direct-coupled electroretinogram recording of the retinal pigment epithelium in a mouse

Direct-Coupled Electroretinogram Recording of the Retinal Pigment Epithelium in a Mouse

To position the mouse and electrodes for an experiment, anesthetize the cornea of a sedated mouse with a drop of 0.5% Proparacaine Hydrochloric acid before dilating the cornea with a drop of 2.5% Phenylephrine HCl and 0.5% tropicamide.
Use step one of six to turn on a dim red light inside the dome and place the mouse on a heated recording table. Use forceps to carefully tent the skin of the rear leg, and using one hand to firmly hold the needle electrode, use the other hand to insert the electrode subcutaneously into the rear leg to secure the electrode into place.
Place the reference silver/silver chloride electrode into the animal&#39;s mouth so that the centered pellet rests along the back cheek, and is held in place behind the teeth.
Before placing the capillary electrodes onto the eye, position the electrode holder with the glass capillaries oriented vertically, and flick the electrode holder with an index finger to remove any bubbles that may have been introduced.
Use a 25-gauge needle to fill the capillary tips with HBSS and inspect the capillaries to ensure that there are no air bubbles trapped in the tips. Position the electrode holder stand so that the open tips of the HBSS-filled capillaries are in gentle contact with the cornea, and taking care to avoid introducing bubbles, invert the lubricant eye gel dispenser to discard the initial drops. Then, place a drop of lubricant eye gel onto each eye to maintain conductivity and to prevent desiccation during the recording.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Mouse and electrode positioning
<ol>
	<li>Keep the mice overnight in a well-ventilated, light-tight box for dark adaptation.</li>
	<li>The animals were anesthetized by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (8 mg/kg).</li>
	<li>Apply a drop of 0.5% proparacaine HCl topically to anesthetize the cornea, as well as a drop of 2.5% phenylephrine HCl and 0.5% tropicamide to dilate the pupils.</li>
	<li>Trim the mouse whiskers with scissors to prevent inadvertent twitching from disturbing the glass capillary electrodes during the recording. 
	NOTE: The Direct-Coupled Electroretinogram (DC-ERG) stimulus protocol within the ERG system has several built-in stimulus routines that can be selected by clicking on the Step (forward arrow) or Step (backward arrow). Only Steps 1, 4, and 5 in the software are required to prepare and perform the DC-ERG recording.</li>
	<li>In the ERG system, the software verifies that the correct patient has been selected. Click on the green Protocols button. Under Protocol Description, select DC-ERG. Then click on Run. Verify that this is the correct test being performed by clicking on Yes.</li>
	<li>Use Step 1/6 designated Red Light stimulus, to turn on the red light inside the dome to help position the mouse and electrodes while observing the changes in impedance.</li>
	<li>Place the mouse on a heated recording table and carefully tent the skin of the rear leg using forceps. Hold the needle electrode firmly in one hand and insert it subcutaneously into the rear leg to secure it into place.</li>
	<li>Place the reference Silver/Siverchloride(Ag/AgCl) electrode (Table of Materials) inside the mouth so that the sintered pellet rests along the back cheek and is held in place behind the teeth. 
	NOTE: Gold wire electrodes should not be used as the mouth reference electrode as they have different impedance characteristics and increase the peak-to-peak noise.</li>
	<li>Prior to placing the capillary electrodes to the mouse&#39;s eye, hold the electrode holder with the glass capillaries vertically and flick the electrode holder with the index finger to remove any bubbles that may have been introduced. Fill the tip with Hanks&#39; Balanced Salt Solution (HBSS) using a 25 G needle attached to a syringe and inspect to ensure that there is no air bubble trapped at the tip. Position the electrode holder stand so that the open tip of the HBSS-filled capillaries are in gentle contact with the cornea.</li>
	<li>Use special precautions to avoid introducing air bubbles by holding the lubricant eye gel dispenser inverted and discarding the initial drops. Place a drop of lubricant eye gel on each eye to maintain conductivity and prevent desiccation during the recording.</li>
</ol>
2. DC-ERG recording
<ol>
	<li>Click on Step (forward arrow) to select Step 4/6 &#34;Long Flash No-Li.&#34;</li>
	<li>Click on Impedance. Use the Impedance Checking screen to examine the resistances of the left and right eyes. The impedance values for the recording electrodes at each eye are expected to be similar (~39 k&#8486;). The impedance values for the ground and the reference electrodes are expected to be less than 10 k&#8486;).</li>
	<li>Click on Preview to view the traces for the left and right eye. Wait for a stable baseline to be achieved (&#60;10 min). Click on Stop to exit the trace preview. 
	NOTE: Abrupt changes in drift direction or aberrant noise in the recording will not improve with time and will require identifying the capillary electrode that needs attention. The most likely cause is a bubble introduced to the tip of the electrode. Refill the tip with HBSS. Click on Preview again to check the baseline.</li>
	<li>Click on Step (forward arrow) to select Step 5/6 &#34;Long Flash 10 cd 7 min.&#34; Click Run to start the recording.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ag/AgCl (mouth) Electrode</td>
			<td>WPI Inc</td>
			<td>EP1</td>
			<td>Mouth reference electrode for mouse</td>
		</tr>
		<tr>
			<td>Ceramic Tile</td>
			<td>Sutter Instrument</td>
			<td>CTS</td>
			<td>Used to cut the glass capillary tube to an appropriate size</td>
		</tr>
		<tr>
			<td>Cotton Tipped Cleaning Stick</td>
			<td>Puritan Medical Products</td>
			<td>867-WC No Glue</td>
			<td>To be used as a spacer to improve the fit of the electrode holder assembly</td>
		</tr>
		<tr>
			<td>Electroretinogram (ERG) System</td>
			<td>Diagnosys LLC</td>
			<td>E3 System</td>
			<td>Visual electrophysiology system to diagnose ophthalmic conditions in vision research and drug trials</td>
		</tr>
		<tr>
			<td>Bunsen Burner</td>
			<td>Argos Technologies</td>
			<td>BW20002460</td>
			<td>Or equivlaent to shape glass under flame</td>
		</tr>
		<tr>
			<td>Glass Capillary Tube (1.5 mm)</td>
			<td>Sutter Instruments</td>
			<td>BF150-75</td>
			<td>For filling with HBSS and making contact to the cornea</td>
		</tr>
		<tr>
			<td>Hank&#8217;s Buffered Salt Solution (HBSS)</td>
			<td>Thermo Fisher Scientific Inc</td>
			<td>14175-095</td>
			<td>Commercially available. Maintain at RT</td>
		</tr>
		<tr>
			<td>In-Line Filter</td>
			<td>Whatman</td>
			<td>6722-5001</td>
			<td>To protect vacuum pump from aerosols</td>
		</tr>
		<tr>
			<td>Low Noise Cable for Microelectrode Holders</td>
			<td>WPI Inc</td>
			<td>5372</td>
			<td>Suggested for improving the length and placement of the cables and electrode holder assemblies</td>
		</tr>
		<tr>
			<td>Magnetic Ball Joint</td>
			<td>WPI Inc</td>
			<td>500871</td>
			<td>For magnetically positioning the electrode holder assembly on the stage</td>
		</tr>
		<tr>
			<td>MatLab</td>
			<td>Mathworks</td>
			<td></td>
			<td>MatLab: For editing the analysis software</td>
		</tr>
		<tr>
			<td>Matlab Curvefit Toolbox</td>
			<td>Mathworks</td>
			<td></td>
			<td>Toolbox for MatLab (only required for editing the analysis software)</td>
		</tr>
		<tr>
			<td>MatLab Compiler</td>
			<td>Mathworks</td>
			<td></td>
			<td>Toolbox for MatLab (only required for editing and re-releasing the analysis software)</td>
		</tr>
		<tr>
			<td>MatLab Runtime version 9.5</td>
			<td>Mathworks</td>
			<td>R2018b (9.5)</td>
			<td>Required to run the analysis software: https://www.mathworks.com/products/compiler/matlab-runtime.html</td>
		</tr>
		<tr>
			<td>Microelectrode Holders (45 degrees)</td>
			<td>WPI Inc</td>
			<td>MEH345-15</td>
			<td>For holding the capillaries</td>
		</tr>
		<tr>
			<td>Needle (25 ga)</td>
			<td>Covidien</td>
			<td>8881250313</td>
			<td>For filling the capillary tubes with HBSS</td>
		</tr>
		<tr>
			<td>needle (ground) electrode</td>
			<td>Rhythmlink</td>
			<td>13mm - one elctrode</td>
			<td>Subdermal needle electrode (ground) for mouse (13mm long, 0.4mm diameter needle, 1.5m leadwire)</td>
		</tr>
		<tr>
			<td>Regulator/Power Conditioner</td>
			<td>Furman</td>
			<td>P-1800</td>
			<td>Or equivalent to remove DC-offset from noise introduced through power line</td>
		</tr>
		<tr>
			<td>Syringe (12 mL)</td>
			<td>Monoject</td>
			<td>1181200777</td>
			<td>For filling the capillary tubes with HBSS</td>
		</tr>
		<tr>
			<td>T-clip</td>
			<td>Cole-Parmer</td>
			<td>06852-20</td>
			<td>For electrode holder assembly</td>
		</tr>
		<tr>
			<td>Vacuum Desiccator</td>
			<td>Bel-Art</td>
			<td>420120000</td>
			<td>Clear polycarbonate bottom &#38; cover</td>
		</tr>
		<tr>
			<td>Pharmacological treatment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lubricant eye gel</td>
			<td>Alcon</td>
			<td>0078-0429-47</td>
			<td>Helps lubricates corneal surface and maintain electrical contact with capillary electrodes</td>
		</tr>
		<tr>
			<td>Phenylephrine Hydrochloride 2.5%</td>
			<td>Akorn</td>
			<td>17478-201-15</td>
			<td>Short acting mydriatic eye drops (for pupil dilation)</td>
		</tr>
		<tr>
			<td>Proparacaine Hydrochloride 0.5%</td>
			<td>Akorn</td>
			<td>17478-263-12</td>
			<td>Local anesthetic for ophthalmic instillation</td>
		</tr>
		<tr>
			<td>Tropicamide 0.5%</td>
			<td>Akorn</td>
			<td>17478-101-12</td>
			<td>Short acting mydriatic eye drops (for pupil dilation)</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>AnaSed</td>
			<td>sc-362949Rx</td>
			<td>Analgesic and muscle relaxant</td>
		</tr>
		<tr>
			<td>Zetamine (Ketamine HCl)</td>
			<td>VetOne</td>
			<td>501072</td>
			<td>Anesthetic for intramuscular injections</td>
		</tr>
	</tbody>
</table>

Source: Miyagishima, K. J., et al. <a href="https://app.jove.com/v/61491">Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium.</a> J. Vis. Exp. (2020).
This video demonstrates the recording of a direct-coupled electroretinogram (DC-ERG) on a dark-adapted, anesthetized mouse retinal pigment epithelium (RPE). After applying dilating drops, the animal is placed in a recording setup, and the capillary electrode filled with a salt buffer is placed on the cornea. After establishing the baseline, DC-ERG is recorded.

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

Begin with a dark-adapted, anesthetized mouse.
Apply anesthetic and dilating drops to the cornea.
Place the mouse on a raised platform in front of a dome with a dim red light.&#160;
Secure a ground electrode subcutaneously on the hind leg and a reference electrode inside the mouth.
Take an electrode holder with a capillary filled with salt solution. Flick to remove air bubbles.
Fill the capillary tip fully with the salt solution.
Position the capillary on the cornea to record a direct-coupled electroretinogram or DC-ERG.
The DC-ERG measures subtle electrical responses of the retinal pigment epithelium or RPE.
RPE is a specialized cell monolayer that maintains the health and structural integrity of photoreceptor cells.
Apply a drop of eye lubricant to maintain conductivity and prevent desiccation.
Check the resistance values for each eye to ensure they are similar and within range.
Establish a stable baseline, and then start recording the DC-ERG.

27 Görüntüleme. Bir farede retina pigment epitelinin doğrudan bağlı elektroretinogram kaydı

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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.

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JoVE Journal

Nörobilim

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.

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.

Ex Vivo Elektroretinogramın Küçük ve Büyük Gözlerde Retina Fonksiyonunu İncelemesi için Kurulum ve Koşulların Optimize Edilmesi

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Electroretinogram Recording for Infants and Children under Anesthesia to Achieve Optimal Dark Adaptation and International Standards

Electroretinogram (ERG) is the only clinical objective test available to assess retinal function. Full-field ERG (ffERG) measures the panretinal rod and cone photoreceptor function as well as inner retinal function and is an important measure in the diagnosis and management of inherited retinal diseases as well as inflammatory, toxic, and nutritional retinopathies. Adhering to international standards and maintaining retinal dark adaptation are critical to acquire valid and reliable dark-adapted (scotopic) and light-adapted (photopic) ffERG responses. Performing ffERG in infants and children is challenging and often requires general anesthesia in the operating room. However, maintaining retinal dark adaptation in the operating room is becoming increasingly difficult given the numerous light sources from anesthesiology monitoring systems and other equipment. A practical and widely applicable method for ffERG testing is described in the operating room that optimizes retinal dark adaptation. The method reduces operating room time by dark-adapting the patient before general anesthesiology is instituted. The operating room is modified for dark adaptation and any remaining light source in the darkened operating room is minimized with the use of a modified portable foldable darkroom that encloses the patient&#8217;s head and the ERG examiner during ffERG scotopic recordings. The simple method adheres to ffERG international standards and provides valid reliable scotopic and photopic ffERG recordings that are critical to assess objective retinal function in this young age group where subjective assessment of visual function such as visual acuity and visual fields are not possible. Furthermore, the ffERG is the gold standard clinical test in detecting early onset inherited retinal diseases including Leber congenital amaurosis where approved gene therapy has become available. In sedated conditions, very low amplitude ffERG signals can be detected due to minimal orbicularis muscle activity interference, which is particularly relevant in patients after gene therapy to detect improved amplitude responses.

Adhering to international standards and maintaining retinal dark adaptation are critical to acquire valid full-field electroretinogram responses in the diagnosis and management of inherited retinal diseases. A practical protocol using a portable darkroom is provided to obtain full-field&#160;electroretinogram for infants and children under sedation or general anesthesia in the operating room setting.

Optimal Karanlık Adaptasyon ve Uluslararası Standartlara Ulaşmak İçin Anestezi Altındaki Bebekler ve Çocuklar Için Elektroretinogram Kaydı

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Direct-Coupled Electroretinogram Recording of the Retinal Pigment Epithelium in a Mouse

TRANSKRIPT

Direct-Coupled Electroretinogram Recording of the Retinal Pigment Epithelium in a Mouse