143 Synaptic loss in the MS spinal cord Introduction Multiple sclerosis (MS) is an inflammatory demyelinating and degenerative disease of the central nervous system and a major cause of chronic disability in young adults1. Although 85% of people with MS (pwMS) initially experience a relapsing phenotype, most will develop chronic neurological dysfunction (progressive MS). Magnetic resonance imaging (MRI) is commonly used to support diagnosis and monitoring of pwMS. Although MRI lesion volume has been shown to be a useful predictor of disease activity, its value as an outcome is limited in people with progressive MS2. Volumetric indices of the whole brain, or segmented proportions thereof3, as well as the spinal cord4 appear to provide better outcome prediction. Given the reported correlation between indices of spinal cord cross-sectional area (CSA) and disability5-7, and earlier evidence suggesting axonal loss as the key driver of chronic disability8,9, we were surprised to find no correlation between axonal loss and CSA in a recent study of whole postmortem spinal cords10. Because synaptic damage has been recognized as an important feature of MS in the brain11,12, we investigated whether such damage also occurs in the spinal cord, thereby potentially providing an additional substrate of chronic disease deterioration, and of CSA shrinkage. We addressed the following 3 questions: (1) What is the extent of synaptic loss in the MS spinal cord? (2) Does synaptic loss correlate with neuronal loss? (3) Do CSA and/or spinal cord gray matter (GM) area provide noninvasive substrates of synaptic damage? Materials and methods The protocol for this study received prior approval by the review board of the Multiple Sclerosis and Parkinson’s Tissue Bank covered by Ethical Approval 08/MRE09/31+5 (Research Ethics Committee for Wales; Integrated Research Application System project ID 126869). Tissue sampling and immunohistochemistry Two blocks each of cervical, thoracic, and lumbar cord levels were selected, and serial 10μmthick sections were cut using a Shandon Finesse ME+ microtome (Thermo Fisher Scientific, Dartford,UK),mountedonSuperfrost+slides (VWR, Lutterworth,UK) and leftat 60°Covernight. Sections were stained with hematoxylin and eosin (H&E), and immunohistochemistry was undertaken using primary antibodies against myelin basic protein (MBP; SMI-94, mouse monoclonal, 1:100; Covance, Princeton, NJ), synapsin-1 (polyclonal rabbit, 1:100, NB300104; Novus Biologicals, Littleton, CO), and synaptophysin (Dako Omnis FLEX Ready-to-Use monoclonal mouse anti-human, clone DAK-SYNAP; Agilent, Stockport, UK). Synapsin-1 immunostaining was done manually; for synaptophysin immunostaining, a Dako (Carpinteria, CA) autostainer was used (Figs 1 and 2). Area measurements The area of GM demyelination (if any) within the anterior horn boundaries was measured on MBP sections, whereas H&E sections were used to measure CSA and anterior horn GM area at low magnification (objective ×4). The total number of neurons within each anterior horn was counted at higher magnification (objective ×20) on H&E sections (see Fig 2). Quantification of synapses Sections immunostained for synaptophysin and synapsin-1 were scanned using a SlideScanner (C12000 NanoZoomer-XR Digital slide scanner; Hamamatsu Photonics, Welwyn Garden City, UK) and stored as Nanozoomer Digital Pathology image files. To assess transmittance (T),
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