Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Summary

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Abstract

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Introduction

Electroretinogram (ERG) is a well-established technique that can be used to record the electrical activity of the retina triggered by light. The ERG signal is generated mainly by voltage changes caused by radial currents (along the axis of photoreceptors and bipolar cells) flowing in the resistive extracellular space of the retina. The first ERG signal was recorded in 1865 by Holmgren from the surface of a fish eye¹. Einthoven and Jolly 1908² divided the ERG response to the onset of light into three different waves, called a-, b-, and c-waves, that are now known to reflect mainly the activity of photoreceptors, ON bipolar cells, and pigment epithelium cells, respectively^3-8. ERG can be recorded from the eyes of anesthetized animals or humans (in vivo), from isolated eye preparation⁹, across isolated intact retina (ex vivo)^3,10-15 or across specific retina layers with microelectrodes (local ERG)^4,16. Of these, in vivo ERG is currently the most widely used method to assess retinal function. It is a noninvasive technique that can be used for diagnostic purposes or to follow the progression of retinal diseases in animals or patients. However, in vivo ERG recordings produce a complicated signal with several overlapping components, often contaminated by extraocular physiological noise (e.g., breathing and cardiac activity).

Local ERG can be used to record the signal across specific layers of the retina but it is the most invasive and has the lowest signal-to-noise ratio (SNR) as compared to the other ERG recording configurations. Local ERG is also technically demanding and requires expensive equipment (e.g., microscope and micromanipulators). Transretinal ERG from the intact, isolated retina (ex vivo ERG) offers a compromise between in vivo and local ERG methods allowing stable and high SNR recordings from intact retinas of animals or humans¹⁷. Recently, this method has been used successfully to study rod and cone photoreceptor function in mammalian, primate and human retinas^18-20. In addition, due to absence of pigment epithelium in the ex vivo retina, the positive c-wave component of the ERG signal is removed and a prominent negative slow PIII component is revealed in the ex vivo recordings. The slow PIII component has been shown to originate from the activity of Müller glia cells in the retina^21-23. Thus, ex vivo ERG method could also be used to study Müller cells in the intact retina. Several studies have also shown that ex vivo ERG recordings could be used to measure concentration of pharmacological agents around the retina²⁴ and test the safety and efficacy of drugs^25-27.

Multiple commercial in vivo systems are available and used in many laboratories that do not necessarily have extensive electrophysiology background. In contrast, ex vivo devices have not been available until recently¹⁷ and as a result only very few laboratories are currently taking advantage of this powerful technique. It would be beneficial to make ex vivo ERG recordings available to more laboratories in order to advance our knowledge about retinal physiology and pathology, and to develop new therapies for blinding diseases. We demonstrate here a simple and affordable ex vivo ERG device¹⁷ and show how it can be used in combination with several commercially available in vivo ERG systems to record rod- and cone-mediated signaling (a- and b-waves) and the function of Müller cells (slow PIII) from intact wild-type mouse retinas.

Protocol

All experimental protocols were in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the institutional Animal Studies Committee at Washington University.

1. Setting Up Perfusion and Specimen Holder

1. Prepare solution for retina perfusion fresh on the day of the experiment. Use distilled and deionized water. Use one of the following three solutions.
   1. Prepare Bicarbonate-containing Ames’ solution (1 L): 1 bottle of Ames’ media and 1.9 g of NaHCO₃,
   2. Prepare Locke’s solution (in mM): 112.5 NaCl, 3.6 KCl, 2.4 MgCl₂, 1.2 CaCl₂, 10.0 HEPES, 20.0 NaHCO₃, 3.0 Na succinate, 0.5 Na glutamate, 0.02 EDTA, and 10.0 glucose, 0.1% MEM vitamins and amino acids
   3. Prepare HEPES-buffered Ringer solution (in mM): 133.3 NaCl, 3.3 KCl, 2.0 MgCl₂, 1.0 CaCl₂, 10.0 glucose, 0.01 EDTA, 12.0 HEPES, pH adjusted to 7.5 with ~5.8 ml of 1 M NaOH, add 0.72 g/L Leibovitz culture medium L-15. Use 20 - 50 μM DL-AP4 and 50 – 100 μM BaCl₂ to isolate the photoreceptor response with any perfusion media.
2. Prepare solution for electrodes²⁸ (in mM): 140.0 NaCl, 3.6 KCl, 2.4 MgCl₂, 1.2 CaCl₂, 3 HEPES, 0.01 EDTA and adjust pH to 7.4 – 7.5 with NaOH. Electrode solution can be stored at RT for several months.
3. Prepare and test the specimen holder.
   1. Glue black/grey filter paper on top of the domes of the specimen holder’s bottom part (see Figure 1A). Spread two-component 5 min epoxy glue carefully around the edges of the flat tops of the domes. If necessary, do it under dissection microscope.
   2. Wait until the glue is almost dry (about 4 min) and press the filter paper on the domes by using a flat item. Glue filter paper at least one day before the experiment. The filter paper can be used multiple times but should be replaced after a month of recordings. Use 70% ethanol to clean domes carefully from any glue residue before installing the replacement paper.
   3. Fill the electrode channels with electrode solution trying to avoid any air bubbles and screw pellet electrodes enclosed with a threaded adapter into the electrode channels (see Figures 1A and B). Connect the top and bottom pieces of the specimen holder with four screws and fill the perfusion lines with electrode solution.
   4. Measure the resistance and voltage between the leads of each electrode pair using a multimeter (Figure 1B). Resistance should be below 100 Ωk and the voltage below 10 mV if the channels are bubble-free and the electrodes are in good conditions.
4. Pour 400 - 700 ml of perfusion media in the glass bottle. Separate another 300 ml to be used in the dissection of the retinas and store it in a fridge. Set the perfusion tubing in the heat exchanger block and place the preheated block on the heating plate (see Figure 1D).
5. Enclose the bottle with a cap that has connections to CO₂/O₂ gas (if Ames’ or Locke’s is used) and for perfusion tubing (see Figure 1D). Preheat the bottle with the media to 37 ^oC and place it on the heating plate or on the top of the light stimulation unit in water bath set to 37 – 39 ^oC¹⁷.
   1. Prime the perfusion lines by filling them with perfusion media to initiate the gravity-driven flow.
      1. In LKC system, place the bottle on the top of the stimulator unit¹⁷ to provide a significant gravitational flow that is then adjusted by flow-rate regulators without being affected by the lowering level of the perfusion solution in the bottle during the experiment.
      2. In Ocuscience system, place the perfusion bottle inside the Faraday cage in order to minimize noise and place long perfusion output lines from the specimen holder (see step 2.8) well below the level of the specimen holder (and perfusion bottle) to increase gravitational drive of the solution. Shield these output lines and connect the shield to the amplifier’s ground to prevent coupling of the electromagnetic noise to the ERG signal.
6. Adjust the perfusion to 3 – 5 ml/min by using flow-rate regulators. Connect 5% CO₂ / 95% O₂ gas from a cylinder with proper regulator and adjust the flow rate to ensure steady bubbling of the media in the bottle through an air stone.

2. Sample Preparation

1. Assemble clean and sharp dissection instruments including straight-bladed microscissors, one or two 45^o tweezers, razor blade and a rectangular piece of filter paper.
2. Pour about 200 ml of cold perfusion solution in a large Petri dish so that the whole bottom part of the specimen holder (including the domes) can be immersed into the solution. This step becomes important when mounting the retinas on the domes (see step 2.6). Although some solutions are designed to be saturated with carbogen (5% CO₂ / 95% O₂), this is not essential for the dissection purposes and was not done in the experiments described here.
3. For a typical experiment, keep animals in 12/12 hrs dark/light cycle and dark-adapt them for 6 – 12 hrs before the recordings. Euthanize the animal by CO₂ inhalation followed by cervical dislocation under dim red light and do all the following procedures under dim red or IR light (use e.g., red filter in front of the microscope light source).
   1. Pull the eyes out by using tweezers and put them in the media. Place one eye at a time on a small piece of paper (e.g., some regular filter paper) and make a slit approximately on the level of ora serrata while holding the eye with tweezers (this is done outside of the solution here).
4. Cut along the ora serrata (or closer to the equator of the eye) with microscissors and remove the cornea and the lens. Place the eye cup in the cold media in the large Petri dish and repeat the same procedure with the other eye.
5. Cut a small incision from the top of the eye cup towards the optic nerve by keeping the scissors between the retina and sclera in order to keep the retina as intact as possible. Grip the sclera from both sides of the incision by using two tweezers and pull the tweezers away from each other to detach the retina.
   1. Cut the optic nerve and try to isolate the retina with a minimum amount of physical contact to the distal surface. RPE will mostly detach automatically from the retina during the dissection process. It is more important to avoid mechanical disturbance of the retina than to perform the dissection quickly, generally retinas can be incubated at least 30 min in the solution without significant effects on the response properties.
6. Mount the retinas on the domes of the specimen holder (Figure 1C). Immerse the bottom part of the specimen holder in the Petri dish with the dissected retinas. Slide the retina, photoreceptor side (the convex surface of the isolated retina) upwards, above the dome and lift the specimen holder so that the retina attaches on the filter paper. Repeat the procedure for the other retina.
7. Dry the holder plate carefully to prevent electrical crosstalk between the retinas as well as noise and signal shunting. Specimen holder has O-rings around the bottom part domes to prevent solution spill between the bottom and top parts of the holder as well as to help electrical isolation of the photoreceptor and ganglion cell sides of the individual retinas. Attach the top part of the holder with the four screws (see Figure 1B) and fill the perfusion channels with perfusion solution by using a syringe and a needle.
8. Transfer the specimen holder next to the heat exchanger block and connect the input and output perfusion lines to the specimen holder (Figure 1D). Connect the electrodes to the ERG amplifier (top electrodes connect to the ground/minus pin in the amplifier) and attach the stimulator/control unit on the specimen holder by using an adaptor or slide the heating pad into the stimulator unit depending on which in vivo ERG system is used.

3. Recordings

1. Configure the amplifier and stimulus settings by using the in vivo system’s software. Set the acquisition frequency to a value between 1 and 10 kHz and low-pass filtering to 300 Hz. Do not use high-pass filtering. Use 60 Hz (or 50 Hz in Europe) notch filter if necessary.
2. Record baseline without light stimulation and with dim stimulation (e.g., green light of -35 dB or 0.3 mCd sm^-2) to test for good electrical connection between the electrodes and retina sample. Wait 10 – 20 min before starting the data collection so that retinal temperature and function reach a stable state.
   1. Choose stimulus parameters according to any specific experiment and start data collection. E.g., use green light from -40 up to 0 dB or from 0.1 up to 1,000 mCd sm^-2 for a scotopic response family. Use about 2 to 3 log-units brighter flashes in photopic recordings where rods have to be suppressed by background light (3 – 30 Cdm^-2) or a probe flash (about 1.3 Cd sm^-2 = 1 dB, see Figure 3).
   2. However, remember that the exact light intensity values might be somewhat dependent on your system calibration and experimental conditions. As a rule of thumb, scale intensities down by 5 – 10-fold as compared to in vivo recordings when using green light¹⁷. A black cover with apertures above the retinas (included in the commercial adapter system) can be used to reduce light scattering in the specimen holder and facilitate homogenous and equal stimulation of both retinas by the Ganzfeld sphere of the commercial in vivo systems.
      NOTE: For recordings from dark-adapted retina, the intensities of test flashes used in a typical experiment bleach only a negligible fraction of the pigment so that the lack of RPE-driven regeneration is not an issue. In addition, it has been previously shown that cone pigment regeneration can still take place in the isolated retina via the Müller cell visual cycle²⁰.
3. As experiments last typically from 30 min up to several hours, monitor baseline drift, noise level and response stability during the experiment as changes in these might indicate technical problems.
   ​NOTE: Increased noise or decreased response amplitudes may indicate air bubbles in the perfusion or electrode channels, too low or high temperature, solution leak/spill or displacement of the retina.

4. Cleaning

1. Detach the specimen holder from the perfusion lines, open it and flush the retinas from the filter paper. Remove the electrodes and rinse them with distilled water (do not use ethanol). Clean the specimen holder (including the perfusion channels) with ethanol and/or distilled water. Flush perfusion tubing carefully with >70% ethanol.
2. Rinse the perfusion bottle with distilled water (do not use detergents). Ethanol can be also used to clean the bottle.

Results

We recorded flash responses from dark-adapted wild-type (WT) C57BL/6 mouse retinas by following the experimental protocols described above and illustrated in Figure 1 by using different standard perfusion solutions (Figure 2). The response waveforms and kinetics as well as sensitivity of rod photoreceptors appeared similar in Ames’ and Locke’s media (Figure 2A and B). On the other hand, under HEPES-buffered Ringer solution (no bicarbonate or ...

Discussion

We demonstrate here the critical steps for obtaining high-quality ex vivo ERG recordings simultaneously from two isolated mouse retinas by using in vivo ERG system components together with an ex vivo ERG adapter. In this study we perfused both retinas from the animal with the same solution (either Ames’, Locke’s or Ringer) but it is also possible to perfuse each retina with a different solution e.g., for drug testing purposes. The most important steps for obtaining high qua...

Materials

Name	Company	Catalog Number	Comments
In vivo ERG system	OcuScience	HMsERG	www.ocuscience.us/id77.html
In vivo ERG system	LKC Technologies	UTAS-E 3000	www.lkc.com/products/UTAS/bigshot.html
Ex vivo adapter	OcuScience	Ex VIVO ERG adapter	www.ocuscience.us/id107.html
Dissection microscope	North Central Instruments	Leica M80	May use any brand
IR emitter	Opto Diode Corp.	OD-50L	www.optodiode.com
Prowler Night Vision Scopes	B.E. Meyers Electro Optics	D4300-I	Military grade product.
Red filter	Rosco Laboratories	Roscolux #27 Medium Red	May be used instead of IR system
Red head light	OcuScience	ERGX011	www.ocuscience.us/catalog/i29.html
Microscissors	WPI, Inc.	500086	www.wpiinc.com/
Dumont tweezers #5	WPI, Inc.	14101
Razor blades	Electron Microscopy Sciences	72000	www.emsdiasum.com
Scale	Metler Toledo	AB54-S/FACT	May use any brand
pH meter and electrode	Beckman Coulter	pHI 350	May use any brand
NaCl	Sigma-Aldrich	S7653	May use any brand
KCl	Sigma-Aldrich	60129	May use any brand
MgCl2	Sigma-Aldrich	63020	1.0 M solution
CaCl2	Sigma-Aldrich	21114	1.0 M solution
EDTA	Sigma-Aldrich	431788	May use any brand
HEPES	Sigma-Aldrich	H3375	May use any brand
Sodium Bicarbonate	Sigma-Aldrich	S6297	May use any brand
Ames medium	Sigma-Aldrich	A1420	May use any brand
BaCl2	Sigma-Aldrich	B0750	May use any brand
DL-AP4	Tocris Bioscience	101	May use any brand
Succinic acid disodium salt	Sigma-Aldrich	224731	May use any brand
L-Glutamic acid	Sigma-Aldrich	G2834	May use any brand
D-(+)-Glucose	Sigma-Aldrich	G7528	May use any brand
Leibovitz culture medium L-15	Sigma-Aldrich	L4386	May use any brand
MEM vitamins	Sigma-Aldrich	M6895
MEM amino acids	Sigma-Aldrich	M5550
Carbogen	Airgas	UN3156	5% CO2