Fig 2: Axonal regeneration and synaptic integration in canine with hemisection SCI.(A) Diagram of canine animal experimental model. (B) Immunofluorescence image with NF200 and Ln in the HADA/HRR + NT3/Cur hydrogel group at 18 wpi. (B1) High-resolution image of the dashed box in (B). (C) Immunofluorescence images with NF200 and Ln in the CTRL group. (C1) High-resolution image of the dashed box in (C). (D1 and D2) High-resolution images with Map2, NeuN, and SYN. (E) Representative electrophysiological traces in the left sciatic nerves with quantitative statistics of the latency and action potential of MEPs, respectively. (F) Immunofluorescence image with Map2, 5HT, and Syn in the HADA/HRR + NT3/Cur group. (F1) High-resolution image of the dashed box in (F). (G) High-resolution image with Map2, vGLUT, and Syn in the HADA/HRR + NT3/Cur group. (H) Hindlimb joint movements of beagles in the HADA/HRR + NT3/Cur hydrogel group and the CTRL group and Obly scoring statistics (* indicate statistical significance in left leg between the HADA/HRR + NT3/Cur group and CTRL group, *P < 0.05; # indicate statistical significance in right leg between the HADA/HRR + NT3/Cur group and CTRL group, #P < 0.05). (I1 and I2) High-resolution images with Map2, TH, and Syn in the HADA/HRR + NT3/Cur group. (J1 and J2) High-resolution images with Map2, CGRP, and Syn in the HADA/HRR + NT3/Cur group. (K1 and K2) High-resolution images of Map, vGAT, and Syn in the HADA/HRR + NT3/Cur group.

Fig 3: Neuronal relaying connections of phenotypically appropriate target synapses across lesion at 8 wpi.(A) Immunofluorescence images with Map2, 5HT, and CST in the HADA/HRR + NT3/Cur group. (B) Schematic illustration of resident interneurons to bridge descending tract. (C) High-resolution images with CST, 5HT, and Syn in the rostral border in the HADA/HRR + NT3/Cur group. (D) High-resolution images with Map2, 5HT, and Syn in the caudal border in the HADA/HRR + NT3/Cur group. (E) Quantization of 5HT+ axon number. (F) BBB scoring statistics for different groups. (G) High-resolution images with Map2, vGLUT, and Syn in the borders in the HADA/HRR + NT3/Cur group. (H) Distance of glutamatergic axon terminals from the lesion center. (I) TEM image of type I synapses in the boarder. Red, presynaptic membrane; green, postsynaptic membrane. (J) Immunofluorescence images with CGRP and TH in the HADA/HRR + NT3/Cur group. (K) Schematic illustration of regeneration of axons related to sensory and bladder function recovery. (L) High-resolution imaging with Map2, TH, and Syn. (M) Quantitative analysis of TH+ axons in the caudal border. (N) Immunofluorescence images with α-SMA. (O) High-resolution images with Map2, CGRP, and Syn. (P) Quantitative analysis of CGRP+ axons at the lesion center. (Q) High-resolution images of Map2, vGAT, and Syn. (R) TEM image of type II synapses in the border. (R1) High-resolution image of (R). (S) Statistical analysis of pain threshold. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Statistical methods include one-way ANOVA and two-way ANOVA. SA, serotoninergic axons; GN, glutamatergic neurons.

Fig 4: The HADA/HRR-based hydrogels manipulated ECM deposition in the complete transection site.(A) Representative images of longitudinal spinal cord sections obtained at 2 wpi in the CTRL and HADA/HRR + NT3/Cur hydrogel group. Dotted lines demarcated astrocyte border around the lesion. Boxed border regions were magnified with NF200 and different ECM [laminin (Ln), collagen I (Col 1), and fibronectin (Fn)] immunoreactivity intermingled in the region. (B) Corollary histogram of Ln fiber angle for each magnified region in (A). (C) Schematic illustration of Ln immunoreactivity at borders. (D) Quantification of Ln density at borders (n = 3 per group). (E) Axon contact length with Ln (n = 6 per group) at borders. (F) High-resolution confocal images of NF200 with Gap43. (G) Representative Western blot and (H) quantitative densitometry results of NF200 (h1), Tuj1 (h2), and Gap43 (h3) protein expressions at 2 wpi (n = 3 per group). (I) Immunostainings for glial fibrillary acidic protein (GFAP) and CSPG of the HADA/HRR + NT3/Cur hydrogel group at 2 wpi. The boxed border regions were magnified and immunostained with Ln and NF200 (i1), CSPG and NF200 (i2) or CSPG, Ln, and NF200 (i3). (J) Images of immunostaining for CSPG, Ln, and NF200 at borders at 2 wpi without treatments. (K) Images of immunostaining for Ln and NF200 in the HADA/HRR + NT3/Cur hydrogel group at 8 wpi. The boxed border regions were magnified in different groups. Values were plotted as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with Bonferroni.

Fig 5: Regulation of inflammatory response after SCI.(A) Representative images of longitudinal spinal cord sections at 1 wpi in the CTRL group and (B) HADA/HRR + NT3/Cur group. Boxed border regions were magnified with CD68 and CD206 immunoreactivity. The white dotted lines indicated the border between host spinal cord and lesion. (C) Representative images of longitudinal spinal cord sections at 2 wpi in the CTRL group and (D) HADA/HRR + NT3/Cur group. Boxed border regions were magnified with CD68 and CD206 immunoreactivity. The white dotted lines indicated the boundary between host spinal cord and lesion. (E) Quantification of CD68 immunoreactive intensity (normalized to the intact region) and (F) the proportion of CD206 to CD68 immunoreactive intensity at rostral and caudal borders at 1 wpi. Five regions of 0.5 mm × 0.5 mm were randomly selected and measured (n = 3 per group). (G) Quantification of CD68 immunoreactive intensity (normalized to the intact region) and (H) the proportion of CD206 to CD68 immunoreactive intensity at rostral and caudal borders at 2 wpi. Five regions of 0.5 mm × 0.5 mm were randomly selected and measured (n = 3 per group). (I) mRNA expression levels of various proinflammatory factors and (J) anti-inflammatory factors in different groups at 1 wpi determined by quantitative PCR (n = 3 per group). (K) mRNA expression levels of various proinflammatory factors and (L) anti-inflammatory factors in different groups at 2 wpi determined by quantitative PCR (n = 3 per group). Values were plotted as means + SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with Bonferroni.