Ahmet Guven

Medical ozone therapy reduces oxidative stress and intestinal damage in an experimental model of necrotizing enterocolitis in neonatal rats

PURPOSE Necrotizing enterocolitis (NEC) remains a major cause of morbidity and death in neonates. Evidence suggests that an imbalance between activated proinflammatory response with inadequate antiinflammatory protection results in NEC. Ozone has been proposed as an antioxidant enzyme activator, immunomodulator, and cellular metabolic activator. Therefore, this study was designed to investigate whether medical ozone therapy is effective on neonatal rat model of NEC. MATERIALS AND METHODS Thirty-eight newborn Sprague-Dawley pups were randomly divided into 3 groups of NEC, NEC + ozone, and control (left to breast feed). Necrotizing enterocolitis was induced by enteral formula feeding and exposure to 100% carbon dioxide inhalation for 10 minutes after +4 degrees C cold exposures for 5 minutes and 97% oxygen for 5 minutes 2 times daily. The NEC + ozone group received 0.7 mg/kg per day ozone/oxygen mixture intraperitoneally for a total of 3 days after first day of NEC procedure. The pups were killed at fourth day, and their intestinal tissues were harvested for biochemical and histopathologic analysis. Blood sample from pups were also obtained. RESULTS The mortality rate and the weight loss were significantly higher in NEC group than control and treatment groups. Oxidative stress markers (malondialdehyde and protein carbonyl content) significantly increased and antioxidant enzyme activities (superoxide dismutase and glutathione peroxidase) were significantly decreased in NEC group. All these biochemical changes were ameliorated in NEC + ozone group. Nitrate plus nitrite levels and serum tumor necrosis factor alpha were elevated in NEC group and reduced in treatment group. In addition, histopathologic injury score of NEC group was significantly higher than NEC + ozone group. CONCLUSION Ozone treatment significantly reduced the severity of NEC by modulating antioxidative defense and antiinflammatory protection in our experimental animal model.

Scavenging of peroxynitrite reduces renal ischemia/reperfusion injury.

Introduction. Nitric oxide (NO) and peroxynitrite (OONO—) are implicated in the pathophysiology of renal ischemia/reperfusion (I/R). The aim of this study was to investigate and compare the efficiency of S-methylisothiourea (SMT), an iNOS inhibitor, and mercaptoethylguanidine (MEG), a scavenger of peroxynitrite, on renal dysfunction and injury induced by I/R of rat kidney. Materials and Methods. Thirty-two male Sprague-Dawley rats were divided into four groups: sham-operated, I/R, I/R+SMT, and I/R+MEG. Rats were given SMT (10 mg/kg ip) or MEG (10 mg/kg ip) 6 h prior to I/R and at the beginning of reperfusion. All rats except sham-operated underwent 60 min of bilateral renal ischemia followed by 6 h of reperfusion. After reperfusion, kidneys and blood were obtained for evaluation. Superoxide dismutase, glutathione peroxidase, malondialdehide, protein carbonyl content, and nitrite/nitrate level (NOx) were determined in the renal tissue. Serum creatinine (SCr), blood urea nitrogen (BUN), and aspartate aminotransferase (AST) were determined in the blood. Additionally, renal sections were used for histological grade of renal injury. Results. SMT and MEG significantly reduced the I/R-induced increases in SCr, BUN, and AST. Both SMT and MEG attenuated the tissue NOx levels, indicating reduced NO production. In addition, SMT and MEG markedly reduced elevated oxidative stress product, restored decreased antioxidant enzymes, and attenuated histological alterations. Interestingly, MEG exerted a greater renoprotective effect than SMT. Conclusions. These data support the finding that iNOS and peroxynitrite are involved in the renal I/R injury, and suggest that a scavenger of peroxynitrite might be more effective than iNOS inhibitors as a therapeutic intervention.

Efficacy of hyperbaric oxygen therapy and medical ozone therapy in experimental acute necrotizing pancreatitis.

Objectives: Our aims were to evaluate the efficacy of ozone therapy (OT) in an experimental rat model of acute necrotizing pancreatitis (ANP) and to compare its effects with hyperbaric oxygen (HBO) therapy in this entity. Methods: Forty Sprague-Dawley rats were divided into sham-operated, ANP, ANP + HBO, and ANP + OT groups. Acute necrotizing pancreatitis was induced by infusing 1-mL/kg 3% sodium taurocholate into the common biliopancreatic duct. Hyperbaric oxygen was administered twice daily at a 2.8-atm pressure for 90 minutes. Ozone therapy was set as daily intraperitoneal injections of 0.7-mg/kg ozone/oxygen gas mixture. Hyperbaric oxygen and OT were continued for 3 days after the induction of ANP. The surviving animals were killed at the fourth day, and their pancreases were harvested for biochemical, microbiological, and histopathologic analyses. Results: Serum amylase/lipase and neopterin levels and tissue oxidative stress parameters were similar to shams values in both the ANP + HBO and the ANP + OT groups. Histopathologic injury scores were significantly lower in the treatments groups than in the ANP group. When compared with the ANP group, the number of infected rats was significantly lesser in the ANP + HBO and the ANP + OT groups. Conclusions: Hyperbaric oxygen and OT reduce the severity and the mortality in the experimental rat model of ANP, and a greater benefit was received for OT comparing with HBO.

The efficacy of ozone therapy in experimental caustic esophageal burn.

INTRODUCTION Ozone has been proposed as an antioxidant enzyme activator, immunomodulator and cellular metabolic activator. This study was designed to investigate the efficacy of ozone therapy in the prevention of esophageal damage and stricture formation developed after esophageal caustic injuries in the rat. MATERIALS AND METHODS Forty-five rats were allocated into three groups; sham-operated, un-treatment and treatment groups. Caustic esophageal burn was created by instilling 15% NaOH in the distal esophagus. The rats were left untreated or treated with 1 mg/kg/day ozone intraperitoneally. All rats were sacrificed at 28 days. Efficacy of the treatment was assessed by measuring the stenosis index (SI) and histopathologic damage score, and biochemically by determining tissue hydroxyproline content (HP), superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA) and protein carbonyl content (PCC) in esophageal homogenates. RESULTS Whereas seven (47%) rats died in the un-treatment group, all rats in the sham-operated and the treatment group survived during the study. SI, the histopathologic damage score, was significantly lower in the ozone-therapy group than the un-treatment group. HP levels were significantly higher in the un-treatment group than the group treated with ozone. Caustic esophageal burn increased MDA and PCC levels and also decreased SOD and GPx enzyme activities. In contrast, ozone therapy decreased the elevated MDA and PCC levels and also increased the reduced SOD and GPx enzyme activities. CONCLUSION Ozone has a preventive effect in the development of fibrosis by decreasing tissue damage and increasing the antioxidant enzyme activity in an experimental model of corrosive esophageal injury.

A prospective, randomized, double-blind, placebo-controlled trial of glyceryl-trinitrate ointment in the treatment of children with anal fissure

BACKGROUND Anal fissure in children usually is treated by sitz baths, stool softeners, and analgesic ointments. However, some cases are intractable to the treatment. In recent years, it has been reported that nitric oxide donors such as local glyceryl-trinitrate (GTN) ointment causes a reversible chemical sphincterotomy. Although the GTN ointment can be an alternative therapy for adult cases, it has not yet been studied in the children who suffer from anal fissure. METHODS Sixty-five children with anal fissure were divided randomly into 3 groups. Each group received double-blinded a topical ointment that contained either 0.2% GTN, 10% lidocaine, or placebo. These ointments were applied to the lowest part of the anal canal twice daily. Patients were periodically reviewed, and the study was ended after 8 weeks. RESULTS Complete healing of the fissure occurred in 26 of 31 (83.9%) patients treated with GTN, 7 of 14 (50%) patients treated with lidocaine, and 6 of 17 (35.2%) treated with placebo. In 29 of 31 (93.5%) GTN-treated patients, a total relief of symptoms was observed, whereas this occurred in 7 of 14 (50%) treated with lidocaine and 6 of 11 (35.3%) in the placebo group. The differences between the study group and control groups were highly statistically significant (P < .001). CONCLUSION The majority of children suffering from anal fissure will be cured and have relief of symptoms after topical application of GTN ointment to the anal canal.

Erdosteine and Ebselen As Useful Agents in Intestinal Ischemia/ Reperfusion Injury

BACKGROUND Reactive oxygen and nitrogen species generated during reperfusion of the tissue are characteristic of ischemia/reperfusion (I/R) injury. The purpose of the present study was to investigate whether erdosteine and ebselen, molecules with antioxidant properties and peroxynitrite scavenging capability, respectively, can reduce oxidative stress and histological damage in the rat small bowel subjected to mesenteric I/R injury. MATERIALS AND METHODS Forty Sprague-Dawley rats were divided into five groups equally: sham, I/R, I/R plus erdosteine, I/R plus ebselen, and I/R plus erdosteine and ebselen. Intestinal ischemia for 45 min and reperfusion for 3 d were carried out. Ileal specimens were obtained to determine the tissue levels of malondialdehide (MDA), protein carbonyl content (PCC), superoxide dismutase (SOD), glutathione peroxidase (GPx), nitrite/nitrate (NO(x)) level and histological changes. RESULTS Intestinal I/R resulted in increased tissue MDA, PCC, and NO(x) levels and decreased SOD and GPx activities. Both erdosteine and ebselen alone significantly decreased MDA, PCC, and NO(x) levels and increased antioxidant enzymes activities, but all values were different from control. These changes almost returned to control values in the group treated with erdostein and ebselen. Histopathologically, the intestinal injury in rats treated with erdosteine and ebselen as well as combination were less than I/R group. CONCLUSIONS Both erdosteine and ebselen were able to attenuate I/R injury of the intestine via inhibition of lipid peroxidation and protein oxidation, maintenance of antioxidant, and free radical scavenger properties. Nevertheless, combination treatment showed more promising results, suggesting that scavenging peroxynitrite nearby antioxidant activity is important in preventing intestinal I/R injury.

Melatonin and 1400 W Ameliorate both Intestinal and Remote Organ Injury Following Mesenteric Ischemia/Reperfusion

OBJECTIVE Acute intestinal ischemia reperfusion (I/R) injury affects not only the intestines but also remote organs due to pro-inflammatory and tissue injurious factors. Thus, we aimed to investigate the roles of melatonin (a powerful antioxidant) and 1400W (a strong inhibitor of inducible nitric oxide) in a rat intestinal I/R injury model, since oxidative and nitrosative injury are believed to be the major causes. METHODS A total of 56 Wistar albino rats were used, with seven rats in each group. After I/R induction in the intestines by clamping/unclamping the superior mesenteric artery, we measured malondialdehyde, superoxide dismutase, glutathione peroxidase, nitric oxide, and 3-nitrotyrosine levels in lung, kidney, and liver tissues (to evaluate remote organ injury) as well as in the intestines. Study groups received melatonin, 1400W or both to examine the roles of these molecules in the pathogenesis of injury following I/R. RESULTS Melatonin and 1400W had an ameliorating effect on both oxidative and nitrosative stress in the intestine and the lung against mesenteric I/R injury in rats. Moreover, each of these two agents had an inhibitory effect on oxidative injury and histopathological changes in the intestine and the lung. Furthermore, the combination of both agents (melatonin and 1400W) was more effective than either of the agents alone (P < 0.05). CONCLUSION Melatonin and 1400W, either alone or in combination, were efficient in ameliorating experimental I/R injury of the intestines.

α-Lipoic Acid and Ebselen Prevent Ischemia/Reperfusion Injury in the Rat Intestine

PurposeReactive oxygen species (ROS) and reactive nitrogen species (RNS), generated during tissue reperfusion, are characteristic of ischemia/reperfusion (I/R) injury. We conducted this study to evaluate the protective effect of α-lipoic acid (α-LA) and ebselen against intestinal I/R injury.MethodsForty Sprague-Dawley rats were divided into five groups: a sham-operated group; an I/R group, subjected to intestinal ischemia for 45 min and reperfusion for 3 days; an I/R+α-LA group; an I/R+ebselen group; and an I/R+α-LA+ebselen group. We collected ileal specimens, to measure the tissue levels of malondialdehyde (MDA), protein carbonyl content (PCC), superoxide dismutase (SOD), and glutathione peroxidase (GPx), and to evaluate the histologic changes.ResultsThere was a significant decrease in SOD and GPx levels, with an increase in MDA and PCC levels and intestinal mucosal injury in the intestinal I/R group (P < 0.05). Superoxide dismutase and GPx levels were significantly higher, MDA and PCC levels were significantly lower, and intestinal injury was significantly less severe in the I/R+α-LA+ebselen group than in the I/R group (P < 0.05). Although shortened villi and epithelial lifting were seen in the I/R group, only slight mucosal injury was seen in the treatment groups.Conclusionα-Lipoic acid and ebselen played an important role in attenuating I/R injury of the intestine by scavenging ROS and RNS.

Hyperbaric oxygen therapy reduces the severity of necrotizing enterocolitis in a neonatal rat model

INTRODUCTION Hyperbaric oxygen (HBO) therapy is known to increase oxygen concentration in tissues leading to induction of an adaptive increase in antioxidants, stimulation of angiogenesis, improvement of white blood cell action, and regulation of inflammatory process. Therefore, we tested the potential beneficial effect of HBO in neonatal rat model of necrotizing enterocolitis (NEC). MATERIALS AND METHODS Thirty newborn Sprague-Dawley rats, provided by the Experimental Research Council, Gulhane Military Medical Academy, Ankara,Turkey, were randomly divided into 3 groups as follows: NEC, NEC + HBO, and control. Necrotizing enterocolitis was induced by enteral formula feeding and exposure to hypoxia after cold stress at 4 degrees C and oxygen. The NEC + HBO group received HBO at 2.8 atmosphere absolute (ATA) for 90 minutes daily for 3 days. The pups were killed on the fourth day, and their intestinal tissues were harvested for biochemical and histopathologic analysis. Blood samples were also obtained from the pups. RESULTS The mortality rate was highest in the NEC group (3 pups in the NEC group vs 1 pup in the NEC + HBO group). Malondialdehyde and protein carbonyl content were significantly increased, whereas superoxide dismutase and glutathione peroxidase were significantly decreased in the NEC group. All these changes were similar to control levels in the NEC group by HBO treatment. Nitrate plus nitrite (NO(x)) levels and serum tumor necrosis factor alpha were increased in the NEC group and histopathologic injury score and apoptosis index in the NEC group were significantly higher than in the NEC + HBO group. CONCLUSION Hyperbaric oxygen significantly reduced the severity of NEC in our study.

Comparison of the Efficacy of Melatonin and 1400W on Renal Ischemia/Reperfusion Injury: A Role for Inhibiting iNOS

Introduction. We investigated the roles of melatonin (a powerful antioxidant, iNOS inhibitor, and a scavenger of peroxynitrite) and 1400W (a strong and selective inhibitor of inducible nitric oxide) on renal dysfunction and injury induced by ischemia/reperfusion (I/R) of rat kidney, since oxidative and nitrosative injury are believed to be the major causes. Materials and methods. Thirty-two male Sprague-Dawley rats were divided into four groups of sham-operated, I/R, I/R + Melatonin and I/R + 1400W. Rats were given either melatonin (10 mg/kg) or 1400W (10 mg/kg) in the I/R + Melatonin and I/R + 1400W groups respectively at 6 h prior to ischemia and at the beginning of reperfusion via intraperitoneal route. I/R injury was induced by 60 min of bilateral renal ischemia followed by 6 h of reperfusion. After reperfusion, kidneys and blood were obtained for histopathologic and biochemical evaluation. Results. Melatonin and 1400W had an ameliorative effect on both oxidative and nitrosative stress in the kidneys against renal I/R injury in rats. In addition, melatonin significantly reduced elevated nitro-oxidative stress product, restored decreased antioxidant enzymes and attenuated histological alterations when compared with 1400W. Conclusions. Both Melatonin and 1400W were efficient in ameliorating experimental I/R injury of the kidneys. Moreover, melatonin was more effective than 1400W possibly through inhibiting iNOS as well as scavenging free oxygen radicals and peroxynitrite.