Figure legends
Figure 1. Compound 11 treatment reduces AEP activity in injured nerves. Sciatic nerves were cut and surgically repaired in wild type mice. Beginning on the third post-injury day, animals were administered either CP11 or vehicle, orally, once daily. Repaired nerves were harvested and assayed for AEP activity on the third (3d) or seventh (7d) after the onset of treatment. A set of intact nerves was assayed similarly for controls. Mean (+ SEM) AEP activity is scaled to the maximum activity found in all samples.
Figure 2. Treatments with CP11 result in larger restored neuromuscular responses. A. Outline of experimental scheme. Mice were treated with CP11 or vehicle daily, five days per week for two weeks, beginning on the third day following sciatic nerve transection and repair. After two more weeks, neuromuscular reinnervation was tested. B. Examples of maximal amplitude M responses (Mmax) recorded four weeks after sciatic nerve transection and repair from a vehicle-treated (top) and a CP11-treated (bottom) mouse. C. Mean (+ SEM) amplitudes of Mmax recorded from tibialis anterior (TA) and lateral gastrocnemius (LG) in vehicle-treated and CP11-treated mice, either via i.p. injection or oral gavage, four weeks after sciatic nerve transection and repair. Significance of differences between CP11-treated and vehicle-treated mice was evaluated for each muscle using ANOVA and post hoc paired testing. P values are shown to indicate the significance of differences in paired comparisons.
Figure 3. Treatments with CP11 increase the number of motoneurons whose axons have regenerated successfully. A. Image of a horizontal section through the right side of the lumbar spinal cord of a mouse in which the sciatic nerve was cut and repaired four weeks earlier. Motoneurons were labeled by application of different fluorescent retrograde tracers into the reinnervated gastrocnemius (GAST, red) and tibialis anterior (TA, green) muscles. Three neurons in this image contained both tracers (yellow arrows). This mouse was treated with intraperitoneal injections of CP11. B. Mean (+ SEM) number of motoneurons retrogradely labeled from tracer injection into GAST and TA four weeks after sciatic nerve transection and repair and two weeks after treatment, either by intraperitoneal injection (i.p.) or orally (oral), with compound 11 (CP11, 10 mg/Kg), or vehicle. Numeric values indicate p values from pairwise comparisons (ns = not significant).
Figure 4. Treatments with compound 11 (CP11) increased the number of muscle sensory neurons whose axons regenerated successfully. A. In a section from the L4 DRG of a mouse four weeks after sciatic nerve transection and repair and two weeks after treatment with CP11, neurons retrogradely labeled from tracer injections into GAST (red) and TA (green) are shown. Three cells in this image were labeled by both tracers (yellow arrows). B. Mean (+ SEM) numbers of L4 DRG neurons retrogradely labeled from TA, GAST, or Both in vehicle-treated and CP11-treated mice are shown. Each symbol in each graph represents the number of labeled neurons in a single animal in the series. Significance of differences between groups are displayed as p values above comparison brackets.
Figure 5. Neurite elongation is enhanced by CP11 treatments. A. Example of an adult DRG neuron after 48 hours in culture, marked by immunoreactivity to β3 tubulin. This culture was treated for 24 hours with CP11. The arrows indicate the longest neurite measured in this cell. B. Cumulative frequency distributions of neurite lengths in four treatment groups. Each cumulative histogram represents the average of the distributions of neurite lengths from cultures of DRG neurons from six mice. The horizontal dashed line at the 50thpercentile marks the median neurite length, shown at the vertical dashed lines extending to the X axis. C. Average (+ SEM) median neurite lengths in six cultures of the four treatment groups. Each symbol represents the neurite length measured from cells derived from a single animal. P values are shown only for significant differences between groups. All other paired comparisons were not statistically significant.
Figure 6. Enhancement of neurite outgrowth produced by CP11 treatment is TrkB-independent. A. Distributions of neurite lengths of cultured DRG neurons without treatments (Media) and after treatments with CP11 or 7,8-DHF in the presence or absence of the TrkB inhibitor, ANA-12 are displayed as violin plots. The solid white lines in each violin mark their medians. The horizontal dashed line running through the figure marks the median neurite length from the control group (Media). Numbers above brackets indicate p values associated with statistical comparisons between groups. B. Image of two β3 tubulin-immunoreactive DRG neurons (red) in a culture treated for 24 hours with CP11. Cultures also were reacted with an antibody to the extracellular domain of the TrkB receptor, and a green fluorescing secondary antibody. The lower cell in the image was scored as TrkB+ and the upper cell as TrkB-. C. Cumulative frequency distributions of neurite lengths in cultured adult DRG neurons identified as TrkB+ (top) and TrkB- (bottom) in control (Media) cultures and in cultures treated with either CP11 or 7,8-DHF. Neurite lengths in cultures from different mice were scaled to the median neurite length in the corresponding control (Media only) group. Each cumulative histogram represents the average of the distributions of these scaled neurite lengths from cultures of DRG neurons from six mice. Medians for each group are represented by the projection of the dashed line at the 50th percentile to the horizontal. D. Average (+ SEM) median scaled neurite lengths from the same cultures are shown for TrkB+ (top) and TrkB- (bottom) neurons. Numbers above brackets are p values resulting from statistical comparisons made between groups.
Figure 7. Neurite length is not increased by CP11 treatments in AEP knockout mice. Average median neurite lengths (+ SEM), scaled to the median length found in untreated cultures derived from WT mice, are shown for cultures from AEP KO mice and those derived from WT mice. Data from untreated cultures (Media), cultures treated with CP11 (red bars), cultures treated with 7,8-DHF (green bars) are shown. Numbers above brackets indicate p values for all significant differences between groups. All other paired comparisons were not statistically significant.