Yoo M , Khaled M , Gibbs KM , Kim J , Kowalewski B , Dierks T , Schachner M .

	Figure 1. Thermostability of ARSB and ChaseABC.Stock solutions of each enzyme were incubated at 37°C/pH 6.8 for up to 125 hours. At the indicated time points aliquots were diluted into pre-warmed assay buffer (pH 8.0 for ChaseABC, pH 5.6 for ARSB) and subjected to activity determination (see Methods). Asterisks indicate significant differences between the groups *p<0.001 as assessed by t-Test. Data represent means ± SEM (n = 4).
	Figure 2. Chondroitin 4-O-sulfate (C4S) immunoreactivity is reduced at five days after ARSB injection after moderate compression-injury.Immediately after spinal cord injury, one µl of ARSB (10 U/ml) was injected at the injury site (arrow) and 0.5 mm rostral and caudal to this site (arrowheads). After 5 days, the mice were perfused, and sagittal spinal cord sections were analyzed by immunofluorescence using an antibody specific for C4S. C4S immunoreactivity is higher at the injury site in the buffer treated control mice (A) versus ARSB treated mice (B). (C) and (D) are higher magnifications of the insets in (A) and (B), respectively. Diamidino-phenylindole (DAPI, blue) was used for nuclear staining and merged with C4S immunoreactivity (C,D). Arrows and arrowheads indicate the injury site and injection sites, respectively.
	Figure 3. C4S immunoreactivity is reduced at 9 weeks after enzyme injection in severe compression injury.Immediately after spinal cord injury, one µl ChaseABC (10 U/ml), ARSB (10 U/ml) or buffer was injected at the injury site and 0.5 mm rostral and caudal to this site. After 9 weeks, the mice were perfused, and sagittal spinal cord sections were analyzed by immunofluorescence using an antibody specific for C4S. C4S immunoreactivity is more intense at the injury site in the buffer treated control mice (A) versus the ChaseABC (B) and ARSB (C) treated mice. Immunoreactivities of the entire images were quantified above threshold using Image J software (D). Mean fluorescence intensities of the area at 0.4 mm equidistant rostral and caudal to the center of the injury site show significantly less C4S immunoreactivity in ChaseABC and ARSB treated mice versus buffer treated control mice. Reduction of immunoreactivity is not significantly different between applications of ChaseABC versus ARSB. Arrows indicate the injury site. Asterisks indicate significant differences between the groups *p<0.05 and **p<0.01 as assessed by one-way ANOVA followed by Tukey’s post-hoc analysis. Data represent means ± SEM (n = 4 mice).
	Figure 4. C4S and C6S immunoreactivities are reduced at 6 and 9 weeks after ARSB and ChaseABC treatment.Immediately after moderate (A,B) and severe (C–E) spinal cord injury, one µl ChaseABC (10 U/ml), ARSB (10 U/ml) or buffer was injected at the injury site and 0.5 mm rostral and caudal to this site. After 6 (A,B) and 9 weeks (C–E), the mice were perfused, and sagittal spinal cord sections were analyzed by immunofluorescence using the CS56 antibody reacting with C4S and C6S. Immunoreactivity is more intense at the injury site in the buffer treated control mice (A,C) versus the ChaseABC (D) and ARSB (B,E) treated mice. Immunoreactivities of the entire images were quantified above threshold using Image J software (F). Mean fluorescence intensities of the area at 0.4 mm equidistant rostral and caudal to the center of the injury site show significantly less expression of CSs in ChaseABC and ARSB treated mice versus buffer treated control mice. Reduction of immunoreactivity is not significantly different between applications of ChaseABC versus ARSB. Arrows indicate the injury site. Asterisks indicate significant differences between the groups *p<0.05 (ARSB) and **p<0.01 (ChaseABC) as assessed by one-way ANOVA followed by Tukey’s post-hoc analysis. Data represent means ± SEM, (n = 3 mice).
	Figure 5. Functional recovery of ChaseABC or ARSB treated mice after moderate (A) and severe (B) compression.One µl ChaseABC (10 U/ml), ARSB (10 U/ml) or buffer was injected at the injury site and 0.5 mm rostral and caudal to this site. The Basso Mouse Scale for analysis of locomotor activity was used to score functional recovery up to 6 (A) and 9 (B) weeks after moderate and severe spinal cord injury (SCI), respectively. Asterisks indicate significant differences between the enzyme treated groups versus the buffer treated control group at the same time points (*p<0.05, **p<0.01). No significant difference is detectable between the ChaseABC and ARSB treated groups by one-way ANOVA followed by Tukey’s post-hoc analysis. Data represent means ± SEM. Number of mice are indicated in brackets.
	Figure 6. GFAP immunoreactivity is not different at 6 or 9 weeks after enzyme treatment.One µl of the ChaseABC (10 U/ml), ARSB (10 U/ml) or buffer was injected at the injury site and 0.5 mm rostral and caudal to this site after moderate and severe compression injury. After 6 (A,B) and 9 weeks (C–E), the mice were perfused, and sagittal spinal cord sections were analyzed by immunofluorescence for glial fibrillary acidic protein (GFAP). Photomicrographs show GFAP immunoreactivity at the injury site in the buffer treated control mice (A,C) versus ChaseABC (D) and ARSB (B,E) treated mice. Immunoreactive areas were quantified above threshold using Image J software (F). Mean fluorescence intensities of the area at 0.4 mm equidistant rostral and caudal to the center of the injury site are similar in all groups. Arrows indicate the injury site. One-way ANOVA followed by Tukey’s post-hoc analysis indicates no difference between the groups (p>0.9). Data represent means ± SEM (n = 3 mice).
	Figure 7. 5-HT immunoreactive axons are more abundant 9 weeks after enzyme treatment than in control mice.One µl ChaseABC (10 U/ml), ARSB (10 U/ml) or buffer was injected at the injury site and 0.5 mm rostral and caudal to this site in mice with severe compression injury. After 9 weeks, the mice were perfused, and sagittal spinal cord sections were analyzed by immunofluorescence. Double immunostaining for serotonin (5-HT) and neurofilament-M (NF-M) shows higher immunoreactivities caudal injury site in the ChaseABC (D,E,F) and ARSB (G,H,I) treated mice versus buffer treated control mice (A,B,C). (A,D,G) Immunostainings for 5-HT and (B,E,H) NF-M, and (C,F,I) merged for 5-HT with NF-M. 5-HT immunoreactive axons are seen beyond the injury site in the ChaseABC and ARSB injected mice. Immunoreactive areas were quantified above threshold using Image J software (J). Mean fluorescence intensity of the area between the injury site and 1 mm caudal to it is significantly higher in ChaseABC treated versus buffer treated control mice. Asterisk indicates significant differences between the groups *p<0.05 by one-way ANOVA followed by Tukey’s post-hoc analysis. Data represent means ± SEM, (n = 5 mice).
	Figure 8. Tyrosine hydroxylase (TH) immunoreactive axons are more abundant 9 weeks after enzyme treatment than in control mice.One µl ChaseABC (10 U/ml), ARSB (10 U/ml) or buffer was injected at the injury site and 0.5 mm rostral and caudal of this site in mice with severe compression injury. After 9 weeks, the mice were perfused, and sagittal spinal cord sections were analyzed by immunofluorescence. Double immunostaining for TH and neurofilament-M (NF-M) shows higher immunoreactivities caudal to the injury site in the ChaseABC (D,E,F) and ARSB (G,H,I) treated mice versus the buffer treated control mice (A,B,C). (A,D,G) Immunostainings for TH and (B,E,H) NF-M, and (C,F,I) merged for TH with NF-M. Immunoreactive areas were quantified above threshold using Image J software (J). Mean fluorescence intensity of the area between the injury site and 1 mm caudal to it shows significantly higher immunofluorescence intensity in ARSB versus buffer treated control and ChaseABC treated mice. Asterisks indicate significant differences between the groups *p<0.05 and **p<0.01 as assessed by one-way ANOVA followed by Tukey’s post-hoc analysis. Data represent means ± SEM, (n = 4 mice).
	Figure 9. Iba1 immunoreactive microglia/macrophages are more abundant 9 weeks after ChaseABC treatment than in control mice.One µl ChaseABC (10 U/ml), ARSB (10 U/ml) or buffer was injected at the injury site and 0.5 mm rostral and caudal to this site in mice after severe compression. After 9 weeks, the mice were perfused, and sagittal spinal cord sections were analyzed by immunofluorescence. Immunoreactivity for Iba1 is less intense at the injury site in the buffer treated control mice (A) versus ChaseABC (B) or ARSB (C) treated mice. (D) (E) and (F) are higher magnifications of the insets in (A) (B) and (C), respectively, showing more microglia/macrophages with activated, amoeboid morphology in the area between the injury site and 1 mm caudal to it of mice treated with ChaseABC (E) or ARSB (F) versus the buffer treated control mice (D). Immunoreactive areas were quantified above threshold using Image J software (G). Mean fluorescence intensities at 0.4 mm equidistant rostral and caudal to the center of the injury site shows significantly higher immunoreactivity for Iba1 in ChaseABC treated mice versus buffer treated control mice. Arrows indicate the injury site. Diamidino-phenylindole (DAPI, blue) was used for nuclear staining and merged with Iba1 immunostaining (D,E,F). Asterisk indicates a significant difference between the groups *p<0.05 as assessed by one-way ANOVA followed by Tukey’s post-hoc analysis. Data represent means ± SEM, (n = 3 mice).

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