The PPAR alpha agonist gemfibrozil is an ineffective treatment for spinal cord injured mice

Abstract

Peroxisome Proliferator Activated Receptor (PPAR)-α is a key regulator of lipid metabolism and recent studies reveal it also regulates inflammation in several different disease models. Gemfibrozil, an agonist of PPAR-α, is a FDA approved drug for hyperlipidemia and has been shown to inhibit clinical signs in a rodent model of multiple sclerosis. Since many studies have shown improved outcome from spinal cord injury (SCI) by anti-inflammatory and neuroprotective agents, we tested the efficacy of oral gemfibrozil given before or after SCI for promoting tissue preservation and behavioral recovery after spinal contusion injury in mice. Unfortunately, the results were contrary to our hypothesis; in our first attempt, gemfibrozil treatment exacerbated locomotor deficits and increased tissue pathology after SCI. In subsequent experiments, the behavioral effects were not replicated but histological outcomes again were worse. We also tested the efficacy of a different PPAR-α agonist, fenofibrate, which also modulates immune responses and is beneficial in several neurodegenerative disease models. Fenofibrate treatment did not improve recovery, although there was a slight trend for a modest increase in histological tissue sparing. Based on our results, we conclude that PPAR-α agonists yield either no effect or worsen recovery from spinal cord injury, at least at the doses and the time points of drug delivery tested here. Further, patients sustaining spinal cord injury while taking gemfibrozil might be prone to exacerbated tissue damage.

Fig. 1

Locomotor assessment of SCI animals treated with gemfibrozil (GEM) or vehicle in Study I. Mice were tested after SCI (75 kdyn) using the BMS locomotor scale starting at 1 day post-injury (dpi) until 28 dpi. Vehicle controls had significantly higher BMS scores starting at 5 dpi (A). BMS sub-scores were also significantly lower in the gemfibrozil treatment group (B). Specific behavioral categories were compared between groups. Gemfibrozil treated animals had a trend for reduced stepping (C), significantly reduced coordination at 21–28 dpi (D), worse paw position at 14 dpi (E), and reduced trunk stability at 24 dpi (F). *p<0.05, **p<0.01 and ***p<0.001.

Fig. 2

Spared tissue analysis of SCI animals in Study I. Spinal cord cross-sections spanning 0.75 mm rostral and caudal to the lesion epicenter (Epi) were stained for eriochrome cyanine to label myelin (blue) and neurofilament for axons (brown) (A, B). Black lines demarcate the border between intact spared tissue and pathological tissue. Stereologic analysis of spared white matter and gray matter revealed that gemfibrozil (GEM) treatment did not significantly change tissue sparing but did cause reduced white matter compared to vehicles at every distance examined (C, D).

Fig. 3

Analysis of the area of tissue occupied by activated microglia and macrophages in cross-sections from Study I. (A, B) Images of spinal cord sections immunolabeled for CD68 were used to outline the frank lesion core and tissue outside the lesion core (delineated by dotted line). Areas demarcated by black boxes are shown in high power adjacent to cross-sections. The area of immunoreactivity was measured separately for tissue inside (C) and outside (D) the lesion core at 28 dpi. Gemfibrozil (GEM) treated animals had significantly reduced CD68 immunoreactivity in tissue outside the lesion core (Two-way ANOVA with repeated measures, pb0.05). Low power image scale bar=100 µm, high power image scale bar=50 µm.

Fig. 4

Quantification of T cells inside and outside lesion core in Study I. Spinal cord sections were immunolabeled for CD3, a pan T cell marker (A, B), and the number of CD3 positive cells inside and outside the lesion was counted in vehicle and gemfibrozil (GEM) treated animals. Tissue sections and lesion cores are outlined. Areas in boxes are shown at high power below cross-sections. There were significantly more CD3+ cells outside the lesion in gemfibrozil treated mice compared to controls (C) (p<0.001). Low power image scale bar=100 µm, high power image scale bar=25 µm.

Fig. 5

BMS locomotor analysis of mice in Study II that received gemfibrozil (GEM) or vehicle (VEH) beginning either before (A, B) or after SCI (C, D). Two-way repeated measures ANOVA revealed no differences between the groups for BMS scores or sub-scores.

Fig. 6

Behavioral and histological analyses of mice in Study III that received gemfibrozil (GEM), fenofibrate (FEN) or vehicle beginning 2 h post-injury. (A, B) Locomotor recovery was tested using the BMS locomotor scale starting at 1 day post-injury (dpi). No differences were detected in the BMS scores or sub-scores. Stereological analysis of spared white matter (C) and gray matter (D) revealed no significant differences between vehicle and drug groups. However, the fenofibrate group had significantly more gray matter sparing at 0.45 mm caudal to epicenter (Epi) compared to gemfibrozil treated mice (p<0.01).

Fig. 7

Behavioral and histological analyses of SCI animals in Study IV receiving post-SCI vehicle or a higher dose of gemfibrozil (GEM) compared Studies I–III. (A, B) The BMS locomotor scale was used starting at 1 day post-injury; no significant differences were detected between groups. (C, D) Stereological analysis of tissue sparing revealed gemfibrozil-treated mice had significantly less spared gray matter (C) and greater lesion size (D) caudal to the lesion epicenter (Epi). Two-way repeated measures ANOVA followed by Bonferroni post-hoc analysis, *p<0.05, **p<0.01.