the detergent like action of the toxin upon myelin lipids rather than immune-mediated inflammatory reaction, which is usually seen in the real disease scenarios16. If the study aims to investigate the mechanisms of demyelination/axonal damage in multiple sclerosis, an immune-mediated demyelination model, such as experimental autoimmune encephalomyelitis (EAE)17, should be considered. The advantage of using the toxin-induced demyelination model is that a focal lesion in the optic nerve can be produced and monitored longitudinally. In contrast to the brain lesions, the effects of demyelinated lesions on the optic nerve are more clinically apparent and measureable (lesion size can be accurately determined by VEPs or MRI imaging). Although remyelination in the optic nerve appeared to be not as pronounced as that observed previously in the case of spinal cord18, this optic nerve injection model, in conjunction with the VEP recording technique, is able to provide a superior tool to monitor the process of demyelination and remyelination in vivo, and to potentially evaluate new remyelinating therapies.

Optic neuritis is one of the most common form of optic neuropathy, causing complete or partial loss of vision1. Histologically, it is featured by inflammatory demyelination, retinal ganglion cell axonal loss and varying degrees of remyelination in the optic nerve2. Optic neuritis is usually the manifest onset of multiple sclerosis. The visual evoked potential (VEP) is a non-invasive tool for investigating the function of the visual system. It reflects the post-retinal function from the retina to the primary visual cortex and is affected in many optic nerve disease conditions3. The VEP has been predominantly used in optic neuritis patients to assess the integrity of the visual pathway4. 
The latency of VEP, which reflects the velocity of signal conduction along the visual pathway, is considered to be an accurate measurement of the level of myelin associated changes in the optic nerve5; while the amplitude of VEP is believed to be closely correlated with axonal damage of the retinal ganglion cells (RGC)6. This hypothesis has been fairly well established using the rat model of lysolecithin-induced optic nerve demyelination5. 
Here, we explicate a comprehensive protocol of optic nerve microinjection technique in rodents, which can minimise the surgical manipulation-related damage to the nerve per se as well as to the adjacent tissues such as extraocular muscles and blood vessels. Also, the skull electrode implantation surgery has been described for VEP recording in animals7. The VEP recordings can be repeatedly carried out on animals over a period of time to assess demyelination/remyelination related changes as well as impact on axonal integrity in the optic nerve.

<table><tbody><tr><td>Ketamine 100 mg/ml (Ketamil)</td><td>Troy Laboratories</td><td>AC 116</td><td></td></tr><tr><td>Medetomidine 1 mg/ml (Domitor)</td><td>Pfizer</td><td>sc-204073</td><td></td></tr><tr><td>Tropicamide 1.0% (Mydriacyl)</td><td>Alcon</td><td>sc-202371</td><td></td></tr><tr><td>Homoeothermic blanket system</td><td>Harvard Apparatus</td><td>NC9203819</td><td></td></tr><tr><td>Impedance meter&amp;nbsp;</td><td>Grass</td><td>F-EZM5</td><td></td></tr><tr><td>Screw electrodes&amp;nbsp;</td><td>Micro Fasteners</td><td>M1.0&amp;times;3mm Csk Slot M/T 304 S/S</td><td></td></tr><tr><td>Subdermal needle electrodes&amp;nbsp;</td><td>Grass</td><td>F-E3M-72</td><td></td></tr><tr><td>Rapid Repair&amp;nbsp;</td><td>DeguDent GmbH</td><td></td><td></td></tr><tr><td>Light-emitting diode&amp;nbsp;</td><td>Nichia</td><td>NSPG300A</td><td></td></tr><tr><td>Bioamplifier</td><td>CWE, Inc.</td><td>BMA-400</td><td></td></tr><tr><td>CED system</td><td>Cambridge Electronic Design, Ltd.</td><td>Power1401</td><td></td></tr><tr><td>Hamilton syringe&amp;nbsp;</td><td>Hamilton</td><td>87930</td><td></td></tr><tr><td>Lysolecithin</td><td>Sigma</td><td>L4129</td><td></td></tr><tr><td>Evan&amp;rsquo;s blue&amp;nbsp;</td><td>Sigma</td><td>E2129</td><td></td></tr></tbody></table>

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.

再现的帧内期间VEP迹线示于图1和N 1延迟一个显著延迟视神经注射后可以看到。脱髓鞘局部视神经病变可使用Luxol坚牢蓝染色5的组织学切片中观察到。 图2示出了代表性的部分有一个小焦点脱髓鞘病变视神经的中心。需要注意的是横截面并不代表病变的总体积。脱髓鞘区域可对神经推断病灶体积通过使用三维重建的每个连续的横截面来测量。我们已经证明了使用该模型在我们以前的研究延迟延迟和病灶体积之间有很强的相关性，也没有VEP延迟延迟在注射盐水的对照5。 据认为，以下的闪光照明早VEP组分是更稳定的7 </sup&gt;和主要受通过眼膜，膝状纤维初级视觉皮层的兴奋。我们的研究已经表明，N1延迟在视神经5与脱髓鞘最强的线性关系。因此，我们建议N1延迟应该用于数据分析和为在评估髓鞘再生疗法的影响纵向VEP监控。 VEP的振幅，虽然相比于延迟更多变，更表示在视神经6轴突的功能。脑电图为基础的缩放可以考虑幅度分析9。 <img alt="图1" src="/files/ftp_upload/52934/52934fig1.jpg" /> 视神经注射后图1的VEP延迟。代表VEP痕迹从之前的个体大鼠和视神经显微注射（0.8微升溶血卵磷脂）后2天。 VEP记录重复的每一天（会期内痕迹秀中的n相同的颜色），用于演示本VEP记录协议的可重复性。 （垂直比例尺:10μV;水平比例尺:10毫秒）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52934/52934fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="见图2" src="/files/ftp_upload/52934/52934fig2.jpg" /> 图2.脱髓鞘在视神经。视神经从溶血卵磷脂显微注射后的大鼠代表性横截面。髓鞘成分被染成蓝色利用luxol蓝快。脱髓鞘的小病灶可以看出，在该截面的中心。脱髓鞘区域可以在纵向连续截面来估算三维尺度病变体积来衡量。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52934/52934fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination at JoVE.com

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

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 ...

visual-evoked-potential-recording-rat-model-experimental-optic-nerve

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

12.2K 次观看.  Macquarie University. 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. 

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

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

单机在体内</em从大鼠视交叉>录音

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

科研

神经科学

Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both laboratory and clinical settings. Currently, preclinical ERG and VEP measurements are performed with anesthesia to ensure stable electrode placements. However, the very presence of anesthesia has been shown to contaminate normal physiological responses. To overcome these anesthesia confounds, we develop a novel platform to assay ERG and VEP in conscious rats. Electrodes are surgically implanted sub-conjunctivally on the eye to assay the ERG and epidurally over the visual cortex to measure the VEP. A range of amplitude and sensitivity/timing parameters are assayed for both the ERG and VEP at increasing luminous energies. The ERG and VEP signals are shown to be stable and repeatable for at least 4 weeks post surgical implantation. This ability to record ERG and VEP signals without anesthesia confounds in the preclinical setting should provide superior translation to clinical data.

We show surgical implantation and Recording procedures to measure visual electrophysiological signals from the eye (electroretinogram) and brain (visual evoked potential) in conscious rats, which is more analogous to the human condition where recordings are conducted without anesthesia confounds.

植入和无线电图记录和视觉诱发电位清醒大鼠

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both ...

行为学

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history have&#160;remained poorly understood. Recently, we developed a reliable, reproducible animal model of PION and tested the treatment effect of some neurotrophic factors in this model1. The purpose of this video is to demonstrate our photochemically induced model of posterior ischemic optic neuropathy, and to evaluate its effects with retrograde labeling of retinal ganglion cells. Following surgical exposure of the posterior optic nerve, a photosensitizing dye, erythrosin B, is intravenously injected and a laser beam is focused onto the optic nerve surface. Photochemical interaction of erythrosin B and the laser during irradiation damages the vascular endothelium, prompting microvascular occlusion mediated by platelet thrombosis and edematous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback, owing to a loss of axonal input - a remote, injury-induced and clinically relevant outcome. Thus, this model provides a novel platform to study the pathophysiologic course of PION,&#160;and can be further optimized for testing therapeutic approaches for optic neuropathies as well as other CNS ischemic diseases.

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

光化学诱导后部缺血性视神经病变的大鼠模型

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history ...

医学

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

电图同时记录和麻醉大鼠视觉诱发电位

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...

Visual Evoked Potential Recordings in Mice Using a Dry Non-invasive Multi-channel Scalp EEG Sensor

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable because of the plunger-spring-barrel structural facet and mechanical strengths resulting from metal materials. The whole process for acquiring the VEP responses in vivo from a mouse consists of four steps: (1) sensor assembly, (2) animal preparation, (3) VEP measurement, and (4) signal processing. This paper presents representative measurements of VEP responses from multiple mice with a submicro-voltage signal resolution and sub-hundred millisecond temporal resolution. Although the proposed method is safer and more convenient compared to other previously reported animal EEG acquiring methods, there are remaining issues including how to enhance the signal-to-noise ratio and how to apply this technique with freely moving animals. The proposed method utilizes easily available resources and shows a repetitive VEP response with a satisfactory signal quality. Therefore, this method could be utilized for longitudinal experimental studies and reliable translational research exploiting non-invasive paradigms.

We designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable. This paper describes the whole process from manufacturing the proposed EEG electrode to signal processing of visual evoked potential (VEP) signals measured on a mouse scalp using a dry non-invasive multi-channel EEG sensor.

干无创多通道头皮脑电传感器在小鼠视觉诱发电位记录中的应用

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable ...

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury are essential for translating novel therapeutic strategies from rodent models to clinical application due to their closer similarities to humans in size and anatomy. Here we describe some in vivo methods to evaluate the function and structure of the retinal ganglion cells (RGCs) and optic nerve (ON) in large animals, including visual evoked potential (VEP), pattern electroretinogram (PERG) and optical coherence tomography (OCT). Both goat and non-human primate were employed in this study. By presenting these in vivo methods step by step, we hope to increase experimental reproducibility among different labs and facilitate the usage of large animal models of optic neuropathies.

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

Here we demostrate several in vivo tests (flash visual evoked potential, pattern electroretinogram and optic coherence tomography) in goat and rhesus macaque to understand the structure and function of the optic nerve and its neurons.

体内 大型动物视网膜神经节细胞及视神经功能及结构的评估方法

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury ...

Inducing Local Demyelination in the Optic Nerve Using Lysolecithin

Source: You, Y. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/52934">Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination.</a> J. Vis. Exp. (2015)
This video demonstrates the procedure of surgically accessing the optic nerve in an anesthetized animal and precisely delivering lysolecithin to induce focal demyelination, creating neuropathological conditions for further study.

使用 Lysolecithin 诱导视神经局部脱髓鞘

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. ...

JoVE Encyclopedia of Experiments

Neuropathology

Epilepsy and Seizure Disorders

Preparing a Rat Model to Assess Visual Pathway Integrity Using an Electrophysiology Setup

Source: Nguyen, C. T. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54158">Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats.</a>&#160;J. Vis. Exp. (2016)
This video demonstrates the preparation of a rat model to study retinal health and visual pathway integrity. Visual evoked potential and electroretinography reference and active electrodes are connected to the rat, followed by aligning the platform with the rat in front of the Ganzfeld bowl for even retinal illumination and closing the Faraday cage to avoid external interference.

准备大鼠模型以使用电生理学设置评估视觉通路完整性

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Neurophysiology

Electrophysiology Techniques

Electron Microscopic Analysis of Myelin Damage in a Rat Model of Optic Nerve Injury

Take fixed optic nerve sections from a control rat and a rat with optic nerve injury.
The control section exhibits axons with tightly packed myelin sheaths containing concentric plasma membrane layers. The injured section exhibits decompacted myelin with large gaps.
Incubate with an osmium-ferrocyanide complex that binds to lipids and enhances the myelin sheath&#39;s contrast.
Treat with thiocarbohydrazide to create additional binding sites for osmium.
Reapply osmium to further enhance lipid contrast.
Stain with heavy metals, which bind to macromolecules and enhance structural contrast.
Dehydrate using increasing alcohol concentrations and rinse with acetone for resin embedding.
Embed the tissue in resin, and coat the resin with gold to improve image quality.
Use serial block-face scanning electron microscopy to simultaneously slice and image. Focus the electron beam on the surface and detect the backscattered electrons to generate images.
Myelin sheaths appear dark, while spaces within damaged sheaths appear lighter, allowing differentiation between normal and damaged myelin.

大鼠视神经损伤模型中髓鞘损伤的电子显微镜分析

Neuromuscular Disorders

Recording of Visual Evoked Potentials via the Skull Electrodes in a Rat Model

Source: You, Y., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/52934">Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination.</a> J. Vis. Exp. (2015).
This video demonstrates the process of measuring visual evoked potentials or VEPs in a dark-adapted, anesthetized rat with pre-implanted skull electrodes, which record the electrical signals from the brain&#39;s visual cortex in response to light stimuli.

通过大鼠模型中的颅骨电极记录视觉诱发电位

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. VEP ...

在实验视神经脱髓鞘模型大鼠视觉诱发电位记录

摘要

探索更多视频

此视频中的章节

单机在体内录音

植入和无线电图记录和视觉诱发电位清醒大鼠

光化学诱导后部缺血性视神经病变的大鼠模型

电图同时记录和麻醉大鼠视觉诱发电位

干无创多通道头皮脑电传感器在小鼠视觉诱发电位记录中的应用

体内大型动物视网膜神经节细胞及视神经功能及结构的评估方法

使用 Lysolecithin 诱导视神经局部脱髓鞘

准备大鼠模型以使用电生理学设置评估视觉通路完整性

大鼠视神经损伤模型中髓鞘损伤的电子显微镜分析

通过大鼠模型中的颅骨电极记录视觉诱发电位

在实验视神经脱髓鞘模型大鼠视觉诱发电位记录

摘要

探索更多视频

此视频中的章节

单机在体内录音

植入和无线电图记录和视觉诱发电位清醒大鼠

光化学诱导后部缺血性视神经病变的大鼠模型

电图同时记录和麻醉大鼠视觉诱发电位

干无创多通道头皮脑电传感器在小鼠视觉诱发电位记录中的应用

体内 大型动物视网膜神经节细胞及视神经功能及结构的评估方法

使用 Lysolecithin 诱导视神经局部脱髓鞘

准备大鼠模型以使用电生理学设置评估视觉通路完整性

大鼠视神经损伤模型中髓鞘损伤的电子显微镜分析

通过大鼠模型中的颅骨电极记录视觉诱发电位

体内大型动物视网膜神经节细胞及视神经功能及结构的评估方法