More than 64 million people in the world are affected by glaucoma, and it is the leading cause of blindness in the world. The exact cause of glaucoma is yet to be determined, but it is believed to be due to increased intraocular pressure, leading to glutamate-induced excitotoxicity within the eye specifically in normaltension glaucoma. Acetylcholine (ACh) is a neurotransmitter that is found in the eye and it plays an important role in retinal development by causing bursts of stimulation in the neurons known as retinal waves. A growing body of evidence suggests that ACh provides neuroprotection against glutamate-induced excitotoxicity in the nervous system. An increase in acetylcholine could protect neurons and reduce the excitotoxicity within the eye.
Our experiment focuses on the porcine eye, since it is an inexpensive resource that is readily available. Since the brain and the eye both use the same neurotransmitters, it has been hypothesized that ACh can provide the same neuroprotection in the retina, thus preventing glutamate-induced excitotoxicity from damaging the nerves within the eye. Our goal is to find the best electrode and eye dissection combination to test acetylcholine levels using Fast Scan Cyclic Voltammetry (FSCV).
10,10-Bis[(2-fluoro-4-pyridinyl) methyl]-9(10H)-anthracenone (DMP- 543) is an effective compound for increasing acetylcholine release and is both inexpensive and takes little time to synthesize. This drug has been shown to produce increased acetylcholine levels in brain tissue. Therefore, researchers have tested DMP-543 within retinal tissue as a result since the brain and eyes use the same neurotransmitters.
FSCV is an electrochemical technique that is beneficial for measuring electroactive neurotransmitters due to its high temporal resolution and its selectivity. However, ACh is commonly thought to be non-electroactive, so enzyme layers of acetylcholinesterase and choline oxidase were needed. These enzymes allow acetylcholine to be broken down into the electroactive compound, hydrogen peroxide.
Four different electrode types were tested: a carbon fiber cylindrical microelectrode, a carbon fiber flat disc electrode, a copper wire flat disc electrode, and a platinum wire flat disc electrode. Many problems were faced in the creation of the electrodes, the biggest of which being the inability to calibrate the electrodes with repeatable results. This lack of repeatability with all electrode types tested prevented us from actually testing DMP-543 on porcine retina. However, we were able to conclude that flat disc electrode shapes are the best shape for enzyme coatings and that platinum electrodes seem to be our best chance of getting readings. Additionally, we had some success with eye preparation. We will be continuing forward with an eyecup preparation for future experiments due to its ability to measure collective levels of neurotransmitters and have the greatest longevity out of the flat mount, isolated retina, and eyecup preparations.
Further research on platinum flat disc electrodes and silver wire flat disc electrodes will be completed to try and consistently calibrate them for ACh recording. Once we are able to characterize ACh using one of those electrodes, we will be able to quantify the ACh release in porcine retina in response to DMP-543.
*This scholar and faculty mentor have requested that only an abstract be published.