The DC-ERG technique can be used for the non-evasive evaluation of retinal pigment epithelium function to monitor age-related changes or disease progression and to assess pharmacological intervention effects. This technique improves upon DC-ERG reproduce ability and the easy of use by simplifying the capillary electrode preparation. It can also be supplemented with a standard ohm software application to facilitate analysis.
Begin by carefully sliding a 25-gauge syringe needle through the silicone rubber gasket to the back wall of the electrode holder. Fill the base of the electrode holder with degassed HBSS while slowly retracting the needle, taking care not to introduce bubbles, and replace the threaded cap without tightening. Reinsert the syringe needle to fill the empty space within the gap with HBSS and hold the capillary horizontally while filling to prevent solution escaping from the other end.
Grasping the filled capillary at the bent end, slowly insert the other end through the loosened cap and tighten the screw cap into place. Tilt the electrode holders with the capillary electrodes facing up to allow any bubbles to flow out and degas the capillaries in a vacuum chamber for five to 10 minutes. Then, slowly release the vacuum and refill the electrode holders and glass capillaries, as demonstrated.
To make a custom stand for the microelectrode holder, remove the black polyacetal clips from one side of a number eight T-clip and use a cylinder base with magnetic ball joints machined in half to adjust the height. Secure the modified T-clips to the magnetic ball mounting screws with M3-size nuts and a slide in approximately one-inch tapered wooden handle, modified from a cotton tip cleaning stick, into the stand at an angle to secure the microelectrode holder into the custom-made T-clip magnetic ball joint stand. Then, use the rare-earth magnets cylinder base to securely position the customized electrode holder stand onto the metal plate of the stage, enabling a 360-degree rotation on a 180-degree axis.
To perform an electrode test, first, gently lower the fully assembled HBSS-filled capillary microelectrodes into a small container of HBSS. Place the needle ground electrode and the silver, silver chloride centered pellet reference electrode in the same HBSS to complete the circuit and select or create an appropriate identifier to describe the mouse to be tested. To select the direct coupled electroretinogram protocol to be performed, click protocols and select DC-ERG.
Click run. A dialog box will pop up with the patient information. If the information is correct, click yes and proceed to steps one of six.
Close the doors to the Faraday cage and click impedance to display the impedance mode. Verify that the values for the mouth reference, tail ground and recording electrodes are acceptable and click step to proceed to step four of six to test the baseline stability. To begin viewing the traces, click preview.
The traces should be low noise with a peak-to-peak amplitude of less than 200 microvolts. To position the mouse and electrodes for an experiment, anesthetize the cornea of a sedated mouse with a drop of 5%proparacaine hydrochloric acid before dilating the cornea with a drop of 2.5%phenylephrine HCL and 5%tropicamide. In the ERG system software, verify that the correct patient is selected.
Click protocols. Under protocol description, select DC-ERG and click run. Click yes to verify that the correct test is being performed.
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'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.
To record the DC-ERGs, click step to select step five of six and click impedance to 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 should be similar. And the impedance values for both the ground and reference electrodes should be less than 10 kilohms.
Click preview to view the traces for the left and right eyes and wait up to 10 minutes for a stable baseline to be achieved. Then, click stop to exit the trace preview and click run to start the recording. Here, a sample dataset from conditional knockout and wild-type mice is shown.
In this analysis, the trace suffered from minute bubbles in the electrode. It increased the peak-to-peak noise in the trace. In a separate analysis, the bubbles were eliminated using the vacuum chamber prior to assembling the microelectrodes within the electrode holder stands.
The best fit lines to the initial 25 seconds can be calculated, allowing the drift-corrected responses to be replotted and the amplitudes of the DC-ERG components to be identified. As expected, reduced expression of cure 7.1 potassium channels in the RPE greatly attenuates the c-wave and fast oscillation, indicating a significant impairment of the retinal pigment epithelium electrical properties. Here, the relative amplitudes of the DC-ERG components, normalized to wild-type and plotted against the relative two largest light-evoked a-wave amplitudes, are shown.
This form of analysis can determine whether the reduced electrical responses of the RPE can be accounted for by the reduction of photoreceptor activity alone. Or if there is an underlying defect that originates in the RPE. Remember to achieve a stable baseline prior to moving on to the mouse recording and to reinspect the electrodes periodically for bubbles that may have been introduced.
Dark-adapted ERGs can be recorded prior to the DC-ERG to measure the cone-driven retinal function. Light-adapted ERGs can also be performed after the DC-ERG to evaluate the cone-driven retinal responses.
