Journal: Nature

Article Title: Required growth facilitators propel axon regeneration across complete spinal cord injury

doi: 10.1038/s41586-018-0467-6

Figure Lengend Snippet: a, Schematics show demarcation of SCI lesion center (Cn) and evenly spaced lines beyond the Cn placed by image analysis software (Neurolucida®, Microbrightfield) for quantification of axon intercepts in horizontal tissue sections of mice with SCI and one (D 1 ) or two (D 1 +D 2 ) hydrogel depots. b, Multi-fluorescent, survey images show BDA-labeled axons and GFAP-labelled astrocytes that demarcate astrocyte scar proximal borders (PB) and distal borders (DB) around the non-neural lesion core (LC) after SCI. The hydrogel of the empty depot (left) was tagged with a blue fluorescent label for visualization. Note the essential absence of axons passing the astrocyte scar (AS) proximal border (PB) to reach the lesion center (Cn) or beyond in the mouse with SCI plus empty depot (left), in striking contrast with the large number of axons that regrew through the lesion core (LC) and passed beyond the distal astrocyte scar border (DB) into spared grey matter (gm) in the mouse with full treatment of stimulatory AAV plus growth factors (right). GFAP staining shows that the SCI lesions are anatomically complete across the entire width of the spinal cord in both cases. Note that the second depot was placed at 9 days after SCI, by which time the distal astrocyte scar border was essentially formed . Note also that astrocytes do not migrate into the depots, potentially giving the mistaken impression of cavity formation when looking only at the GFAP channel alone. Nevertheless, examination of other fluorescence channels shows that depot sites clearly contain DAPI-stained stromal cells and BDA-positive axons. c, Large area survey images of BDA-labeled axons in composite mosaic scans of horizontal sections. In a control mouse (top) that received SCI plus empty depot, few axons reach the lesion center, almost none pass beyond, and no axons are present at 3mm. In the a treated mouse (middle) that received stimulatory AAV plus growth factors, many axons regrow through the lesion core and reach or pass 1.5mm beyond the lesion center, which is the equivalent length of a full thoracic spinal segment in mice . Note also that there are no axons present at 3mm, demonstrating that the SCI lesion was complete and that axons that are found past the lesion center represent axon regrowth after SCI in response to the experimental manipulations. In an uninjured mice (bottom), there are many labeled axons at the distance equivalent to 3mm beyond the location of SCI in injured mice. d, Graph shows mean±SEM numbers of axon intercepts at lesion centers for all experimental groups (dots in graphs show numbers and distribution of individual mice per group). (ns not significant versus SCI-only, # P <0.01 versus SCI-only and ns versus each other, ** P <0.01, *** P <0.001 versus all other groups, one-way ANOVA/Bonferroni, F (12, 57) = 22.3).

Article Snippet: Cell counts were performed using stereological image analysis software (StereoInvestigator®, 9.14.5 32 Bit, and NeuroLucida®, 9.14.5 32 Bit, MicroBrightField, Williston, VT) operating a computer-driven microscope regulated in the x, y and z axes (Zeiss).

Techniques: Software, Labeling, Staining, Fluorescence, Control