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Myelin swelling is dynamic in LPC-treated cortical slices and is mitigated by TTX (A) Schematic of experimental set-up for the slice culture preparation and LPC application in ex vivo mouse cortical organotypic slices. (B) Exemplar two photon images of myelin swelling after LPC. Yellow asterisks indicate swellings which increase in size, orange asterisks indicate swellings which decreased in size. See also Movie S3. Scale bar = 10 µm. (C) Quantification of percent of swellings which increased in size, remained stable or reduced following application of LPC, n = 72 swellings from N = 3 slices. (D)Z-projected confocal image of a slice fixed 2.5 hours after LPC application showing selective myelin swelling. Marker NF (neurofilament), MBP antibody, AAV-MBP:mem-tdTom and merged image. Scale bar = 5 µm. (E) Exemplar two photon images of an internode that persists after LPC induced swelling (left) and an internode which fragments after swelling (right). End of internodes are marked with orange arrows. Yellow asterisks indicate swellings. Red arrows indicate areas of myelin fragmentation. Scale bar, 20 µm. (F) Quantification of number of swellings per 100 µm at 1 hour post-LPC treatment that subsequently either persisted or fragmented by 2.5 hours. Unpaired t-test with Welch’s correction. n = 18 persistent and n = 18 damaged sheaths. Error bars are mean ± 95% CI. (G) Schematic of experimental set-up for TTX application in the LPC model. (H) Exemplar two photon images of oligodendrocytes from slices treated with vehicle, vehicle+TTX, LPC, LPC+TTX. Yellow asterisks indicate swellings. Scale bar, 50 µm. (I) Quantification of swellings/oligodendrocyte at 2.5 hours in slices treated with vehicle, LPC, LPC+TTX, and vehicle+TTX. Two-way ANOVA with Tukey’s multiple comparisons tests. n = 26 oligodendrocytes from N = 3 slices vehicle treated, n = 20 oligodendrocytes from N = 3 slices vehicle+TTX treated. n = 22 oligodendrocytes from N = 3 slices LPC treated, n = 21 oligodendrocytes from N = 3 slices, LPC+TTX treated. Clear circles represent swelling number per individual oligodendrocyte, filled circles represent average number of swellings per oligodendrocyte per slice. Error bars are mean ± 95% CI.
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