在实验视神经脱髓鞘模型大鼠视觉诱发电位记录

摘要

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.

摘要

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.

引言

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.

研究方案

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

1. 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（> 12周）中使用的实验。
2. 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。
3. 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。
4. 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。
5. 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）。
6. 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。
7. 允许至少1周为动物从手术VEP记录之前恢复。

2.视神经注射

1. 麻醉动物，备皮和应用悬垂如上（1.1和1.2）。
2. 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。
3. 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。
4. 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。
5. 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。
6. 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。
7. 上放置一个变暖垫动物从麻醉中恢复。

3. VEP记录

1. 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）。
2. 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。
3. 保持体温在37±0.5℃，由homoeothermic毯系统与直肠温度计探针。
4. 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。
5. 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。
6. 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。
7. 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。
8. 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。
9. 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。

4，组织准备和组织学

1. 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。
2. 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。
3. 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。

结果

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?...

讨论

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’ 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 ...

材料

Name	Company	Catalog Number	Comments
Ketamine 100 mg/ml (Ketamil)	Troy Laboratories	AC 116
Medetomidine 1 mg/ml (Domitor)	Pfizer	sc-204073
Tropicamide 1.0% (Mydriacyl)	Alcon	sc-202371
Homoeothermic blanket system	Harvard Apparatus	NC9203819
Impedance meter	Grass	F-EZM5
Screw electrodes	Micro Fasteners	M1.0×3mm Csk Slot M/T 304 S/S
Subdermal needle electrodes	Grass	F-E3M-72
Rapid Repair	DeguDent GmbH
Light-emitting diode	Nichia	NSPG300A
Bioamplifier	CWE, Inc.	BMA-400
CED system	Cambridge Electronic Design, Ltd.	Power1401
Hamilton syringe	Hamilton	87930
Lysolecithin	Sigma	L4129
Evan’s blue	Sigma	E2129