Effects of zileuton and montelukast in mouse experimental spinal cord injury

Abstract

Background and purpose: 5-lipoxygenase (5-LO) is the key enzyme in leukotriene (LT) biosynthesis from arachidonic acid (AA). Here, we examined the role of the 5-LO-product, cysteinyl-LT (Cys-LT), with a 5-LO inhibitor (zileuton) and a Cys-LT, receptor antagonist (montelukast), in the inflammatory response and tissue injury associated with spinal cord injury (SCI).

Experimental approach: SCI was induced in mice by the application of vascular clips to the dura via a two-level T6 to T7 laminectomy for 1 min. Cord inflammation was assessed histologically and by measuring inflammatory mediators (ELISA) and apoptosis by annexin V, TUNEL, Fas ligand staining and Bax and Bcl-2 expression (immunohistochemistry and western blots). Motor function in hindlimbs was assessed by a locomotor rating scale, for 10 days after cord injury.

Key results: SCI in mice resulted in tissue damage, oedema, neutrophil infiltration, apoptosis, tumour necrosis-alpha (TNF-alpha) and cyclooxygenase-2 (COX-2) expression, prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)) production, and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in injured tissue. Treatment of the mice with zileuton or montelukast reduced the spinal cord inflammation and tissue injury, neutrophil infiltration, TNF-alpha, COX-2 and pERK1/2 expression, PGE(2) and LTB(4) production, and apoptosis. In separate experiments, zileuton or montelukast significantly improved the recovery of limb function over 10 days.

Conclusions and implications: Zileuton and montelukast produced a substantial reduction of inflammatory events associated with experimental SCI. Our data underline the important role of 5-LO and Cys-LT in neurotrauma.

Figure 1

Effect of zileuton and montelukast on histological alterations of the spinal cord tissue 24 h after injury. (a) Twenty-four hours after trauma, a significant damage to the spinal cord from SCI+vehicle-treated mice at the perilesional area was assessed by the presence of oedema and alteration of the white matter. A significant protection from the SCI was observed in the tissue collected from SCI+zileuton (b) or montelukast-treated mice (c). This figure is representative of at least three experiments performed on different experimental days. wm, white matter; gm, grey matter. No histological alteration was observed in the spinal cord tissues from sham-operated mice (data not shown). SCI, spinal cord injury.

Figure 2

Effect of zileuton and montelukast on hindlimb motor dysfunction after SCI. The degree of motor dysfunction of (a) Sham groups and (b) SCI groups was assessed every day for 10 days after SCI induction by Basso–Beattie–Bresnahan criteria (Basso et al., 1995). Treatment with zileuton or montelukast reduces the loss of motor function after SCI. Values shown are mean±s.e.mean of 10 mice for each group. ^*P<0.01 versus SCI+vehicle. SCI, spinal cord injury.

Figure 3

Effect of zileuton and montelukast on immunohistochemical localization and activity of MPO. A significant positive staining and a significant increase of MPO activity was observed in the spinal cord tissues collected from SCI+vehicle-treated mice (a, d). In mice treated with zileuton (b, d) or montelukast (c, d), the MPO staining and activity was visibly and significantly reduced in comparison with the SCI+vehicle-treated mice. No positive staining for MPO was observed in spinal cord tissues collected from sham-operated mice (data not shown). Image is representative of at least three experiments performed on different experimental days. Data are means±s.e.mean of 10 mice for each group. ^*P<0.01 versus sham-operated mice; °P<0.01 versus SCI+vehicle-treated mice. wm, white matter; gm, grey matter; MPO, myeloperoxidase; SCI, spinal cord injury.

Figure 4

Typical densitometry evaluation. Densitometry analysis of immunocytochemistry photographs (n=5 photos from each sample collected from all mice in each experimental group) for MPO, TNF-α, FAS-L, Bax and Bcl-2 from spinal cord tissues was assessed. The assay was carried out by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266). Data are expressed as % of total tissue area. ^*P<0.01 versus Sham; °P<0.01 versus SCI. ND, not detectable; MPO, myeloperoxidase; SCI, spinal cord injury; TNF-α, tumour necrosis-α.

Figure 5

Effect of zileuton and montelukast on spinal cord levels and immunohistochemical localization of TNF-α. SCI caused, at 24 h, an increase in the release of TNF-α in spinal cord tissues (a, d). Treatment with zileuton (b, d) or montelukast (c, d) significantly inhibited the SCI-induced increase of TNF-α. No positive staining for TNF-α was observed in spinal cord tissues obtained from the sham group of mice (data not shown). Figure is representative of at least three experiments performed on different experimental days. wm, white matter; gm, grey matter. Data are means±s.e.mean of 10 mice for each group. ^*P<0.01 versus sham-operated mice; °P<0.01 versus SCI+vehicle-treated mice. SCI, spinal cord injury; TNF-α, tumour necrosis-α.

Figure 6

Effect of zileuton and montelukast on apoptosis. At 24 h after SCI, spinal cord tissues obtained from SCI+vehicle-treated mice demonstrated a marked appearance of positive stain for annexin V FITC (a), an index of cells that are induced to undergo apoptosis. Some cells showed a positive intracellular staining to PI (a1), an index of cells in the late stage of apoptosis. On the contrary, spinal cord tissues section from mice treated with zileuton or montelukast demonstrate no cells in the earlier stage (b and c, respectively) of apoptosis and fewer cells (positive to PI) in the later stages of apoptosis (b1 and c1, respectively). (a2, b2, and c2) Staining combination of a and a1, b and b1, and c and c1, respectively. The figure is representative of at least three experiments performed on different experimental days. FITC, fluorescein isothiocyanate; PI, propidium iodide SCI, spinal cord injury.

Figure 7

Effect of zileuton and montelukast on TUNEL staining in perilesional spinal cord tissue. At 24 h after the trauma, tissues obtained from SCI+vehicle-treated mice demonstrated a marked appearance of dark-brown apoptotic cells and intercellular apoptotic fragments (a). In contrast, tissues obtained from mice treated with zileuton (b) or montelukast (c) demonstrated a small number of apoptotic cells or fragments. Almost no apoptotic cells were detectable in the spinal cord tissue of sham-operated mice (data not shown). Panel d demonstrates the positive staining in the Kit-positive control tissue. The figure is representative of at least three experiments performed on different experimental days. SCI, spinal cord injury; TUNEL, terminal deoxynucleotidyltransferase-mediated UTP end labelling assay.

Figure 8

Effect of zileuton and montelukast on immunohistological staining for FAS Ligand. At 24 h after the trauma spinal cord sections obtained from SCI+vehicle-treated mice exhibited positive staining for FAS Ligand (a) mainly localized in inflammatory cells as well as in nuclei of Schwann cells in the white (wm) and grey (gm) matter of the spinal cord tissues. Treatment of mice subjected to SCI with zileuton (b) or montelukast (c) reduced the degree of positive staining for FAS Ligand in the spinal cord. There was no staining for FAS Ligand in spinal cord tissues obtained from the sham group of mice (data not shown). SCI, spinal cord injury.

Figure 9

Effect of zileuton and montelukast on Bax levels. At 24 h, SCI caused an increase of Bax expression in vehicle-treated mice (a). On the contrary, the degree of positive staining for Bax was reduced in spinal cord tissues collected from mice treated with zileuton (b) or montelukast (c). No positive staining for Bax was observed in the tissue section from sham-operated mice (data not shown). Figure is representative of at least three experiments performed on different experimental days. (d) Western blot analysis of extracts of spinal cord tissue collected at 24 h after injury. Basal levels of Bax was present in the tissue from sham-operated mice. Bax band is more evident in the tissue from SCI+vehicle-treated mice. The treatment with zileuton or montelukast reduced Bax levels. Data were normalized on the basis of β-actin levels. Densitometric analysis of protein expression represents the mean±s.e.mean of 10 spinal cord tissues. °P<0.01 versus sham-operated mice; ^*P<0.05 versus SCI+vehicle. wm, white matter; gm, grey matter; SCI, spinal cord injury.

Figure 10

Effect of zileuton and montelukast on the Bcl-2 levels. At 24 h, SCI caused a decrease of the release of Bcl-2 expression in vehicle-treated mice (a). The degree of positive staining for Bcl-2 was increased in spinal cord tissues collected from mice treated with zileuton (b) or montelukast (c). Figure is representative of at least three experiments performed on different experimental days. (d) Western blot analysis of extracts of spinal cord tissue collected at 24 h after injury. Basal levels of Bcl-2 were present in the tissue from sham-operated mice. Bcl-2 band is less evident in the tissue from SCI+vehicle-treated mice. The treatment with zileuton or montelukast increased Bcl-2 levels. Data were normalized on the basis of β-actin levels. Densitometric analysis of protein expression represents the mean±s.e.mean of 10 spinal cord tissues. °P<0.01 versus sham; ^*P<0.05 versus SCI+vehicle. wm, white matter; gm, grey matter; SCI, spinal cord injury.

Figure 11

Effect of zileuton and montelukast on the COX-2 expression. Western blot analysis of extracts of spinal cord tissue collected at 24 h after injury. SCI caused an increase of COX-2 expression in vehicle-treated mice. The treatment with zileuton or montelukast reduced COX-2 levels. Densitometric analysis of protein expression represents the mean±s.e.mean of 10 spinal cord tissues. Data were normalized on the basis of β-actin levels. °P<0.01 versus sham; ^*P<0.05, ^**P<0.01 versus SCI+vehicle. COX-2, cyclooxygenase-2; SCI, spinal cord injury.

Figure 12

Effect of zileuton and montelukast on ERK1/2 activation. Western blot analysis of extracts of spinal cord tissue collected at 24 h after injury. SCI caused an increase of the ERK1/2 phosphorylation in vehicle-treated mice. The treatment with zileuton or montelukast reduced pERK1/2 levels. Densitometric analysis of protein expression represents the mean±s.e.mean of 10 spinal cord tissues. Data were normalized on the basis of ERK-2 levels. °P<0.01 versus sham; ^*P<0.05, ^**P<0.01 versus SCI+vehicle. ERK1/2, extracellular signal-regulated kinase 1/2; SCI, spinal cord injury.

Figure 13

Effect of zileuton and montelukast on levels of PGE₂ and LTB₄ in spinal cord. SCI caused, at 24 h, an increase in the release of PGE₂ (a) and LTB₄ (b) in spinal cord tissues. Treatment with zileuton or montelukast significantly inhibited the SCI-induced increase of PGE₂ (a) and LTB₄ (b). Data are means±s.e.mean of 10 mice for each group. °P<0.01 versus sham-operated mice; ^*P<0.05; ^***P<0.01 versus SCI+vehicle-treated mice. LTB₄, leukotriene B₄; PGE₂, prostaglandin E₂; SCI, spinal cord injury.