Sensorimotor behavioural tests after CSP-TTK21 treatment in chronic SCI with severe disability.

(A) BMS quantification of CSP (n = 15) or CSP-TTK21 (n = 14) treated mice after chronic SCI (Treatment: f(1) = 0.002, p = 0.96 (TOST: given equivalence bounds of −0.21 and 0.21, 90% confidence intervals fall −0.37 and 0.32 and p = 0.307, thus H0 is undecided); Time: f(12) = 192.1, p p = 0.215, thus H0 is rejected); Interaction (treatment × time): f(12) = 6.6, p = 0.82 (TOST: given equivalence bounds of −0.21 and 0.21, 90% confidence intervals fall 4.19 and 4.99 and p > 0.999, thus H0 is rejected). Two-way repeated measures ANOVA with Sidak post hoc test. (B) Gridwalk quantification of the percentage of slips per total number of steps per run in CSP (n = 4) or CSP-TTK21 (n = 6) treated mice with a BMS score greater than 3 after chronic SCI (Treatment: f(1) = 0.2, p = 0.69 (TOST: given equivalence bounds of −1.78 and 1.78, 90% confidence intervals fall −7.96 and 2.63 and p = 0.693, thus H0 is undecided); Time: f(12) = 7.1, p p p = 0.82 (TOST: given equivalence bounds of −1.78 and 1.78, 90% confidence intervals fall −28.59 and 41.29 and p > 0.999, thus H0 is rejected). Two-way repeated measures ANOVA with Sidak post hoc test). (C) Bar graph indicating the number of mice with a BMS score greater than 3 across postinjury time points. (D) Hargreaves test indicating average paw withdrawal latency in CSP or CSP-TTK21 (Welch two-tailed t test: CSP: 6.7 ± 1.1, n = 4; CSP-TTK21: 7.6 ± 0.5, n = 6, p = 0.51, TOST: t(8) = −0.3, p = 0.63 given equivalence bounds of −0.5 and 0.5 on a raw scale and an alpha of 0.05) treated mice with a BMS score greater than 3. (E) Von Frey test indicating average paw withdrawal threshold in CSP or CSP-TTK21 (Welch two-tailed t test: CSP: 5.5 ± 1.6, n = 4; CSP-TTK21: 6.4 ± 0.7, n = 6, p = 0.64, TOST: t(4.3) = −0.1, p = 0.52 given equivalence bounds of −0.8 and 0.8 on a raw scale and an alpha of 0.05) treated mice with a BMS score greater than 3. All data are given as mean ± SEM. n = biologically independent animals. The data can be found in S1 Data. BMS, Basso Mouse Scale; CSP, carbon nanosphere; SCI, spinal cord injury; TOST, two one-sided tests.</p

S1 Fig -

(A) Twelve weeks old mice were housed in SH or EE 1 week after a spinal cord transection. (B and C) Animals in SH remained impaired unable to step until day 42 after injury as shown by BMS (B) and Gridwalk (C). EE significantly enhanced locomotion (mean ± SEM, two-way ANOVA, Fisher LSD post hoc ** P P P S1 Data. BMS, Basso Mouse Scale; EE, enriched environment; SH, standard housing. (PDF)</p

S4 Fig -

(A) Representative micrographs of GFAP intensity (red) around the SCI site (white asterisks) and cavity size (white dotted line) in CSP or CSP-TTK21 mice. (B) Quantification of cavity size in CSP or CSP-TTK21-treated mice (CSP: 471,574.0 ± 76,631.0, n = 8; CSP-TTK21: 486,466.0 ± 45,491.0, n = 14; p = 0.87; TOST: t(20.0) = 0.5, p = 0.31 given equivalence bounds of −56,344.2 and 56,344.2 on a raw scale and an alpha of 0.05). (C) Quantification of GFAP intensity in CSP or CSP-TTK21-treated mice (CSP: 1,272.0 ± 31.3, n = 3; CSP-TTK21: 1,339.0 ± 20.8, n = 4, p = 0.12, TOST: t(3.7) = −1.4, p = 0.88 given equivalence bounds of −14.5 and 14.5 on a raw scale and an alpha of 0.05). Mean ± SEM; unpaired two-tailed Student t test or Welch t test. n = biologically independent animals. The data can be found in S1 Data. CSP, carbon nanosphere; SCI, spinal cord injury; TOST, two one-sided tests. (PNG)</p

Graphical diagram summarizing the experimental design. <i>Made with BioRender</i>.

(PNG)</p

S5 Fig -

(A) Representative micrographs of CD68 immunofluorescence (red) and DAPI (blue) around the SCI site (white asterisks) in CSP or CSP-TTK21-treated mice. Lesion site (white dotted line). (B) Quantification of CD68 intensity in CSP or CSP-TTK21-treated mice (CSP: 472.0 ± 8.1, n = 3 CSP-TTK21: 434.5 ± 37.8 n = 4, p = 0.44, TOST: t(3.3) = 0.5, p = 0.69 given equivalence bounds of −16.3 and 16.3 on a raw scale and an alpha of 0.05). Mean ± SEM; unpaired two-tailed Student t test. n = biologically independent animals. The data can be found in S1 Data. CSP, carbon nanosphere; SCI, spinal cord injury; TOST, two one-sided tests. (PNG)</p

Histone acetylation in DRG, raphe, and layer 5 cortical neurons in CSP-TTK21-treated mice in a chronic SCI with severe disability.

(A) Representative micrographs of H3K9ac immunostaining (green, white arrows) in DRG neurons. (B) Quantification of H3K9ac immunostaining in DRG neurons from CSP or CSP-TTK21-treated mice (CSP: 3,505.0 ± 399.8; CSP-TTK21: 6,549.0 ± 122.1, p n = 4). (C) Representative micrographs of H3K27ac immunostaining (green, white arrows) in DRG neurons. (D) Quantification of H3K27ac immunostaining in DRG neurons in CSP or CSP-TTK21-treated mice (CSP: 7,738.0 ± 472.0, n = 5; CSP-TTK21: 9,779.0 ± 195.4, p n = 4). (E) Representative micrographs of H3K9ac staining (green, white arrows) in raphe neurons. (F) Quantification of H3K27ac immunostaining in raphe neurons from CSP or CSP-TTK21-treated mice (CSP: 15.9 ± 0.2; CSP-TTK21: 27.1 ± 1.4, p n = 4). (G) Representative micrographs of H3K9ac immunostaining (green, white arrows) in layer 5 cortical neurons. (H) Quantification of H3K9ac immunostaining in layer 5 cortical neurons from CSP or CSP-TTK21-treated mice (CSP: 18.2 ± 1.8; CSP-TTK21: 39.0 ± 4.1, p n = 4). (I) Representative micrographs of H3K27ac staining (green, white arrows) in layer 5 cortical neurons. (J) Quantification of H3K27ac immunostaining in layer 5 cortical neurons from CSP or CSP-TTK21-treated mice (CSP: 22.9 ± 2.9; CSP-TTK21: 42.1 ± 1.3, p n = 4). Mean ± SEM; unpaired two-tailed Student t test; ** p p n = biologically independent animals. The data can be found in S1 Data. CSP, carbon nanosphere; DRG, dorsal root ganglion; SCI, spinal cord injury.</p

Axonal growth and synaptic plasticity in CSP-TTK21-treated mice in a chronic SCI.

(A) Representative micrographs of BDA-traced CST axons (green, white arrows) after chronic SCI in CSP or CSP-TTK21-treated mice, GFAP (magenta) was used to determine the lesion site. (B) Average distance between the lesion border and the furthest rostral (CSP: −329.8 ± 41.4, n = 6; CSP-TTK21: 438.3 ± 40.4, p n = 9) or caudal axons (CSP: −548.6 ± 58.7, n = 7; CSP-TTK21: −366.2 ± 54.6, p n = 9) in CSP and CSP-TTK21-treated mice. (C) Representative micrographs of Dextran-traced sensory axons (green, white arrows) after chronic SCI in CSP or CSP-TTK21-treated mice. GFAP (magenta) was used to determine the lesion site. (D) Average distance between the lesion border and furthest rostral (CSP: −361.2 ± 47.3, n = 6; CSP-TTK21: 301.6 ± 35.8, p n = 5) or caudal axons (CSP: −610.5 ± 25.2, n = 6; CSP-TTK21: −227.8 ± 22.7, p n = 7) in CSP and CSP-TTK21-treated mice. (E) Representative micrographs of 5-HT-positive axons (green) sprouting around ChAT-positive motoneurons (magenta) in the lumbar ventral horn (white dotted lines) below the lesion site in CSP or CSP-TTK21-treated mice. (F) Quantification of 5-HT intensity in the lumbar ventral horn (CSP: 2,410.0 ± 549.3; CSP-TTK21: 5,189.0 ± 662.2, p n = 4). (G) Orthogonal 3D confocal images of VGlut1+ boutons (green) from group-1a afferents in proximity to motoneurons (ChAT+, magenta) in the lumbar ventral horn below the lesion site. Intersection of dotted lines indicates example of apposition. (H) Quantification of Vglut1+ puncta per 1 mm2 in CSP or CSP-TTK21 mice (CSP: 6,380.0 ± 483.1, n = 3, CSP-TTK21: 11,615.0 ± 1,337.0, n = 4, p I) Quantification of VGlut1+ boutons in proximity to motoneurons (CSP: 1.7 ± 0.2; CSP-TTK21: 4.5 ± 0.3, p n = 4). (J) Orthogonal 3D confocal images of VGat+ boutons (green) in proximity to motoneurons (ChAT+, magenta) in the lumbar ventral horn below the lesion site. Intersection of dotted lines indicates example of apposition. (K) Quantification of VGat+ puncta per 1 mm2 (CSP: 66,366.0 ± 2,970.0; CSP-TTK21: 66,996.0 ± 2,069.0, p = 0.87, TOST: t(5.4) = 0.2, p = 0.41 given equivalence bounds of −1,535.7 and 1,535.7 on a raw scale and alpha of 0.05, n = 4). (L) Quantification of VGlut1+ boutons in proximity to motoneurons (CSP: 7.0 ± 1.1; CSP-TTK21: 9.3 ± 0.9, p = 0.15, TOST: t(5.7) = −1.2, p = 0.86 given equivalence bounds of −0.6 and 0.6 on a raw scale and alpha of 0.05, n = 4). All data are given as mean ± SEM; unpaired two-tailed Student t test; * p p p n = biologically independent animals. The data can be found in S1 Data. CSP, carbon nanosphere; CST, corticospinal tract; SCI, spinal cord injury; TOST, two one-sided tests.</p

Table summarizing the experimental design and data analysis.

Table summarizing the experimental design and data analysis.</p

Excel spreadsheets containing the quantitative data for each experiment as described in the results and figure legends.

(XLSX)</p

S3 Fig -

(A) Representative micrographs of cJUN immunostaining (green, white arrows) in layer 5 cortical neurons from CSP or CSP-TTK21-treated mice. (B) Quantification of cJUN immunostaining in layer 5 cortical neurons (CSP: 2,539.0 ± 295.4; CSP-TTK21: 2,355.0 ± 206.7, p = 0.63, TOST: t(0.1) = 0.1, p = 0.5 given equivalence bounds of −153.0 and 153.0 on a raw scale and an alpha of 0.05, n = 4). (C) Representative micrographs of SPRR1a immunostaining (green, white arrows) in layer 5 cortical neurons. (D) Quantification of SPRR1a immunostaining in layer 5 cortical neurons in CSP or CSP-TTK21 (CSP: 3,940.0 ± 491.6, n = 3; CSP-TTK21: 3,636.0 ± 350.3, n = 4; p = 0.08, TOST: t(4.8) = −1.9, p = 0.94 given equivalence bounds of −344.4 and 344.4 on a raw scale and an alpha of 0.05). Mean ± SEM; unpaired two-tailed Student t test or Welch t test. n = biologically independent animals. The data can be found in S1 Data. CSP, carbon nanosphere; TOST, two one-sided tests. (PNG)</p