Epothilones Improve Axonal Growth and Motor Outcomes after Stroke in the Adult Mammalian CNS

Abstract

Stroke leads to the degeneration of short-range and long-range axonal connections emanating from peri-infarct tissue, but it also induces novel axonal projections. However, this regeneration is hampered by growth-inhibitory properties of peri-infarct tissue and fibrotic scarring. Here, we tested the effects of epothilone B and epothilone D, FDA-approved microtubule-stabilizing drugs that are powerful modulators of axonal growth and scar formation, on neuroplasticity and motor outcomes in a photothrombotic mouse model of cortical stroke. We find that both drugs, when administered systemically 1 and 15 days after stroke, augment novel peri-infarct projections connecting the peri-infarct motor cortex with neighboring areas. Both drugs also increase the magnitude of long-range motor projections into the brainstem and reduce peri-infarct fibrotic scarring. Finally, epothilone treatment induces an improvement in skilled forelimb motor function. Thus, pharmacological microtubule stabilization represents a promising target for therapeutic intervention with a wide time window to ameliorate structural and functional sequelae after stroke.

Keywords: axon regeneration; fibrotic scar; ischemia; neuroplasticity; stroke.

Graphical abstract

Figure 1

Axonal Re-mapping after Cortical Stroke (A) We induced photothrombotic stroke through the thinned skull, resulting in an infarct surrounded by scar tissue (stained with glial fibrillary acidic protein [GFAP], scale bar, 300 μm). At day 21, fluorescent cholera toxin B (CTb) and AAV9 encoding for hSyn.eGFP were injected into the primary motor cortex (M1) and the premotor cortex (PMC). (B) Whole-brain organic solvent-based brain clearing (arrowhead, infarct area; scale bars, 1 mm). (C) Retrograde CTb labeling was analyzed in 4 regions of interest (ROIs): the injection site (1) and ROIs anterior (2), lateral (3), and medial (4) to the infarct. (D and E) No differences were seen at the injection site, whereas stroke led to a reduction in retrograde labeling in all other areas (n = 10 stroke versus n = 10 sham animals, Mann-Whitney test; scale bars, 50 μm; images in D show CTb-labeled cells in ROI 2 in sham versus stroke). (F–H) Anterograde EGFP labeling was assessed in PMC and somatosensory cortex (S1) anterior and medial to the infarct, and these areas were divided into ROIs I–IV (summation images of all animals, depicted as color-coded maximal intensity z projections). Stroke led to a reduction in anterograde connections in ROIs I and IV, but also induced new connections in ROIs II and III (i.e., in anterolateral motor and somatosensory cortex; n = 10 stroke versus n = 10 sham animals, Mann-Whitney test; scale bars, 300 μm). See also Figure S1.

Figure 2

Epothilone B and D Promote Stroke-Induced Axonal Neuroplasticity (A) Experimental timeline. (B) EpoB (n = 15) and EpoD (n = 15) treatments both induced significantly more retrograde connections from regions lateral and anterior to the infarct compared to animals treated with vehicle (n = 9) after stroke (Kruskal-Wallis test followed by Dunn’s multiple comparisons test). (C–F) Analysis of anterograde axonal connections showed that EpoB also induced significantly more connections emanating from areas anterolateral from the stroke region (summation images depict color-coded maximal intensity z projections; stroke and vehicle, n = 9 mice; stroke and EpoB, n = 15 mice; stroke and EpoD, n = 15 mice; Kruskal-Wallis test followed by Dunn’s multiple comparisons test; scale bars, 300 μm). See also Figure S2.

Figure 3

Epothilones Modulate Peri-infarct Scarring after Cortical Stroke and Increase Long-Range Connectivity (A–D) EpoB strongly reduced peri-infarct scarring, assessed by laminin and fibronectin immunohistochemistry, 14 days after stroke induction (n = 15 mice per group; Mann-Whitney test for each time point; drawing indicate areas of investigation; images show representative examples; scale bars, 200 μm). (E and F) Long-range projections from peri-infarct motor cortex were assessed by quantifying EGFP⁺ axons in the red nucleus within the ipsilateral brainstem. Stroke induced a strong reduction of these connections, but EpoB significantly increased axonal area coverage (n = 10 mice per group; Kruskal-Wallis test followed by Dunn’s multiple comparisons test; images show representative examples; scale bars, 50 μm). See also Figures S3, S4A, and S4B.

Figure 4

Epothilones Improve Motor Outcomes after Cortical Stroke (A and B) Images show representative examples of successful grid walk ambulation in a sham-treated animal and a foot fault in an animal after stroke (arrowhead). Motor outcome was assessed for 14 days after stroke induction by testing skilled forelimb function using the elevated grid walk test. Whereas mice performed similarly before infarct induction (day −1), cortical stroke worsened skilled motor function of the contralateral forelimb, assessed by the foot fault frequency, until 14 days after stroke (n = 10 mice per group; repeated-measures 2-way ANOVA followed by Bonferroni’s multiple comparisons test). (C) EpoB and EpoD both improved skilled motor function of the contralateral forelimb compared to vehicle-treated animals after stroke (stroke and vehicle, n = 9; stroke and EpoB, n = 16; stroke and EpoD, n = 14; repeated-measures 2-way ANOVA followed by Bonferroni’s multiple comparisons test). See also Figures S4E and S4F.