1. Systemic administration of epothilone B promotes axon regeneration after spinal cord injury
by Jörg Ruschel, Farida Hellal, Kevin C. Flynn, Sebastian Dupraz, David A. Elliott, Andrea Tedeschi, Margaret Bates, Christopher Sliwinski, Gary Brook, Kristina Dobrindt, Michael Peitz, Oliver Brüstle, Michael D. Norenberg, Armin Blesch, Norbert Weidner, Mary Bartlett Bunge, John L. Bixby, and Frank Bradke
Science
Volume 348(6232):
April 17, 2015
Published by AAAS

2. Fig. 1 EpoB reduces inhibitory fibrotic scarring after SCI by abrogating meningeal fibroblast polarization and migration.
EpoB reduces inhibitory fibrotic scarring after SCI by abrogating meningeal fibroblast polarization and migration. (A) Mass spectrometric analysis of CNS tissue and blood after a single i.p. injection of epoB; n = 4 rats per time point. (B) Immunoblots (IB) of indicated proteins in lesion extracts; n = 3 rats. (C) Human spinal cord after injury (asterisk); laminin immunolabeling. (D) Immunolabeling for laminin, glial fibrillary acidic protein (GFAP), or chondroitin sulfates (CS-56) after rat spinal cord hemisection. (E) Laminin-immunopositive (+) area at the lesion; n = 7 to 8 rats per group. (F) Glycosaminoglycan amounts in spinal cord lesion extracts; n = 8 rats per group. (G) Rat meningeal fibroblasts (RMFs) in wound-healing assays. (H) Percentage of the area shown in (G) occupied with RMFs after 48 hours, n = 3 experiments. (I and J) Immunolabeling of tyrosinated (TyrTub) and detyrosinated tubulin (DetyrTub, arrowheads). (K) IB of indicated proteins in RMFs 24 hours after treatment. (L) Immunolabeling for fibronectin, detyrosinated and tyrosinated tubulin [4′,6-diamidino-2-phenylindole (DAPI); nuclear staining] in the rat meninges at the lesion. Bottom panel, magnification of fibroblasts (arrowheads) in top panel. dpi, days postinjury. Scale bars, 50 μm. Schemes in (D) and (L) indicate lesion and displayed region (red box). Values are plotted as means + SEM. *P < 0.05, ***P < by Student’s t test.
Jörg Ruschel et al. Science 2015;348:
Published by AAAS

3. Fig. 2 EpoB promotes microtubule protrusion and axon elongation in neurons while dampening microtubule dynamics in scar-forming fibroblasts.
EpoB promotes microtubule protrusion and axon elongation in neurons while dampening microtubule dynamics in scar-forming fibroblasts. (A) Beta-3 tubulin (Tuj-1) immunolabeling of neurons on inhibitory substrates (CSPGs, chondroitin sulfate proteoglycans; Sema 3A, Semaphorin 3A). (B) Neurite length of cortical neurons after 48 hours under indicated conditions; n = 3 to 4 experiments. (C) EB3-mCherry time-lapse projections in Nogo-A exposed neuron before and after epoB treatment (asterisks, stable landmarks). Bottom panels, high magnification of boxed areas in top panels. (D) EB3-mCherry fluorescence intensity in neurites under indicated conditions; n = 9 to 16 neurons (from three experiments). (E) Neurite growth on Nogo-A. Black arrowhead, time of indicated treatment. n = 12 to 15 neurons (from three experiments). (F and G) EB3-mCherry time-lapse projections of nocodazole-treated neuron (F) and epoB-treated meningeal fibroblast (G). Bottom panels, high magnification of boxed areas in top panels. (H) EB3-mCherry time-lapse projections before and after epoB treatment in cultured meningeal fibroblasts with (arrowhead) or without Tau-expression. Bottom panels, magnification of boxed areas in top panels. (I) EB3-mCherry fluorescence intensity in fibroblast periphery under indicated conditions; n = 20 cells per condition (from four experiments). Scale bars, 25 μm. Values are plotted as means [+ SEM in (B) and (E)]. *P < 0.05, **P < 0.01 by Student’s t test. n.s., not significant.
Jörg Ruschel et al. Science 2015;348:
Published by AAAS

4. Fig. 3 EpoB reduces dystrophy and promotes regeneration of injured spinal cord axons.
EpoB reduces dystrophy and promotes regeneration of injured spinal cord axons. (A) Electron microscope images of human SCI. (Top) Undamaged axon containing microtubules (black arrowheads). (Bottom) Retraction bulb (indicated by white arrowheads) without microtubules. (Middle) Magnification of boxed area in bottom panel. Scale bars, 500 nm. (B) Beta-3 tubulin (Tuj-1) immunolabeling of retraction bulbs in chronic human SCI. Scale bar, 10 μm. (C) Lesioned GFP-positive spinal cord axons in mice forming retraction bulbs (yellow arrowheads), dying back (red arrowheads), or regenerating (green arrowheads). Boxed area in top panels, displayed region in panels below. Scale bars, 100 μm. (D and E) Percentage of injured axons forming retraction bulbs (D) and distance between injured axons and injury site (E); n = 8 mice per group. Values are plotted as means + SEM. (F) Microruby-traced mouse dorsal column axons after injury (white arrowheads), laminin and GFAP immunolabeling (dashed line, lesion border). Scale bar, 100 μm. (G) Average distance between caudal lesion margin and injured axons in individual animals (circles) and group means (vertical bars) ± SEM. *P < 0.05, **P < 0.01 by Student’s t test.
Jörg Ruschel et al. Science 2015;348:
Published by AAAS

5. Fig. 4 EpoB promotes regrowth of raphespinal axons and improves walking after spinal cord contusion injury.
EpoB promotes regrowth of raphespinal axons and improves walking after spinal cord contusion injury. (A) Serotonin (5HT) immunolabeling (dashed line, lesion border) and (B) number of 5HT-labeled (+) fibers caudal to a spinal dorsal hemisection; n = 7 to 8 rats per group. (C) Coronal sections of the lumbar spinal cord after contusion injury. (Left panel) Coimmunostaining of 5HT, synaptophysin (Syn), and choline acetyltransferase (ChAT). (Right panels) Magnification of each marker in boxed area (left panel) visualizing serotonergic innervation of motor neurons (arrowheads). (D) Total length of 5HT-immunopositive fibers in the ventral horn (5,7-DHT, 5,7-dihydroxytryptamine); n = 4 (uninjured), 6 (7 dpi), 11 to 12 rats (56 and 70 dpi) per group. (E) Number of footfalls on the horizontal ladder; n = 10 to 11 rats per group. dpi, days postinjury. Scale bars, 50 μm. Schemes in (A) and (C) indicate lesion and displayed region (red box). Values are plotted as means + SEM. *P < 0.05; n.s., not significant by Student’s t test.
Jörg Ruschel et al. Science 2015;348:
Published by AAAS