Date of Defense

4-13-2023

Date of Graduation

4-2023

Department

Biological Sciences

First Advisor

Cindy Linn

Second Advisor

Hope Katherine Vanzo-Sparks

Third Advisor

Andrew Thompson

Abstract

Irreversible vision loss can be caused by retinal damage, disease, or aging. In recent years, the prevalence of ocular damage due to blast trauma has been increasing due to the modernization of warfare. Adult mammalian retinal neurons were previously thought to lack the capacity for regeneration to reverse vision loss, which deemed the effects of many ocular damages permanent. However, recent studies from this lab have shown that PNU-282987, an agonist to the alpha 7 subtype of neural nicotinic acetylcholine receptors, will result in neural regeneration in adult mammalian cells. For this research project, I will be using Methyllycaconitine (MLA), an antagonist to the alpha 7 subtype of neural nicotinic acetylcholine receptors to inhibit the effect of PNU-282987 in blast-exposed mouse retinas. The objective of this experiment is to verify that PNU-282987’s regenerative effect in the adult mammalian retina is mediated through the alpha 7 subtype of neural nicotinic acetylcholine receptors. We expect that methyllycaconitine (MLA) will inhibit the effect of PNU-282987 in blast-exposed mouse retinas. The hypothesis of this experiment is that PNU-282987 will act on the alpha 7 subtype of neural nicotinic acetylcholine receptors to induce neurogenesis. For this study, a blast exposure was introduced to adult mouse eyes to mimic ocular damage that typically occurs in soldiers after landmine blasts during combat. The blasts were introduced using a modified paintball gun at 35 psi, which caused a significant loss of cells in the INL, ONL, and GCL. This was followed by PNU treatment or MLA treatment before the introduction of PNU. After treatment, the animals were euthanized, and the retinas were then removed, fixed, and then immunostained with primary antibodies RFP to label the Müller glial cells in tdTomato transgenic mice, as well as with antibodies against recoverin to label photoreceptors. Secondary antibodies including Alexa Fluor 594 (red) and 488 (green) were used for visualization using retinal sections with a Nikon confocal microscope. Photoreceptor cell counts increased in PNU-282987 treated blast-exposed retina, while retinas in the presence of MLA and PNU-282987 showed cell loss due to blast. Meanwhile, tdTomato-positive photoreceptors were observed in blast-exposed retinas after 3 weeks of PNU treatments. However, if eyes were treated with MLA before PNU-282987 treatment, no tdTomato-positive photoreceptors were seen. These results support the hypothesis that PNU-282987 works through alpha7 nACh receptors. If vision loss can be regained via PNU-282987, it can improve the quality of life for many people that have experienced damage to their eyesight due to blast trauma as well as in neurodegenerative diseases.

Access Setting

Honors Thesis-Open Access

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