(808.10) Myo/Nog Cells Migrate to Areas of Cell Death and are Phagocytic in a Murine Model of Retinitis Pigmentosa

Poster Board Number: A306

Courtney Helm (Philadelphia College of Osteopathic Medicine), Rachel Souza (Philadelphia College of Osteopathic Medicine), Scott Serpico (Philadelphia College of Osteopathic Medicine), Mark Martin (Philadelphia College of Osteopathic Medicine), Eric Sugarman (Philadelphia College of Osteopathic Medicine), Carlos Font (Philadelphia College of Osteopathic Medicine), Diana Crowley (Philadelphia College of Osteopathic Medicine), Rushil Brahmbhatt (Philadelphia College of Osteopathic Medicine), E-Jine Tsai (Philadelphia College of Osteopathic Medicine), Sarah Coughlan (Philadelphia College of Osteopathic Medicine), Mary Woodruff (Philadelphia College of Osteopathic Medicine), Paul Lecker (Philadelphia College of Osteopathic Medicine), Grzegorz Gorski (Philadelphia College of Osteopathic Medicine), John Benalcazar (Philadelphia College of Osteopathic Medicine), Lindsay Gugerty (Philadelphia College of Osteopathic Medicine), Jacqueline Gerhart (Philadelphia College of Osteopathic Medicine), Mindy George-Weinstein (Philadelphia College of Osteopathic Medicine), Arturo Bravo Nuevo (Philadelphia College of Osteopathic Medicine)

"Introduction

Retinitis Pigmentosa (RP) is a set of diseases that leads to progressive visual impairment, affecting over one million people worldwide. Despite the heterogeneity of the disease course and mechanism, the various forms of RP all involve progressive loss of retinal rod and cone cells. Myo/Nog cells have been shown to play a crucial role in ocular development. They have been implicated in neuroprotection in response to acute injuries and stress. In this study, we examined the behavior of Myo/Nog cells in a murine model of congenital retinitis pigmentosa (RP), determined the anatomical location of these cells as the degeneration progresses, and assessed their possible role of clearing out the debris caused by the degeneration.

Methods

C3H/Hej (C3H) mice, which are homozygous for the cGMP phosphodiesterase 6B (PDE6) mutation (also known as the rd1 mutation), were used as the animal model of RP in this study. C3H and C57Bl/6J (C57) control mice were assessed at weeks 2.5, 3, 4, 5, and 6 using scotopic electroretinographs (ERG), ocular computer topography (OCT), and immunofluorescence microscopy. Enucleated eyes were fixed in paraformaldehyde and cryo-sectioned. Sections were incubated in TUNEL reagent to detect and quantify cell death. Cryosections were labeled with a Myo/Nog specific monoclonal antibody to brain-specific angiogenesis inhibitor (BAI1) . Myo/Nog cell counts were performed by identifying the total number of BAI1+ cells, TUNEL+ cells, and the number of cells that were positive for both BAI1 and TUNEL. Myo/Nog cell counts, photoreceptor counts, outer nuclear layer (ONL), and inner nuclear layer (INL) thicknesses were all obtained using NIS elements software on a FluorScope microscope. 

Results

ERG showed decreased amplitudes of the A and B waves in C3H mice when compared to the C57 group. OCTs demonstrated a significant difference in retinal thickness between the two groups. Progressive thinning of the retina in the C3H mice was confirmed by microscopy. We observed early degeneration of the retina of C3H mice in the central and mid-peripheral areas before week 2.5, which then progressed more significantly in the edge of the retina in later weeks. Myo/Nog cells were significantly more numerous in C3H mice compared to C57 mice at all time points. Most Myo/Nog cells of C3H mice were found in the outer nuclear layer (ONL) and within the neighboring choroid. Data obtained from ERG and histological demonstrated progressive photoreceptor loss over time, and a rapid decline in ONL/INL thickness between weeks 2.5-4 in C3H mice. Myo/Nog cells appeared to have phagocytosed cell corpses as evidenced by the overlap in Myo/Nog cell labeling with the labeling of cellular debris (the depleted photoreceptors). 

Conclusions

These findings are consistent with the behaviors of Myo/Nog cells in other forms of retinal stress and injury. Future studies will examine the importance of Myo/Nog cells in clearance in retinopathies.

Philadelphia College of Osteopathic Medicine, Philadelphia, PA





The overall number of Myo/Nog cells were counted in all layers of the retina in C3H and C57 mice and averaged at each time point; This image illustrates retinal thickness in C57 vs C3H Mice; changes in cell density and thickness of the ONL over time in week 2.5, week 3, week 4, week 5 and week 6. The first column demonstrates these changes from week 2.5 to week 6 in the edge of the retinas of C57 mice while the second column shows these changes from the mid-periphery. The third column depicts these changes in the edge of C3H retinas over time while the last column shows the changes in the mid periphery of the C3H retinas."