Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

The overall goal of this procedure is to test the visual function in normal light injured and remote ischemic preconditioned and light injured rats using the ERG and thereby demonstrating the protective effects of remote limb ischemia on the retina. This is accomplished by first dark, adapting the rats for 12 hours. The second step is to record the baseline dark adapted ERG using LED light stimuli and a digital amplifier.

Next, subject the rats to a retinal stress by exposing it to a bright continuous light. The final step is to compare the ERG recordings of the light damage rats and the ones preconditioned with remote limb ischemia, and measure the photoreceptor responses as seen in the A wave and the inner retinal responses as seen in the B wave. Ultimately, the dark adapted ERG is used to assess the scotopic visual function.

So this method can provide insights into remote ischemic based neuroprotection. It can also be applied to other forms of retinal injury and disease pharmacological testing, and for the screening of animals with visual defects. To begin this procedure, adapt the rat in the dark for 12 hours, then anesthetize it with the injection of ketamine xylazine cocktail.

Once the animal is unconscious, check the depth of anesthesia by slightly pinching its foot to make sure the reflex response is absent. Next, apply a drop of atropin and a drop of prox methane to its cornea. Cut to black thread of 10 centimeters long.

Then make a loop with the thread and slip the loop over the equator of the eye, slightly tight knit in order to draw the eyeball slightly forward and keep the cornea clear from the eyelid. Subsequently, apply carmer eyedrops to the corneas. Position the rat on the absorbent bedding on top of the heated platform with its head placed in the opening of the ganzfeld.

Then insert the internal temperature probe into the rectum to ensure that the body temperature is maintained at 37 degrees Celsius, and secure the temperature probe in position by taping its cord to the tail of the rat. Next, insert the reference electrode subcutaneously into the hind limb. Place the negative electrode securely in the mouth to prevent the electrode from slipping out of the mouth.

Affix the connecting lead to a stable surface. Then position the positive electrode over the center of the cornea and ensure that the electrode touches the cornea gently using a microm manipulator. Once the electrodes are in place, drape the hole set up, including the ganzfeld and the animal with an opaque material to preserve dark adaptation.

In the acquisition software. Set two kilohertz as a sampling rate with five milliseconds for pre-collection sampling and a collection time of 100 to 1000 milliseconds. Then set the band pass filters to one to 1000 hertz and ensure that the sampling is triggered for a period of 100 milliseconds following a flash.

Now, check the recording baseline. It should be free of extraneous noise, but show the amplifier noise and respiratory oscillation. Then run a test flash.

This is a typical ERG waveform in response to the test flash. When it is done, allow the animal to readapt to the dark for 10 minutes. Then recheck the baseline for stable signal.

Subsequently, begin the recording At the end of the recording session, check the stability of the animal's body temperature. Remove the electrodes, then reapply the carmer polymer to the corneas. Afterward, allow the animal to recover on a heat pad until it is fully mobile and active before returning to the animal housing.

This figure shows the measurement of a wave and B wave from a dark adapted ERG. The trace shown here is recorded from the cornea of a dark adapted eye to a bright flash of light given at the time shown as T zero. The amplitude of a wave is measured from the base line to the first trough.

The amplitude of the B wave is measured from the trough of the A wave to the following positive peak. The latency is measured from the stimulus artifact to the point of interest on the trace, such as the trough of the A wave. The raw dominated ERG signal increases with the increasing light stimulus.

A wave becomes apparent at approximately 0.4 logs scotopic candela seconds per meter squared, and the twin flash paradigm has been used to separate the mixed ERG signal into cone and rod responses. Once mastered. This technique can be done within 30 minutes.