Selective adenosine A(2a) receptor agonists reduce the apoptosis in an experimental model of spinal cord trauma.

Adenosine is an important regulator of inflammatory mechanisms. Functional studies indicate a protective effect of adenosine A2A receptor agonists in spinal cord injury (SCI). The basic molecular mechanisms accounting for their protective effects from spinal cord injury have to be fully elucidated. The aim of this study is to evaluate in vivo protection by two selective A2A receptor agonists, 2-[p-(2-carboxyethyl)phenylethylamino]-50-ethylcarboxamidoadenosine (CGS 21680, 100 microg/kg) and (4-[3-(6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl)prop-2-ynyl] piperidine-1-carboxylic acid methyl ester) (ATL 313, 3 microg/kg) on the degree of apoptosis, in the experimental model of spinal cord injury. Spinal cord trauma was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. Spinal cord trauma in mice was characterised by edema, neutrophilic infiltration and apoptosis. ATL 313, administered by subcutaneously implanted osmotic minipumps after SCI, clearly reduced motor deficit for up to 19 days after operation. The selective A2A receptor agonists ATL 313 and CGS 21680 administered after SCI, reduced tissue damage, TUNEL staining, cytokine (TNF-alpha) expression, Bax, Fas-L and Caspase-3 expression, Annexin-V staining, while increasing Bcl-2 expression. In conclusion, our results demonstrate that treatment with adenosine A2A receptor agonists prevents the apoptotic process that is an important step of secondary damage after SCI.

Effect of PD98059, a selective MAPK3/MAPK1 inhibitor, on acute lung injury in mice.

The aim of the present study is to evaluate the contribution of mitogen-activated protein kinase 1-3 MAPK3/MAPK1) in a model of acute lung inflammation in mice. Injection of carrageenan into the pleural cavity of mice elicited an acute inflammatory response characterized by: accumulation of fluid containing a large number of neutrophils (PMNs) in the pleural cavity, infiltration of PMNs in lung tissues and subsequent adhesion molecule expression (I-CAM and P-selectin), lipid peroxidation, and increased production of tumour necrosis factor-alpha, (TNF-alpha) and interleukin-1beta (IL-1beta). Furthermore, carrageenan induced lung apoptosis (Bax and Bcl-2 expression) as well as nitrotyrosine formation, NF-kB activation, and pJNK expression, as determined by immunohistochemical analysis of lung tissues and the degree of lung inflammation and tissue injury (histological score). Administration of PD98059, an inhibitor of MAPK3/MAPK1 (10 mg/kg) 1 h after carrageenan caused a reduction in all the parameters of inflammation measured. Thus, based on these findings we propose that inhibitors of the MAPK3/MAPK1 signaling pathways, such as PD98059, may be useful in the treatment of various inflammatory diseases.

Role of free radicals and poly(ADP-ribose)polymerase-1 in the development of spinal cord injury: new potential therapeutic targets.

Oxidative stress results from an oxidant/antioxidant imbalance, an excess of oxidants and/or a depletion of antioxidants. A vast amount of circumstantial evidence implicates oxygen-derived free radicals (especially, superoxide and hydroxyl radical) and high energy oxidants (such as peroxynitrite) as mediators of secondary damage associated with spinal cord injury. Reactive oxygen species (ROS) (e.g., superoxide, peroxynitrite, hydroxyl radical and hydrogen peroxide) are all potential reactants capable of initiating DNA single strand breakage, with subsequent activation of the nuclear enzyme poly (ADP ribose) synthetase (PARS), leading to eventual severe energy depletion of the cells, and necrotic-type cell death. Moreover, Poly(ADP-ribosyl)ation is regulated by the synthesizing enzyme poly(ADP-ribose) polymerase-1 (PARP-1) and the degrading enzyme poly(ADP-ribose) glycohydrolase (PARG). Here, we review the roles of ROS, PARP-1 and PARG in spinal cord injury as well as the beneficial effect of the in vivo treatment with novel pharmacological tools (e.g. peroxynitrite decomposition catalysts, selective superoxide dismutase mimetics (SODm), PARP-1 and PARG inhibitors.

Effect of tumour necrosis factor-alpha receptor 1 genetic deletion on carrageenan-induced acute inflammation: a comparison with etanercept.

In the present study, we used tumour necrosis factor-alpha receptor 1 knock-out mice (TNF-alphaR1KO) to evaluate an in vivo role of TNF-alphaR1 on the pathogenesis of inflammatory diseases. We used a murine model of carrageenan-induced acute inflammation (pleurisy), a preclinical model of airway inflammation. The data proved that TNF-alphaR1KO were resistant to carrageenan-induced acute inflammation compared with TNF-alpha wild-type mice. TNF-alphaR1KO 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-alphaR1KO at 4 h and 24 h after carrageenan injection. Moreover, TNF-alpha and interleukin-1beta concentrations were reduced in inflamed areas and in pleural exudates from TNF-alphaR1KO. To support the results generated using pleural inflammation, carrageenan-induced paw oedema models were also performed. In order to elucidate whether the observed anti-inflammatory effects were related to the inhibition of TNF-alpha, we also investigated the effect of etanercept, a TNF-alpha 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 carrageenan-induced pleurisy. Our results showed that administration of etanercept resulted in the same outcome as that of deletion of the TNF-alphaR1 receptor, adding a new insight to TNF-alpha as an excellent target by therapeutic applications.

Effects of zileuton and montelukast in mouse experimental spinal cord injury.

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.

Combination of dexamethasone and etanercept reduces secondary damage in experimental spinal cord trauma.

The aim of our study was to evaluate the therapeutic efficacy of combination therapy with etanercept and dexamethasone (DEX) in vivo in experimental murine model of spinal cord trauma, which was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. Spinal cord injury in mice resulted in severe trauma characterized by edema, neutrophil infiltration, and cytokine production followed by recruitment of other inflammatory cells, production of inflammation mediators, tissue damage, apoptosis and disease. Treatment of the mice with etanercept (1.25 mg/kg) and DEX (0.025 mg/kg) when administered as a combination therapy but not as a single treatment significantly reduced the degree of (1) spinal cord inflammation and tissue injury (histological score), (2) infiltration of neutrophils (MPO evaluation), (3) inducible nitric oxide synthase, nitrotyrosine, and cytokines expression (tumor necrosis factor-alpha and interleukin-1 beta), (4) and apoptosis (Terminal deoxynucleotidyltransferase-mediated UTP end labeling staining, Fas-ligand expression and Bax and Bcl-2 expression). In a separate set of experiments we have also clearly demonstrated that the combination therapy significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly demonstrate for the first time that strategies targeting multiple proinflammatory pathways may be more effective than a single effector molecule for the treatment of spinal cord trauma.

Glycogen synthase kinase-3beta inhibition attenuates the development of bleomycin-induced lung injury.

Glycogen synthase kinase-3 (GSK-3) is an ubiquitous serine-threonine protein kinase that participates in a multitude of cellular processes and has recently been implicated in the pathophysiology of a number of diseases. The aim of this study is to investigate the effects of TDZD-8, a potent and selective GSK-3beta inhibitor, on the development of lung injury caused by administration of bleomycin (BLM). Mice subjected to intra-tracheal administration of BLM developed significant lung injury characterized by marked neutrophil infiltration and tissue edema. An increase in immunoreactivity to nitrotyrosine, iNOS, TNF-alpha and IL-1beta was also observed in the lungs of BLM-treated mice. In contrast, administration of BLM-treated mice with TDZD-8 (1 mg/kg daily) significantly reduced (I) the degree of lung injury, (II) the increase in staining (immunohistochemistry) for myeloperoxidase (MPO), nitrotyrosine, iNOS, TNF-alpha and IL-1beta and (III) the degree of apoptosis, as evaluated by Bax and Bcl-2 immunoreactivity and TUNEL staining. Taken together, these results clearly demonstrate treatment with the GSK-3beta inhibitor TDZD-8 reduces the development of lung injury and inflammation induced by BLM in mice.

Rosiglitazone reduces the evolution of experimental periodontitis in the rat.

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) receptor appears to play a pivotal role in the regulation of cellular proliferation and inflammation. Recent evidence also suggests that rosiglitazone, a PPAR-gamma agonist, reduces acute and chronic inflammation. We hypothesized that rosiglitazone would attenuate periodontal inflammation. In the present study, we investigated the effects of rosiglitazone in a rat model of ligature-induced periodontitis. At day 8, ligation significantly induced an increase in neutrophil infiltration, as well as of gingivomucosal tissue expression of iNOS, nitrotyrosine formation, and poly (ADP-ribose) polymerase activation. Ligation significantly increased Evans blue extravasation in gingivomucosal tissue and alveolar bone destruction. Intraperitoneal injection of rosiglitazone (10 mg/kg 10% DMSO daily for 8 days) significantly decreased all of the parameters of inflammation, as described above. Analysis of these data demonstrated that rosiglitazone exerted an anti-inflammatory role during experimental periodontitis, and was able to ameliorate the tissue damage associated with ligature-induced periodontitis.

Effect of aminoguanidine in ligature-induced periodontitis in rats.

The role of nitric oxide and reactive oxygen species is well-demonstrated in inflammation. In this study, we evaluated the effect of aminoguanidine, a nitric oxide synthase inhibitor, in a rat model of periodontitis. We induced periodontitis in rats by placing a piece of 2/0 braided silk around the lower left 1st molar. At day 8, the gingivomucosal tissue encircling the mandibular 1st molar was removed for biochemical and histological analysis. Ligation significantly increased inducible nitric oxide synthase activity and expression, and damaged tissue revealed increased neutrophil infiltration, lipid peroxidation, and positive staining for nitrotyrosine formation and poly (ADP-ribose) polymerase activation. Ligation significantly increased Evans blue extravasation in gingivomucosal tissue and alveolar bone destruction. Aminoguanidine (100 mg/kg i.p., daily for 8 days) treatment significantly reduced all these inflammatory parameters, indicating that it protects against the tissue damage associated with periodontitis by reducing nitric oxide production and oxidative stress.

Role of inflammation in the secondary injury following experimental spinal cord trauma.

AIM: The primary traumatic mechanical injury to the spinal cord causes the death of a number of neurons that cannot be recovered, neither regenerated. However, neurons continue to die for hours after spinal cord injury (SCI), and this represents a potentially avoidable event. One of mechanisms that have been touted to contribute importantly to the evolution of such secondary cell death is the local inflammatory response in the injured spinal cord. In this report we have used an in vivo model to induce acute SCI and reproduce the acute pathological events associated with inflammation after traumatic SCI in rats. METHODS: Twenty-two adult male Sprague-Dawley rats were used in the study. SCI was produced by extradural clip compression at T5-T9 level. The rats spinal cord was analysed at 1 hour to measure the malonildialdehyde (MDA) levels considered an index of lipid peroxidation. At 4 hours were measured the levels of myeloperoxidase (MPO) activity considered as the index of leukocytes activity. Finally the spinal cord was extracted 12 hours after the trauma to measure the cytoplasmatic levels of IkB-a considered as the index of activity of the transcriptional factor nuclear factor-kB (NF-kB). RESULTS: After the SCI, both the levels of MDA and MPO were significantly higher compared with naive and sham-operated rats (p=0.01). Western blotting analysis demonstrated the disappearance of IkB-alpha in the cytoplasm indicating nuclear translocation of the NF-kB. CONCLUSION: The study confirms the role of inflammation in contributing to the secondary injury after experimental SCI in the rat.

Evaluating breast cancer risk: available models to assess individual breast cancer risk and probability to be a BRCA mutation carrier.

Several models have been generated to estimate breast cancer risk and to predict the probability to identify a BRCA1 or BRCA2 germ line mutation. Each model, based on a slightly different study design and purpose, takes into account a definite number of relevant parameters and ultimately pertains to a specific cohort of women or population. Aim of this work is to underline validity and limits of available tools and their appropriate use in the daily practice.

Neuroprotective effects of almond skins in experimental spinal cord injury.

BACKGROUND & AIMS: Functional deficits following spinal cord injury (SCI) arise from both mechanical injury and from secondary tissue reactions involving inflammation. Natural almond skins (NS) were tested to evaluate anti-inflammatory effects on an animal model of SCI. METHODS: SCI was induced by the application of vascular clips to the dura via a four-level T5-T8 laminectomy. In the present study, to elucidate whether the protective effects of NS are related to the total phenolic content, we also investigated the effect of a blanched (BS) almond skins (industrially obtained by removing bran from the nut) in SCI. NS and BS (30 mg/kg respectively) were administered per os, 1 h and 6 h, after SCI. RESULTS: SCI in mice resulted in severe injury characterized by edema, tissue damage, production of inflammatory mediators and apoptosis (measured by Bax, Bcl-2 and Tunel assay). NS treatment, 1 and 6 h after SCI, reduced all parameters of inflammation as neutrophil infiltration, NF-κB activation, PAR formation, iNOS expression and apoptosis. However, treatment with BS did not exert any protective effect. CONCLUSIONS: Our results suggest that NS treatment, reducing the development of inflammation and tissue injury, may be useful in the treatment of SCI.

Effect of rosiglitazone and 15-deoxy-Delta12,14-prostaglandin J2 on bleomycin-induced lung injury.

Thiazolidinedione rosiglitazone and 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), are two peroxisome proliferator-activated receptor (PPAR)-gamma ligands. The aim of this study was to investigate the effect of rosiglitazone and 15d-PGJ2 on the lung injury caused by bleomycin administration. Mice subjected to intratracheal administration of bleomycin developed significant lung injury. An increase in immunoreactivity to nitrotyrosine, poly(ADP ribose) polymerase (PARP) and inducible nitric oxide synthase as well as a significant loss of body weight and mortality was observed in the lung of bleomycin-treated mice. Administration of the two PPAR-gamma agonists rosiglitazone (10 mg x kg(-1) i.p.) and 15d-PGJ2 (30 microg x kg(-1) i.p.) significantly reduced the: 1) loss of body weight, 2) mortality rate, 3) infiltration of the lung with polymorphonuclear neutrophils (myeloperoxidase activity), 4) oedema formation, and 5) histological evidence of lung injury. Administration of rosiglitazone and 15d-PGJ2 also markedly reduced the nitrotyrosine, PARP and inducible nitric oxide synthase formation. In addition, treatment with the PPAR-gamma antagonist bisphenol A diglycidyl ether (1 mg x kg(-1) i.p. 30 min before the rosiglitazone or 15d-PGJ2) significantly antagonised the effect of the two PPAR-gamma agonists. These results demonstrate that the two peroxisome proliferator-activated receptor-gamma agonists, rosiglitazone and 15-deoxy-Delta12,14-prostaglandin J2, significantly reduce lung injury induced by bleomycin in mice.