Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

The overall goal of the following experiment is to introduce glutamate or hypoxia stress as a potential model to test the effectiveness of neuroprotective agents. This is achieved by first obtaining freshly in nucleated bovine eyes and performing a quick, but still a traumatic explanation of the retina. Next, after fusing the retinas with a special nutrient solution, electrodes are used to record a series of stable baseline electroretinogram or ERGs.

Then glutamate is added to the nutrient solution, or hypoxia is induced by adding 100%nitrogen instead of oxygen. Finally, an ERG is recorded in order to see the effect of glutamate stress or hypoxia on the amplitudes of A and B waves. The results show a change in retinal function when exposed to hypoxia or glutamate stress through ERG recordings.

The main advantage of this technique over existing methods like cell culture or in vivo ERG recordings, is that functional testing can be performed in contrast to cell cultures without having the variability exerted by electrode placement or general anesthesia compared to in vivo experiments. This methods can help answer key questions in the pharmacological field, such as testing neuroprotective agents. The implications of this technique extends to therapy or diagnosis of acute or chronic diseases.

Possible neuroprotective agents can now be tested with this stress model. Though this method can provide insight into neuroprotection in the ophthalmological field, it can also be applied to other medical areas such as neuro degenerative diseases. Generally, individuals new to this method will struggle because of the preparation technique.

The model itself was developed by Professor Sickle in Cologne, and he was a great neurophysiologist. After obtaining bovine eyes from a slaughtered animal, place them in sickle solution at room temperature for transport to the lab using a dim red light under dark adapted conditions. Remove the anterior part of the eye, then perform an equatorial incision about four millimeters posterior to the limbus, and remove the cornea, iris ciliary body and lens in one piece.

Keep the retinas in sickle solution. Next, mechanically loosen the vitreous attachments to the retinal surface and remove the vitreous from the open eye cup. Then divide the eye into four quadrants, and using a TR refine punch out round areas of about seven millimeters in diameter.

Gently separate the retina from the pigment epithelium and to place it on a recording device, transfer the retina onto mesh and use a plastic ring to secure it on the electrodes. Then place the recording device inside a box protected from light. Before taking measurements dark, adapt the retina then with calibrated neutral density filters and a light stimulus of 10 microseconds set At five minute intervals, apply a one hertz single white xenon flash for stimulation with an intensity of 6.3 matching light intensity or MLX at the retinal surface.

After the dark adaptation period under constant perfusion, measure the amplitudes of the electrical signal until stable a wave amplitudes are recorded. To begin testing, replace pure oxygen with either pure nitrogen to test for hypoxia or 250 micromolar glutamate. Record the electrical responses every five minutes for 45 minutes.

Measure the B wave amplitude from the trough of the A wave to the peak of the B wave to investigate the effect of hypoxia or glutamate on the photoreceptor potential. Under scotopic conditions, add one millimolar aspartate to the nutrient solution to suppress the B wave to statistically evaluate the data, use the Coof Smirnoff test to ensure a normal distribution for all data. Calculate the reduction of the A and B wave amplitudes and percentages after the exposure phase.

In comparison to the last measurement before the exposition, compare the reduction of the ERG amplitudes after 45 minutes at the end of the exposition period to ERG measured before application. Compare the a and b wave at the end of the washout phase to the corresponding amplitude before exposition. To examine a possible recovery for statistical analysis, use JMP statistical software or SPSS software.

Calculate data throughout as the mean plus or minus standard deviation. Use the appropriate statistical test to estimate significance as shown here. After a one hour perfusion of the retinal preparations with oxygen saturated standard solution, ERG amplitude stabilized and displayed less variation of amplitudes between single measurements.

pH osmotic pressure temperature and PO two were kept constant for all tests. To isolate the photoreceptor signal from the signal of the inner retina one millimolar aspartate was added to the standard solution to suppress the B wave. Under hypoxic conditions, a decrease in a wave amplitude of 87.0%was noted.

After an exposition time of 45 minutes at the end of the washout, a decrease of 36.5%was noted. That was statistically significant. In addition, a statistically significant initial decrease in B wave amplitudes of 87.23%was recorded in this setting.

A statistically significant reduction of 25.5%was noted. After exposition with 250 micromolar glutamate, a 7.8%non-significant reduction of a wave amplitudes was detected after the defined time interval. This was followed by a non-significant reduction of 1.9%Finally, decreased amplitudes of the ERG by 83.7%were recorded, and at the end of the washout, a BWA recovery was observed, resulting in a non-significant reduction of 2.3%after 75 minutes of perfusion with standard solution.

While attempting this procedure, it's important to remember to keep an eye on your ERG baseline. Following this procedure, neuroprotective or offending agents can be tested in order to answer additional questions like toxicity After its development. Is technique paved the way for researchers in the field of XV with electrophysiology to explore ERG changes in a standardized model.