Neuromuscular Pathology of Extrinsic Tongue Muscles in the Pink1‐/‐ Rat Model of Parkinson Disease

Abstract

IntroductionParkinson disease (PD) affects the central and peripheral nervous systems resulting in widespread pathology. Recently, more research attention has been directed toward peripheral nerves and muscles as potential sources of pathology relating to oromotor control and swallowing. Swallowing impairment (dysphagia) presents during the prodromal phase of the disease, worsens over time, and can result in serious health and quality of life impairment. Post‐mortem human analyses show pathology of nerves and muscles of the pharynx and larynx. However less is known about tongue muscles. To study clinically relevant oromotor and swallow deficits across disease progression, we use the Pink1‐/‐ rat model of early‐onset autosomal dominant PD. We have shown oromotor and swallow dysfunction in the prodromal and early stages of disease (4‐8 months) in Pink1‐/‐ rats. At 6 months there were no differences between Pink1‐/‐ and WT genioglossus (GG) muscle in many measures of muscle biology, nor was there evidence of denervation. However, we speculate this may occur later in disease progression. We hypothesize that Pink1‐/‐ GG muscles will have smaller myofiber sizes with a shift in myofiber heavy chain isoform profiles toward slower profiles compared to WT GG muscles. Additionally, we hypothesize that that Pink1‐/‐ GG NMJs will be fewer in total number with less overlap of pre‐ and post‐synaptic structures and greater motor endplate area and dispersion ratios compared to WT GG NMJs. Data and analyses are ongoing; results with expanded methodology will be presented.MethodsWe evaluated the GG muscles of 10‐month‐old rats, Pink1‐/‐ (n=2) WT (n=2)(Sage Labs, Boyertown, PA, USA). Fixed tissues were flash frozen and sectioned at 20 microns. We labeled pre‐synaptic structures with primary antibodies to NF‐M & ZNP‐1 (red fluorophore secondaries) and post‐synaptic acetylcholine receptors at the motor endplate with alpha‐bungarotoxin‐AF488 (green fluorophore). Images were taken at 20x using a fluorescent microscope (Olympus, Bx53, USA) and analyzed with ImageJ (imagej.nih.gov). Then we counted the total number of motor endplates (green) in each tissue section. We also counted how many motor endplates co‐localized with nerve terminals (red) to quantify the number of structurally intact NMJs. Here we report effects sizes due to an incomplete data set.ResultsPreliminary results demonstrated modest effect size with trends towards WT rats having more total NMJs (Cohen’s D=0.359) which was compatible with our hypothesis, but a lower proportion of structurally intact NMJs (Cohen’s D=0.454) contrary to our hypothesis.DiscussionPreliminary results suggest differences in total NMJs and NMJ structure between WT and Pink1‐/‐ rats. We will report data sets of myofiber cross‐sectional area, myofiber type composition, and the NMJ parameters of total number, overlap of pre‐ and post‐synaptic structures, motor endplate area, and dispersion ratio. These results will provide a substantial presentation of the neuromuscular phenotype present at mid‐disease timepoints in Pink1‐/‐ rats aged 10 months. Investigating neuromuscular biology of extrinsic tongue muscles and swallow‐related structures is vital for understanding of the onset, sequelae, and underlying pathology of dysphagia in PD. These results are important to an ongoing effort to develop novel therapies targeted at the devastating swallowing deficits in PD.