Francesco Fulia

Increased levels of malondialdehyde and nitrite/nitrate in the blood of asphyxiated newborns : reduction by melatonin

Free radicals have been implicated in the pathogenesis of neonatal asphyxia and its complications. This study measured a product of lipid peroxidation, malondialdehyde, and the nitrite/nitrate levels in the serum of 20 asphyxiated newborns before and after treatment with the antioxidant melatonin given within the first 6 hr of life. Ten asphyxiated newborns received a total of 80 mg of melatonin (8 doses of 10 mg each separated by 2‐hr intervals) orally. One blood sample was collected before melatonin administration and two additional blood samples (at 12 and 24 hr) were collected after giving melatonin. A third group of healthy newborn children served as controls. Serum malondialdehyde and nitrite+nitrate concentrations in newborns with asphyxia before treatment were significantly higher than those in infants without asphyxia. In the asphyxiated newborns given melatonin, there were significant reductions in malondialdehyde and nitrite/nitrate levels at both 12 and 24 hr. Three of the 10 asphyxiated children not given melatonin died within 72 hr after birth; none of the 10 asphyxiated newborns given melatonin died. The results indicate that the melatonin may be beneficial in the treatment of newborn infants with asphyxia. The protective actions of melatonin in this study may relate to the antioxidant properties of the indole as well as to the ability of melatonin to increase the efficiency of mitochondrial electron transport.

Individual and synergistic antioxidative actions of melatonin: studies with vitamin E, vitamin C, glutathione and desferrrioxamine (desferoxamine) in rat liver homogenates

The pharmacological effects of melatonin, vitamin E, vitamin C, glutathione and desferrioxamine (desferoxamine) alone and in combination on iron‐induced membrane lipid damage in rat liver homogenates were examined by estimating levels of malondialdehyde and 4‐hydroxyalkenals (MDA + 4‐HDA). Individually, melatonin (2.5–1600 μM), vitamin E (0.5–50 μM), glutathione (100–7000 μM) and desferrioxamine (1–8 μM) inhibited lipid peroxidation in a concentration‐dependent manner. Vitamin C had both a pro‐oxidative (25–2000 μM) and an antioxidative (2600–5000 μM) effect. The IC50 (concentration that reduces damage by 50%) values were 4, 10, 426, 2290 and 4325 μM for vitamin E, desferrioxamine, melatonin, glutathione and vitamin C, respectively. The synergistic actions of melatonin with vitamin C, vitamin E, and glutathione were systematically investigated. When melatonin was combined with vitamin E, glutathione, or vitamin C, the protective effects against iron‐induced lipid peroxidation were dramatically enhanced. Even though melatonin was added at very low concentrations, it still showed synergistic effects with other antioxidants at certain concentrations. Furthermore, melatonin not only reversed the pro‐oxidative effects of vitamin C, but its efficacy in reducing lipid peroxidation was improved when it was combined with prooxidative concentrations of vitamin C. The results provide new information in terms of the possible pharmacological use of the combination of melatonin and classical antioxidants to treat free radical‐related conditions.

Protective effects of melatonin in ischemic brain injury

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.

Effects of tempol, a membrane-permeable radical scavenger, in a gerbil model of brain injury.

There is evidence that the excessive generation of reactive-oxygen radicals contributes to the brain injury associated with transient, cerebral ischemia. This study investigates the effects of tempol, a small, water-soluble molecule, that crosses biological membranes, on the brain injury caused by bilateral occlusion and reperfusion of both common carotid arteries in the gerbil (BCO). Treatment of gerbils with tempol (30 mg/kg i.p. at 30 min prior to reperfusion and at 1 and 6 h after the onset of reperfusion) reduced the formation of post-ischemic brain oedema. Tempol also attenuated the increase in the cerebral levels of malondialdehyde (MDA) and the hippocampal levels of myeloperoxidase (MPO) caused by cerebral ischemia and reperfusion. The immunohistochemical analysis of the hippocampal region of brains subjected to ischemia-reperfusion exhibited positive staining for nitrotyrosine (an indicator of the generation of peroxynitrite) and poly(ADP-ribose) synthetase (PARS) (an indicator of the activation of this nuclear enzyme secondary to single strand breaks in DNA). In gerbils subjected to BCO, which were treated with tempol, the degree of staining for nitrotyrosine and PARS was markedly reduced. Tempol increased survival and reduced the hyperactivity (secondary to the ischemia-induced neurodegeneration) caused by cerebral ischemia and reperfusion. The loss of neurons from the pyramidal layer of the CA1 region caused by ischemia and reperfusion was also attenuated by treatment of gerbils with tempol. This is the first evidence that the membrane-permeable, radical scavenger tempol reduces the cerebral injury caused by transient, cerebral ischemia in vivo.

Effect of melatonin on cellular energy depletion mediated by peroxynitrite and poly (ADP-ribose) synthetase activation in an acute model of inflammation.

DNA single‐strand breakage and activation of the nuclear enzyme poly (ADP‐ribose) synthetase (PARS) triggers an energy‐consuming, inefficient repair cycle, which contributes to peroxynitrite‐induced cellular injury. Recently, we proposed that during an acute model (pleurisy), cellular injury is mediated by peroxynitrite formation and consequent PARS activation. Here, we investigated whether in vivo melatonin treatment inhibits cellular injury induced by peroxynitrite production and PARS activation in macrophages collected from rats subjected to carrageenan‐induced pleurisy. Macrophages harvested from the pleural cavity exhibited a significant production of peroxynitrite, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123. Furthermore, carrageenan‐induced pleurisy caused a suppression of macrophage mitochondrial respiration, DNA strand breakage, activation of PARS, and reduction of cellular levels of NAD+. In vivo treatment with melatonin [12.5 or 25 or 50 mg/kg, intraperitoneally (i.p.), 30 min before carrageenan] significantly reduced peroxynitrite formation in a dose‐dependent manner and prevented the appearance of DNA damage, decrease in mitochondrial respiration, loss of cellular levels of NAD+, and PARS activation. Our study supports the view that the antioxidant and anti‐inflammatory effect of melatonin is also correlated with the inhibition of peroxynitrite production and PARS activation. In conclusion, melatonin may be a novel pharmacological approach to prevent cell injury in acute inflammation.

Effects of calpain inhibitor I on multiple organ failure induced by zymosan in the rat

ObjectiveZymosan enhances the formation of reactive oxygen species, which contributes to the pathophysiology of multiple organ failure. We investigated the effects of calpain inhibitor I (5, 10, or 20 mg/kg) on the multiple organ failure caused by zymosan (500 mg/kg, administered intraperitoneally as a suspension in saline) in rats. SettingUniversity research laboratory. SubjectsMale Sprague-Dawley rats. InterventionsMultiple organ failure in rats was assessed 18 hrs after administration of zymosan and/or calpain inhibitor I and was monitored for 12 days (for loss of body weight and mortality rate). Measurement and Main ResultsTreatment of rats with calpain inhibitor I (5, 10, or 20 mg/kg intraperitoneally, 1 and 6 hrs after zymosan) attenuated the peritoneal exudation and the migration of polymorphonuclear cells caused by zymosan in a dose-dependent fashion. Calpain inhibitor I also attenuated the lung, liver, and intestinal injury (histology) as well as the increase in myeloperoxidase activity and malondialdehyde concentrations caused by zymosan in the lung, liver, and intestine. Immunohistochemical analysis for nitrotyrosine and for poly(adenosine-disphosphate-ribose) revealed positive staining in lung, liver, and intestine from zymosan-treated rats. The degree of staining for nitrotyrosine and poly(adenosine-disphosphate-ribose) was reduced markedly in tissue sections obtained from zymosan-treated rats administered calpain inhibitor I (20 mg/kg intraperitoneally). Furthermore, treatment of rats with calpain inhibitor I significantly reduced the expression of inducible nitric oxide synthase and cyclooxygenase-2 in lung, liver, and intestine. ConclusionThis study provides the first evidence that calpain inhibitor I attenuates the degree of zymosan-induced multiple organ failure in the rat.

La conjonctivite à Chlamydia trachomatis du nouveau-né

C.D. Salpietro, G. Bisignano, F. Fulia, A. Marino, I. Barberi Infections of the conjunctiva are frequent in the neonatal period. While Neisseria gonorrhoea and chemical agents were considered as the main etiologies of ophtalmiae neonatorum in the past, Chlamydia trachomatis is today a major cause of neonatal conjunctivitis. Thus in a study of 180 unior bilateral neonatal conjunctivitis the authors found a prevalence of Chlamydia trachomatis infection of 41 %. The importance of the etiological diagnosis of neonatal conjunctivitis is emphasized, in order to define a specific treatment. Etiological diagnosis of Chlamydia trachomatis infection is based upon immunofluorescence and molecular diagnosis techniques (PCR, LCR). Prevention of neonatal Chlamydia trachomatis conjunctivitis relies upon screening and treatment of Chlamydia trachomatis infections in pregnant women and their partners. Treatment requires oral macrolides, the topical treatment being ineffective.

Protective effects of M40401, a selective superoxide dismutase mimetic, on zymosan-induced nonseptic shock

ObjectiveZymosan enhances formation of reactive oxygen species, which contributes to the pathophysiology of organ failure during nonseptic shock. Here we have investigated the effects of M40401, a new superoxide dismutase mimetic, on the organ failure associated with nonseptic shock caused by zymosan in rats. DesignExperimental study. SettingLaboratory. SubjectsMale Sprague-Dawley rats. InterventionsWe investigated the effects of M40401 on the organ failure associated with nonseptic shock caused by zymosan (500 mg/kg, administered intraperitoneally as a suspension in saline) in rats. Measurements and Main ResultsOrgan failure and systemic inflammation in rats were assessed 18 hrs after administration of zymosan and/or M40401 and were monitored for 12 days (for loss of body weight and mortality). Treatment of rats with M40401 (10 mg/kg intraperitoneally, 1 and 6 hrs after zymosan) attenuated the peritoneal exudation and the migration of polymorphonuclear cells caused by zymosan. M40401 administration also attenuated the lung and intestinal injury (histology) as well as the increase in myeloperoxidase activity and malondialdehyde concentrations caused by zymosan in lung and intestine. Immunohistochemical analysis for nitrotyrosine and for poly(adenosine 5′-diphosphate-ribose) revealed positive staining in lung and intestine from zymosan-treated rats. The degree of staining for nitrotyrosine and poly(adenosine 5′-diphosphate-ribose) was markedly reduced in tissue sections obtained from zymosan-treated rats administered with M40401. ConclusionThis study provides the first evidence that M40401 attenuates the degree of zymosan-induced nonseptic shock in the rat.