Name: optimizing the setup and conditions for ex vivo electroretinogram to study retina function in small and large eyes
Uploaded: 2022-06-27T13:00:00
Description: Watch this Scientific Journal Video about Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes at JoVE.com

This work was supported by National Eye Institute grants EY02665 and EY031706 and International Retinal Research Foundation to Dr. Vinberg, National Institutes of Health Core Grant (EY014800), and an Unrestricted Grant from Research to Prevent Blindness, New York, NY, to the Department of Ophthalmology &#38; Visual Sciences, University of Utah. Dr. Frans Vinberg is also a recipient of a Research to Prevent Blindness/Dr. H. James and Carole Free Career Development Award, and Dr. Silke Becker of an ARVO EyeFind grant. We thank Dr. Anne Hanneken from The Scripps Research Institute for providing the donor eye used for recordings shown in Figure 2E.

All experiments using mice were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Studies Committee at the University of Utah. Pig eyes for demonstration of this video were obtained postmortem from the slaughterhouse (Sustainable Swine Resources, Johnsonville). Eyes were obtained from human donors after brain or cardiac death with consent for research use through the Utah Lions Eye Bank, the San Diego Eye Bank or Lifesharing, which are full...

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Originally developed in 1865 by Holmgren to measure retinal light responses from the amphibian retina10, technical constraints initially prevented the ERG from being widely used. Nevertheless, seminal studies by Ragnar Granit and others identified the cellular origins of the ERG and measured photoreceptor and ON-bipolar cell responses ex vivo11,12,13. Since then, refined methods have allowed more...

Electroretinography measures retinal function in response to light1. It is integral to studying retinal physiology and pathophysiology, and measuring the success of therapies for retinal diseases. The in vivo ERG is widely used to assess retinal function in intact organisms, but it has significant limitations2,3. Amongst these, the quantitative analysis of individual retinal cell types in the in vivo ERG is hampered, since it records the sum of potential changes, and therefore overlaying responses, from all retinal cells to light stimuli4. Furthermore, it does not readily allow addition of drugs to the retina, is vulnerable to systemic influences, and has a relatively low signal-to-noise ratio. These disadvantages are eliminated in the ex vivo ERG that investigates the function of the isolated retina2,3,5,6. The ex vivo ERG allows the recording of large and stable responses from specific retinal cell types by addition of pharmacological inhibitors and easy evaluation of therapeutic agents, which can be added to the superfusate. At the same time, it removes influences of systemic effects and eliminates physiological noise (e.g., heartbeat or breathing).
In the ex vivo ERG, retinas or retinal samples are isolated and mounted photoreceptor-side up on the dome of the specimen holder3,5. The specimen holder is assembled, connected to a perfusion system that supplies the retina with heated, oxygenated media, and placed onto the stage of a microscope, which has been modified to deliver computer-controlled light stimuli. To record the responses elicited by light, the specimen holder is connected to an amplifier, digitizer, and recording system (Figure 1). This technique allows isolation of responses from rod and cone photoreceptors, ON-bipolar cells, and M&#252;ller glia by changing the parameters of the light stimuli and adding pharmacological agents.
An existing patch clamp or multi-electrode array (MEA) setup can be converted to record ex vivo ERG, either in conjunction with a commercially available ex vivo ERG adapter or a custom polycarbonate computer numerical control (CNC)-machined specimen holder, to measure light responses in retinas from small animal models, such as mice. This modification increases the accessibility of ex vivo ERG while minimizing the need for specialized equipment. The design of the specimen holder simplifies the mounting technique and integrates electrodes, eliminating the need for manipulation of microelectrodes compared to previously reported transretinal ex vivo ERG methods7. The perfusion rate and temperature inside the specimen holder are important factors that affect the response properties from photoreceptors and ON-bipolar cells. By adjusting these conditions, the ex vivo ERG can be reliably recorded from the isolated mouse retina over prolonged periods of time. Optimized experimental conditions allow ex vivo ERG recordings in retinal punches from larger retinas, including large animal eyes and human donor eyes8.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2 mm socket</td>
			<td>WPI</td>
			<td>2026-10</td>
			<td>materials to prepare electrode</td>
		</tr>
		<tr>
			<td>Ag/AgCl Electrode</td>
			<td>World Precision Instruments</td>
			<td>EP1</td>
			<td>materials to prepare electrode</td>
		</tr>
		<tr>
			<td>Ames&#39; medium</td>
			<td>Sigma Aldrich</td>
			<td>A1420</td>
			<td>perfusion media</td>
		</tr>
		<tr>
			<td>barium chloride</td>
			<td>Sigma Aldrich</td>
			<td>B0750</td>
			<td>potassium channel blocker</td>
		</tr>
		<tr>
			<td>DL-AP4</td>
			<td>Tocris</td>
			<td>0101</td>
			<td>broad spectrum glutamatergic antagonist</td>
		</tr>
		<tr>
			<td>OcuScience Ex Vivo ERG Adapter</td>
			<td>OcuScience</td>
			<td>n/a</td>
			<td>ex vivo ERG specimen holder</td>
		</tr>
		<tr>
			<td>Threaded luer connector</td>
			<td>McMaster-Carr</td>
			<td>51525K222 or 51525K223</td>
			<td>materials to prepare electrode</td>
		</tr>
	</tbody>
</table>

optimizing the setup and conditions for ex vivo electroretinogram to study retina function in small and large eyes

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in retinal diseases, and testing therapeutic interventions. The ex vivo electroretinogram (ERG) allows the quantification of contributions from individual cell types in the isolated retina by addition of specific pharmacological agents and evaluation of tissue-intrinsic changes independently of systemic influences. Retinal light responses can be measured using a specialized ex vivo ERG specimen holder and recording setup, modified from existing patch clamp or microelectrode array equipment. Particularly, the study of ON-bipolar cells, but also of photoreceptors, has been hampered by the slow but progressive decline of light responses in the ex vivo ERG over time. Increased perfusion speed and adjustment of the perfusate temperature improve ex vivo retinal function and maximize response amplitude and stability. The ex vivo ERG uniquely allows the study of individual retinal neuronal cell types. In addition, improvements to maximize response amplitudes and stability allow the investigation of light responses in retina samples from large animals, as well as human donor eyes, making the ex vivo ERG a valuable addition to the repertoire of techniques used to investigate retinal function.

Ex vivo ERG enables recording of reproducible and stable photoreceptor and ON-bipolar cell light responses, for example, from the mouse retina (Figure 2A-C). Recording of photoreceptor responses from human donor retinas is possible with up to 5 h postmortem delay of enucleation (Figure 2D) and of ON-bipolar cell responses with a &#60;20 min enucleation delay (Figure 2E). Important parameter...

Watch this Scientific Journal Video about Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes at JoVE.com

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Modification of existing multielectrode array or patch clamp equipment makes the ex vivo electroretinogram more widely accessible. Improved methods to record and maintain ex vivo light responses facilitate the study of photoreceptor and ON-bipolar cell function in the healthy retina, animal models of eye diseases, and human donor retinas.

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in ...

This protocol demonstrates the conditions needed to maintain retinal function in ex vivo ERG, particularly the extremely sensitive B wave produced by ON-bipolar cells. With ex vivo ERG, we can measure the function of different types of retinal neurons with a high signal to noise ratio while permitting the easy introduction of pharmacological agents. This method is particularly amenable to quantifying the efficacy of pharmacological agents on retinal function and disease.To begin, convert a multi-electrode array setup by connecting the ex vivo ERG specimen holder to a differential amplifier via a headstage. The amplifier must be plugged into the analog input of the interface board of the multi-electrode array system. Use the recording software for the multi-electrode array to record and store the input data from the ex vivo ERG.Set the gain of the differential amplifier to 100 and add an additional 10X voltage amplification, depending on the digitizer specifications. Set the low pass filter to 100 hertz. Alternatively, to convert a patch clamp setup, connect the ex vivo ERG specimen holder via a headstage to a differential amplifier, which must be connected to the headstage of the patch clamp amplifier.Use the patch clamp system software and the digitizer to record and store the input data from the ex vivo ERG. Set the parameters as shown earlier. Connect LEDs with the appropriate wavelengths to the microscope.To control light stimuli, use an LED driver controlled by analog outputs from a digitizer. Control the LEDs with recording software that will enable the triggering of light stimuli. Then, calibrate the light output of the LEDs at the position of the retina in the specimen holder using a photodiode.If necessary, insert neutral density filters to dim the light intensity in the light path. To prepare the electrode, insert a silver silver chloride pellet electrode into a threaded lure connector. Fill the inside of the lure connector with hot glue, and insert a two millimeter socket into the hot glue from the non-threaded side.Solder a silver wire of the EP-1 electrode to the two millimeter socket. Screw the finished electrode with an O-ring on the thread into the electrode ports of the ex vivo ERG specimen holder. For large eyes, including human donor eyes, clean the globe of the remaining connective tissue and remove the anterior segment and lens.Use a scalpel to make a cut approximately three millimeters from the limbus. Insert curved dissection scissors into the incision and cut along the limbus to remove the anterior portion of the eye with the lens. Obtain retinal specimens for ex vivo electroretinography with a three to six millimeter disposable biopsy punch.To mount the tissue on the specimen holder, place the lower half of the specimen holder into a large Petri dish, and fill it with oxygenated Ames'medium such that the dome of the specimen holder is just submerged. Carefully grasp the edge of the retina with fine forceps and transfer the retina onto the dome of the ex vivo specimen holder, photoreceptor side facing up. Lift the specimen holder from the Ames'solution, taking care that the retina stays in place.Completely dry the plate of the specimen holder to minimize noise, electrical crosstalk, and signal shunting. Then assemble both halves of the specimen holder with four screws and connect the perfusion line. Drive the electrode in the lower half of the specimen holder and connect the anode cable to the inner retinal side and the cathode cable to the photoreceptor side.Perfuse the specimen holder with at least one milliliter per minute of oxygenated Ames'medium for 10 to 20 minutes to allow light responses to stabilize. Ex vivo ERG enabled the recording of photoreceptor and ON-bipolar cell light responses from the mouse retina. Recording of photoreceptor responses from human donor retinas was possible with up to five hours postmortem delay of enucleation and less than 20 minutes delay for ON-bipolar cell responses.Under ideal conditions, response amplitudes and kinetics in both cell types were relatively stable over time, but showed a slow decline 40 to 45 minutes after retinas were mounted on the specimen holder. Reduced temperature in the specimen holder greatly slowed the kinetics of both photo receptors and ON-bipolar cells. However, it decreased the amplitude of the B wave but not the A wave.Conversely, slowing the perfusion rate reduced the amplitudes of both photo receptor and ON-bipolar cell responses but did not affect the implicit time of either the A or the B wave. Cessation of perfusion for 10 minutes followed by reperfusion resulted in a complete loss of ON-bipolar cell function with preserved photoreceptor responses. Sufficient perfusion speed and physiological temperature are critical to obtaining stable responses, in particular, from ON-bipolar cells in the ex vivo ERG.This protocol allows the in-depth exploration of the differences in photoreceptor function in the human macular and periphery, answering questions that could previously only be carried out in non-human primates.

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