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

The overall goal of this procedure is to assess the function of cells from an isolated mouse retina using a commercial in vivo, electroretinogram, or ERG system together with an ex vivo adapter. This is accomplished by first dissecting the retinas from both eyes of a mouse. The second step is to mount the retinas photoreceptor side up into a specimen holder.

Next, the specimen holder is placed on a heating plate in the in vivo ERG system and connected to the gravity controlled perfusion. The final step is to connect the electrodes of the specimen holder to an amplifier and stimulate the retinas with light to obtain ERG responses. Ultimately, ex vivo ERG is used to show how rod and cone mediated signaling pathways can be dissected using pharmacology and specialized light stimulation protocols.

The main advantage of this technique over other existing methods, such as in vivo, ERGs, or single cell recordings, is that it provides higher signal to noise ratio longer and more stable recordings, as well as possibility for easy and quantitative pharmacological manipulations. In addition, this method can be used to screen efficacy and safety of drugs designed to treat retinal diseases in primate and human donor retinas At least one day in advance, modify the specimen holder's bottom part by gluing some black gray filter paper to the top of the domes. Use two component five minute epoxy along the edges of the domes, and if needed, use a dissection microscope After the glue dries, press the filter paper flat to the dome.

It will be good for about a month of recording. Then remove the paper and clean the dome with 70%ethanol. Now to prepare the electrode first, fill the electrode channels with electrode solution.

Try to avoid introducing air bubbles. Then screw pellet electrodes into the electrode channels. Connect the top and bottom pieces of the specimen holder with four screws and fill the perfusion lines with electrode solution.

Next, measure the resistance and voltage between the leads of the electrode pairs. The resistance should be below 100 kilo ohms and the voltage below 10 millivolts. If the channels are bubble free and the electrodes are in good condition now pour 400 to 700 milliliters of perfusion media into a glass bottle for the sample perfusion.

Also, pour out 300 milliliters for the dissection and store it in a fridge. Then put the perfusion tubing through the heat exchanger block and place the preheated block on the heating plate. Now enclos the bottle with a cap that has a gas inlet and a perfusion tubing inlet.

The gas inlet should be attached to an airstone. Put the preheated bottle where the perfusion media will be maintained at 37 degrees Celsius. Then prime the perfusion lines by filling them with perfusion media to initiate the gravity driven flow in an ocus science system.

Place the bottle inside the Faraday cage to minimize noise and use long perfusion output lines from the specimen holder. Positioned well below the level of the specimen holder and perfusion bottle shield these output lines and connect the shield to the amplifier's ground through the faraday cage. To prevent coupling of the electromagnetic noise to the ERG signal, attach the temperature probe to the heating stage.

Now using regulators, adjust the solution flow to three to five milliliters per minute. Lastly, if needed, introduce car and gas into the solution and adjust the regulator for a steady stream of bubbles from the airstone. Begin with pouring out about 200 milliliters of cold perfusion solution in a large Petri dish so that the whole bottom part of the specimen holder can be fully immersed.

Euthanize an animal raised under a 12, 12 hours dark light cycle and then dark adapted for six to 12 hours before the recordings. All of the following procedures must be performed under dim red or infrared light. After isolating each eye, place them into the eye medium.

Then transfer one eye to a small piece of filter paper on the paper. Grip it with tweezers and cut a slit into it at about the same level as the aura serrata from the slit. Cut along the aura serrata or somewhere closer to the equator to remove the cornea and lens.

Then remove the cornea and the lens ends and transfer the eye cup back to the cold media. Repeat this same procedure with the other eye. Now working with an eye cup, make a small incision from the top towards the optic nerve.

Keep the scissors between the retina and sclera so the retina remains as intact as possible. Next, grip the sclera on both sides of the incision using two tweezers and pull apart the tissue to detach the retina. To isolate the retina, cut the optic nerve and remove the retina with a minimal amount of contact to its distal surface.

Now, mount the isolated retinas on the domes of the specimen holder. First, immerse the bottom part of the specimen holder in the petri dish with the dissected retinas. Next slide.

The retina photoreceptor or convex side upwards over the dome. Then gently lift the specimen holder so that the retina attaches to the filter paper. Then repeat the procedure for the other retina.

Now dry the holder plate carefully to prevent crosstalk noise and signal shunting. After drying the holder, screw on the top part and fill the perfusion channels with perfusion solution from a syringe using a needle. Place the specimen holder next to the heat exchange block.

Then connect the input and output perfusion lines. Complete the setup by connecting the electrodes to the ERG amplifier and attaching the stimulator control unit to the specimen holder. Using an adapter To obtain large and reusable responses, it is important to perform careful dissection and mounting of the retinas, as well as maintain stable and bubble free perfusion above the retinas Before collecting data.

Wait 10 to 20 minutes so that the retinas temperature and function stabilize flash responses were recorded from dark adapted wild type C 57 black six retinas using different standard profusion solutions. The response waveforms and kinetics appeared similar in ames media and in locks media. The sensitivity of Rod photoreceptors was also similar.

On the other hand, in heaps buffered ringer solution without bicarbonate or carbogen response, amplitudes were significantly smaller. BWA stability was also often compromised in heap's ringer. Adding 40 micromolar of A PB efficiently removed the positive B wave.

This revealed a large, slow negative wave that has been attributed to Mueller cell activity, adding 100 micromolar of barium abolished. This component revealing the photoreceptor response of the ex vivo ERG signal. This method can be combined with other tools like pharmacology and genetics to answer questions about the molecular mechanisms of retinal signaling and blinding diseases in mammalian, including human retinas.