Showing papers by "Achille P. Caputi published in 2000"

Effects of n-acetylcysteine in a rat model of ischemia and reperfusion injury.

Abstract: Objective: Splanchnic artery occlusion shock (SAO) causes an enhanced formation of reactive oxygen species (ROS), which contribute to the pathophysiology of shock. Here we have investigated the effects of n -acetylcysteine (NAC), a free radical scavenger, in rats subjected to SAO shock. Methods and results: Treatment of rats with NAC (applied at 20 mg/kg, 5 min prior to reperfusion, followed by an infusion of 20 mg/kg/h) attenuated the mean arterial blood and the migration of polymorphonuclear cells (PMNs) caused by SAO-shock. NAC also attenuated the ileum injury (histology) as well as the increase in the tissue levels of myeloperoxidase (MPO) and malondialdehyde (MDA) caused by SAO shock in the ileum. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine in the plasma of the SAO-shocked rats after reperfusion. Immunohistochemical analysis for nitrotyrosine and for poly(ADP-ribose) synthetase (PARS) revealed a positive staining in ileum from SAO-shocked rats. The degree of staining for nitrotyrosine and PARS were markedly reduced in tissue sections obtained from SAO-shocked rats which had received NAC. Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin, which was mainly localised in the vascular endothelial cells. Ileum tissue section obtained from SAO-shocked rats with anti-intercellular adhesion molecule (ICAM-1) antibody showed a diffuse staining. NAC treatment markedly reduced the intensity and degree of P-selectin and ICAM-1 in tissue section from SAO-shocked rats. In addition, in ex vivo studies in aortic rings from shocked rats, we found reduced contractions to noradrenaline and reduced responsiveness to a relaxant effect to acetylcholine (vascular hyporeactivity and endothelial dysfunction, respectively). NAC treatment improved contractile responsiveness to noradrenaline, enhanced the endothelium-dependent relaxations and significantly improved survival. Conclusion: Taken together, our results clearly demonstrate that NAC treatment exert a protective effect and part of this effect may be due to inhibition of the expression of adhesion molecule and peroxynitrite-related pathways and subsequent reduction of neutrophil-mediated cellular injury.

Protective effects of melatonin in ischemic brain injury

Abstract: Recent studies have demonstrated that melatonin is a scavenger of oxyradicals and peroxynitrite and an inhibitor of nitric oxide (NO) production. NO, peroxynitrite (formed from NO and superoxide anion), and poly (ADP-Ribose) synthetase (PARS) have been implicated as mediators of neuronal damage following focal ischemia. In the present study, we have investigated the effects of melatonin treatment in Mongolian gerbils subjected to cerebral ischemia. Treatment of gerbils with melatonin (10 mg kg−1, 30 min before reperfusion and 1, 2, and 6 hr after reperfusion) reduced the formation of post-ischemic brain edema, evaluated by water content. Melatonin also attenuated the increase in the brain levels malondialdehyde (MDA) and the increase in the hippocampus of myeloperoxidase (MPO) caused by cerebral ischemia. Positive staining for nitrotyrosine was found in the hippocampus of Mongolian gerbils subjected to cerebral ischemia. Hippocampus tissue sections, from Mongolian gerbils subjected to cerebral ischemia, also showed positive staining for PARS. The degrees of staining for nitrotyrosine and for PARS were markedly reduced in tissue sections obtained from animals that received melatonin. Melatonin treatment increased survival and reduced hyperactivity linked to neurodegeneration induced by cerebral ischemia and reperfusion. Histological observations of the pyramidal layer of CA-1 showed a reduction of neuronal loss in animals that received melatonin. These results show that melatonin improves brain injury induced by transient cerebral ischemia.

Inducible nitric oxide synthase-knockout mice exhibit resistance to pleurisy and lung injury caused by carrageenan.

Abstract: In the present study, we investigated the role of inducible (or type 2) nitric oxide synthase (iNOS) in the development of acute inflammation by comparing the responses in wild-type mice (WT) and mice lacking (knockout [KO]). When compared with carrageenan-treated iNOS-WT mice, iNOS-KO mice that had received carrageenan exhibited a reduced degree of pleural exudation and polymorphonuclear cell migration. Lung myeloperoxidase (MPO) activity and lipid peroxidation were significantly reduced in iNOS-KO mice in comparison with iNOSWT mice. Immunohistochemical analysis for nitrotyrosine revealed positive staining in lungs from carrageenan-treated iNOS-WT mice. Lung tissue sections from carrageenan-treated iNOS-WT mice showed positive staining for poly adenosine diphosphate (ADP)-ribose synthetase that was mainly localized in alveolar macrophages and in airway epithelial cells. The intensity and degree of staining for nitrotyrosine and poly-ADP-ribose synthetase were markedly reduced in tissue sections from carrageenan-treated iNOS-KO mice. The inflamed lungs of iNOS-KO mice also showed an improved histologic status. Furthermore, a significant reduction in the suppression of energy status, in DNA strand breakage, and in decreased cellular levels of nicotinamide adenine dinucleotide (NAD(+)) was observed ex vivo in macrophages harvested from the pleural cavity of iNOS-KO mice subjected to carrageenan-induced pleurisy. Taken together, our results clearly show that iNOS plays an important role in the acute inflammatory response.

Beneficial effects of peroxynitrite decomposition catalyst in a rat model of splanchnic artery occlusion and reperfusion

Abstract: The aim of the present study was to investigate the protective effect of the peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)-porphyrinato iron (III) (FeTMPS) in a model of splanchnic artery occlusion shock (SAO). SAO shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed by release of the clamp (reperfusion). At 60 min after reperfusion, animals were killed for histological examination and biochemical studies. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine (a marker of peroxynitrite-induced oxidative processes) in the plasma of the SAO-shocked rats after reperfusion, but not during ischemia alone. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, an index of nitrogen species such as peroxynitrite, in the necrotic ileum in shocked rats. SAO-shocked rats developed a significant increase of tissue myeloperoxidase...

Beneficial effects of n-acetylcysteine on ischaemic brain injury.

Abstract: Nitric oxide (NO), peroxynitrite, formed from NO and superoxide anion, poly (ADP-ribole) synthetase have been implicated as mediators of neuronal damage following focal ischaemia. Here we have investigated the effects of n-acetylcysteine (NAC) treatment in Mongolian gerbils subjected to cerebral ischaemia. Treatment of gerbils with NAC (20 mg kg−1 30 min before reperfusion and 1, 2 and 6 h after reperfusion) reduced the formation of post-ischaemic brain oedema, evaluated by water content. NAC also attenuated the increase in the brain levels of malondialdehyde (MDA) and the increase in the hippocampus of myeloperoxidase (MPO) caused by cerebral ischaemia. Positive staining for nitrotyrosine was found in the hippocampus in Mongolian gerbils subjected to cerebral ischaemia. Hippocampus tissue sections from Mongolian gerbils subjected to cerebral ischaemia also showed positive staining for poly (ADP-ribose) synthetase (PARS). The degree of staining for nitrotyrosine and for PARS were markedly reduced in tissue sections obtained from animals that received NAC. NAC treatment increased survival and reduced hyperactivity linked to neurodegeneration induced by cerebral ischaemia and reperfusion. Histological observations of the pyramidal layer of CA1 showed a reduction of neuronal loss in animals that received NAC. These results show that NAC improves brain injury induced by transient cerebral ischaemia. Keywords: Cerebral ischaemia, gerbil, neuronal death, brain injury, brain oedema, lipid peroxidation, NAC Introduction Neural damage following stroke and other neurodegenerative processes is thought the stem from overexcitation attributable to a massive release of excitatory neurotransmitter glutamate acting on the N-methyl-D-aspartate (NMDA) receptor and other receptor subtypes (Lipton & Rosenberg, 1994; Meldrum & Garthwaite, 1990; Choi et al., 1956). Evidence includes findings in many animal species that glutamate receptor antagonists block neuronal damage following vascular stroke and reduce neurotoxicity elicited by treatment of cerebral cortical cultures with glutamale or NMDA (Dawson & Dawson, 1997). Neurotoxicity elicited by stimulation of NMDA receptors is mediated, at least in part, by augmentation of nitric oxide (NO) formation, as NMDA receptor activation stimulates neuronal NO synthase (nNOS) activity. Protection against NMDA neurotoxicity occurs following treatment of primary brain cultures with NOS inhibitors (Dawson et al., 1991, 1993) and cortical cultures from mice with targeted disruption of nNOS (Dawson et al., 1996). Neuronal damage following vascular stroke is markedly diminished in animals treated with NOS inhibitors (Dawson & Dawson, 1997; Iadecola, 1997) or in mice with nNOS gene distruption (Huang, 1994). Nitric oxide is a free radical that chemically reacts with its cellular target. There are multiple potential cellular targets that NO can modify to elicit a range of activities from cellular signalling to cell death. The majority of the toxic effect of NO appear to be a result of the reaction of NO with superoxide to form the very toxic compound peroxynitrite (Beckman et al., 1990). Peroxynitrite is cytotoxic via a number of independent mechanisms including (i) the initiation of lipid peroxidation, (ii) the inactivation of a variety of enzymes (most notably, mitochondrial respiratory enzymes and membrane pumps) (Crow & Beckman, 1995) and (iii) depletion of glutathione (Phelps et al., 1995). Moreover, peroxynitrite can also cause DNA damage (Inoue & Kawanishi, 1995; Salgo et al., 1995) resulting in the activation of the nuclear enzyme poly (ADP-Ribose) synthetase (PARS), depletion of NAD and ATP and ultimately cell death (Szabo et al., 1997). Interventions, which reduce the generation or the effects of peroxynitrite and PARS activation exert beneficial effects in a variety of models of inflammation and shock including the model of cerebral ischaemia used here. These therapeutic interventions include NOS inhibitor (Iadecola, 1997), a vitamin E-like antioxidant (Cuzzocrea et al., 1999a; Calapai et al., 1993), a SOD-mimetic (Cuzzocrea et al., 1999b), a peroxynitrite decomposition catalyst (Salvemini et al., 1998), and PARS inhibitors (Cuzzocrea et al., 1997; Eliasson et al., 1997). NAC has antioxidant property (Aruoma et al., 1989) and as a sulphydryl donor, may contribute to the regeneration of endothelium-derived relaxing factor and glutathione (Harrison et al., 1991). Increasing evidence indicates that the action of NAC is pertinent to microcirculatory blood flow and tissue oxygenation. NAC was shown to enhance oxygen consumption via increased oxygen extraction in patients 18 h after the onset of fulminant liver failure (Harrison et al., 1991). It was speculated that NAC could also exert beneficial effects on impaired nutritive blood flow in patients with severe sepsis (Harrison et al., 1991). In the present study, we examined the protective effect of NAC against oxidative stress during brain ischaemia and reperfusion injury using both biochemical and morphological parameters as follows: the levels of lipid peroxidation, the number of polymorphonuclear leukocytes (PMNs) that infiltrate the oxidatively damaged region of the brain were detected.

Beneficial effects of melatonin in a rat model of splanchnic artery occlusion and reperfusion.

Abstract: The aim of the present study was to investigate the protective effect of the pineal secretary product melatonin in a model of splanchnic artery occlusion shock (SAO). SAO shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed thereafter by release of the clamp (reperfusion). At 60 min after reperfusion, animals were sacrificed for tissue histological examination and biochemical studies. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine (a marker of peroxynitrite-induced oxidative processes) in the plasma of the SAO-shocked rats after reperfusion, but not during ischemia alone. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, an index of nitrogen species such as peroxynitrite, in the necrotic ileum in shocked rats. SAO-shocked rats developed a significant increase of tissue myeloperoxidase and malondialdehyde activity, and marked histological injury to the distal ileum. SAO shock was also associated with a significant mortality (0% survival at 2 hr after reperfusion). Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin, which was mainly localized in the vascular endothelial cells. Ileum tissue sections obtained from SAO-shocked rats with anti-intercellular adhesion molecule (ICAM-1) antibody showed a diffuse staining. Melatonin (applied at 3 mg/kg, 5 min prior to reperfusion, followed by an infusion of 3 mg/kg per hr), significantly reduced ischemia reperfusion injury in the bowel as evaluated by histological examination. This prevented the infiltration of neutrophils into the reperfused intestine, is evidenced by reduced myeloperoxidase activity and reduced lipid peroxidation. This was evaluated by malondialdehyde activity which reduced the production of peroxynitrite during reperfusion, markedly reduced the intensity and degree of P-selectin and ICAM-1 in tissue section from SAO-shocked rats and improved their survival. Taken together, our results clearly demonstrate that melatonin treatment exerts a protective effect and part of this effect may be due to inhibition of the expression of adhesion molecule and peroxynitrite-related pathways and subsequent reduction of neutrophil-mediated cellular injury.

17β-Estradiol Antiinflammatory Activity in Carrageenan-Induced Pleurisy

Abstract: We have recently demonstrated that 17beta-estradiol (E2) opposes cytokine-dependent increase of inducible nitric oxide synthase (iNOS) activity in rat smooth muscle cells and proposed that this effect might be associated to an antiinflammatory activity of this hormone. In the present study, we examine the E2 effects on a well-known in vivo model of inflammation. We show that, in carrageenan treatment of ovariectomized rats, prior exposure to E2 significantly attenuated inflammatory response as measured by histological examination and exudate production. The effect was visible with a single injection of a physiological dose of E2 1 h before the carrageenan treatment and was blocked by coadministration of the estrogen receptor antagonists ICI 182,780 or tamoxifen. This latter observation suggests that the effect is receptor mediated. The mechanisms by which estradiol has beneficial effects in this model of inflammation are unclear: we show that in hormonally treated rats there is a decrease in polymorphonuclear cells migration as shown by cell counting and myeloperoxidase measurement. In addition, E2 pretreatment opposes carrageneen-induced high lipid peroxidation maintaining malondialdehyde activity at control levels. E2 treatment decreases NO production and the activity of iNOS with consequent diminished nitrite synthesis and nitrosine accumulation. Finally, immunohistochemical analysis for poly (ADP-ribose) synthase revealed a positive staining in lungs from carrageenan-treated rats that was blocked by estradiol treatment. We conclude that E2 attenuates the degree of inflammation and tissue damage associated with carrageenan-induced pleurisy in the rat.

IRFI 042, a novel dual vitamin E-like antioxidant, inhibits activation of nuclear factor-κB and reduces the inflammatory response in myocardial ischemia–reperfusion injury

Abstract: Background: Nuclear factor-κB (NF-κB) is a ubiquitous rapid response transcription factor involved in inflammatory reactions which exerts its effect by expressing cytokines, chemokines, and cell adhesion molecules. Oxidative stress causes NF-κB activation. IRFI 042 is a novel dual vitamin E-like antioxidant and we, therefore, investigated its ability to protect the heart from oxidative stress and to halt the inflammatory response in a model of myocardial ischaemia–reperfusion injury. Methods: Anaesthetized rats were subjected to total occlusion (45 min) of the left main coronary artery followed by 5-h reperfusion (MI/R). Sham myocardial ischaemia rats (sham-operated rats) were used as controls. Myocardial necrosis, cardiac output, cardiac and plasma vitamin E levels, myocardial malondialdehyde (MAL), cardiac SOD activity and elastase content (an index of leukocyte infiltration), hydroxyl radical (OH⋅) formation, cardiac amount of mRNA codifying for ICAM-1 (evaluated by the means of reverse transcriptase polymerase chain reaction) and ICAM-1 immunostaining in the at-risk myocardium were investigated. NF-κB activation and the inhibitory protein of NF-κB, I-κBα, were also studied in at-risk myocardium by using electrophoretic mobility shift assay (EMSA) and Western blot analysis, respectively. Results: The ischaemia–reperfusion model produced wide heart necrosis (area at risk−necrotic area=52±5%; necrotic area−left ventricle=28±3%), increased cardiac MAL, an index of lipid peroxidation (area at risk=62.5±3.9 nmol/g tissue; necrotic area=80.3±5.1 nmol/g tissue), induced tissue neutrophil infiltration, and caused a marked decrease in endogenous antioxidants. Furthermore, myocardial ischaemia plus reperfusion caused in the area at risk peak message for ICAM-1 at 3 h of reperfusion and increased cardiac ICAM-1 immunostaining at 5 h of reperfusion. NF-κB activation was also evident at 0.5 h of reperfusion and reached its maximum at 2 h of reperfusion. I-κBα was markedly decreased at 45 min of occlusion; peak reduction was observed at 1 h of reperfusion and thereafter it was gradually restored. Intraperitoneal administration of IRFI 042 (5, 10, 20 mg/kg, 5 min after reperfusion) reduced myocardial necrosis expressed as a percentage either of the area at risk (18±4%) or the total left ventricle (11±2%), and improved cardiac output. This treatment also limited membrane lipid peroxidation in the area at risk (MAL=14.3±2.5 nmol/g tissue) and in the necrotic area (MAL=26.5±3.7 nmol/g tissue), restored the endogenous antioxidants vitamin E and superoxide dismutase, and inhibited detrimental hydroxyl radical formation. Finally, IRFI 042 blocked the activation of NF-κB, reduced cardiac ICAM-1 expression, and blunted tissue elastase content, an index of leukocytes accumulation at the site of injury. Conclusions: Our data suggest that IRFI 042 is cardioprotective during myocardial infarction by limiting reperfusion-induced oxidative stress and by halting the inflammatory response.

Calpain Inhibitor I Reduces the Development of Acute and Chronic Inflammation

Abstract: There is limited evidence that inhibition of the activity of the protease calpain I reduces inflammation. Here we investigate the effects of calpain inhibitor I in animal models of acute and chronic inflammation (carrageenan-induced pleurisy and collagen-induced arthritis). We report here for the first time that calpain inhibitor I (given at 5, 10, or 20 mg/kg i.p. in the pleurisy model or at 5 mg/kg i.p every 48 hours in the arthritis model) exerts potent anti-inflammatory effects (eg, inhibition of pleural exudate formation, mononuclear cell infiltration, delayed the development of the clinical signs and histological injury) in vivo. Furthermore, calpain inhibitor I reduced (1) the staining for nitrotyrosine and poly (ADP-ribose) polymerase (immunohistochemistry) and (2) the expression of inducible nitric oxide synthase and cyclooxygenase-2 in the lungs of carrageenan-treated rats and in joints from collagen-treated rats. Thus, prevention of the activation of calpain I reduces the development of acute and chronic inflammation. Inhibition of calpain I activity may represent a novel therapeutic approach for the therapy of inflammation.

Beneficial effects of tempol, a membrane-permeable radical scavenger, in a rodent model of collagen-induced arthritis.

Abstract: Objective To investigate the effects of tempol, a membrane-permeable radical scavenger, in rats with collagen-induced arthritis (CIA). Methods CIA was induced in Lewis rats by intradermal injection of 100 μl of an emulsion of 100 μg of bovine type II collagen (CII) in complete Freund's adjuvant (FCA) at the base of the tail. On day 21, a second injection of CII in FCA was administered. Results Lewis rats developed an erosive arthritis of the hind paws when immunized with CII in FCA. Macroscopic evidence of CIA first appeared as periarticular erythema and edema in the hind paws. The incidence of CIA was 100% by day 27 in the CII-challenged rats, and the severity of CIA progressed over a 35-day period. Radiographs revealed focal resorption of bone, with osteophyte formation in the tibiotarsal joint, and soft tissue swelling. The histopathologic features included erosion of the cartilage at the joint margins. Treatment of rats with tempol (10 mg/kg/day intraperitoneally) starting at the onset of arthritis (day 23) delayed the development of the clinical signs on days 24–35 and improved the histologic status of the knee and paw. Immunohistochemical analysis for nitrotyrosine and poly(ADP-ribose) synthetase (PARS) revealed positive staining in the inflamed joints of CII-treated rats. The degree of nitrotyrosine and PARS staining was markedly reduced in tissue sections obtained from CII-treated rats that had received tempol. Furthermore, radiographs revealed protection against bone resorption and osteophyte formation in the joints of tempol-treated rats. Conclusion This study is the first to provide evidence that tempol, a small molecule that permeates biologic membranes and scavenges reactive oxygen species, attenuates the degree of chronic inflammation and tissue damage associated with CIA in the rat.

Cloricromene, a coumarine derivative, protects against collagen-induced arthritis in Lewis rats

Abstract: The aim of the present study was to investigate the effects of cloricromene, a coumarine derivative, in rats subjected to collagen-induced arthritis. Collagen-induced arthritis (CIA) was induced in Lewis rats by an intradermal injection of 100 μl of the emulsion (containing 100 μg of bovine type II collagen) (CII) and complete Freund's adjuvant (CFA) at the base of the tail. On day 21, a second injection of CII in CFA was administered. Lewis rats developed an erosive hind paw arthritis when immunized with CII in CFA. Macroscopic clinical evidence of CIA first appeared as peri-articular erythema and oedema in the hind paws. The incidence of CIA was 100% by day 27 in the CII challenged rats and the severity of CIA progressed over a 35-day period with radiographic evaluation revealing focal resorption of bone together with osteophyte formation in the tibiotarsal joint and soft tissue swelling. The histopathology of CIA included erosion of the cartilage at the joint margins. Treatment of rats with cloricromene (10 mg kg−1 i.p. daily) starting at the onset of arthritis (day 23), delayed the development of the clinical signs at days 24–35 and improved histological status in the knee and paw. Immunohistochemical analysis for iNOS, COX-2, nitrotyrosine and for poly (ADP-ribose) synthetase (PARS) revealed a positive staining in inflamed joints from collagen-treated rats. The degree of staining for iNOS, COX-2, nitrotyrosine and PARS were markedly reduced in tissue sections obtained from collagen-treated rats, which had received cloricromene. Radiographic signs of protection against bone resorption and osteophyte formation were present in the joints of cloricromene-treated rat. This study provides the first evidence that cloricromene, a coumarine derivative, attenuates the degree of chronic inflammation and tissue damage associated with collagen-induced arthritis in the rat. British Journal of Pharmacology (2000) 131, 1399–1407; doi:10.1038/sj.bjp.0703695

Role of free radicals and poly(ADP-ribose) synthetase in intestinal tight junction permeability

Abstract: Small intestine permeability is frequently altered in inflammatory bowel disease and may be caused by the translocation of intestinal toxins through leaky small intestine tight junctions (TJ) and adherence (1,2). The role of hydrogen peroxide (H2O2), and nitric oxide (NO) and PARS in the permeability and structure of small intestine TJ is not clearly understood. In vitro study, MDCK (Madin-Darby Canine Kidney) cells were exposed to H2O2 (100 µM for 2h), or zymosan (200 µl of stock solution 1 mg/ml for 4h), in the presence or absence of a treatment with poly(ADP-ribose) synthetase (PARS) inhibitor 3-aminobenzamide (3-AB: 3 mM) or with n-acetylcysteine (NAC 10 mM). In vivo study, wild-type mice (WT) and mice lacking (KO) of the inducible (or type 2) nitric oxide synthase (iNOS) were treated with zymosan (500 mg/kg, suspended in saline solution, i.p.). In addition INOSWT mice were treated with 3-AB (10 mg/kg, i.p.) or with NAC (40 mg/kg, i.p.) 1 hour and 6 h after zymosan administration. Exposure of MDCK cells to hydrogen peroxide caused a significant impairment in mitochondrial respiration that was associated with a reduction of cells adherence as well as derangement of the junctional proteins. A significant increase of nitrate and nitrite levels, stable metabolites of nitric oxide (NO), were found in MDCK supernatant after zymosan incubation. NO production was associated with a significant reduction of cell adherence and impairment of occludin protein. Pre-treatment of the cells with 3-AB or with NAC caused a significant prevention of H2O2-mediated occludin junctional damage as well as reduced the NO-induced occludin damage. In addition, H2O2 and NO are able to induce a significant derangement of β-catenin and Zonula Ocludence-1 (ZO-1). We found an increase of tight junctional permeability to lanthanum nitrate (molecular weight, 433) in the terminal ileal TJs in zymosan-treated iNOSWT mice compared with permeableTJ in the control animals. Zymosan-treated iNOSKO mice showed a significant increase of tight junctional permselectivity. There were no differences in strand count or strand depth in the ilea from control or treated animals. In addition, a significant disrupted immunofluorescence signal for occludin, ZO-1 and β-catenin was observed in the terminal ilea of zymosantreated iNOSWT mice. In ileal fragments from zymosan-treated iNOSKO mice, we found less irregular distribution patterns of occludin, ZO-1 and β-catenin. Similarly NAC or 3-AB treatments were able to prevent zymosan-induced damage of junctional proteins in iNOSWT mice. In conclusion, this study demonstrates that the alteration of permselectivity is most likely induced by ROS and PARS activation.

Role of hyperbaric oxygen exposure in reduction of lipid peroxidation and in multiple organ failure induced by zymosan administration in the rat.

Abstract: The aim of the present study was to evaluate the effects of hyperbaric oxygen (HBO) therapy on multiple organ failure induced by zymosan. Administration of zymosan (500 mg/kg) in the rat induced neutrophil infiltration in the lung, liver, and intestine as evaluated by increase in myeloperoxidase (MPO) activity. Therefore, lipid peroxidation was significantly increased in zymosan-treated rats. This inflammatory process coincided with the damage of lung, liver, and small intestine. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine in the lung, liver, and small intestine of zymosan-shocked rats. HBO (2 absolute Atmosphere) exposure attenuates the increase in the tissue levels of MPO and malondialdehyde (MDA) caused by zymosan in the lung, liver, and intestine. In addition, HBO (2 absolute Atmosphere) was effective in preventing the development of lung, liver, and intestine injury. Taken together, the present results demonstrate that HBO may also be an efficacious treatment in multiple organ failure induced by zymosan.

Beneficial effects of tempol, a membrane-permeable radical scavenger, in a rodent model of splanchnic artery occlusion and reperfusion.

Abstract: The aim of the present study was to investigate the effects of tempol, a membrane-permeable radical scavenger, in rats subjected to splanchnic artery occlusion shock (SAO). Rats subjected to SAO developed a significant decrease in mean arterial blood pressure, a significant increase in tissue myeloperoxidase activity, and a marked injury to the distal ileum. SAO shock resulted in 100% mortality at 2 h after reperfusion. At 60 min after reperfusion, a marked increase in the immunoreactivity to nitrotyrosine and to poly (ADP-ribose) synthetase was observed in the necrotic ileum of rats with SAO. Staining of sections of the ileum obtained from SAO-shocked rats with anti-intercellular adhesion molecule (ICAM-1) and anti-P-selectin antibodies resulted in diffuse staining. Tempol (30 mg/kg bolus injection 5 min prior to reperfusion, followed by an infusion of 30 mg/kg/h intravenously) attenuated 1) the infiltration of the reperfused intestine with neutrophils, 2) the lipid peroxidation, 3) the production of peroxynitrite, 4) the degree of P-selectin and ICAM-1 staining in tissue sections from SAO-shocked rats, 5) histological signs of bowel injury, and 6) mortality at 2 h after reperfusion. Taken together, our results clearly demonstrate that the intracellular radical scavenger tempol reduces the intestinal injury of rats subjected SAO shock.

The Tyrosine Kinase Inhibitor Tyrphostin AG126 Reduces the Development of Acute and Chronic Inflammation

Abstract: Protein tyrosine kinases help to regulate the expression of many genes that play important roles in inflammation. Here we investigate the effects of the tyrosine kinase inhibitor tyrphostin AG126 in two animal models of acute and chronic inflammation, carrageenan-induced pleurisy and collagen-induced arthritis. We report here that tyrphostin AG126 (given at 1, 3, or 10 mg/kg i.p. in the pleurisy model or 5 mg/kg i.p. every 48 hours in the arthritis model) exerts potent anti-inflammatory effects in animal models of acute and chronic inflammation in vivo. These include the inhibition of pleural exudate formation and mononuclear cell infiltration (pleurisy model) and the development of clinical signs and tissue injury (arthritis model). Furthermore, tyrphostin AG126 reduced the staining for nitrotyrosine and poly (ADP-ribose) polymerase (by immunohistochemistry) and the expression of inducible nitric oxide synthase and cyclooxygenase-2 in the lungs of carrageenan-treated rats and in the joints from collagen-treated rats. Thus, we provide the first evidence that prevention of the activation of protein tyrosine kinases reduces the development of acute and chronic inflammation, and that inhibition of the activity of certain tyrosine kinases may represent a novel approach for the therapy of inflammation.

Protective effects of Cyclosporin-A in splanchnic artery occlusion shock

Abstract: Cyclosporin A (CsA) is an immunosuppressant drug that inhibits nitric oxide (NO) synthase induction in vascular smooth muscle cells. Splanchnic artery occlusion (SAO) shock is a lethal type of shock characterized by a marked vascular dysfunction in which the L-arginine/nitric oxide pathway plays an important role. We investigated whether CsA exerts protective effects in SAO shock by interfering with the L-arginine/nitric oxide pathway. Male anaesthetized rats (n=156) were subjected to clamping of the splanchnic arteries for 45 min. This surgical procedure resulted in an irreversible state of shock (SAO shock). Sham operated animals were used as controls. SAO shocked rats had a decreased survival (86+/-6 min, while sham shocked rats survived more than 240 min), marked hypotension, increased serum levels of TNF-alpha, enhanced plasma nitrite/nitrate concentrations (75+/-7.1 microM; sham shocked rats=1.6+/-0.5 microM) and enhanced inducible NO synthase (iNOS) protein induction and activity in the aorta. Moreover aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (PE, 1 nM - 10 microM). CsA (0.25, 0.5 and 1 mg kg(-1), 5 min after reperfusion) increased survival rate (SAO+CsA=236+/-9 min following the highest dose), reverted the marked hypotension, reduced plasma nitrite/nitrate concentration (11+/-5.2 microM following the highest dose), restored to control values the hyporeactivity to PE, and blunted iNOS protein induction and activity in aortic rings. The present data indicate that in an experimental rat model CsA may have antishock properties related to inhibition of L-arginine/nitric oxide pathway.