Interleukin‐10 (IL‐10) exerts a wide spectrum of regulatory activities in the immune and inflammatory response. The aim of this study was to investigate the role of endogenous IL‐10 on the modulation of the secondary events in mice subjected to spinal cord injury induced by the application of vascular clips (force of 24 g) to the dura via a four‐level T5–T8 laminectomy. IL‐10 wild‐type mice developed severe spinal cord damage characterized by oedema, tissue damage and apoptosis (measured by Annexin‐V, terminal deoxynucleotidyltransferase‐mediated UTP end labeling staining, Bax, Bcl‐2, and Fas‐L expression). Immunohistochemistry demonstrated a marked increase of localization of TNF‐α, IL‐1β and S100β, while western blot analysis shown an increased immunoreactivity of inducible nitric oxide synthase in the spinal cord tissues. The absence of IL‐10 in IL‐10 KO mice resulted in a significant augmentation of all the above described parameters. We have also demonstrated that the genetic absence of IL‐10 worsened the recovery of limb function when compared with IL‐10 wild‐type mice group (evaluated by motor recovery score). Taken together, our results clearly demonstrate that the presence of IL‐10 reduces the development of inflammation and tissue injury events associated with spinal cord trauma.

Myrtucommulone (MC), a nonprenylated acylphloroglucinol contained in the leaves of myrtle (Myrtus communis), has been reported to suppress the biosynthesis of eicosanoids by inhibition of 5-lipoxygenase and cyclooxygenase-1 in vitro and to inhibit the release of elastase and the formation of reactive oxygen species in activated polymorphonuclear leukocytes. Here, in view of the ability of MC to suppress typical proinflammatory cellular responses in vitro, we have investigated the effects of MC in in vivo models of inflammation. MC was administered to mice intraperitoneally, and paw edema and pleurisy were induced by the subplantar and intrapleural injection of carrageenan, respectively. MC (0.5, 1.5, and 4.5 mg/kg i.p.) reduced the development of mouse carrageenan-induced paw edema in a dose-dependent manner. Moreover, MC (4.5 mg/kg i.p. 30 min before and after carrageenan) exerted anti-inflammatory effects in the pleurisy model. In particular, 4 h after carrageenan injection in the pleurisy model, MC reduced: 1) the exudate volume and leukocyte numbers; 2) lung injury (histological analysis) and neutrophil infiltration (myeloperoxidase activity); 3) the lung intercellular adhesion molecule-1 and P-selectin immunohistochemical localization; 4) the cytokine levels (tumor necrosis factor-␣ and interleukin-1␤) in the pleural exudate and their immunohistochemical localization in the lung; 5) the leukotriene B 4 , but not prostaglandin E 2 , levels in the pleural exudates; and 6) lung peroxidation (thiobarbituric acid-reactant substance) and nitrotyrosine and poly (ADP-ribose) immunostaining. In conclusion, our results demonstrate that MC exerts potent anti-inflammatory effects in vivo and offer a novel therapeutic approach for the management of acute inflammation.

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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-a (TNF-a) and cyclooxygenase-2 (COX-2) expression, prostaglandin E 2 (PGE 2 ) and leukotriene B 4 (LTB 4 ) production, and extracellular signalregulated 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-a, 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.

Permanent functional deficits following spinal cord injury (SCI) arise from both mechanical injury and from secondary tissue reactions involving inflammation. The mitogen-activated protein kinases (MAPKs) play a critical role in cell signaling and gene expression. MAPK family includes three major members: extracellular signal regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), representing three different signaling cascades. Moreover, various studies have clearly shown that high-mobility group box 1 (HMGB1) protein is implicated as a putative danger signal involved in the pathogenesis of a variety of inflammatory conditions including autoimmunity, cancer, trauma and hemorrhagic shock, and ischemia-reperfusion injury. Recently, we have reported that the pineal secretory product melatonin exerts important anti-inflammatory effects in an experimental model of SCI induced by the application of vascular clips (force of 24 g) to the dura after a four-level T5-T8 laminectomy. However, no reports are available on the effect of melatonin on MAPK signaling pathways and HMGB1 expression in SCI. The aim of the present study was to evaluate whether the melatonin protective effect observed in SCI is related to the regulation of MAPK signaling pathways and HMGB1 in mice. In this study we demonstrate the efficacy of treatment with the melatonin in SCI in mice in reducing (a) motor recovery, (b) activation of MAPKs p38, JNK and ERK1/2, (c) tumor necrosis factor-alpha expression, and (d) expression of HMGB1. We propose that melatonin's ability to reduce SCI in mice is also related to a reduction in MAPK signaling pathways and HMGB1 expression.

Matrix metalloproteinases (MMPs) are associated with matrix turnover in both physiological and pathological conditions. Several data indicate that MMPs play an important role in the pathogenesis of colitis. Various evidence has documented that the pineal secretory product melatonin exerts an important anti-inflammatory effect in different experimental models including colitis. However, no reports are available on the relationship between the activity and expression of MMPs and anti-inflammatory effect of melatonin. The aim of the present study was to evaluate whether melatonin prevents the experimental colitis in rats by regulating MMP-9 and MMP-2 activity and expression. Colitis was induced by intracolonic instillation of dinitrobenzene sulphonic acid (DNBS). Four days after DNBS administration, colon TNF-alpha production was associated with colon damage. Biochemical methods and zymography were used to analyse MMP-9 and MMP-2 activities in colon tissues from DNBS-injured rats. Our studies reveal that melatonin prevented colon injury and lipid peroxidation in rats at 4 days after DNBS-induced colitis. Melatonin also reduced proMMP-9 and MMP-2 activities that were induced in the colon tissues by DNBS administration. Reduced MMP-9 and MMP-2 activities were associated with reduced expression of TNF-alpha. We conclude that melatonin's ability to reduce DNBS-induced colon injury in rats is related to a reduction in proMMP-9 and MMP-2 activities and expression.

SummaryIn the present study, we used tumour necrosis factor-a receptor 1 knock-out mice (TNF-aR1KO) to evaluate an in vivo role of TNF-aR1 on the pathogenesis of inflammatory diseases. We used a murine model of carrageenaninduced acute inflammation (pleurisy), a preclinical model of airway inflammation. The data proved that TNF-aR1KO were resistant to carrageenan-induced acute inflammation compared with TNF-a wild-type mice. TNF-aR1KO showed a significant reduction in accumulation of pleural exudate and in the number of inflammatory cells, in lung infiltration of polymorphonuclear leucocytes and lipid peroxidation and showed a decreased production of nitrite/nitrate in pleural exudates. Furthermore, the intensity and degree of the adhesion molecule intercellular adhesion molecule-1 and P-selectin, Fas ligand (FasL), inducible nitric oxide sythase and nitrotyrosine determined by immunohistochemical analysis were reduced markedly in lung tissues from TNF-aR1KO at 4 h and 24 h after carrageenan injection. Moreover, TNF-a and interleukin-1b concentrations were reduced in inflamed areas and in pleural exudates from TNF-aR1KO. To support the results generated using pleural inflammation, carrageenaninduced paw oedema models were also performed. In order to elucidate whether the observed anti-inflammatory effects were related to the inhibition of TNF-a, we also investigated the effect of etanercept, a TNF-a soluble receptor construct, on carrageenan-induced pleurisy. The treatment with etanercept (5 mg/kg subcutaneously 2 h before the carrageenan injection) reduces markedly both laboratory and histological signs of carrageenaninduced pleurisy. Our results showed that administration of etanercept resulted in the same outcome as that of deletion of the TNF-aR1 receptor, adding a new insight to TNF-a as an excellent target by therapeutic applications.

The matrix metalloproteinases (MMPs) are important enzymes that regulate developmental processes, maintain normal physiology in adulthood and have reparative roles at specific stages after an insult to the nervous system. MMPs, particularly MMP-9/gelatinase B, promote early inflammation and barrier disruption after spinal cord injury (SCI). Recently, we have reported that the pineal secretory product melatonin exerts important anti-inflammatory effects in an experimental model of SCI induced by the application of vascular clips (force of 24 g) to the dura after a four-level T5-T8 laminectomy. However, no reports are available on the relationship between the activity of MMPs and melatonin's anti-inflammatory effects. The aim of the present study was to evaluate whether the protective effect of melatonin observed in SCI is related to the regulation of MMP-9 and MMP-2 in mice. Biochemical and zymographic methods were used to analyze MMP-9 and -2 expression and activities in spinal cord tissue from SCI-treated mice at 24 hr after the trauma. Our studies reveal that melatonin reduced SCI and lipid peroxidation in spinal cord at 24 hr after SCI. Melatonin also diminished proMMP-9 and -2 activities that were induced in the spinal cord tissues at 24 hr after SCI. The reduced activities of MMP-9 and -2 were associated with depressed expression of TNF-alpha. We propose that melatonin's ability to reduce SCI in mice is also related to a reduction in MMP-9 and MMP-2 activity and expression.