Fig. 2: CSPG digestion shifts inflammatory gene co-expression dynamics after spinal cord injury during the resolution phase of inflammation.

a Experimental design of inflammatory gene co-expression analysis. b–e Dynamic analysis of the expression of 29 target genes over time with or without CSPG digestion. Gene expression was measured by qPCR on RNA extracted from the injury epicentre at different time-points after SCI. b Bar graph showing that multivariate pattern detection using dual multiple factor analysis (dMFA) detected 2 dynamic patterns (dimensions) over time explaining ~56% of the total variation in gene co-expression. c Loadings correlation bar graph to show which cytokines contribute to each of the patterns (interpreted as the Pearson r correlation coefficient ranging from −1 to 1). d Bar graph representing the Euclidean distance to the naïve centroid for each group at different time-points, measuring the relative movement of treated animals with respect to naïve in the global inflammatory gene profile. Two-way ANOVA with time and group as factors using Tukey for multiple testing correction. *p < 0.05, **p < 0.01, ***p < 0.001 versus uninjured (naive) group; ^#p < 0.05 versus LV-GFP group. Data are shown as mean ± SEM (n = number of animals/samples, with n = 6 for each group–time combination except LV-GFP at 12 h, LV-ChABC at 6 and 12 h post injury (n = 4); LV-GFP at 3 dpi (n = 5) and LV-ChABC at 7 dpi (n = 7). e Bidimensional plots of the component scores in dimensions 1 and 2. Ellipsoids represent the bivariate standard deviation and the coloured circles the centroid. There is little divergence of LV-ChABC and LV-GFP at any timepoint except 7 dpi, where LV-ChABC becomes highly diverged from LV-GFP and is proximal to naïve, reflecting a gene expression pattern comparable to uninjured animals at 7 dpi after CSPG digestion. f–i Further pattern analysis at 7 dpi was performed using principal component analysis (PCA) for 42 inflammatory-related genes, confirming a reduction of pro-inflammatory genes after CSPG digestion, assessed by qPCR. f Loadings correlation heat map show dimension 1 loadings are positive for almost all cytokines, indicative of a global higher cytokine co-expression in LV-GFP vs. LV-ChABC-treated animals. g Component score bar graphs for each group and dimension at 7 dpi show significant differences between LV-GFP and LV-ChABC in dimension 1. **p < 0.01 versus control (LV-GFP) group. Results were assessed for normality using the Shapiro–Wilk test and analysed using a two-tailed unpaired t test. Data are shown as mean ± SEM. (h) Bidimensional plot of the component scores for each group in dimension 1 and 2 at 7 dpi showing significant differences between LV-GFP and LV-ChABC in dimension 1. **p < 0.01 versus control group (LV-GFP) (n = 5 per treatment). i Heatmap showing gene expression data for 42 key genes in the inflammatory response at 7 dpi. LV-ChABC treatment elicits gene expression patterns closer to naïve than LV-GFP treated animals. j Bar graph showing all significant pro-inflammatory gene expression differences between LV-GFP and LV-ChABC treatments at 7 dpi. *p < 0.05, **p < 0.01 versus control (LV-GFP) group. Results were assessed for normality using the Shapiro–Wilk test and analysed using a two-tailed unpaired t test. Data are shown as mean ± SEM (n = 6 naïve group, n = 5 per treatment). Detailed statistics and exact p values are provided in Supplementary Table 8. Source data are provided as a Source Data file.