Assessment of Murine Retinal Function by Electroretinography
通过视网膜电图评估小鼠视网膜功能   

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本实验方案简略版
PLOS Genetics
Dec 2015

 

Abstract

The electroretinogram (ERG) is a sensitive and noninvasive method for testing retinal function. In this protocol, we describe a method for performing ERGs in mice. Contact lenses on the mouse cornea measure the electrical response to a light stimulus of photoreceptors and downstream retinal cells, and the collected data are analyzed to evaluate retinal function.

Keywords: Electroretinogram (ERG) (视网膜电图(ERG)), Mice (小鼠), Retinal degeneration (视网膜变性), Photoreceptors (光感受器)

Background

Electroretinograms (ERGs) are used by researchers and clinicians to test retinal function by measuring the electrical response of retinal cells to a light stimulus. The ERG is a useful tool for measuring retinal responses in mice due to its high level of sensitivity and noninvasive nature, and can be utilized to assess eye disease and retinal degeneration (Duncan et al., 2003; Phillips et al., 2010; Zhao et al., 2011; Vollrath et al., 2015). In mouse genetic retinal disease models, ERGs can be used to assess retinal degeneration at multiple time points as the disease progresses (Duncan et al., 2003). For studies evaluating the effect of drug treatment on the mouse eye, retinal function can be assessed before and after treatment in the same eye (Zhao et al., 2011). In the following protocol we describe a method for measuring the functional response of photoreceptors and downstream retinal cells in mice that builds on a previously published approach (Phillips et al., 2010). The protocol can be readily applied to animals three weeks of age and older.

ERGs can be used to measure the electrical response to light flashes in either dark-adapted (scotopic) or light-adapted (photopic) mice. In scotopic ERGs, mice are presented with low intensity light flashes to induce rod activation, so the function of rod photoreceptor and downstream retinal cells can be examined (Fu, 2010). A prolonged period of dark-adaptation is critical to achieve maximal rod sensitivity, and to keep cone stimulation minimal (Pepperberg, 2003). In photopic ERGs, mice are presented with high intensity light flashes after a period of light stimulation. Under these conditions, there is high cone activation and the rod response is suppressed. Therefore, photopic ERGs can be used to measure the function of cone photoreceptors and downstream retinal cells (Fu, 2010).

This method of performing ERGs allows for the calculation of amplitude and time-to-peak of two major waves, the a-wave and b-wave. The a-wave is a measure of the initial response of photoreceptors to a brief flash of light (Brown, 1968; Perlman, 2015). The b-wave is a measure of the response of downstream retinal neurons, including bipolar cells, to photoreceptor stimulation (Brown, 1968; Perlman, 2015). Loss of amplitude in either the a-wave or b-wave may be attributed to a number of retinal dystrophies (Creel, 2015), while ‘supernormal’ waves with increased amplitude have been attributed to cone dystrophies (Phillips et al., 2010).

Materials and Reagents

  1. Absorbent pads (Bound Tree Medical, catalog number: 111-16650 )
  2. Syringes and needles (BD, catalog number: 329461 )
  3. Spray/squirt water bottle (Qorpak, catalog number: PLC-03431 )
  4. Cotton swabs (Uline, catalog number: S18985PK )
  5. Two 29 gauge, 12 mm needle electrodes for ground and reference (The Electrode Store, catalog number: GRD-SAF )
  6. Mice
  7. Ketamine hydrochloride (NADA: 045-290)/xylazine hydrochloride (NADA: 139-236) cocktail (80 mg/kg/13 mg/kg)
  8. 1% atropine sulfate ophthalmic solution (NDC: 24208-750-60)
  9. 2.5% phenylephrine hydrochloride ophthalmic solution (NDC: 17478-200-12)
  10. 0.5% proparacaine hydrochloride (NDC: 24208-730-06)
  11. Hydrating eye ointment (Refresh Tears; Allergan, NDC: 0023-0798)
  12. 2.5% hypromellose (NDC: 17238-610-15)
  13. 70% ethanol

Equipment

  1. Dark room
  2. Red filter (ROSCO, catalog number: Roscolux Supergel R27 Medium Red)
  3. Black electrical tape (3M, catalog number: 06132 )
  4. Surgical tape (3M, catalog number: 15270 )
  5. Ganzfeld ColorDome (Diagnosys, catalog number: D125 ) or similar
  6. Espion visual electrophysiology system (Diagnosys, catalog number: D315 ) or similar
  7. Two Bayer-Mittag contact lens electrodes (Mt Sinai, Phil Cook) (Bayer et al., 2001)
  8. Forceps (Fine Scientific Tools, catalog number: 11295-00 )
  9. Elevated mouse platform (Foam or plastic can be used to fit space and size requirements)
  10. Non-electric heating pad (Braintree Scientific, catalog number: DPIP )
  11. Clean mouse cage
  12. Optional: opaque box (Rubbermaid Commercial Products, catalog number: 9S31 ), blackout cloth (Thorlabs, catalog number: BK5 ), red light headlamp (Princeton Tec, model: Sync headlamp ), red light desk lamp (AstroGizmos, catalog number: DSKLMP22 ), lux meter (Fisher Scientific, catalog number: 06-662-63 )

Software

  1. Data analysis software such as Microsoft Excel (v 14.7.1) or GraphPad Prism (v 7.0)

Procedure

Note: This protocol should be performed according to Institutional Animal Care and Use Committee regulations, and is subject to institutional approval.

  1. Day before experiment
    1. Place mice in a light-proof dark room and allow them to dark-adapt overnight.
      1. Check with your institution on regulations regarding overnight housing of animals.
      2. A lux meter can be used to assess if the room is light-proof.
    2. Warm the non-electric heat pad in a 37 °C bath or incubator overnight.

  2. Prepping for the experiment
    1. Set up the dark room
      1. Existing light sources can be used if covered with red filter to make them procedure-safe.
      2. Cover the computer monitor and any miscellaneous lights with red filter or black electrical tape.
      3. Cover the 37 °C heat pad with an absorbent pad. Position the elevated platform with heat pad on top so that the mouse can be easily prepared and moved into the dome.
        Note: The heat pad may need to be reheated to 37 °C if multiple animals are tested.
    2. Move mice to experimental dark room if different from overnight adaption room. This can be done using either a blackout cloth or light-proof box. Mice that are not being tested should be kept in a dark environment.
    3. Set stimulus settings as described below.

  3. Preparing the mice
    Note: From this point on, only red-filtered light sources should be used in order to keep mice dark-adapted.
    1. Anesthetize the mouse by injection of ketamine hydrochloride/xylazine hydrochloride (80 mg/kg/13 mg/kg), and allow mice to become fully sedated (2-5 min). Check for a toe-pinch response to ensure the animal is adequately anesthetized.
    2. Treat both eyes with 1% atropine sulfate, 2.5% phenylephrine hydrochloride, and 0.5% proparacaine hydrochloride, allowing drops to sit on the eyes for ~2 min before wicking with a cotton swab and applying the next drop. After this point the eyes should be kept hydrated with a hydrating eye ointment until the contact lens electrodes are applied (Figure 1).


      Figure 1. Anesthetized mouse with atropine sulfate, phenylephrine hydrochloride, proparacaine hydrochloride, and hydrating eye drops

    3. Position the mouse on the heated platform. Place the ground needle electrode in the base of the tail, and reference needle electrode subdermally between the eyes (Figure 2).


      Figure 2. Electrode placement on the mouse

    4. Wick any excess moisture from the eyes, and add a small drop of 2.5% hypromellose to each eye.
    5. Use fingers or forceps to position the contact lens electrodes onto the corneas. The hypromellose should keep the lenses in place. It may be helpful to tape down the electrode wires to the pad using surgical tape so they do not shift during the experiment (Figure 3, Video 1).


      Figure 3. Contact lens electrodes on the mouse eye

      Video 1. Applying the contact lens electrodes to the mouse eye

    6. Slide the heated platform into the dome so that the mouse’s eyes are inside of the dome (Figures 4 and 5).


      Figure 4. Mouse with electrodes on the heated, elevated platform


      Figure 5. Mouse with eyes inside of the Ganzfeld ColorDome

    7. Before beginning and throughout the scotopic and photopic assays, periodically check that impedance levels are acceptably low (within the green range). A low impedance level indicates that the electrodes have been properly installed.
      Note: The impedance may be high if any of the electrodes have shifted and the circuit has been broken. The Espion visual electrophysiology system displays impedance using green, yellow and red bars to signify the level of resistance in the circuit.

  4. Running the scotopic (dark-adapted) ERG
    1. Settings to record scotopic ERGs
      Step
      1
      2
      3
      4
      5
      6
      7
      8
      Pulse intensity
      (cd sec/m2)
      0.001
      0.002
      0.05
      0.1
      0.78
      1
      5
      10

    2. Start with the following settings
      5 trials/step
      Pulse frequency: 0.2 Hz
      Sample frequency: 1,000 Hz
      Trial pre-trigger time: 20 msec
      Trial post-trigger time: 250 msec
    3. Run through each step of light intensity using the Espion E2 system.
      Note: Check the impedance every 2-3 steps or as needed to ensure that the electrode circuit has not been broken.

  5. Running the photopic (light-adapted) ERG
    1. Light adapt the mouse for at least 10 min at a light intensity of 20 cd sec/m2. Light adaptation saturates the rod response so that the cones, which account for only 3% of the photoreceptor population in the mouse eye, can be stimulated and recorded without rod interference (Fu, 2010).
      Note: It may be necessary to give the mouse a second, reduced dose of ketamine/xylazine if the animal begins to wake up.
    2. Settings to record photopic ERGs
      Step
      1
      2
      3
      4
      5
      6
      Pulse Intensity
      (cd sec/m2)
      0.78
      1    
      2.25
      5    
      10  
      20  

    3. Start with the following settings:
      25 trials/step
      Pulse frequency: 1.0 Hz
      Sample frequency: 1,000 Hz
      Trial pre-trigger time: 20 msec
      Trial post-trigger time: 250 msec
    4. Run through each step of light intensity using the Espion E2 system.

  6. Post-experiment
    1. Remove electrodes from the mouse and gently clean contact electrodes with a cotton swab and water. Needle electrodes can be wiped with 70% ethanol.
    2. Place mouse in a clean cage on top of a heat pad until it recovers. Keep the mouse’s eyes moist with a hydrating eye ointment (Figure 6).


      Figure 6. Mouse recovering in a clean, heated cage

Data analysis

  1. Processing ERG data (Figures 7 and 8)
    1. Data from two eyes can either be averaged for a single animal, or analyzed separately if one eye is experimental and one is control.
    2. Use a data analysis software such as Microsoft Excel or GraphPad Prism to generate traces.
    3. a-wave:
      1. The a-wave amplitude can be measured by calculating the difference in trace amplitude between time (t) = 0 and the lowest point on the trace.
        Note: The tail of the trace may have points lower than the bottom of the a-wave, which is the first wave to be recorded. Exclude these tail points if this is the case.
      2. The a-wave latency of response (time-to-peak) can be measured by calculating elapsed time between t = 0 and the time point at the lowest point on the trace.
    4. b-wave:
      1. The b-wave amplitude can be measured by calculating the difference in trace amplitude between the bottom of the a-wave and the top of the tallest curve.
        Note: There may be oscillating potentials that rise above the top of the b-wave curve. Exclude these points, as they will give an inaccurate b-wave if included. Oscillating potentials are thought to reflect cell activity in the inner retina, and can be isolated using a digital filter (Asi and Perlman, 1992).
      2. The b-wave implicit time (time-to-peak) can be measured by calculating elapsed time between the bottom of the a-wave and the top of the b-wave.


        Figure 7. Representative ERG traces of C57BL/6J adult mice. A pulse intensity of 1 cd sec/m2 was used for measurement of the scotopic ERG, and a pulse intensity of 5 cd sec/m2 was used for measurement of the photopic ERG.


        Figure 8. Defining the a-wave and b-wave on an ERG trace. The a-wave is measured by calculating the difference in trace amplitude between time (t) = 0 and the lowest point on the trace. The b-wave amplitude is measured by calculating the difference in trace amplitude between the bottom of the a-wave and the top of the tallest curve.

  2. Statistical analysis of ERG data
    1. a-wave and b-wave amplitude:
      Note: Statistical analysis of a-wave and b-wave amplitude shown is based on data in Vollrath et al. (2015), and may not be appropriate for every data set.
      1. Compile calculated a-wave amplitudes at a single light intensity for scotopic ERGs in each experimental group and perform a two-way ANOVA with Bonferroni’s correction for multiple comparisons (Figure 9) (Vollrath et al., 2015).
      2. Repeat the statistical analysis for each light intensity.
      3. The following notation can be used to show p-value significance: ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05 (Figure 9).
        Note: We recommend using at least 5 animals per group to achieve statistical significance, although more may be necessary.
      4. Repeat statistical analysis for every light intensity for b-wave amplitude of scotopic ERGS, a-wave amplitude of photopic ERGs, and b-wave amplitude of photopic ERGs.
      5. A significant difference in a-wave amplitude at one or more light intensities may indicate a difference in photoreceptor function between the two experimental groups. A significant difference in b-wave amplitudes may indicate a difference in the function of retinal cells downstream of the photoreceptors, including bipolar cells (Vollrath et al., 2015).
    2. a-wave and b-wave time-to-peak:
      1. Compile calculated time-to-peak at a single light intensity for each experimental group and perform a two-way ANOVA with Bonferroni’s correction for multiple comparisons.
      2. Repeat the statistical significance at each light intensity for a-wave and b-wave time-to-peak for both scotopic and photopic ERGs.
      3. A significant difference in a-wave or b-wave time at one or more light intensities may indicate a delay in photoreceptor response or downstream retinal signaling (Perlman, 2015).


        Figure 9. Statistical analysis of ERG data comparing two mouse genotypes (Vollrath et al., 2015). Comparison of mean amplitudes ± SD of scotopic a-wave and photopic b-wave at various light intensities for two mouse genotypes (n = 5 males at 10-11 weeks for each). Statistical analysis by two-way ANOVA with Bonferroni’s correction for multiple comparisons.

Acknowledgments

Supported by grants from the Foundation Fighting Blindness, the Macular Degeneration Research Program of the BrightFocus Foundation, the Thome Memorial Foundation, and NIH grants R01 EY025790 and T32 EY20485.
This protocol should be cited as Benchorin G., Calton M.A., Beaulieu M.O., Vollrath D. Assessment of murine retinal function by electroretinography. Bio Protoc. 2017.

References

  1. Asi, H. and Perlman, I. (1992). Relationships between the electroretinogram a-wave, b-wave and oscillatory potentials and their application to clinical diagnosis. Doc Ophthalmol 79(2): 125-139.
  2. Bayer, A. U., Cook, P., Brodie, S. E., Maag, K. P. and Mittag, T. (2001). Evaluation of different recording parameters to establish a standard for flash electroretinography in rodents. Vision Res 41(17): 2173-2185.
  3. Brown, K. T. (1968). The electroretinogram: its components and their origin. Vision Res. 8:633-677.
  4. Creel, D. J. (2015). The electroretinogram and electro-oculogram: clinical applications by Donnell J. Creel. In: Kolb, H. E., Nelson, R., Fernandez, E. and Jones, B. (Eds.). The Organization of the Retina and Visual System.
  5. Duncan, J. L., LaVail, M. M., Yasumura, D., Matthes, M. T., Yang, H., Trautmann, N., Chappelow, A. V., Feng, W., Earp, H. S., Matsushima, G. K. and Vollrath, D. (2003). An RCS-like retinal dystrophy phenotype in mer knockout mice. Invest Ophthalmol Vis Sci 44 (2): 826–838.
  6. Fu, Y. (2010). Phototransduction in Rods and Cones. In: Kolb, H. E., Nelson, R., Fernandez, E. and Jones, B. (Eds.). The Organization of the Retina and Visual System.
  7. Perlman, I. (2015). The electroretinogram: ERG by Ido Perlman. In: Kolb, H. E., Nelson, R., Fernandez, E. and Jones, B. (Eds.). The Organization of the Retina and Visual System.
  8. Pepperberg, D. R. (2003). Bleaching desensitization: background and current challenges. Vision Res 43(28): 3011-3019.
  9. Phillips, M. J., Webb-Wood, S., Faulkner, A. E., Jabbar, S. B., Biousse, V., Newman, N. J., Do, V. T., Boatright, J. H., Wallace, D. C. and Pardue, M. T. (2010). Retinal function and structure in Ant1-deficient mice. Invest Ophthalmol Vis Sci 51(12): 6744-6752.
  10. Vollrath, D., Yasumura, D., Benchorin, G., Matthes, M. T., Feng, W., Nguyen, N. M., Sedano, C. D., Calton, M. A. and LaVail, M. M. (2015). Tyro3 modulates Mertk-associated retinal degeneration. PLoS Genet 11(12): e1005723.
  11. Zhao, C., Yasumura, D., Li, X., Matthes, M., Lloyd, M., Nielsen, G., Ahern, K., Snyder, M., Bok, D., Dunaief, J. L., LaVail, M. M. and Vollrath, D. (2011). mTOR-mediated dedifferentiation of the retinal pigment epithelium initiates photoreceptor degeneration in mice. J Clin Invest 121(1): 369-383.

简介

视网膜电图(ERG)是一种用于测试视网膜功能的敏感和无创性方法。 在本协议中,我们描述了一种在小鼠中进行ERGs的方法。 小鼠角膜上的隐形眼镜测量对光感受器和下游视网膜细胞的轻度刺激的电响应,并且收集的数据被分析以评估视网膜功能。
【背景】研究人员和临床医生使用视网膜电图(ERGs)通过测量视网膜细胞对光刺激的电响应来测试视网膜功能。 ERG是一种用于测量小鼠视网膜反应的有用工具,由于其灵敏度高,无创性,可用于评估眼部疾病和视网膜变性(Duncan et al。,2003; Phillips et al。,2010; Zhao et al。,2011; Vollrath et al。,2015)。在小鼠遗传性视网膜疾病模型中,随着疾病的进展,ERG可以用于评估多个时间点的视网膜变性(Duncan等,2003)。对于评估药物治疗对小鼠眼睛的影响的研究,可以在同一只眼睛中治疗前后评估视网膜功能(Zhao et al。,2011)。在以下协议中,我们描述了一种测量基于以前发表的方法建立的小鼠中光感受器和下游视网膜细胞功能反应的方法(Phillips等,2010)。该方案可以方便地应用于三周龄以上的动物。
ERG可以用于测量暗适应(scotopic)或光适应(photopic)小鼠中的光闪烁的电响应。在阴性ERGs中,小鼠呈现低强度光闪烁以诱导杆活化,因此可以检查棒状感光器和下游视网膜细胞的功能(Fu,2010)。长时间的暗适应对于实现最大杆敏感性是至关重要的,并且保持锥体刺激最小(Pepperberg,2003)。在明亮的ERG中,经过一段时间的光刺激后,老鼠呈现高强度的闪光。在这些条件下,有高的锥体激活和棒响应被抑制。因此,明胶ERGs可用于测量锥形光感受器和下游视网膜细胞的功能(Fu,2010)。
执行ERG的这种方法允许计算两个主波(a波和b波)的幅度和时间峰值。 a波是光感受器对短暂闪光的初始响应的测量(Brown,1968; Perlman,2015)。 b波是测量下游视网膜神经元(包括双极细胞)对感光细胞刺激的反应(Brown,1968; Perlman,2015)。 a波或b波的振幅损失可归因于许多视网膜营养不良(Creel,2015),而具有增加幅度的“超常”波已归因于锥体营养不良(Phillips等,2010) 。

关键字:视网膜电图(ERG), 小鼠, 视网膜变性, 光感受器

材料和试剂

  1. 吸收垫(Bound Tree Medical,目录号:111-16650)
  2. 注射器和针头(BD,目录号:329461)
  3. 喷水壶(Qorpak,目录号:PLC-03431)
  4. 棉签(Uline,目录号:S18985PK)
  5. 两个29针,12 mm针电极,用于接地和参考(电极店,目录号:GRD-SAF)
  6. 小鼠
  7. 盐酸氯胺酮(NADA:045-290)/盐酸赛拉嗪(NADA:139-236)混合物(80mg/kg/13mg/kg)
  8. 1%硫酸阿托品眼科溶液(NDC:24208-750-60)
  9. 2.5%盐酸去氧肾上腺素眼科溶液(NDC:17478-200-12)
  10. 0.5%盐酸普拉卡因(NDC:24208-730-06)
  11. 保湿眼膏(Refresh Tears; Allergan,NDC:0023-0798)
  12. 2.5%羟丙甲纤维素(NDC:17238-610-15)
  13. 70%乙醇

设备

  1. 黑暗的房间
  2. 红色滤光片(ROSCO,目录号:Roscolux Supergel R27 Medium Red)
  3. 黑色电子胶带(3M,目录号:06132)
  4. 手术胶带(3M,目录号:15270)
  5. Ganzfeld ColorDome(诊断,目录号:D125)或类似的
  6. Espion视觉电生理系统(Diagnosys,目录编号:D315)或类似的
  7. 两台Bayer-Mittag隐形眼镜电极(西奈山,Phil Cook)(拜耳等人,2001)
  8. 镊子(精细科学工具,目录号:11295-00)
  9. 高架鼠标平台(泡沫或塑料可用于适应空间和尺寸要求)
  10. 非电加热垫(Braintree Scientific,目录号:DPIP)
  11. 清洁鼠标笼
  12. 可选:不透明盒(Rubbermaid商品,目录号:9S31),遮光布(Thorlabs,目录号:BK5),红光前照灯(普林斯顿Tec,型号:Sync头灯),红灯台灯(AstroGizmos,目录号:DSKLMP22 ),lux仪表(Fisher Scientific,目录号:06-662-63)

软件

  1. 数据分析软件如Microsoft Excel(v 14.7.1)或GraphPad Prism(7.0)

程序

注意:本协议应根据"机构动物保护和使用委员会条例"进行,并经机构许可。

  1. 实验前一天
    1. 将小鼠放在防晒黑暗的房间里,让它们在夜间变暗。
      1. 请向贵机构查询有关动物过夜住房的规定。
      2. 可以使用勒克斯表来评估房间是否防光。
    2. 在37°C的浴室或孵化器中将非电热垫温热过夜。

  2. 进行实验
    1. 设置黑暗的房间
      1. 现有的光源可以用红色滤光片覆盖,使其安全。
      2. 盖上电脑显示器和带红色滤光片或黑色电子胶带的任何杂项灯。
      3. 用吸收垫覆盖37°C的热垫。将升高的平台放置在顶部,使鼠标可以轻松准备并移动到圆顶中。
        注意:如果测试了多只动物,热垫可能需要重新加热至37°C。
    2. 如果与过夜适应室不同,将小鼠移至实验室。这可以使用遮光布或防光盒来完成。未经测试的小鼠应保持在黑暗环境中。
    3. 按如下所示设置刺激设置。

  3. 准备老鼠
    注意:从这一点开始,只能使用红色滤光的光源,以保持老鼠适应黑暗。
    1. 通过注射氯胺酮/盐酸赛拉嗪(80mg/kg/13mg/kg)麻醉小鼠,并使小鼠变得完全镇静(2-5分钟)。检查脚趾反应以确保动物充分麻醉。
    2. 用1%阿托品硫酸盐,2.5%盐酸去氧肾上腺素和0.5%盐酸普拉卡因治疗双眼,允许滴在眼睛上静置约2分钟,然后用棉签吸干并应用下一滴。在这一点之后,眼睛应该用水合眼膏软膏保持水分直到接触透镜电极被施用(图1)。


      图1.硫酸阿托品麻醉小鼠,盐酸苯肾上腺素,盐酸普拉卡因和水合滴眼剂

    3. 将鼠标放在加热的平台上。将接地针电极放置在尾部的基部,并将参考针电极细眼放置在眼睛之间(图2)。


      图2.鼠标上的电极放置

    4. 从眼睛中吸收多余的水分,并向每只眼睛加入一小滴2.5%的羟丙甲纤维素。
    5. 使用手指或镊子将隐形眼镜电极定位在角膜上。羟丙甲纤维素应保持镜片到位。使用手术胶带将电极线粘贴到垫可能有帮助,因此在实验过程中不会发生移位(图3,视频1)。


      图3.鼠标眼睛上的隐形眼镜电极

      Video 1. Applying the contact lens electrodes to the mouse eye

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    6. 将加热的平台滑入穹顶,使鼠标的眼睛在圆顶内(图4和图5)。


      图4.加热,升高的平台上的电极鼠标


      图5. Ganzfeld ColorDome 中的眼睛内的鼠标

    7. 在开始之前和整个深色和深色测定中,定期检查阻抗水平是否可接受(在绿色范围内)。低阻抗电平表示电极已正确安装。
      注意:如果任何电极发生偏移,电路已损坏,阻抗可能较高。 Espion视觉电生理系统使用绿色,黄色和红色条显示阻抗,以表示电路中的电阻水平。

  4. 运行scotopic(暗适应)ERG
    1. 记录暗疮ERG的设置
      步骤
      1
      2
      3
      4
      5
      6
      7
      8
      脉冲强度
      (cd sec/m 2
      0.001
      0.002
      0.05
      0.1
      0.78
      1
      5
      10

    2. 从以下设置开始
      5次试验/步骤
      脉冲频率:0.2 Hz 采样频率:1000 Hz 试用预触发时间:20 msec
      试用触发后时间:250毫秒
    3. 使用Espion E2系统运行光强度的每个步骤。
      注意:每隔2-3个步骤或根据需要检查阻抗,以确保电极电路未损坏。

  5. 运行照片(光适应)ERG
    1. 光以20 cd sec/m 2的光强度使小鼠适应至少10分钟。光适应使杆反应饱和,使得仅在小鼠眼中只有感光体群体的3%的锥体可以被刺激和记录而没有杆干扰(Fu,2010)。
      注意:如果动物开始醒来,可能需要给予老鼠第二次减少剂量的氯胺酮/赛拉嗪。
    2. 记录显示ERG的设置
      步骤
      1
      2
      3
      4
      5
      6
      脉冲强度
      (cd sec/m 2
      0.78
      1    
      2.25
      5    
      10  
      20  

    3. 从以下设置开始:
      25次试验/步骤
      脉冲频率:1.0Hz 采样频率:1000 Hz 试用预触发时间:20 msec
      试用触发后时间:250毫秒
    4. 使用Espion E2系统运行光强度的每个步骤。

  6. 实验后
    1. 从鼠标中取出电极,用棉签和水轻轻地清洁接触电极。针电极可以用70%乙醇擦拭。
    2. 将鼠标放在加热垫顶部的干净的笼子中,直至恢复。用保湿眼膏软膏保持鼠标的眼睛(图6)

      图6.鼠标在干净,加热的笼中恢复

数据分析

  1. 处理ERG数据(图7和8)
    1. 来自两只眼睛的数据可以对于单个动物进行平均,或者单独分析,如果一只眼睛是实验的,另一只眼睛是对照的。
    2. 使用数据分析软件(如Microsoft Excel或GraphPad Prism)生成跟踪。
    3. 一波:
      1. a波幅度可以通过计算时间(t)= 0和迹线上最低点之间的迹线幅度差来测量。
        注意:轨迹的尾部可能具有低于a波底部的点,这是要记录的第一波。如果是这种情况,请排除这些尾点。
      2. 可以通过计算t = 0和跟踪最低点的时间点之间的经过时间来测量响应(时间到峰值)的a波延迟。
    4. b波:
      1. b波幅度可以通过计算a波的底部和最高曲线的顶部之间的迹线幅度差来测量。
        注意:可能有振荡电位升高到b波曲线顶部以上。排除这些点,因为它们会给出不准确的b波。振荡电位被认为反映细胞在内部视网膜的活动,并且可以使用数字滤波器(Asi和Perlman,1992)进行分离。
      2. b波隐含时间(时间到峰值)可以通过计算a波的底部和b波的顶部之间的时间来测量。


        图7. C57BL/6J成年小鼠的代表性ERG踪迹。使用1cd sec/m 2的脉冲强度用于测量阴道ERG,并且脉冲强度5 cd sec/m 2 用于测量明视ERG。


        图8.在ERG轨迹上定义a波和b波。通过计算时间(t)= 0和最小点之间的跟踪幅度差,测量a波跟踪。通过计算a波的底部和最高曲线的顶部之间的迹线幅度差来测量b波幅度。

  2. ERG数据统计分析
    1. a波和b波振幅:
      注意:所显示的a波和b波振幅的统计分析基于Vollrath等人的数据。 (2015),并且可能不适用于每个数据集。
      1. 在每个实验组中编制单个光强度下的单一光强度的计算的a波振幅,并对Bonferroni的多重比较校正进行双因素ANOVA(图9)(Vollrath等,2015) 。
      2. 对每个光强度重复统计分析。
      3. 以下符号可用于显示p值意义: **** P ≤0.0001, *** /em>≤0.001, ≤0.01,< em> * P ≤0.05(图9)。
        注意:我们建议每组使用至少5只动物达到统计学意义,但可能需要更多。
      4. 重复统计学分析,观察各向异性ERGS的b波振幅,光敏ERG的a波振幅以及明视ERG的b波振幅。
      5. 在一个或多个光强度下的a波幅度的显着差异可以指示两个实验组之间的感光器功能的差异。 b波幅度的显着差异可以指示感光器下游的视网膜细胞的功能差异,包括双极细胞(Vollrath等人,2015)。
    2. a波和b波时间到峰值:
      1. 在每个实验组的单个光强度下编译计算的时间峰值,并执行带有Bonferroni校正多重比较的双向ANOVA。
      2. 在每个光强度下重复统计学显着性,用于暗波和明镜ERG的a波和b波时间峰值。
      3. 在一个或多个光强度下,a波或b波时间的显着差异可能表明感光器反应或下游视网膜信号传导的延迟(Perlman,2015)。


        图9.比较两种小鼠基因型的ERG数据的统计分析(Vollrath等人,2015)。两种小鼠基因型(n = 5个男性,10-11周龄)的两种小鼠基因型的平均幅度±SD和不同光强度下的明视b波的平均幅度±SD。通过双因素ANOVA统计分析,Bonferroni对多次比较的校正。

致谢

支持基金会失败盲人,BrightFocus基金会,Thome纪念基金会和NIH授予R01 EY025790和T32 EY20485的黄斑变性研究计划。
本协议应引用为Benchorin G.,Calton M.A.,Beaulieu M.O.,Vollrath D. Assessement of murine retinal function by eletroretinography。生物样品2017年。

参考文献

  1. Asi,H.和Perlman,I。(1992)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/1591967"target ="_ blank" >视网膜电图a波,b波和振荡电位之间的关系及其在临床诊断中的应用 Doc Ophthalmol 79(2):125-139。
  2. Bayer,AU,Cook,P.,Brodie,SE,Maag,KP and Mittag,T。(2001)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih评估不同的记录参数,以建立啮齿动物中闪光视网膜电图的标准。 41(17):2173-2185。
  3. Brown,KT(1968)。  视网膜电图:其组成部分及其起源。 Vision Res 。 8:633-677。
  4. Creel,DJ(2015)。  在:Kolb,HE,Nelson,R.,Fernandez,E.and Jones,B.(Eds。)。视网膜和视觉系统组织。
  5. Duncan,JL,LaVail,MM,Yasumura,D.,Matthes,MT,Yang,H.,Trautmann,N.,Chappelow,AV,Feng,W.,Earp,HS,Matsushima,GK和Vollrath,D。(2003 )。一个RCS样的视网膜营养不良表型在mer敲除小鼠。 Invest Ophthalmol Vis Sci 44(2):826-838。
  6. Fu,Y.(2010)。< 光杆和锥体中的光转导。:Kolb,HE,Nelson,R.,Fernandez,E.and Jones,B。(Eds。 )。视网膜和视觉系统组织。
  7. Perlman,I.(2015)。  视网膜电图:Ido Perlman的ERG。在:Kolb,HE,Nelson,R.,Fernandez,E.and Jones,B.(Eds。)。视网膜与视觉系统组织。
  8. Pepperberg,DR(2003)。  漂白脱敏:背景和目前的挑战。 Vision Res 43(28):3011-3019。
  9. Phillips,MJ,Webb-Wood,S.,Faulkner,AE,Jabbar,SB,Biousse,V.,Newman,NJ,Do,VT,Boatright,JH,Wallace,DC and Pardue,MT(2010)在Ant1缺陷小鼠中,class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/20671283"target ="_ blank">视网膜功能和结构。 em> Invest Ophthalmol Vis Sci 51(12):6744-6752。
  10. Vollrath,D.,Yasumura,D.,Benchorin,G.,Matthes,MT,Feng,W.,Nguyen,NM,Sedano,CD,Calton,MA和LaVail,MM(2015)。< a class = ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26656104"target ="_ blank"> Tyro3 调制Mertk 相关视网膜退化。 PLoS Genet 11(12):e1005723。
  11. Zhao,C.,Yasumura,D.,Li,X.,Matthes,M.,Lloyd,M.,Nielsen,G.,Ahern,K.,Snyder,M.,Bok,D.,Dunaief,JL,LaVail ,MM和Vollrath,D。(2011)。  mTOR介导的视网膜色素上皮的去分化引发小鼠中的光感受器变性。 J Clin Invest 121(1):369-383。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Benchorin, G., Calton, M. A., Beaulieu, M. O. and Vollrath, D. (2017). Assessment of Murine Retinal Function by Electroretinography. Bio-protocol 7(7): e2218. DOI: 10.21769/BioProtoc.2218.
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