145 Synaptic loss in the MS spinal cord Results Clinicopathological data A total of n = 154 tissue blocks from 26 subjects, 8 healthy controls (mean age = 82±6years), and 18 pwMS (mean age = 65±11years, disease duration = 29±11years) were included in the study. A total of n = 122 of 154 tissue blocks from 22 of 26 subjects, 7 healthy controls (mean age = 82±7years), and 15 pwMS (mean age = 69±11years, disease duration = 29±11years) were included for analysis of synaptophysin immunostaining. To explore whether synaptic pathology is detectable using a different epitope, a total of n = 51 of 154 tissue blocks from 20 subjects, 7 healthy controls, and 13 pwMS were immunostained for synapsin. To explore the possible impact of the duration of fixation on the immunostaining in our samples, n = 9 tissue blocks from 9 subjects, 3 healthy controls, and 6 pwMS were used where tissue had been fixed in 10% neutral buffered formalin for 3weeks, cryoprotected in 30% sucrose for 2weeks, and then snap-frozen in isopentane cooled over dry ice. Mean time in these cases between death and tissue fixation was 18±5hours. All remaining blocks used in this study were from samples that had been fixed, following a mean time between death and tissue fixation of 36±17hours, in 4% formalin solution for a mean of 30±24months before processing. In 3 of 154 blocks, mononuclear cells suggestive of inflammation were detected. For the remainder of tissue blocks, cellularity was decreased in areas of demyelination compared to the surrounding nonlesional tissue. However, cellularity in nonlesional tissue was indistinguishable from control tissue, with no changes suggestive of inflammation. Against this backdrop, no systematic assessment of inflammatory cells was undertaken. Synaptic pathology is extensive in MS Visual inspection of synaptophysin and synapsin immunostained sections revealed significant difference in staining intensity between MS and controls; 1/T was reduced by 54% in nonlesional GM (p <0.001) and 58% in GM lesions (p <0.001). Synaptophysin intensity in GM lesions was significantly less pronounced than in the nonlesional GM (9%, p =0.004; Fig 3A, B, E). Using synaptophysin immunostained sections, the synaptic bouton area in MS was reduced by 92% in nonlesional GM and 96% in GM lesions (p <0.001) compared to controls, with a moderate difference between nonlesional and lesional GM (42%, p <0.001; see Fig 3C, D, F). In synapsin-1 immunostained sections, we detected a 58% lower synaptic bouton area in MS compared to controls (p <0.001; see Fig 2J–L). In synaptophysin stained sections, short fixation time was associated with a nonsignificantly (7%) lower synaptic bouton area compared to sections of tissue that underwent a long fixation time. In synapsin-1 stained sections, short fixation time was associated with a nonsignificantly (2%) lower synaptic bouton area compared to sections of tissue that underwent a long fixation time. Loss of synapses does not correlate with anterior horn area, or with the number of neurons Using synaptophysin stained sections, strong correlation was detected between synaptic bouton and anterior horn GM area in controls (p <0.001), but not in MS. Similarly, the
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