Delayed atorvastatin delivery promotes recovery after experimental spinal cord injury

Abstract

Spinal cord injury (SCI) significantly alters gene expression, potentially impeding functional recovery. This study investigated the effects of atorvastatin, a widely prescribed cholesterol-lowering drug, on gene expression and functional recovery in a chronic murine SCI model. Female C57BL/6J mice underwent moderate 0.25 ​mm lateral compression SCI and received daily atorvastatin (10 ​mg/kg) or vehicle-only injections from two weeks post-injury for four weeks. Sensorimotor functions were assessed using the Basso Mouse Scale (BMS), its subscore, and the inclined plane test. RNA sequencing of spinal cord tissues identified robust transcriptomic changes from SCI and a smaller subset from atorvastatin treatment. Atorvastatin enhanced sensorimotor recovery within two weeks of treatment initiation, with effects persisting to the experimental endpoint. Pathway analysis showed atorvastatin enriched neural regeneration processes including Fatty Acid Transport, Axon Guidance, and the Endocannabinoid Developing Neuron Pathway; improved mitochondrial function via increased TCA Cycle II and reduced Mitochondrial Dysfunction; and decreased Inhibition of Matrix Metalloproteases. Key gene drivers included Fabp7, Unc5c, Rest, and Klf4. Together, these results indicate atorvastatin's potential in chronic SCI recovery, especially where already indicated for cardiovascular protection.

Keywords: Chronic spinal cord injury; Locomotor recovery; Pathway analysis; RNA sequencing; Spinal cord transcriptomics.

Graphical abstract

Fig. 1

Experimental design for delayed atorvastatin or vehicle control administration following lateral compression SCI. (A) Experimental timeline for evaluating the effects of atorvastatin on SCI in C57BL/6J female mice. (B) Table of experimental groups (sex, n included in table) for assessing the therapeutic effect of atorvastatin delivery initiated 2 ​wk post-SCI. Experimental mice underwent a laminectomy at vertebral T8-T9 followed by lateral compression SCI using forceps with a 0.25 ​mm gap. Subsequently mice were randomized across two groups: SCI ​+ ​Atorvastatin or SCI ​+ ​Vehicle, receiving, respectively, daily intraperitoneal (i.p.) injections of either atorvastatin in a vehicle, or the vehicle alone, starting 14 ​d post-injury. Daily injections continued for 4 ​wk, with sensorimotor recovery evaluated using BMS and inclined plane tests at specified intervals (1A, green ticks). SCI was induced in the experimental groups when mice were 12 ​wk. Spinal cords from Uninjured Control mice were isolated at 14 ​wk of age. In the SCI groups, treatment (atorvastatin or vehicle) commenced when mice were 14 ​wk old and continued until spinal cord isolation at 18 ​wk of age for downstream RNA-seq analyses.

Fig. 2

Delayed atorvastatin treatment enhances functional recovery following SCI. Measures of sensorimotor function and body mass were conducted pre-injury and at multiple post-injury time points. (A) Basso Mouse Scale (BMS) scores highlight improved locomotor recovery in atorvastatin-treated mice, indicating significant enhancements in movements such as ankle motion and tail position starting from 2 ​wk post-treatment, with elevated scores at 28, 35, and 42 ​d post-injury. (B) BMS subscores indicate enhanced recovery of additional motor measures, including stepping frequency, coordination, and trunk stability, at 35 and 42 ​d post-injury. (C) Inclined plane test results demonstrate increased strength in atorvastatin-treated mice, with notable improvements in ability to maintain a grip at greater angles observed at 28, 35, and 42 ​d post-injury. (D) Body mass measurement, an indirect measure of health and recovery, showed no significant differences between groups throughout the recovery period. Statistical analysis utilized two-way repeated measures ANOVA and Student-Newman-Keuls post-hoc tests. Values represent mean ​± ​SEM. ∗∗∗p ​< ​0.001; ∗∗p ​< ​0.01; ∗p ​< ​0.05.

Fig. 3

Gene expression profiling of spinal cord shows a common transcriptomic response across SCI treatment conditions and an effect of delayed delivery of atorvastatin. (A) Spinal cord sample processing workflow depicting spinal cord isolation to RNA sequencing at 44 ​d post-injury. A 5 ​mm segment at the injury epicenter (vertebral levels T8-T9) was collected from the SCI-treated mice and the corresponding region from Uninjured Controls. The samples underwent RNA extraction, quality assessment, library construction, and high-throughput Illumina paired-end sequencing. “RIN” ​= ​RNA integrity number. (B) Volcano plots compare gene expression of SCI ​+ ​Vehicle with Uninjured Controls or (C) SCI ​+ ​Atorvastatin with Uninjured Controls, highlighting differentially expressed genes surpassing the expression fold change and statistical significance thresholds (|log2(fold change)| ​> ​0.322 [>25 ​% change]; FDR <0.05), with top 10 genes by fold change and p value in each quadrant encircled and listed. (D) Heatmap of gene expression by replicate in transcripts per million (TPM) details genes commonly changed in SCI across both treatment groups, indicating a core set of SCI-responsive genes. The Z-score for each replicate was calculated across groups per gene. “UI_Ctl” ​= ​Uninjured Control; “SCI_Veh” ​= ​SCI ​+ ​Vehicle; “SCI_Statin” ​= ​SCI ​+ ​Atorvastatin. (E) PCA plot delineates the groups based on gene expression, with the SCI ​+ ​Atorvastatin group showing tighter clustering.

Fig. 4

Delayed atorvastatin delivery modulates differential gene expression and pathway engagement after SCI. (A) Venn diagram depicting overall expression changes following SCI, with comparisons of genes commonly and uniquely affected by atorvastatin or vehicle treatments relative to the Uninjured Control. (B) Genes upregulated or (C) downregulated by SCI, illustrating the unique and shared transcriptional changes elicited by atorvastatin vs. vehicle treatment when compared to uninjured control. Abbreviation in (B) and (C): “SCI ​+ ​Statin” ​= ​SCI ​+ ​Atorvastatin. These analyses quantify the scale of transcriptional modifications induced by SCI and the extent to which atorvastatin treatment influences gene expression, utilizing a |log₂(fold change)| threshold >0.322 (>25% absolute change) and FDR <0.05. (D and E) Ingenuity Pathway Analysis (IPA) of pathways significantly affected by atorvastatin treatment, comparing “SCI-shared” pathways (common differentially expressed genes (DEGs) from both SCI ​+ ​Atorvastatin and SCI ​+ ​Vehicle conditions vs. Uninjured Control) to "Atorvastatin-unique" pathways (DEGs specific to SCI ​+ ​Atorvastatin vs. Uninjured Control). Pathways listed showed significantly different activation between SCI-shared and Atorvastatin-unique gene sets, ranked by Z-score difference. (D) Top 10 upregulated pathways sorted by descending Z-score difference. (E) Top 10 downregulated pathways sorted by ascending Z-score difference. ∗∗∗Δ|Z-score| > 3.29: p ​< ​0.001; ∗∗Δ|Z-score| > 2.58: p ​< ​0.01.

Fig. 5

Delayed atorvastatin delivery after SCI modulates a subset of genes beyond vehicle treatment. (A) Venn diagram analysis of gene expression patterns after SCI, demonstrating unique and shared transcriptional changes due to atorvastatin and vehicle treatment. Pattern 1: Genes activated after SCI and further activated by atorvastatin treatment. Pattern 2: Genes activated after SCI with activation abrogated by atorvastatin treatment. Pattern 3: Genes downregulated after SCI and further downregulated by atorvastatin treatment. Pattern 4: Genes downregulated after SCI with downregulation abrogated by atorvastatin treatment. (B) Expression profiles of the 13 DEGs of interest from overlapping Venn Diagram analyses in (A), presented in transcripts per million (TPMs), illustrating the modulatory effects of atorvastatin on genes altered by SCI. Inset shows the four relative TPM expression patterns observed for the 13 genes identified in (A). Abbreviations in (A) and/or (B): “SCI ​+ ​Veh” ​= ​SCI ​+ ​Vehicle; “SCI ​+ ​Statin” ​= ​SCI ​+ ​Atorvastatin; “UI Ctl” ​= ​Uninjured Controls. Significance thresholds for comparisons: FDR <0.05 for SCI ​+ ​Atorvastatin vs. Uninjured Control and SCI-Vehicle vs. Uninjured Control; “ns” ​= ​not significant at FDR <0.05; p ​< ​0.001 for SCI ​+ ​Atorvastatin vs. SCI-Vehicle. Values represent mean ​± ​SEM. ^#FDR <0.05; ∗∗∗p ​< ​0.001.