Extended Data Fig. 3: Procedures for quantification of BDA-labelled propriospinal axons after SCI. | Nature

Extended Data Fig. 3: Procedures for quantification of BDA-labelled propriospinal axons after SCI.

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

Extended Data Fig. 3

a, Schematics show demarcation of SCI lesion centre (Cn) and evenly spaced lines beyond the lesion centre placed by image analysis software (Neurolucida, MicroBrightField) for quantification of axon intercepts in horizontal tissue sections of mice with SCI and one (D1) or two (D1 + D2) hydrogel depots. b, Multi-fluorescent, survey images show BDA-labelled axons and GFAP-labelled astrocytes that demarcate astrocyte scar proximal borders and distal borders around the non-neural lesion core 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 to reach the lesion centre (Cn) or beyond in the mouse with SCI plus empty depot (left), in contrast to the large number of axons that regrew through the lesion core and passed beyond the distal astrocyte scar border into spared grey matter 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 nine days after SCI, by which time the distal astrocyte scar border was essentially formed35. 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-labelled axons in composite mosaic scans of horizontal sections. In a control mouse (top) that received SCI plus empty depot, few axons reach the lesion centre, almost none pass beyond and no axons are present at 3 mm. In the treated mouse (middle) that received stimulatory AAV plus growth factors, many axons regrow through the lesion core and reach or pass 1.5 mm beyond the lesion centre, which is the equivalent length of a full thoracic spinal segment in mice46. Note also that there are no axons present at 3 mm, demonstrating that the SCI lesion was complete and that axons that are found past the lesion centre represent axon regrowth after SCI in response to the experimental manipulations. In an uninjured mouse (bottom), there are many labelled axons at the distance equivalent to 3 mm beyond the location of SCI in injured mice. d, Numbers of axon intercepts at lesion centres for all experimental groups. Data are mean ± s.e.m., 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 not significant versus each other; **P < 0.01; ***P < 0.001, versus all other groups; one-way ANOVA with Bonferroni, F(12, 57) = 22.3.

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