Licofelone modulates neuroinflammation and attenuates mechanical hypersensitivity in the chronic phase of spinal cord injury

Abstract

Inflammation is a major factor shaping outcome during the early, acute phase of traumatic spinal cord injury (SCI). It is known that pro-inflammatory signaling within the injured spinal cord drives pathological alterations in neurosensory processing and shapes functional outcome early after injury. However, it is unclear whether inflammation persists into the chronic phase of injury or shapes sensory processing long after injury. To investigate these possibilities, we have performed biochemical and behavioral assessments 9 months after moderate thoracic spinal contusion injury in the rat. We have found that levels of the pro-inflammatory lipid mediators leukotriene B4 and prostaglandin E2 are elevated in the chronic spinal cord lesion site. Additionally, using metabolomic profiling, we have detected elevated levels of pro-oxidative and inflammatory metabolites, along with alterations in multiple biological pathways within the chronic lesion site. We found that 28 d treatment of chronically injured rats with the dual COX/5-LOX inhibitor licofelone elevated levels of endogenous anti-oxidant and anti-inflammatory metabolites within the lesion site. Furthermore, licofelone treatment reduced hypersensitivity of hindpaws to mechanical, but not thermal, stimulation, indicating that mechanical sensitivity is modulated by pro-inflammatory signaling in the chronic phase of injury. Together, these findings provide novel evidence of inflammation and oxidative stress within spinal cord tissue far into the chronic phase of SCI, and demonstrate a role for inflammatory modulation of mechanical sensitivity in the chronic phase of injury.

Figure 1.

Levels of PGs and LTs are elevated in the lesion site of spinal cords 9 months following SCI. PGE₂ levels (A, n = 3 per group) and LTB₄ levels (B, n = 6 per group) in T10 spinal cord segments of age-matched, uninjured controls (undetectable) and rats 9 months post-SCI (black bars). *p < 0.001, Student's t test. All data are mean ± SEM.

Figure 2.

Metabolic alterations in the spinal cord lesion site 9 months post-SCI. Heat map illustrates the relative change in levels of 126 metabolites with increased (yellow) or decreased (blue) expression in chronic SCI, vehicle-treated animals (n = 8) versus uninjured, age-matched controls (n = 9). Changes in concentration, relative to the median metabolite level, are indicated by intensity of color (see color scale, upper right). Metabolites are arranged by hierarchical clustering, with representative clusters of metabolites highlighted (A–C). Scale bar (upper left) indicates Pearson correlation coefficient (r). Colored bars (right) indicate super class of each metabolite (amino acids, carbohydrates, cofactors and vitamins, energy metabolites, lipids, nucleotides, peptides, and xenobiotics).

Figure 3.

Biochemical markers of oxidative stress and inflammation in the chronically injured spinal cord. Box plots illustrate changes in abundance of select metabolites in spinal cord tissue of uninjured, age-matched controls (white, n = 9) and chronically injured animals (gray, n = 8). For each metabolite, concentration values have been rescaled so that samples across all groups have a median value equal to 1. Box legend: horizontal bar inside box represents group median; upper and lower box boundaries represent 75th percentile and 25th percentile, respectively; upper and lower whiskers represent maximum and minimum of distribution; open circles represent extreme data points. A, Several metabolites significantly increased in chronic SCI spinal cords are associated with oxidative stress. B, Levels of anti-inflammatory and anti-oxidative metabolites are decreased in chronic SCI tissue. *p < 0.05, Welch's two-sample t test. 13- + 9-HODE, 13- and 9-hydroxyoctadecadienoic acid; 24-OH-Chol, 24(S)-hydroxycholesterol; α-Toco, α-tocopherol; Cys-S-SG, cysteine-glutathione disulfide; DHA, dehydroascorbate; GSSG, oxidized glutathione; OEA, oleic ethanolamide; PEA, palmitoyl ethanolamide.

Figure 4.

Changes in the metabolomic profile of the chronically injured spinal cord with licofelone treatment. A, Heat map showing 26 metabolites with altered concentrations in chronic SCI, licofelone-treated animals (SCI + L, n = 9), compared with chronic SCI, vehicle-treated animals (SCI + V, n = 8). Metabolites are arranged by hierarchical clustering. Scale bar (upper left) indicates Pearson correlation coefficient (r). Colored bars (right) indicate super class of each metabolite (e.g., amino acids, carbohydrates). Increases (yellow) and decreases (blue) in concentration, relative to the median metabolite level, are indicated by intensity of color (see color scale). B, Box plots illustrating changes in levels of select compounds within the chronic SCI, vehicle-treated (gray) or licofelone-treated (checkered) groups. For box legend, see Figure 3. *p < 0.05, Welch's two-sample t test. 15-HETE, 15-hydroxyeicosatetraenoic acid; 24-OH-Chol, 24(S)-hydroxycholesterol; 7-β-OH-Chol, 7-β-hydroxycholesterol; HC, homocarnosine; I1P, inositol 1-phosphate.

Figure 5.

Licofelone attenuates mechanical sensitivity of hindpaws in chronic SCI. Hindpaw mechanical withdrawal thresholds (g) of chronic SCI animals before treatment (Pre) and following 28 d of treatment (Post) with vehicle (n = 8) or licofelone (n = 9). Data points connected by lines represent pretreatment and post-treatment scores for individual subjects. Paired Student's t test.

Figure 6.

Licofelone does not affect thermal hypersensitivity of hindpaws in chronic SCI. Hindpaw thermal withdrawal latency (s) of chronic SCI animals before treatment (Pre) and following 28 d of treatment (Post) with vehicle (triangles, n = 8) or licofelone (circles, n = 9). Connected data points represent pretreatment and post-treatment scores for individual subjects. Paired Student's t test.

Figure 7.

Licofelone does not affect hindlimb locomotor function in chronic SCI rats. BBB hindlimb locomotor scores beginning at 8 months post-SCI, before treatment (Pre) and weekly throughout the treatment period. Uninjured, uninjured, age-matched controls (n = 9); SCI + V, chronic SCI, vehicle-treated (n = 8); SCI + L, chronic SCI, licofelone-treated (n = 9). All data are mean ± SEM.