INTRODUCTION
Spinal cord injury (SCI) usually leads to devastating neurological deficits and disabilities. Recently, the National Spinal Cord Injury Statistical Center reported that the annual incidence of SCI in the United States is estimated to be 40 cases per million people (1). People with traumatic SCI are at greater risk than the general population throughout their lifetime for medical complications that may necessitate rehospitalization.
The neurological damage that is incurred at the time of mechanical trauma to the spinal cord is called "primary injury." The primary injury provokes a cascade of cellular and biochemical reactions, which lead to further damage. This provoked cascade of reactions is called "secondary injury." Several mechanisms are involved in secondary injury, including microvascular dysfunction at the site of injury; free radicals formation and lipid peroxidation; accumulation of excitatory neurotransmitters, for example, glutamate (acting on N-methyl-d-aspartate and non-N-methyl-d-aspartate receptors), leading to neural damage due to excessive excitation (excitotoxicity) (2); loss of intracellular balance of sodium, potassium, calcium, and magnesium ions and subsequent increased intracellular ionized calcium concentration (3); increased level of endogenous opioid-like compounds, especially dynorphins, at the site of injury, which contribute to the pathophysiology of secondary injury (4); depletion of high-energy metabolites, leading to anaerobic metabolism at the site of injury (5); provocation of an inflammatory response and recruitment and activation of inflammatory cells associated with secretion of cytokines, which contribute to further tissue damage (6); and activation of calpains and caspases, leading to cellular apoptosis (7).
The contemporary management of SCI consists of supportive care and stabilization of the spine. Numerous pharmacological therapies have been evaluated for the treatment of SCI, although none has met with substantial success. In most human SCIs, the mechanism of the primary injury is acute compression or laceration of the spinal cord due to displacement of bone or disk into the spinal cord during fracture-dislocation or burst fracture of the spine. To mimic most mechanical events that lead to various forms of human SCI, several experimental models have been developed. The development of a rational approach to the treatment of acute traumatic SCI in humans requires a standardized and reproducible animal model in which a quantifiable trauma stimulus can be correlated with functional recovery and morphology of the cord lesion. A model used in SCI research is the compression model; in this model, injury is induced by applying either a weight or an aneurysm clip to the spinal cord (8). This model aims to add to that of the contusion model by replicating the persistence of cord compression that is commonly observed in human SCI (9). In the present study, we used this model to investigate the effect of ethyl pyruvate (EP) on the degree of SCI.
Ethyl pyruvate is a simple aliphatic ester derived from the endogenous metabolite, pyruvic acid. In previous studies performed in our laboratory and that of others, EP has been shown to ameliorate intestinal, renal, or hepatic injury when it is used as a therapeutic agent to treat rodents subjected to mesenteric ischemia and reperfusion, hemorrhagic shock (10), endotoxemia (11), or polymicrobial bacterial sepsis (12). Treatment with EP also ameliorates organ dysfunction in murine models of acute pancreatitis and alcoholic hepatitis (13).
The initiation of inflammatory responses in the central nervous system (CNS) is related to activation of mitogen-activated protein kinases (MAPKs), especially extracellular signal-regulated kinase (ERK) 1/2. ERK proteins are regulated by dual phosphorylation at tyrosine 204 and 187 and threonine 177 and 160 residues. Phosphorylation at both the threonine 202 and tyrosine 204 residues of ERK1 and threonine 185 and tyrosine 187 residues of ERK2 is required for full enzymatic activation. The structural consequences of dual phosphorylation in ERK2 include active site closure, alignment of key catalytic residues that interact with ATP, and remodeling of the activation loop. ERK activation leads to dimerization with other ERKs and subsequent localization to the nucleus. Active ERK dimers phosphorylate serine and threonine residues on nuclear proteins and influence a host of responses that include proliferation, differentiation, transcription regulation, and development. Consequently, in this study, we investigated whether EP reduced inflammatory events associated with experimental SCI through sequential activation of p42/p44 MAPK and nuclear factor κB (NF-κB) in SCI.
To gain a better insight into the mechanism(s) of action of EP, we evaluated the following end points of the inflammatory response: (a) histological damage, (b) motor recovery, (c) neutrophil infiltration, (d) NF-κB activation, (e) ERK1/2 MAPK phosphorylation, (f) nitrotyrosine and poly(ADP-ribose) (PAR) formation, (g) proinflammatory cytokine production, (h) iNOS expression, (i) apoptosis as TUNEL staining, and (j) Bax and Bcl-2 expression.
MATERIALS AND METHODS
Animals
Male adult CD1 mice (25-30 g; Harlan Nossan, Milan, Italy) were housed in a controlled environment and provided with standard rodent chow and water. Mice were housed in stainless steel cages in a room kept at 22°C ± 1°C with a 12-h-light/12-h-dark cycle. The animals were acclimated to their environment for 1 week and had ad libitum access to tap water and rodent standard diet. The study was approved by the University of Messina Review Board for the care of animals. All animal experiments complied with regulations in Italy (D.M. 116192), Europe (O.J. of E.C. L 358/1 12/18/1986), and United States (Animal Welfare Assurance No A5594-01, US Department of Health and Human Services).
Spinal cord injury
Mice were anesthetized using chloral hydrate (400 mg/kg body weight). A longitudinal incision was made on the midline of the back, exposing the paravertebral muscles. These muscles were dissected away, exposing T5-T8 vertebrae. The spinal cord was exposed via a four-level T5-T8 laminectomy, and SCI was produced by extradural compression of the spinal cord using an aneurysm clip with a closing force of 24 g. In all injured groups, the spinal cord was compressed for 1 min. Sham animals were subjected only to laminectomy. After surgery, 1 mL of normal saline was administered subcutaneously to replace the blood volume lost during the surgery. During recovery from anesthesia, the mice were placed on a warm heating pad and covered with a warm towel. The mice were individually housed in a temperature-controlled room at 27°C for a survival period of 10 days. Food and water were provided to the mice ad libitum. During experimental procedure, the animals' bladders were manually voided twice a day until the mice were able to regain normal bladder function. Spinal cord tissues were collected at 24 h after laminectomy or injury and were collected for histological analysis, immunohistochemical studies, Western blot analysis, myeloperoxidase (MPO) activity, and thiobarbituric acid-reactant substances measurement.
Experimental design
A dose-response effect was performed investigating the effect of EP (75, 25, or 8.5 mg/kg) on the development of SCI. The dose was chosen in agreement with our precedent study (14).
Mice were randomly allocated into the following groups: (a) SCI + vehicle group: mice were subjected to SCI plus administration of saline (administered i.p., 1 and 6 h, after SCI) (n = 40 total); (b) EP group: same as the SCI + vehicle group but in which EP (75, 25, or 8.5 mg/kg) was administered i.p., 1 and 6 h, after SCI (n = 40 total); (c) sham + vehicle group: mice were subjected to the surgical procedures as the above groups except that the aneurysm clip was not applied and to these mice was administered saline (administered i.p., 1 and 6 h, after SCI) (n = 40 total); (d) sham + EP group: identical to sham + vehicle group except for the administration of EP (75, 25, or 8.5 mg/kg administered i.p., 1 and 6 h, after SCI (n = 40 total). As described below, mice (n = 10 from each group for each parameters) were killed at 24 h after SCI to evaluate the various parameters. In a separate set of experiments, other 10 animals for each group were observed until 10 days after SCI to evaluate the motor score.
Light microscopy
Spinal cord biopsies were taken at 24 h after trauma. Tissue segments containing the lesion (1 cm on each side of the lesion) were paraffin embedded and cut into 5-μm-thick sections. Tissue sections were deparaffinized with xylene, stained with hematoxylin-eosin, and studied using light microscopy (Dialux 22; Leitz, Wetzlar, Germany).
The segments of each spinal cord were evaluated by an experienced histopathologist. Damaged neurons were counted, and the histopathologic changes of the gray matter were scored on a six-point scale: 0, no lesion observed, 1, gray matter contained 1 to 5 eosinophilic neurons; 2, gray matter contained 5 to 10 eosinophilic neurons; 3, gray matter contained more than 10 eosinophilic neurons; 4, small infarction (less than one third of the gray matter area); 5, moderate infarction; (one third to one half of the gray matter area); and 6, large infarction (more than half of the gray matter area). The scores from all the sections from each spinal cord were averaged to give a final score for an individual mouse. All of the histological studies were performed in a blinded fashion.
Grading of motor disturbance
The motor function of mice subjected to compression trauma was assessed once a day for 10 days after injury. Recovery from motor disturbance was graded using the modified murine Basso, Beattie, and Bresnahan (BBB) hindlimb locomotor rating scale (15).
MPO activity
Myeloperoxidase activity, an indicator of polymorphonuclear leukocyte accumulation, was determined in the spinal cord tissues as previously described (16) at 24 h after SCI. Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 μmol of peroxide per minute at 37°C and was expressed in units per gram of wet tissue.
Measurement of TNF-α and IL-1Symbol concentration
Portions of spinal cord tissues, collected 24 h after SCI, were homogenized, as previously described, in phosphate-buffered saline (PBS) containing 2 mmol/L of phenylmethylsulfonyl fluoride (PMSF; Sigma Chemical Co, Milan, Italy), and tissue TNF-α and IL-1β levels were evaluated. The assay was carried out by using a colorimetric, commercial kit (Calbiochem-Novabiochem Corp, Milan, Italy), according to the manufacturer's instructions. All TNF-α and IL-1β determinations were performed in duplicate serial dilutions.
Western blot analysis for IκB-α, phospho-NF-κB p65 (serine 536), NF-κB p65, Bax, Bcl-2, pERK1/2, and ERK2
Cytosolic and nuclear extracts were prepared. Briefly, spinal cord tissues from each mouse were suspended in extraction buffer A containing 0.2 mM PMSF, 0.15 μM pepstatin A, 20 μM leupeptin, and 1 mM sodium orthovanadate, homogenized at the highest setting for 2 min, and centrifuged at 1,000g for 10 min at 4°C. Supernatants represented the cytosolic fraction. The pellets, containing enriched nuclei, were resuspended in buffer B containing 1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl pH 7.4, 1 mM EGTA, 1 mM EDTA, 0.2 mM PMSF, 20 μM leupeptin, and 0.2 mM sodium orthovanadate. After centrifugation for 30 min at 15,000g at 4°C, the supernatants containing the nuclear protein were stored at −80°C for further analysis. The levels of IκB-α, phospho-NF-κB p65 (serine 536), ERK, pERK, Bax, and Bcl-2 were quantified in cytosolic fractions from spinal cord tissue collected 24 h after SCI, whereas NF-κB p65 levels were quantified in nuclear fractions. The membranes were blocked with 1× PBS, 5% (wt/vol) nonfat dried milk (PM) for 40 min at room temperature, and subsequently probed with specific Abs IκB-α (1:1,000; Santa Cruz Biotechnology, Milan, Italy), phospho-NF-κB p65 (serine 536) (1:1,000; Cell Signaling, Milan, Italy), anti-Bax (1:500; Santa Cruz Biotechnology), anti-Bcl-2 (1:500; Santa Cruz Biotechnology), anti-ERK2, (1:1,000 Santa Cruz Biotechnology), anti-pERK1/2 (1:1,000 Santa Cruz Biotechnology), or anti-NF-κB p65 (1:1,000; Santa Cruz Biotechnology) in 1× PBS, 5% wt/vol nonfat dried milk, 0.1% Tween-20 (PMT) at 4°C, overnight. Membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2,000, Jackson ImmunoResearch, West Grove, Pa) for 1 h at room temperature.
To ascertain that blots were loaded with equal amounts of protein lysates, they were also incubated in the presence of the antibody against β-actin (1:10,000; Sigma-Aldrich Corp, Milan, Italy). The relative expression of the protein bands of IκB-α (∼37 kd), phospho-NF-κB (75 kd), NF-κB p65 (65 kd), Bax (∼23 kd), and Bcl-2 (∼29 kd) was quantified by densitometric scanning of the radiographic films using the GS-700 Imaging Densitometer (GS-700; Bio-Rad Laboratories, Milan, Italy) and a computer program (Molecular Analyst; IBM, Milan, Italy). The dual-phosphorylated form of ERK (pERK1/2) antibody identified two bands of approximately 44 and 42 kd (corresponding to pERK1 and pERK2, respectively). The anti-ERK2 antibody detects total ERK2 (i.e., detects both phosphorylated and nonphosphorylated forms of ERK2).
Immunohistochemical localization of iNOS, nitrotyrosine, PAR, Fas ligand, Bax, and Bcl-2
At 24 h after SCI, the tissues were fixed in 10% (wt/vol) PBS-buffered formaldehyde, and 8-μm sections were prepared from paraffin-embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (vol/vol) hydrogen peroxide in 60% (vol/vol) methanol for 30 min. The sections were permeabilized with 0.1% (wt/vol) Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the section in 2% (vol/vol) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin, respectively. Sections were incubated overnight with anti-iNOS (1:500 in PBS, vol/vol; Transduction Laboratories, Milan, Italy), antinitrotyrosine rabbit polyclonal antibody (1:500 in PBS, vol/vol; Upstate, Milan, Italy), anti-PAR antibody (1:200 in PBS, vol/vol; BioMol, Milan, Italy), anti-Fas ligand antibody (1:500 in PBS, vol/vol; Santa Cruz Biotechnology), anti-Bax antibody (1:500 in PBS, vol/vol; Santa Cruz Biotechnology), or with anti-Bcl-2 polyclonal antibody (1:500 in PBS, vol/vol; Santa Cruz Biotechnology). Sections were washed with PBS and incubated with secondary antibody. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex (DBA; Vector Laboratories, Milan, Italy). To verify the binding specificity for nitrotyrosine, PAR, iNOS, Fas ligand, Bax, and Bcl-2, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations, no positive staining was found in the sections, indicating that the immunoreactions were positive in all the experiments carried out.
Thiobarbituric acid-reactant substances measurement
Thiobarbituric acid-reactant substances measurement, which is considered a good indicator of lipid peroxidation, was determined, as previously described (17), in the spinal cord tissue at 24 h after SCI. Thiobarbituric acid-reactant substances were calculated by comparison with OD650 of standard solutions of 1,1,3,3-tetramethoxypropan 99% malondialdehyde bis(dimethylacetal) 99% (MDA) (Sigma-Aldrich Corp). The absorbance of the supernatant was measured by spectrophotometry at 650 nm.
Annexin V evaluation
The binding of annexin V (Ann-V)-fluorescein isothiocyanate (FITC) to externalized phosphatidylserine was used as a measurement of apoptosis in spinal cord tissue sections with an Ann-V-propidium iodide (PI) apoptosis detection kit (Santa Cruz Biotechnology), according to the manufacturer's instructions. Briefly, normal viable cells in culture will stain negative for Ann-V FITC and negative for PI. Cells that are induced to undergo apoptosis will stain positive for Ann-V FITC and negative for PI as early as 1 h after stimulation. Both cells in later stages of apoptosis and necrotic cells will stain positive for Ann-V FITC and PI. Sections were washed as before, mounted with 90% glycerol in PBS, and observed with a LSM 510 Zeiss laser confocal microscope (Arese, Milan, Italy) equipped with a 40× oil objective.
Materials
All compounds were obtained from Sigma-Aldrich. All other chemicals were of the highest commercial grade available. All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl; Baxter, Italy, UK).
Statistical evaluation
All values in the figures and text are expressed as mean ± SEM of n observations. For the in vivo studies, n represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments performed on different experimental days. The results were analyzed by one-way ANOVA followed by a Bonferroni post hoc test for multiple comparisons. A P < 0.05 was considered significant. BBB scale data were analyzed by the Mann-Whitney U test and considered significant if P < 0.05.
RESULTS
EP reduces the severity of spinal cord trauma
To evaluate if the damage to the spinal cord was associated with a loss of motor function, the modified BBB hindlimb locomotor rating scale score was evaluated. Whereas motor function was only slightly impaired in sham mice, mice subjected to SCI had significant deficits in hindlimb movement (Fig. 1A). Ethyl pyruvate (75, 25, or 8.5 mg/kg) reduced the functional deficits induced by SCI in a dose-dependent manner (Fig. 1A). The severity of the trauma at the level of the perilesional area was assessed by the presence of edema as well as alteration of the white matter, and infiltration of leukocytes (Fig. 1, B and C) was evaluated at 24 h after injury. Significant damage to the spinal cord was observed in the spinal cord tissue from SCI mice when compared with sham-operated mice (Fig. 1, B and C). Notably, significant protection against the SCI was observed in EP-treated (75 mg/kg) mice (Fig. 1, B and C).
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Fig. 1: Effect of EP treatment on histological alterations of the spinal cord tissue 24 h after injury and motor function. The degree of motor disturbance was assessed every day until 10 days after SCI by BBB criteria *P < 0.01 vs. SCI + vehicle (A). Treatments with EP (75 mg/kg) enhanced the recovery after SCI. wm indicates white matter; gm, gray matter. This figure is representative of at least three experiments performed on different experimental days. Significant damage to the spinal cord in mice subjected to SCI, at the perilesional area, was apparent, as evidenced by the presence of edema as well as alteration of the white matter 24 h after injury (B) when compared with sham groups (B). Notably, a significant protection from SCI-associated damage was observed in the tissue samples collected from EP-treated mice (B). The histological score (C) was made by an independent observer.
Effects of EP on neutrophil infiltration
The above mentioned histological pattern of SCI seemed to be correlated with the influx of leukocytes into the spinal cord. Therefore, we investigated the effect of EP on neutrophil infiltration by measuring tissue MPO activity. Myeloperoxidase activity was significantly elevated in the spinal cord at 24 h after injury in mice subjected to SCI when compared with sham-operated mice (Fig. 2A, P < 0.01 vs. sham + vehicle). Treatment with EP attenuated neutrophil infiltration into the spinal cord at 24h after injury in a dose-dependent fashion (Fig. 2A, P < 0.01 vs. SCI).
Fig. 2: Effects of EP on MPO activity and TNF-α and IL-1β levels. After the injury, MPO activity in spinal cord from SCI mice was significantly increased at 24 h after the damage in comparison to sham groups (A). In addition, a substantial increase in TNF-α (B) and IL-1β (C) production was found in spinal cord tissues from SCI mice 24 h after SCI. Treatment with EP (8.5, 25, or 75 mg/kg) significantly attenuated neutrophil infiltration, TNF-α and IL-1β levels, as well as the thiobarbituric acid-reactant substances in the spinal cord. Data are means ± SEM of 10 mice for each group. *P < 0.01 vs. sham; °P < 0.01 vs. SCI + vehicle.
EP modulates expression of TNF-α and IL-1β after SCI
To test whether EP may modulate the inflammatory process through the regulation of the secretion of proinflammatory cytokines, we analyzed the spinal cord levels of TNF-α and IL-1β. A substantial increase in TNF-α and IL-1β production was found in spinal cord tissues collected from SCI mice at 24 h after SCI (Fig. 2, B and C, respectively). Spinal cord levels of TNF-α and IL-1β were significantly attenuated in a dose-dependent manner by the EP treatment (Fig. 2, B and C, respectively).
EP modulates expression of iNOS after SCI
To determine the role of NO produced during SCI, iNOS expression was evaluated by immunohistochemical analysis of spinal cord sections prepared 24 h after SCI. Spinal cord sections from sham-operated mice did not stain for iNOS (Fig. 3A, see densitometry analysis B), whereas spinal cord sections obtained from mice subjected to SCI exhibited positive staining for iNOS (Fig. 3A, see densitometry analysis B) mainly localized in various inflammatory cells in the gray matter. Ethyl pyruvate (75 mg/kg) treatment reduced the degree of positive staining for iNOS in the spinal cord tissues (Fig. 3A, see densitometry analysis B).
Fig. 3: Effects of EP on iNOS expression and nitrotyrosine formation. iNOS expression was evaluated by immunohistochemical analysis in the spinal cord sections 24 h after SCI. Spinal cord sections from sham-operated mice did not stain for iNOS (A), whereas spinal cord sections obtained from SCI-operated mice exhibited positive staining for iNOS (A, see densitometry analysis B) mainly localized in various inflammatory cells in the gray matter. Ethyl pyruvate (75 mg/kg) treatment reduced the degree of positive staining for iNOS in the spinal cord tissues (A, see densitometry analysis B). In addition, sections obtained from SCI animals demonstrated positive staining for nitrotyrosine mainly localized in inflammatory cells and in the nuclei of Schwann cells in the white and gray matter (C, see densitometry analysis D). Ethyl pyruvate treatment (75 mg/kg, 1 and 6 h after SCI induction) reduced the degree of positive staining for nitrotyrosine (C, see densitometry analysis D) in the spinal cord. Spinal cord sections from sham-operated mice did not stain for nitrotyrosine. This figure is representative of at least three experiments performed on different experimental days. Data are expressed as percentage of total tissue area. *P < 0.01 vs. sham; *°P < 0.01 vs. SCI.
Effects of EP on nitrotyrosine formation
Twenty-four hours after SCI, nitrotyrosine, a specific marker of nitrosative stress, was measured by immunohistochemical analysis in the spinal cord sections to determine the localization of various reactive nitrogen species produced during SCI. Spinal cord sections from sham-operated mice did not stain for nitrotyrosine (Fig. 3C, see densitometry analysis D), whereas spinal cord sections obtained from SCI mice exhibited positive staining for nitrotyrosine (Fig. 3C, see densitometry analysis D). The positive staining was mainly localized in inflammatory cells as well as in nuclei of Schwann cells in the white and gray matter of the spinal cord tissues. Ethyl pyruvate (75 mg/kg) reduced the degree of positive staining for nitrotyrosine (Fig. 3C, see densitometry analysis D) in the spinal cord.
Effect of EP on IκB-α degradation, phosphorylation of p65 on Ser536, NF-κB p65 activation
We evaluated IκB-α degradation, phosphorylation of Ser536 on the NF-κB subunit p65, nuclear NF-κB p65 expression by Western blot analysis to investigate the cellular mechanisms whereby treatment with EP attenuates the development of SCI. Basal expression of IκB-α was detected in spinal cord samples from sham-operated animals, whereas IκB-α levels were substantially reduced in SCI mice. Ethyl pyruvate (75 mg/kg) treatment prevented SCI-induced IκB-α degradation (Fig. 4A). In addition, SCI caused a significant increase in the phosphorylation of Ser536 at 24 h (Fig. 4B). Treatment with EP (75 mg/kg) significantly reduced phosphorylation of p65 on Ser536 (Fig. 3, B and B1). Moreover, NF-κB p65 levels in the spinal cord nuclear fractions were also significantly increased at 24 h after SCI compared with the sham-operated mice (Fig. 4C). Ethyl pyruvate treatment significantly reduced the levels of NF-κB p65, as shown in Figure 4C.
Fig. 4: Effects of EP treatment on IκB-α degradation, phosphorylation of NF-κB subunit p65 on the Ser536, nuclear NF-κB p65, and on activation of ERK1/2 MAPK. By Western blot analysis, a basal level of IκB-α was detected in the spinal cord from sham-operated animals, whereas IκB-α levels were substantially reduced in SCI mice. Ethyl pyruvate treatment (75 mg/kg, 1 and 6 h after SCI induction) prevented the SCI-induced IκB-α degradation (A). In addition, SCI caused a significant increase in the phosphorylation of Ser536 at 24 h (B) and in nuclear NF-κB p65 compared with the sham-operated mice (C). Ethyl pyruvate treatment significantly reduced the phosphorylation of p65 on Ser536 (B) and NF-κB p65 levels (C). The intensity of bands was measured using a phosphoimager in all the experimental groups. β-Actin was used as internal control. A representative blot of lysates obtained from each group is shown; the relative expression of the protein bands from three separated experiments was standardized for densitometric analysis to β-actin levels, and densitometric analysis of all animals is reported in A, B, and C (n = 5 mice from each group). *P < 0.01 vs. sham; °P < 0.01 vs. SCI. Spinal cord extracts were immunoblotted for active ERK1/2 (pERK1/2) and total ERK2 (ERK2). pERK1/2 is upregulated in injured mice as compared with sham-operated mice (D). Spinal cord levels of pERK1/2 were significantly attenuated in EP-treated (75 mg/kg) mice subjected to SCI in comparison to SCI animals.
EP modulates the activation of MAPK pathways after SCI
To investigate the cellular mechanisms by which treatment with EP attenuates the development of SCI, we evaluated phosphorylation of ERK1/2, a signaling molecule implicated in the regulation of proinflammatory gene expression. Activation of ERK1/2 was investigated by Western blotting of spinal cord obtained 24 h after SCI. A significant increase in pERK1/2 levels were observed in SCI mice (Fig. 6, A and A1). The treatment of mice with EP (75 mg/kg) significantly reduced the level of pERK1/2 (Fig. 4D).
Effect of EP on lipid peroxidation and PAR formation after SCI
In addition, at 24 h after SCI, thiobarbituric acid-reactive substances were also measured in the spinal cord tissue as an indicator of lipid peroxidation. As shown in Figure 5A, thiobarbituric acid-reactive substances were present at significantly higher levels in spinal cord tissue collected from mice subjected to SCI when compared with sham-operated mice. Lipid peroxidation was significantly attenuated in a dose-dependent fashion by treatment with EP (Fig. 5A). Activation of the enzyme, poly(ADP-ribosyl) polymerase (PARP), has been implicated in the pathogenesis of SCI. Accordingly, we used an immunohistochemical approach to assess the presence of PAR, as an indicator of in vivo PARP activation. As shown Figure 5B, there was positive staining for PAR localized in the nuclei of Schwann cells in the white and gray matter of the spinal cord tissues from mice subjected to SCI (see particles B and densitometry analysis C). Ethyl pyruvate (75 mg/kg) reduced the degree of positive staining for PAR (Fig. 5, see densitometry analysis C) in the spinal cord. No positive staining for PAR has been found in the spinal cord from sham-operated mice (Fig. 5B, see densitometry analysis C).
Fig. 5: Effects of EP on PAR, as an indicator of in vivo PARP activation and lipid peroxidation in spinal cord tissue. After the injury, thiobarbituric acid-reactant substances in spinal cord from SCI mice was significantly increased at 24 h after the damage in comparison to sham groups (A). Treatment with EP (8.5, 25, or 75 mg/kg) significantly attenuated these levels. Data are means ± SEM of 10 mice for each group. *P < 0.01 vs. sham; °P < 0.01 vs. SCI + vehicle. Immunohistochemistry for PAR revealed the occurrence of positive staining for PAR localized in the nuclei of Schwann cells in the white and gray matter of the spinal cord tissues from mice subjected to SCI (B, see particles and densitometry analysis C). Ethyl pyruvate (75 mg/kg) reduced the degree of positive staining for PAR (B, see particles and densitometry analysis C) in the spinal cord. No positive staining for PAR was found in spinal cord sections from sham-operated mice (B, see particles and densitometry analysis C). This figure is representative of at least three experiments performed on different experimental days. Data are expressed as percentage of total tissue area. *P < 0.01 vs. sham; *°P < 0.01 vs. SCI.
EP modulates expression of Fas ligand after SCI
Immunohistological staining for Fas ligand in the spinal cord was also determined 24 h after injury. Spinal cord sections from sham-operated mice did not stain for Fas ligand (Fig. 6A), whereas spinal cord sections obtained from SCI mice exhibited positive staining for Fas ligand (Fig. 6A, see densitometry analysis B) mainly localized in inflammatory cells as well as in nuclei of Schwann cells in the white (wm) and gray (gm) matter of the spinal cord tissues. Ethyl pyruvate (75 mg/kg) treatment reduced the degree of positive staining for Fas ligand in the spinal cord (Fig. 6A, see densitometry analysis B).
Fig. 6: Effect of EP on expression of Fas ligand (a and b) and on apoptosis measured by Ann-V staining (c) after SCI. A substantial increase in Fas ligand expression (A) 24 h after SCI was found in inflammatory cells and in the nuclei of Schwann cells in the white matter (wm) and gray matter (gm) of the spinal cord tissues from SCI mice. Spinal cord levels of Fas-ligand were significantly attenuated in EP-treated, SCI-injured mice in comparison to SCI animals (A). No positive staining for Fas ligand was found in spinal cord sections from sham-operated mice (A). Densitometry analysis of immunocytochemistry photographs (n = 5 photographs from each sample collected from all mice in each experimental group) for Fas ligand (B) from spinal cord tissues was assessed. The assay was carried out by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266, Milan, Italy). Data are expressed as percentage of total tissue area. This figure is representative of at least three experiments performed on different experimental days. *P < 0.01 vs. sham; *°P < 0.01 vs. SCI + vehicle. wm indicates white matter; gm, gray matter. At 24 h after injury, SCI mice demonstrated a marked appearance of stain positive for Ann-V FITC in the perilesional spinal cord tissue (f1). A positive intracellular staining to PI, an index of cells in the late stage of apoptosis, was evident in several cells (f2). On the contrary, spinal cord tissues section from EP-treated SCI mice demonstrated almost no cells in the early (g1) and in the later stages of apoptosis (g2). Almost no apoptotic cells in the early (e1) and in the later stages of apoptosis (e2) were detectable in the spinal cord tissue from sham-operated mice. Please note that e3, f3, and g3 represent the staining combination of e1-e2, f1-f2, and g1-g3, respectively. Sections e, f, and g represent the transmission light.
Effect of EP on apoptosis in spinal cord tissue after injury
To test whether the tissue damage was associated with cell death by apoptosis, we measured Ann-V staining in the perilesional spinal cord tissue at 24 h after SCI. Tissues obtained from SCI mice demonstrated marked staining for Ann-V (Fig. 6C, f1), a finding that indicates the presence of cells undergoing apoptosis. Some cells demonstrated positive intracellular staining with PI, indicating of the later stages of apoptosis (Fig. 6C, f2).In contrast, spinal cord tissue sections from EP-treated mice subjected to SCI demonstrated almost no cells in the early (Fig. 6C, g1) or the later stages of apoptosis (Fig. 6C, g2). Almost no apoptotic cells in the early (Fig. 6C, e1) and in the later stages of apoptosis (Fig. 6C, e2) were detectable in the spinal cord tissue sections from sham-operated mice. (Please note that Figures 6c e3, f3, and g3 represent the staining combination of panels e1-e2, f1-f2, and g1-g3, respectively. Sections e, f, and g represent the transmission light).
Western blot analysis and immunohistochemistry for Bax and Bcl-2
At 24 h after SCI, the appearance of the proapoptic protein, Bax, in spinal cord homogenates was investigated by Western blot. Bax levels were appreciably increased in the spinal cord from mice subjected to SCI (Fig. 7A). Treatment with EP (75 mg/kg) prevented SCI-induced Bax expression (Fig. 7A).
Fig. 7: Western blot analysis for Bax and Bcl-2 and immunohistochemical expression of Bax and Bcl-2. By Western blot analysis, Bax levels were appreciably increased in the spinal cord from SCI mice (A). On the contrary, EP treatment (75 mg/kg, 1 and 6 h after SCI induction) prevented SCI-induced Bax expression (A). Moreover, a basal level of Bcl-2 expression was detected in spinal cord samples from sham-operated mice. Bcl-2 expression was significantly reduced in spinal cord samples from SCI mice (B). Ethyl pyruvate treatment significantly reduced the SCI-induced inhibition of Bcl-2 expression (A). The results are expressed as mean ± SEM from n = 5/6 spinal cord for each group. *P < 0.01 vs. sham; °P < 0.01 vs. SCI + vehicle. Sections of spinal cord from sham-operated mice did not stain for Bax (C), whereas spinal cord sections obtained from SCI mice exhibited positive staining for Bax (C, see densitometry analysis D). Ethyl pyruvate (75 mg/kg) treatment reduced the degree of positive staining for Bax in the spinal cord of mice subjected to SCI (C, see densitometry analysis D). In addition, spinal cord sections from sham-operated mice demonstrated Bcl-2-positive staining (E, see densitometry analysis F), whereas in SCI control mice, the staining significantly reduced (E, see densitometry analysis F). Ethyl pyruvate (75 mg/kg) treatment attenuated the loss of positive staining for Bcl-2 in the spinal cord from SCI- subjected mice (E, see densitometry analysis F). This figure is representative of at least three experiments performed on different experimental days. Data are expressed as percentage of total tissue area. *P < 0.01 vs. sham; °P < 0.01 vs. SCI.
By Western blot analysis, expression of the antiapoptotic protein, Bcl-2, was apparent in spinal cord homogenates from sham-operated mice (Fig. 7B). Twenty-four hours after SCI, Bcl-2 expression was significantly reduced in spinal cord samples from SCI mice (Fig. 7, panels B and B1). Treatment of mice with EP (75 mg/kg) significantly blunted the SCI-induced inhibition of Bcl-2 expression (Fig. 7B).
Immunohistological staining for Bax and Bcl-2 also showed that spinal cord sections from sham-operated mice did not stain for Bax (Fig. 7C), whereas spinal cord sections obtained from SCI mice exhibited positive staining for Bax (Fig. 7C, see densitometry analysis D). Ethyl pyruvate (75 mg/kg) treatment reduced the degree of positive staining for Bax in spinal cord samples from mice subjected to SCI (Fig. 7C, see densitometry analysis D). In addition, spinal cord sections from sham-operated mice demonstrated Bcl-2 positive staining (Fig. 7E, see densitometry analysis F), whereas in SCI mice, the staining significantly decreased (Fig. 7E, see densitometry analysis F). Ethyl pyruvate (75 mg/kg) treatment attenuated the loss of positive staining for Bcl-2 in the spinal cord from SCI-subjected mice (Fig. 7E, see densitometry analysis F).
DISCUSSION
In this report, we demonstrated that EP exerts beneficial effects in a mouse compression model of SCI. We showed that SCI resulted in edema and tissue injury in lateral and dorsal funiculi. This histological damage was associated with a loss of motor function. Spinal cord injury induced an inflammatory response in the spinal cord, characterized by increased IκB-α degradation, enhanced NF-κB activation, upregulated expression of proinflammatory mediators, such as iNOS, and evidence of nitrosative and oxidative stress (nitrotyrosine and thiobarbituric acid-reactive product formation, respectively). Our results indicate that EP reduced (a) the degree of spinal cord damage, (b) neutrophilic infiltration, (c) NF-κB activation, (d) proinflammatory cytokine expression, (e) expression of iNOS, (f) nitrotyrosine and PAR formation, and (g) apoptosis.
All of these findings support the view that EP attenuates the degree of secondary inflammation and improves the motor recovery events. What, then, is the mechanism by which EP protects the spinal cord against this inflammatory injury?
Mitogen-activated protein kinase family members, including ERK1/2 and p38, are thought to be important mediators of signal transduction from cell surface to the nucleus. It was reported that neuroprotection of hypoxic preconditioning in cerebellar granular neurons was related to PI3-K/Akt activation and MEK/ERK phosphorylation. Moreover, Xu et al. (18) demonstrated enhanced activation of ERK1/2 and p38 MAPK in microglia/macrophages in the injured spinal cord after traumatic SCI. Mitogen-activated protein kinase activation was detectable within 1 h after injury and persisted for at least 24 h after injury. Moreover, Ulloa et al. (19) demonstrated that EP inhibited activation of p38 MAPK in LPS-stimulated RAW 264.7 cells. Similarly, in the present study, we demonstrate that EP reduced the activation of pERK1/2 associated with SCI. In addition, experimental evidence suggests that activation of the transcription factor NF-κB plays a central role in the regulation of many genes responsible for the generation of mediators or proteins in secondary inflammation associated to SCI (20). Nuclear factor κB is normally sequestered in the cytoplasm, bound to regulatory proteins IκBs. The exact mechanisms by which EP suppresses NF-κB activation in inflammation are not completely known. However, Han et al. (21) reported that EP has no effect on the degradation of IκB-α or IκB-β in LPS-stimulated RAW 264.7 cells, suggesting that EP acts distally to this step in the activation of NF-κB. In contrast, EP inhibited DNA binding by ectopically overexpressed wild-type p65 homodimers (21). These results suggest that EP inhibits signaling via the NF-κB pathway by covalently modifying p65 (21). We report here that SCI was associated with significant IκB-α degradation as well as increased phosphorylation of Ser536 on p65 in spinal cord tissues at 24 h after injury. Treatment with EP significantly reduced IκB-α degradation as well as the NF-κB translocation.
Taken together, the balance between proinflammatory and prosurvival roles of NF-κB may depend on the phosphorylation status of p65, and MAPK can play a central role in this process. However, the reasons for the apparent discrepancies in the modulatory effects of EP on NF-κB activity remain to be fully clarified. One possibility is suggested by evidence indicating that the activation of NF-κB is under the control of oxidant/antioxidant balance (22).
Nuclear factor κB plays a central role in the regulation of many genes responsible for the generation of mediators or proteins in inflammation. These include the genes for TNF-α, IL-1β, and iNOS, to name a few (23). Herein, we documented increased production of both TNF-α and IL-1β after SCI. Remarkably, there was no increase in the expression of either TNF-α or IL-1β in the spinal cord sections obtained from mice subjected to SCI and treated with EP. This observation is in agreement with a previous study in which EP attenuated the serum levels of TNF-α in an experimental mouse model of septic shock (19). It has also been demonstrated that enhanced formation of NO by iNOS contributes to the inflammatory process (24). This study demonstrates that EP attenuates the expression of iNOS in the tissue from SCI-treated mice when compared with untreated injured mice. These observations are in agreement with previous observations, which showed that the treatment with EP significantly reduced iNOS expression in vitro as well as in vivo (21). Therefore, the inhibition of iNOS expression by EP described in the present study is likely to be due to the inhibition of NF-κB activation by EP.
Moreover, it is well known that pyruvic acid, a compound that is closely related to EP, is an effective scavenger of the reactive oxygen species, hydrogen peroxide (25). In addition, it has been shown that EP inhibits lipid peroxidation, a marker of oxidant stress, in vitro (26) and in vivo (27). In addition, in the present study, we clearly demonstrated that EP treatment fully inhibited the appearance of nitrotyrosine staining, an indication of "increased nitrosative stress" in the inflamed tissue. Moreover, the reduced neutrophil infiltration into the inflamed tissue may be related to these previously described actions of EP. Further study will be needed to determine the relative contribution of EP's multiple modes of action to its anti-inflammatory and cytoprotective effects observed in the current study.
A novel pathway of inflammation, governed by the nuclear enzyme PARP, has been proposed in relation to hydroxyl radical- and peroxynitrite-induced DNA single-strand breakage (28). Recently, it has been demonstrated that PARP activation plays an important role in the central nervous inflammation (29). In addition, it has been shown that various PARP inhibitors exerts protective effects in models of SCI (30). We demonstrate here that EP reduced the increase in PARP activation in the spinal cord in animals subjected to SCI. Thus, we propose that the anti-inflammatory effects of EP may be at least in part due to the prevention of the activation of PARP.
Several studies suggest that glial cells in neurodegenerative diseases (i.e., Alzheimer disease) are affected more than neurons by apoptotic cell death. Apoptosis occurs in two distinct phases: an initial phase, in which apoptosis accompanies necrosis in the degeneration of multiple cell types, and a later phase, which is predominantly confined to white matter and involves oligodendrocytes and microglia (31). Herein, we demonstrated that the treatment with EP attenuated the extent of apoptosis, measured by Ann-V staining, in the spinal cord after SCI. Moreover, various studies have postulated that preserving Bax, a proapoptotic protein, plays an important role in developmental cell death and in CNS injury (32). Similarly, it has been shown that the administration of Bcl-xL fusion protein (Bcl-2 is the most expressed antiapoptotic molecule in adult CNS) into injured spinal cords significantly increased neuronal survival, suggesting that SCI-induced changes in Bcl-xL contribute considerably to neuronal death (33). In our study, we showed that SCI is associated with proapoptotic transcriptional changes, including upregulation of Bax expression and downregulation of Bcl-2 expression. Ethyl pyruvate treatment prevented both of these effects. Fas and TNF-α p75 receptors are expressed on oligodendrocytes, astrocytes, and microglia in the spinal cord after SCI. Fas and p75 colocalize on many TUNEL-positive cells, suggesting that the Fas- and p75-initiated cell death cascades may participate in the demise of some glia after SCI. The endogenous ligand for Fas, FasL, plays a central role in apoptosis induced by a variety of chemical and physical insults (34). Recently, it has been pointed out that Fas/FasL signaling plays a central role in SCI (35). We confirmed here that SCI leads to substantial activation of FasL in the spinal cord tissues, which likely contributes to the evolution of tissues injury. In the present study, we found that EP treatment reduces FasL activation. However, it is not possible to exclude that the antiapoptotic effects of EP treatment may be partially dependent on the attenuation of the inflammatory-induced damage.
After more than 50 years of study in SCIs, there were important developments, the most important of which involved various and numerous approaches at secondary damage. With breakthroughs in the molecular understanding of neural injury and possible repair, recent neuroscience advances have opened the door for hope toward prevention and cure of the devastating effects of SCI. The whole field, ranging from molecular biology to quality-of-life aspects, is too large for any organization to cover adequately, and we needed to concentrate our efforts on problems that would ensure progress in SCI repair was as rapid as possible.
In conclusion, this study demonstrates that EP is able to produce a substantial reduction of inflammatory events associated with experimental SCI through a NF-κB-dependent mechanism via p42/p44 MAPK. Our results imply EP may be useful in the therapy of SCI, trauma, and inflammation.
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