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. 2019 Jan;25(1):86-100.
doi: 10.1111/cns.12984. Epub 2018 May 31.

Feasible Stabilization of Chondroitinase Abc Enables Reduced Astrogliosis in a Chronic Model of Spinal Cord Injury

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Free PMC article

Feasible Stabilization of Chondroitinase Abc Enables Reduced Astrogliosis in a Chronic Model of Spinal Cord Injury

Andrea Raspa et al. CNS Neurosci Ther. .
Free PMC article

Abstract

Aims: Usually, spinal cord injury (SCI) develops into a glial scar containing extracellular matrix molecules including chondroitin sulfate proteoglycans (CSPGs). Chondroitinase ABC (ChABC), from Proteus vulgaris degrading the glycosaminoglycan (GAG) side chains of CSPGs, offers the opportunity to improve the final outcome of SCI. However, ChABC usage is limited by its thermal instability, requiring protein structure modifications, consecutive injections at the lesion site, or implantation of infusion pumps.

Methods: Aiming at more feasible strategy to preserve ChABC catalytic activity, we assessed various stabilizing agents in different solutions and demonstrated, via a spectrophotometric protocol, that the 2.5 mol/L Sucrose solution best stabilized ChABC as far as 14 days in vitro.

Results: ChABC activity was improved in both stabilizing and diluted solutions at +37°C, that is, mimicking their usage in vivo. We also verified the safety of the proposed aqueous sucrose solution in terms of viability/cytotoxicity of mouse neural stem cells (NSCs) in both proliferating and differentiating conditions in vitro. Furthermore, we showed that a single intraspinal treatment with ChABC and sucrose reduced reactive gliosis at the injury site in chronic contusive SCI in rats and slightly enhanced their locomotor recovery.

Conclusion: Usage of aqueous sucrose solutions may be a feasible strategy, in combination with rehabilitation, to ameliorate ChABC-based treatments to promote the regeneration of central nervous system injuries.

Keywords: axonal regeneration; chondroitinase ABC; chronic spinal cord injury; locomotor rehabilitation; thermal stabilization.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Enzymatic activity of ChABC in Sucr solutions in storage and in injection simulation conditions. A, Experimental protocol to assess ChABC activity through the detection of unsaturated disaccharide absorbance at 232 nm (see methods for details). B, Residual ChABC activity at 37°C in sucrose solutions (n = 5). At the beginning (day 0), results point out no significance difference between ChABC in Sal and Sucr 2 mol/L, Sucr 2.5 mol/L, Sucr 3 mol/L. After 1, 2, 3, and 7 d, the activity in Sucr 2.5 mol/L and Sucr 3 mol/L was significantly higher compared with Sal. C, ChABC activity in sucrose solutions expressed as percentage of the initial activity at day 0. D, ChABC in sucrose solutions were diluted in cell culture media (1‐10) to mimic in vivo dilution and interstitial fluid composition (n = 5). E, Results point out that after 1 and 2 d of incubation, ChABC activity in diluted sucrose solutions was importantly decreased but still significantly higher compared to Sal. F, Percentage of residual activity of ChABC in sucrose solutions. (P***<.001 and P** <.01 Sucrose solutions vs Saline solution; P♦♦<.01 Sucr 2.5 mol/L vs Sucr 2 mol/L; P••<.01 Sucr 0.3 mol/L 1 d vs 14 d)
Figure 2
Figure 2
Cytotoxicity effect of sucrose solutions on NSCs in proliferation and differentiation conditions. A, Images show similar mNSC morphologies in cultures with media containing Sal (left), Sucr 0.25 mol/L (middle), and Sucr 0.3 mol/L (right). Live cells (green), dead cells (red‐orange). Scale bars represent 100 μm. B, At 1 d of incubation, values ascribable to viable cells (left) in medium containing Sucr 0.25 mol/L and Sucr 0.3 mol/L were not statistically different to cells cultured in medium containing Sal. In the same way, dead cells (right) in medium containing Sucr 0.25 mol/L and Sucr 0.3 mol/L showed no significant difference compared to medium with Sal. C, Effect of sucrose solutions on mouse differentiated NSCs, qualitative images (1 d incubation) show no density difference in differentiating cultures. Live cells fluorescent bright green, whereas dead cells with compromised membranes are in red‐orange. Scale bars represent 100 μm. D, Histograms represent the percentages of live/dead differentiated NSCs. Cellular quantification reveals a comparable viability between Sal and Sucrose solutions, both at day 1 and day 4
Figure 3
Figure 3
ChABC in Sucr 2.5 mol/L improved hindlimbs locomotor function (BBB scale) and C4S degradation. A, Experimental design showing intraspinal injections into the gliotic scar of ChABC in Sucr 2.5 mol/L at the depth of 100 μm (see methods for details). B, Each animal was subjected to treadmill training. Results point out that at 6 wk after contusion, 2 wk after ChABC treatment, treated animals showed significantly higher BBB score compared to control group (P**<.01 control vs treated) and compared to ChABC (Sal) group (P*<.05 ChABC (Sal) vs ChABC (Sucr 2.5 mol/L) C, quantification of C4S degradation reveals a significant digestion at the epicenter of injury following ChABC (Sucr 2.5 mol/L) treatment (** indicate significant difference compared with both Control and ChABC (Sal); < .01, one‐way repeated‐measures ANOVA, Tukey's post hoc)
Figure 4
Figure 4
Morphological analysis of gliosis. GFAP and NG2 reactivities were decreased in treated group. Images showing immunofluorescence sections of injured spinal cord stained for GFAP (A, F blue = DAPI, red = GFAP) and NG2 (H, M blue = DAPI, red = NG2). These are representative images taken from longitudinal sections. A‐J, Images showing dorsal sections; D‐M, Images showing ventral sections. Scale bars represent 100 μm. N, Results suggest gliosis reactive to GFAP and NG2 was significantly decreased in ChABC (Sucr 2.5 mol/L) if compared to control and ChABC (Sal) groups. (P**<.01 dorsal control vs dorsal treated and P*<.05 dorsal ChABC (Sal) vs dorsal treated; P***<.001 GFAP ventral control vs ventral treated and P**<.01 ventral ChABC (Sal) vs ChABC (Sucr 2.5 mol/L); P*<.05 NG2 dorsal control and dorsal ChABC (Sal) vs NG2 dorsal treated; P*<.05 ventral control vs ventral treated)
Figure 5
Figure 5
Increased reactivity of neural marker in ChABC (Sucr 2.5 mol/L) animals vs control and ChABC (Sal) groups. A‐R, Immunofluorescence images of areas used for quantification of βIIITUB (blue = DAPI, green = βIIITUB), subdivided into dorsal and ventral zones. Histological sections have been further regionalized into rostral, lateral, and caudal areas. Scale bars represent 100 μm. Y, Histological analysis points out the ChABC with sucrose injections significantly increased βIIITUB immunoreactivity. (P**<.01 control vs treated; P*<.05 control vs ChABC (Sal))
Figure 6
Figure 6
A‐R, Immunofluorescence images of areas used for quantification of GAP43 (blue = DAPI, red = GAP43), subdivided into dorsal and ventral zones. Histological sections have been further regionalized into rostral, lateral, and caudal areas. Scale bars represent 100 μm. S, Histological analysis points out the ChABC with sucrose injections significantly increased GAP43 immunoreactivity. (dorsal: P***<.001 rostral, lateral, and caudal control and rostral, caudal ChABC (Sal) vs dorsal treated; P**<.01 lateral ChABC (Sal) vs ChABC (Sucr 2.5 mol/L); ventral: P***<.001 rostral control vs treated; P**<.01 rostral ChABC (Sal) vs treated and P*<.05 caudal control and ChABC (Sal) vs caudal treated)

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