Figure 2

Heparanase inhibition accelerates acute axonal degeneration in ex vivo sciatic nerves. (A) Representative images of SMI31-IR (red) immunostaining of sections from ex vivo SN cultures either uninjured (naïve), after 2 DIV or 2 DIV treated with OGT2115, the Hpse inhibitor. In magnified images; MBP-IR (green) and DAPI-IR (blue). Quantification of SMI31-IR axons of images, *p = 0.02, n = 3. (B) Representative cropped Western blot from one experiment, for ex vivo SN, (representative full length blot is available in supplementary section Fig. S4) after OGT2115 treatment showing a significant decrease in SMI31 expression, quantified by densitometry, ***p = 0.0002, n = 3. Amido black whole protein stain was used for loading control (representative blot available in supplementary section Fig. S4). (C) Semi-thin sections of resin embedded ex vivo SN after 2 DIV or 2 DIV with OGT2115 treatment stained with methylene blue/azurII. Quantification of degenerating axons in the ex vivo SN after 2 DIV treated with OGT2115 showed a significant increase compared to 2 DIV alone, *p = 0.012, n = 4. Red arrow indicates a collapsed myelin sheath which represents a degenerating axon. (D) Representative Western blot of ex vivo SN after 2 DIV with or without OGT2115 showing a significant decrease in HS (3G10) cleaved by Hpse **p = 0.008, n = 3. (E) Representative cropped Western blot from one experiment, of ex vivo SN, (representative full length blot is available in supplementary section Fig. S4) after 2 DIV with or without Heparinase I treatment showing a significant decrease in the loss of SMI31 protein levels *p = 0.026, n = 3. Amido black whole protein stain was used for loading control (representative blot available in supplementary section Fig. S4). (F) Alamar blue analysis for cell viability of ex vivo SN treated with OGT2115. It can be seen that there was no difference in levels between OGT2115 treatment and control (not shown) until 2 DIV where a significant increase was observed after OGT2115 treatment, *p = 0.018. n = 3. The data from the alamar blue assay was normalised to results obtained from cultured media lacking cells containing the alamar blue only, which is defined as the negative control. Students T test was used for all statistical analysis or one-way ANOVA with post hoc Tukey HSD test.