Olga Sonia León Fernández

Effects of ozone oxidative preconditioning on nitric oxide generation and cellular redox balance in a rat model of hepatic ischaemia-reperfusion.

Background: Many studies indicate that oxygen free‐radical formation after reoxygenation of liver may initiate the cascade of hepatocellular injury. It has been demonstrated that controlled ozone administration may promote an oxidative preconditioning or adaptation to oxidative stress, preventing the damage induced by reactive oxygen species and protecting against liver ischaemia–reperfusion (I/R) injury.

Nimesulide limits kainate-induced oxidative damage in the rat hippocampus

Kainate induces a marked expression of cyclooxygenase-2 after its systemic administration. Because cyclooxygenase-2 activity is associated to the production of reactive oxygen species, we investigated the effects of nimesulide, a selective cyclooxygenase-2 inhibitor, on kainate-induced in vivo oxidative damage in the rat hippocampus. A clinically relevant dose of nimesulide (6 mg/kg, i.p. ) was administered three times following kainate application (9 mg/kg, i.p.). After 24 h of kainate administration, the drastic decrease in hippocampal glutathione content and the significant increase in lipid peroxidation were attenuated in nimesulide-treated rats, suggesting that the induction of cyclooxygenase-2 is involved in kainate-mediated free radicals formation.

Ozone oxidative preconditioning is mediated by A1 adenosine receptors in a rat model of liver ischemia/ reperfusion.

The liver is damaged by sustained ischemia in liver transplantation, and the reperfusion after ischemia results in further functional impairment. Ozone oxidative preconditioning (OzoneOP) protected the liver against ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the role of A1 adenosine receptor on the protective actions conferred by OzoneOP in hepatic I/R. By using a specific agonist and antagonist of the A1 subtype receptor (2‐chloro N6 cyclopentyladenosine, CCPA and 8‐cyclopentyl‐1,3‐dipropylxanthine, DPCPX respectively), we studied the role of A1 receptor in the protective effects of OzoneOP on the liver damage, nitiric oxide (NO) generation, adenosine deaminase activity and preservation of the cellular redox balance. Immunohistochemical analysis of nuclear factor‐kappa B (NF‐κB), tumor necrosis factor alpha (TNF‐α) and heat shock protein‐70 (HSP‐70) was performed. OzoneOP prevented and/or ameliorated ischemic damage. CCPA showed a similar effect to OzoneOP + I/R group. A1AR antagonist DPCPX blocked the protective effect of OzoneOP. OzoneOP largely reduced the intensity of the p65 expression, diminished TNF‐α production, and promoted a reduction in HSP‐70 immunoreactivity. In summary, OzoneOP exerted protective effects against liver I/R injury through activation of A1 adenosine receptors (A1AR). Adenosine and .NO produced by OzoneOP may play a role in the pathways of cellular signalling which promote preservation of the cellular redox balance, mitochondrial function, glutathione pools as well as the regulation of NF‐κB and HSP‐70.

Ozone oxidative postconditioning ameliorates joint damage and decreases pro-inflammatory cytokine levels and oxidative stress in PG/PS-induced arthritis in rats.

Rheumatoid Arthritis (RA) is the most prevalent chronic condition present in ~1% of the adult population. Many pro-inflammatory mediators are increased in RA, including Reactive Oxygen Species such as nitric oxide NO, pro-inflammatory cytokines as tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1β) and other molecules. Ozone oxidative postconditioning has regulatory effects on some pathological targets associated with RA. Thus, the aim of this study was to investigate the efficacy of ozone therapy in PG/PS-induced arthritis in rats in point of joints inflammation and morphology. Moreover, cytokines, nitric oxide and oxidative stress levels in spleen homogenates were evaluated. Ozone treatment ameliorated joint damage, reduced TNF-α concentrations as well as TNF-α and IL-1β mRNA levels. Besides, cellular redox balance, nitric oxide and fructolysine levels were reestablished after ozone oxidative postconditioning. It was concluded that pleiotropic ozones effects clarify its therapeutic efficacy in RA. Decreasing inflammation and joint injury, reduction of pro-inflammatory cytokines, TNF-α and IL-1β transcripts and re-establishment of cellular redox balance after ozone treatment were demonstrated.

Ozone oxidative post-conditioning reduces oxidative protein damage in patients with disc hernia.

Abstract Introduction/objectives: Although inflammation in disc hernia (DH) has been recognized and it is a well-known process mediated by loss of the cellular redox balance, only a few studies about the impact of chronic oxidative stress on this neurological disorder have been made. Ozone therapy has been widely used with clinical efficacy in DH. This work aimed at characterizing the systemic redox status of patients with low back pain and neck pain as well as studying if ozone oxidative post-conditioning modified the pathological oxidative stress and protected against oxidative protein damage and if there is any relationship between oxidative changes and pain in both DH. Methods: Redox status of 33 patients with diagnosis of DH by computerized axial tomography, nuclear magnetic resonance, and clinical evaluations was studied. Ozone was administered by paravertebral way. After ozone treatment, plasmatic levels of antioxidant/pro-oxidant markers, pain, and life quality disability parameters were evaluated. Results: One hundred percent of patients showed a severe oxidative stress. Major changes in superoxide dismutase activity, total hydroperoxides, advanced oxidation protein products, fructolysine content, and malondialdehyde were observed. After ozone oxidative post-conditioning, there was a re-establishment of patients’ cellular redox balance as well as a decrease in pain in both DH. A relationship between indicators of oxidative protein damage and pain was demonstrated. Conclusions: Ozone therapy protected against oxidation of proteins and reduced the pain. Relationship between markers of oxidative protein damage, disability parameters, and pain suggests the role of oxidative stress in the pathological processes involved in DH.

Ischaemic and pharmacological preconditionings protect liver via adenosine and redox status following hepatic ischaemia-reperfusion in rats

Although IPC (ischaemic preconditioning) is considered as a protective strategy in HI/R (hepatic ischaemia/reperfusion), the mechanisms for this effect have not been fully elucidated. In the present study we investigate whether PPC (pharmacological preconditioning) by transient activation of A(1)R (adenosine A(1) receptor) protects against long-term HI/R and whether the protective effects of IPC depend on A(1)R activation and whether both preconditionings affect remote organs. Wistar rats underwent IPC and long-term HI/R. Another set of animals were pharmacologically preconditioned with the A(1)R-agonist CCPA [2-chloro-N(6)-cyclopentyladenosine; 0.1 mg/kg of body weight, i.p. (intraperitoneally)] 24 h before HI/R. In other groups, rats received an A(1)R-antagonist, DPCPX (1,3-dipropyl-8-cyclopentylxanthine; 0.1 mg/kg of body weight, i.p.) 24 h before HI/R. Hepatic damage was evaluated by transaminase [AST (aspartate transaminase), ALT (alanine transaminase)] release; inflammation was assessed by hepatic MPO (myeloperoxidase) and serum TNFalpha (tumour necrosis factor alpha) and NO; oxidative stress was estimated by MDA (malondialdehyde) and 4-HDA (4-hydroxyalkenals), SOD (superoxide dismutase) activity, GSH and ADA (adenosine deaminase) as adenosine metabolism. Both preconditionings protected liver and lung against HI/R as indicated by the reduction in transaminases, MPO, MDA+4-HDA, NO, TNFalpha and ADA activity as compared with HI/R (P<0.05). However, pre-treatment with DPCPX abolished the protective effects of IPC and PPC. Preconditionings induced a significant increase in hepatic MnSOD (manganese SOD) activity and NO generation compared with the sham group, and this activity was abolished by DPCPX pre-treatment. A(1)R activation induced hepatic delayed preconditioning and blockade of A(1)R abolished hepatic IPC. IPC, as well as PPC, were able to prevent lung damage. These protective effects are associated with a reduction in oxidative stress, inflammation and endogenous antioxidant preservation.

Ozone protective effects against PTZ-induced generalized seizures are mediated by reestablishment of cellular redox balance and A1 adenosine receptors

Abstract Objectives: Epilepsy is a common seizure disorder affecting approximately 70 million people worldwide. Mitochondrial dysfunction and antioxidant/prooxidant imbalance are emerging as factors that contribute to epileptogenesis. As medical ozone was able to reestablish cellular redox balance and to maintain the protective effects mediated by A1 receptors (A1Rs), the aim of this work was to study ozones effects on antioxidant/prooxidant balance and to clarify if A1Rs play a role in ozones protective actions against pentylenetetrazole (PTZ)-induced convulsions in mice. Methods: Influence of ozones treatments in mice submitted to PTZ-induced seizures was studied. Ozone was administered by rectal insufflation 1 mg/kg (5, 10, 15, 20 treatments), one per day, of 1–1·5 ml at an ozone concentration of 20 μg/ml. Mice received PTZ (90 mg/kg i.p.) 24 hours after the last ozone treatment. Oxygen control groups 26 mg/kg were introduced. Latency to first seizure was determined. Antioxidant/prooxidant balance in brain homogenates was studied. A1 adenosine receptors’ effects on ozones protective actions against seizures were evaluated using 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Results: Highest latency was observed when mice received 15 ozone treatments. Oxygen + PTZ group did not achieve protection against neither convulsions nor brain oxidative injury. Fifteen treatments of ozone protected against biomolecules oxidative damage and the antioxidant systems as well. 8-Cyclopentyl-1,3-dipropylxanthine abolished the ozones protection. Conclusions: Ozone therapy increased the latency for the first seizure and the survival percentage. These effects are discussed in point of ozones capacity to reestablish cellular redox balance, decrease biomolecules damage, and regulate activation of A1 adenosine receptors in PTZ-induced seizures.

Ozone Oxidative Postconditioning Protects Against the Injury Associated with Alcohol Withdrawal Syndrome in Rats

Ethanol withdrawal (EW) increases acetaldehyde accumulation and reactive oxygen species formation that promote damage to the brain. It is necessary to emphasize that while EtOH abuse and dependence are widespread, treatment options are extremely limited. This study aimed at investigating the protective effects of Ozone Oxidative Postconditioning (OzoneOxPost) on brain injury induced by oxidative stress and behavioral changes after 2 weeks of EW in rats. Four groups of rats (n = 10 each one) were settled: (I) Control, (II) Ethanol; (III) Ethanol + Ozone, and (IV) Ethanol + Oxygen. At the end of EW, rats were subjected to behavioral tests followed by brain tissue collection to measure markers of oxidative damage. OzoneOxPost increased food consumption, maintained water intake at the same levels as the control group, and reestablished cellular redox status. Anxiety, locomotor activity, and memory/learning of the rats were improved. These results demonstrated that OzoneOxPost protected the brain against oxidative injury, improving important functions of the Central Nervous Systems (CNS). It is a novel finding that is evidence of ozones effects on CNS.

Ozone in Medicine: Clinical Evaluation and Evidence Classification of the Systemic Ozone Applications, Major Autohemotherapy and Rectal Insufflation, According to the Requirements for Evidence-Based Medicine

ABSTRACT Now that indications are clearly defined, applications have mostly become standardized and the active mechanisms have been well confirmed, medical ozone application in the form of the low-dose concept, is established and proven as a complementary medical method in the treatment of chronic inflammations or diseases associated with chronic inflammatory conditions. More than 11,000 systemic ozone treatments in the form of Major Ozone Autohemotherapy (MAH) in 577 patients and ≥ 47,000 Rectal Insufflations (RI) in 716 patients in various clinical studies are subjected to critical clinical assessment and classification according to the criteria of evidence-based medicine (EBM). Statistically significant clinical and/or pharmacological improvements without side-effects or adverse reactions are found in all studies; special attention is drawn to maintaining hygiene when working with blood and to the use of ozone-resistent and biocompatible materials. On summarizing the evidence classification under RCT + CT (Randomized Controlled Trials + Controlled Trials), i.e., Levels Ib and IIa, 12 studies with 657 ozone-treated patients are obtained for MAH and 6 studies with 227 patients for RI. As a result of the evidence here assessed, the two systemic ozone applications, MAH and RI are part of evidence-based medicine. Both applications are effective, safe and economic.

Medical ozone increases methotrexate clinical response and improves cellular redox balance in patients with rheumatoid arthritis.

Medical ozone reduced inflammation, IL-1β, TNF-α mRNA levels and oxidative stress in PG/PS-induced arthritis in rats. The aim of this study was to investigate the medical ozone effects in patients with rheumatoid arthritis treated with methotrexate and methotrexate+ozone, and to compare between them. A randomized clinical study with 60 patients was performed, who were divided into two groups: one (n=30) treated with methotrexate (MTX), folic acid and Ibuprophen (MTX group) and the second group (n=30) received the same as the MTX group+medical ozone by rectal insufflation of the gas (MTX+ozone group). The clinical response of the patients was evaluated by comparing Disease Activity Score 28 (DAS28), Health Assessment Questionnaire Disability Index (HAQ-DI), Anti-Cyclic Citrullinated (Anti-CCP) levels, reactants of acute phase and biochemical markers of oxidative stress before and after 20 days of treatment. MTX+ozone reduced the activity of the disease while MTX merely showed a tendency to decrease the variables. Reactants of acute phase displayed a similar picture. MTX+ozone reduced Anti-CCP levels as well as increased antioxidant system, and decreased oxidative damage whereas MTX did not change. Glutathione correlated with all clinical variables just after MTX+ozone. MTX+ozone increased the MTX clinical response in patients with rheumatoid arthritis. No side effects were observed. These results suggest that ozone can increase the efficacy of MTX probably because both share common therapeutic targets. Medical ozone treatment is capable of being a complementary therapy in the treatment of rheumatoid arthritis.