Ozone Therapy Clinic and Basic Evidence of its Therapeutic Potential

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Slide 1 : Copyright, 1996 © Dale Carnegie & Associates, Inc. Ozone TherapyClinical and Basic Evidence of its Therapeutic Potential Lamberto Re University of Ancona Clinical Pharmacology & Toxicology
Slide 2 : Our first interest on ozone as a possible therapeutic agent started in 1993 following a discussion on the hypothesis that low ozone doses could induce a positive conditioning against the oxidative stress. Subsequently, the Colleague Leon Fernandez O.S. first proposed the basic evidence of some biochemical effects induced by low ozone doses (Mediators-Inflamm. 7(4): 289-94, 1998). The theory was based upon the fact that the exposure to low, non-toxic, ozone concentrations could potentiate the efficacy of the endogenous antioxidant system by increasing the production or the activity of some enzymatic isoforms. INTRODUCTION
Slide 3 : Similarly to the ischemic preconditioning in which it is scientifically proved that repetitive brief ischemia plays an important role in the acquisition of late-phase cardio protection against ischemia/reperfusion injury in rats (Yamashita et al, Br J Pharmacol, 131(3): 415-422, 2000), it could be speculate that repetitive brief oxidative stress induced with low ozone doses could ameliorate the cell defenses mechanisms against reactive oxygen species (ROS). The hypothesis was supported by other data reported by Rao and Shaha (Free Radic Biol Med, Nov 15; 29 (10): 1015-1027, 2000) demonstrating the formation of multiple isoforms of glutathione S-transferase after the exposure to H2O2.
Slide 4 : Molecular hypotheses Due to our experience as pharmacologist and with previous research devoted mainly to the study of the molecular events underlying the pharmacological action of drugs, our group was initially attracted by a pure scientific curiosity.The main questions addressed to: Why many epidemiological data reported the evidence of a benefit of this gas in different, apparently unrelated, pathologies? Was there a dose-effect relationship? Was this therapy completely safe?Why an agent known for its strong oxidative potential could induce a benefit other than its intrinsic bactericidal action?
Slide 5 : Interaction with Conventional Medicine In the last years Ozone Therapists have been negatively focused for the lacking of rigorous scientific data concerning the use of Medical Ozone. In this context, taking into account the considerations discussed below, we only partially agree with some of the points argued by the critics. Without any doubt, most of the problems arise from the first empiric use and approach to Ozone by the same Ozone Therapists!
Slide 6 : Usually the criticism indicate the Ozone Therapist as a poisoner considering ozone like a venom. In this context however, we can’t forget that every drug (from ancient Greek pharmakon) must be considered a venom! But we can consider aspirin, antibiotic or the same water as venoms? Furthermore, we can’t forget that for every drug the pharmacologists evaluate a parameter to establish the threshold of toxicity and therapeutic (The Therapeutic Index)!
Slide 7 : What is Ozone? Ozone (O3 - PM 48) is an allotropic form of oxygen. It represents an extremely unstable molecule characterized by 3 atoms of oxygen. To temperature environment is a gas, colorless with sharp and prickly odor. It is an extremely important mixture for the life on the earth and one of the fundamental components of the atmosphere.
Slide 8 : As stated above emphasis and attention has been focused on the use of medical ozone. Despite ample clarification confusion persists concerning its potential toxicity as an oxidant agent versus the reported clinical efficacy. This confusion is a major factor preventing a more widespread acceptance.
Slide 9 : Furthermore the use in specialities so diverse as neurology, orthopaedics, internal medicine, sports medicine, endocrinology and others makes it difficult to categorize ozone as a therapeutic agent. This may cause conflicts between the different fields of application and the various medical areas.
Slide 10 : Number of articles per year, relative to “ozone” (Cumulative) Data from MedLine PubMed, October 2007 10 186 6 889 4 762
Slide 11 : Average in Number of articles per year, relative to “ozone in medicine” (Last ten years. Articles not related to ozone pollution or toxicity) Data from MedLine PubMed, October 2007 722 466 Years
Slide 12 : Evolution in Number of articles relative to “Ozone / Clinical Trials” (All records and comparative evolution between 1997 and 2007 Data from MedLine PubMed, October 2007 Includes 243 papers on Clinical Trials 2 articles 98 articles
Slide 13 : Main target diseases / organs of “Ozone Therapy” (Cumulative number or articles up to October 2007) Data from MedLine PubMed, October 2007 Main Target Organs Main Target Diseases
Slide 14 : Ozone is most commonly associated with intervertebral disc herniation (Andreula et al., Am J Neuroradiol. 2003 May;24(5):996-1000) and only recently was introduced in the field of stomatology.
Slide 15 : Regarding disc herniation and the so called disc conflict, most of the ozone actions are bound to its peculiar effects on the bio-humoral environment. The mistaken view to consider ozone a simple mechanistic agent causing the lyses and the reduction of the herniated disc must be, to our opinion, argued and discussed.
Slide 16 : The application of ozone in neuro-radiology is considered scientific and successful based on the possibility to estimate statistically the reduction or the disappearance of the anatomical protrusion. In our opinion this is not proof enough. A follow up considering the status of the patient following the treatment could better indicate the efficacy or not of the more invasive ozone treatments compared to different methodologies based on the neuro-humoral action of ozone such as para-vertebral, percutaneous or others.
Slide 17 : The huge variability of the clinical responsiveness of patients introduces further difficulties in the set up of standardized studies. Recent advances in ozone effects and conditioning, suggest a key role for trace elements. Studies are in progress to evaluate the ratio of Mn2+, Cu2+, Zn2+ and other essential elements for the enzymatic activity of Superoxide Dismutase (SOD) and other enzymes involved in the ozone effect. A dietary insufficiency or impairment either in food supply or metabolic pathways may play a negative role in the efficacy of the ozone treatment.
Slide 18 : Most of the scientific data recently published, and partially ignored by some authors, are indicative of many basic effects of the ozone molecule and could form the basis for future randomized clinical studies. One of the aim of this presentation is to discuss the more recent data published in international journals.
Slide 19 : The pharmacological theory first described by Erlich on drug action: “corpora non agunt nisi fixata” intended as a drug-receptor interaction that following the mass action law promotes and activates a function in the cell, is apparently not applicable to ozone. As pharmacologists and scientists we are naturally drawn to the challenge of questioning the Erlich theory to demonstrate scientifically some pharmacological activities induced by a gas like ozone without a pure drug-receptor interaction.
Slide 20 : In the light of the more recent pharmacological knowledge we can consider ozone as a pro-drug which, at certain non-toxic doses, can induce a rearrangement of the biochemical pathways with the activation of a second messenger in a cascade with a multiple system action. Ischemic Preconditioning (IPC) represents the best similarity in this context.
Slide 21 : Evidence that antioxidant enzymes, nitric oxide pathways and other sub-cellular activities could be modulated by low ozone doses is now proven and could support the surprising effects of ozone in many pathological conditions. Furthermore, in the light of the report of Wentworth et al. (Science 2002; 298: 2195-2199), the scientific data reported here could be scientifically emphasized and pharmacologically indicative. Indeed, the authors demonstrate the physiological presence of an ozone-similar mediator during inflammation, indicating ozone as a new bio-molecule with striking effects which must be considered and studied following new strategies with newly constructed randomized-standardized clinical studies.  
Slide 22 : The first hypothesis of a positive conditioning induced by low Ozone concentrations against the oxidative stress has been proposed by the Cuban Scientist in the earlier 1996. The theory was based upon the fact that low, non-toxic, Ozone doses could raise the efficacy of the endogenous system by increasing the production or the activity of some antioxidant enzymes isoforms. Looking at the IPC in which is scientifically proved that repetitive brief ischaemia plays an important role in the acquisition of late-phase cardio protection against ischaemia/reperfusion injury in rats (Yamashita et al, Br J Pharmacol, 131(3): 415-422, 2000), we can speculate that repetitive brief oxidative stress induced with low Ozone doses could ameliorate the cell defences mechanisms against ROS. Anti-Ageing Hypothesis and Experimental Models
Slide 23 : Other evidences related to the protective action induced by low Ozone concentrations demonstrated the reduction of the intracellular calcium. The cytosolic calcium could be considered as the common final pathway of the cellular activation and an impairment of its intracellular levels could promote damage. A low calcium level represents a further element in supporting the idea of a protection against the oxidative cell damage either in the chronic (physiological) or in the acute (pathological) ageing. Similarly to the IPC the acronyms OzoneOP was introduced to define the effects induced by the Ozone Oxidative Preconditioning.
Slide 24 : Ozone and Diabetes Basic Studies Diabetes produces a large number of changes in vessels that affect the reactivity of smooth muscle and endothelium. Vascular endothelium appears to be a vulnerable target for hyperglycemia-induced metabolic changes. Activation of the polyol pathway, non-enzymatic glycosilation of proteins and the increase of reactive oxygen species (ROS) play an important role in diabetes complications. Ozone has been used as a therapeutic agent and beneficial effects have been observed. However, so far only a few biochemical and pharmacodynamic mechanisms have been elucidated.
Slide 25 : Other studies (Mediators of Inflammation 1998; 7:289-294; Free Radical Research 1999;31:191-196) confirmed that controlled ozone administration may promote an oxidative preconditioning or adaptation to oxidative stress, preventing the damage induced by ROS. Given that diabetes is a disorder associated with oxidative stress, it was postulated that ozone treatment might protect antioxidant systems and maintain at a physiological level other markers of endothelial cell damage associated with diabetic complications. A study using streptozotocin (STZ) as a diabetes inducer was designed to test the protective effect promoted by ozone.
Slide 26 : Ozone treatment improved glycemic control,  the increase of aldose reductase, the fructolysine content, the advanced oxidation protein products, the pancreas integrity and prevented the oxidative damage. Furthermore, increased nitrite and nitrate levels with respect to STZ group occurred, but without changes when compared to non-diabetic controls. The results of this study show that repeated administration of ozone in non-toxic doses might play a role in the control of diabetes and its complications (Pharmacological Research 2001;44 :391-396). In addition, ozone antioxidant properties preserved ?-cells functions and reduced hyper-glycemia. Taken together, these results suggest that this approach may represent a potential complement in the treatment of diabetes and its complications.
Slide 27 : Clinical Studies Because ozone therapy can activate the antioxidant system, influencing the level of glycaemia and some markers of endothelial cell damage at a pre-clinical level, a study to investigate the therapeutic efficacy of ozone treatment in patients with type 2 diabetes and diabetic foot was done aimed at comparing ozone efficacy with respect to the antibiotic therapy. A randomized controlled clinical trial was performed with 101 patients divided into two groups: one (n=52) treated with ozone (local and rectal insufflations of the gas) and the other (n=49) treated with topical and systemic antibiotics.
Slide 28 : The efficacy of the treatments was evaluated by comparing the glycaemic index, the area and perimeter of the lesions, the biochemical markers of oxidative stress and the endothelial damage in both groups after 20 days of treatment. Ozone improved glycaemic control, prevented oxidative stress, normalized levels of organic peroxides and activated superoxide dismutase.
Slide 29 : The pharmacodynamic effect of ozone in the treatment of patients with neuroinfectious diabetic foot can be ascribed to the possibility of its being a superoxide scavenger. Superoxide is considered a link between the four metabolic routes associated with diabetes pathology and its complications. Furthermore, the healing of the lesions improved, resulting in fewer amputations than in the control group. There were no side effects. These results reinforce our opinion that medical ozone treatment could be a complementary therapy in the treatment of diabetes and its complications (European Journal of Pharmacology 2005; 523:151-161).  
Slide 30 : Ozone and SOD Many studies indicate that, after reoxygenation of the liver, oxygen free-radical formation may initiate the cascade of hepatocellular injury, necrosis/apoptosis, and subsequent infiltration of inflammatory cells. Although ROS can arise from a number of sources, xanthine oxidase (XO) is frequently implicated as a significant source of these toxic oxygen species. The IPC is an inducible and potent endogenous mechanism by which repeated episodes of brief ischemia/re-perfusion (I/R) confer a state of protection against subsequent sustained I/R. On the other hand, it has been demonstrated that ozone at low doses is able to promote an OzoneOP through the increase and preservation of antioxidant endogenous systems.
Slide 31 : Superoxide is one of the most relevant radicals in biological regulation. Many regulatory effects are mediated by hydrogen peroxide and other ROS that are chemically derived from superoxide (Physiol Rev 2002; 82: 47-95). Although SOD could protect against liver I/R injury, the administration of SOD does not protect the liver against I/R damage (J Surg Res 1997;73:160-165). The protein SOD degrades rapidly when administered parenterally. Gene delivery has been used to increase protein expression in the cell (Pathology 1998; 30:335-347). Although the benefit of ischemic preconditioning in the liver has already been suggested in a pilot clinical study (Ann Surg 2000;232: 155-162), knowledge of the molecular mechanism is limited. Intermittent clamping is currently is used in practice by many Centers.
Slide 32 : Although the working of the protective mechanism of intermittent clamping still remains unclear, it has been assumed to be a similar mechanism to that described in ischemic preconditioning, mainly by reduction of apoptosis (Ann Surg 2002; 235: 400-407). A large number of pharmacological agents were shown to confer protection against ischemic injury in the liver. These agents include antioxidant, adenosine agonists and nitric oxide (NO.) donors. Nevertheless, only a few drugs are currently at the point of clinical application. However, not only adenosine production but also other mechanisms seem to be involved.
Slide 33 : OzoneOP is able to promote a moderate oxidative stress which, in turn, increases antioxidant endogenous systems protecting against liver damage (J Appl Toxicol 2001;21: 291-301). The protective mechanism mediated by OzoneOP may involve protein synthesis. Elevated ROS concentrations induce in many cells the expression of genes whose products exhibit antioxidatant activity. A major mechanism of redox homeostasis is based on the ROS-mediated induction of redox sensitive signal cascades that lead to increased expression of antioxidants.
Slide 34 : To investigate the influence of the inhibition of protein synthesis on the protective actions conferred by OzoneOP in hepatic I/R rats were treated with cicloheximide in order to inhibit protein synthesis before OzoneOP treatment. Plasma transaminases, malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA) and morphological characteristics were measured as an index of hepatocellular damage; Cu/Zn-SOD, Mn-SOD, catalase (CAT), total hydroperoxides (TH) and reduced glutathione (GSH) levels as markers of endogenous antioxidant system were evaluated. OzoneOP increased Mn-SOD isoform and ameliorated mitochondrial damage. Conversely, cicloheximide abrogated the protection conferred by OzoneOP and decreased Mn-SOD activity. Cellular redox balance disappeared when cicloheximide was introduced. Thus, protein synthesis is involved in the protective mechanisms mediated by OzoneOP and ozone treatment preserved mitochondrial functions and cellular redox balance (Transplant International 2005;18:604–612).
Slide 35 : SOD Linked Pathologies SOD helps and protects cells from DNA damage, lipid peroxidation, ionizing radiation damage, protein denaturation, and many other forms of progressive cell degradation. Mutations in the first SOD enzyme (SOD1) have been linked to familial amyotrophic lateral sclerosis (ALS, a form of motor neuron disease). The other two types have not been linked to any human diseases, however, in mice inactivation of SOD2 causes perinatal lethality and inactivation of SOD1 causes hepatocellular carcinoma. Once more we think that enough evidences are now outlined demonstrating the positive effects induced by Ozone. Taken together, they could represent the key to understand the surprising effects induced by low Ozone doses.
Slide 36 : Ozone and Nitric Oxide (NO) Nitric oxide is a gas. It is highly reactive; that is, it participates in many chemical reactions. (It is one of the nitrogen oxides ("NOx") in automobile exhaust and plays a major role in the formation of photochemical smog.) But NO also has many physiological functions. They share these features: NO is synthesized within cells by an enzyme NO synthase (NOS). The human (and mouse) genome contains 3 different genes encoding NO synthases. nNOS (or NOS-1): found in neurons. iNOS (or NOS-2): found in macrophages, "i" standing for "inducible". eNOS (or NOS-3): found in the endothelial cells that line the lumen of blood vessels. Whereas the levels of nNOS and eNOS are relatively steady, expression of iNOS genes awaits an appropriate stimulus (e.g., ingestion of a parasite).
Slide 37 : Liver transplantation is now accepted as the best treatment for end-stage liver disease. Nevertheless, hepatic I/R injury associated with liver transplantation and hepatic resections are unresolved problems in clinical practice. Many studies indicate that oxygen free-radical formation after reoxygenation of liver may initiate the cascade of hepatocellular injury. The effects of OzoneOP on NO generation and the cellular redox balance have been studied using the inhibitor of the NO synthesis N?-nitro-L-arginine methyl ester (L-NAME) (Liver International 2004;24:55-62).
Slide 38 : All types of NOS produce NO from arginine with the aid of molecular oxygen and NADPH. NO diffuses freely across cell membranes. There are so many other molecules with which it can interact, that it is quickly consumed close to where it is synthesized. The product N?-nitro-L-arginine methyl ester (L-NAME) is an inhibitor of the NO synthesis and it has been used in the experimental study.
Slide 39 : The following parameters were measured: plasma transaminases (aspartate aminotransferase, alanine aminotransferase) as an index of hepatocellular injury; in homogenates of hepatic tissue nitrate/nitrite levels and inducible Nitric Oxide Sintetase (iNOS) by immuno-histochemistry as an index of .NO production; SOD, CAT and GSH levels as markers of the endogenous antioxidant system, and finally MDA + 4-HDA, TH and Tumor Necrosis Factor (TNF-?) as indicators of oxidative stress. A correspondence between liver damage and the increase of .NO, CAT, TH, GSH and MDA + 4HDA concentrations were observed along with a decrease of SOD activity.
Slide 40 : OzoneOP prevented and attenuated hepatic damage in OzoneOP +I/R and OzoneOP+L-NAME+I/R, respectively, in close relation with the above-mentioned parameters. Immuno-histochemistry of iNOS showed that OzoneOP regulated enzymatic activity while TNF-? levels were attenuated in the OzoneOP + I/R group. These results show that OzoneOP protected against liver I/R injury through mechanisms that promote a regulation of endogenous .NO concentrations and the maintenance of cellular redox balance. Ozone treatment may have important clinical implications, particularly in view of the increasing hepatic transplantation programs.
Slide 41 : There are different experimental results and opinions surrounding NO generation and its function in liver I/R injury as well as its protective effects. Nevertheless, the role of NO as a regulator of important processes in liver I/R is unquestionable. OzoneOP regulated NO formation in the OzoneOP + I/R group and decreased the liver damage (increases in AST was prevented and those in ALT were attenuated). L-NAME was able to reduce NO generation in sham operated + L-NAME and NO levels were not detectable in L-NAME + I/R group.
Slide 42 : Nevertheless, OzoneOP promoted NO formation in OzoneOP + L-NAME + I/R in spite of L-NAME´s presence, but lesser than OzoneOP + I/R. There was a concomitant increase in transaminase activities in this group (OzoneOP + L-NAME + I/R). These results suggest that the protection conferred by OzoneOP against the damage in liver I/R seems to be mediated, at least in part, by NO generation.
Slide 43 : The contribution of OzoneOP to NO generation may be a consequence of its actions on gene expression. Punjabi et at (Am J Respir Cell Mol Biol 1994;11:165-172) and Pendino et al (Am J Respir Cell Mol Biol 1996;14:516-525) have shown that exposure to ozone causes NO production in macrophages and type II cells of rats, whereas Haddad et al. (Eur J Pharmacol 1995;293:287-290) demonstrated iNOS induction in rats.
Slide 44 : More recently it has been found that ozone-induced lung hyperpermeability is associated to iNOS and that iNOS mRNA levels are mediated through Tlr-4 which has been identified as the gene that determines susceptibility to endotoxins. There was a correlative patterns of gene expression in two strains (ozone-susceptible and ozone-resistant, respectively) which support a role of Tlr4 in the regulation of iNOS during ozone exposure in the mouse (Am J Physiol Lung Cell Mol Physiol 2001;280: L326-L333).
Slide 45 : NO effects The following reported effects of NO are surprising similar to those induced by the treatments with Ozone at low concentrations. Throughout our clinical experience over more than 15 years we wondered again when our scientific data well complement the clinical effects observed during patients treatment.
Slide 46 : NO relaxes the smooth muscle in the walls of the arterioles. This diffuses into the underlying smooth muscle cells causing them to relax and thus permit the surge of blood to pass through easily. Mice whose genes for the NO synthase found in endothelial cells (eNOS) has been "knocked out" suffer from hypertension. Nitroglycerine, which is often prescribed to reduce the pain of angina, does so by generating nitric oxide, which relaxes the walls of the coronary arteries and arterioles. Three of the pioneers in working out the biological roles of NO shared a Nobel Prize in 1998 for their discoveries. The award to one of them, Ferid Murad, honoured his discovery that nitroglycerine works by releasing NO. This seems particularly appropriate because Alfred Nobel's fortune came from his invention of making dynamite from nitroglycerine! NO also inhibits the aggregation of platelets and thus keeps inappropriate clotting from interfering with blood flow. NO on Blood Flow
Slide 47 : NO on Inflammation The NO produced by NOS-3 inhibits inflammation in blood vessels. It does this by blocking the exocytosis of mediators of inflammation from the endothelial cells. NO may also block exocytosis in other types of cells such as macrophages and cytotoxic T lymphocytes (CTL).
Slide 48 : NO on the Nervous System NO and the Autonomic Nervous System Some motor neurons of the parasympathetic branch of the autonomic nervous system release NO as their neurotransmitter. The actions of NO on penile erection and peristalsis are probably mediated by these nerves. NO and the Medulla Oblongata In severe deoxygenation, NO-sensitive cells in the medulla oblongata respond to this release by increasing the rate and depth of breathing. NO and the Brain In laboratory animals (mice and rats), NO is released by neurons in the CA1 region of the hippocampus and stimulates the NMDA receptors there that are responsible for long-term potentiation (LTP) — a type of memory (and learning). The ease with which NO diffuses away from the synapse where it is generated enables it to affect nearby synapses. Laboratory rats treated with inhibitors of NOS synthesis fail to develop and/or retain learned responses such as the conditioned response.
Slide 49 : NO Killing Pathogens NO aids in the killing of engulfed pathogens (e.g., bacteria) within the lysosomes of macrophages. Mice whose genes for the NO synthase found in macrophages (iNOS) have been knocked out are more susceptible to infections by intracellular bacteria like Listeria monocytogenes. Th1 cells, the ones responsible for an inflammatory response against invaders, secrete NO. Harmless bacteria, living as commensals at the rear of our throat, convert nitrates in our food into nitrites. When these reach the stomach, the acidic gastric juice (pH ~1.4) generates NO from them. This NO kills almost all the bacteria that have been swallowed in our food. (Since the dawn of recorded human history, nitrites have been used to preserve meat from bacterial spoilage.)
Slide 50 : NO and Longevity Mice whose genes for eNos have been knocked out; show signs of premature ageing; have a shortened life span; fail to benefit from the life-extending effect of a calorie-restricted (CR) diet.
Slide 51 : At this purpose we remember that after a conference at Imperial College in London some Colleagues showed us the following press article “Queen Mother uses ozone-therapy against ageing”. So we can’t forget that since 1985, more of 15 Countries all over the world integrate conventional medicine with ozone-therapy. More than 5 millions patients have been submitted to this integrative approach and the adverse effects, mostly from malpractice, are included in a number after the comma preceded by 5 zeros!. Sunday Mirror, May 2001. A news that must be secret!
Slide 52 : Ozone and Purinergic Receptors The liver is damaged by sustained ischemia in liver transplantation, and the reperfusion after ischemia results in further functional impairment as widely showed before. In view that OzoneOP protects the liver against ischemia/reperfusion (I/R) injury, a recent study was conducted to investigate the role of A1 adenosine receptor on the protective actions conferred by OzoneOP in hepatic I/R (Leon-Fernandez et al, Transpl Int, 2008 Jan;21(1):39-48. Epub 2007 Oct 10). 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), the authors studied the role of A1 receptor in the protective effects of OzoneOP on the liver damage, NO generation, adenosine deaminase activity and preservation of the cellular redox balance.
Slide 53 : Immunohistochemical analysis of Nuclear Factor-kappa B (NF-?B), Tumor Necrosis Factor alpha (TNF-a) 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 NF-?B (p65 subunit), diminished TNF-a production, and promoted a reduction in HSP– 70 immunoreactivity.
Slide 54 : The work demonstrated that 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 signaling which promote preservation of the cellular redox balance, mitochondrial function, glutathione pools as well as the regulation of NF-?B and HSP-70.
Slide 55 : The effects of OzoneOP on adenosine and NO have a particular importance in cellular signaling processes. Carini et al (Gastroenterology 2003; 125: 1480) have proposed that both biomolecules are involved in the mechanisms leading to the development of hepatocyte resistance to I/R injury following early and late hepatic preconditioning. These protective mechanisms include: preservation of energy sources, mitochondrial functions, pH, ion homeostasis as well as to reduce oxidative injury and caspase activation. OzoneOP actions preserved mitochondrial integrity (Transpl Int 2005; 18: 604) and reduced generations of protons and lactate concentrations by anaerobic glycolysis (J Appl Toxicol, 2001; 21: 297).
Slide 56 : In summary, OzoneOP exerts protective effects against liver I/R injury through activation of A1AR. In analogy to IPC, adenosine and NO produced by OzoneOP may play a role in the cell signalling pathways which promote preservation of cellular redox balance, mitochondrial function, glutathione pools, regulation of NF-?B and HSP-70, among other effects. OzoneOP may be considered as a pharmacologic liver preconditioning which might be particularly relevant for improving liver transplantation. The finding that the effects of OzoneOP are mediated by A1AR allows considering other potential medical applications for the ozonetherapy mainly in cardiovascular and central nervous systems.
Slide 57 : Ozone and Parkinson`s Disease A recent work evaluated the effects of OzoneOP on an in vivo model of rotenone-induced neuro-degeneration in rats. Oxidative stress has been implicated in numerous pathophysiological situations (J Neurosci 2004;24:7779-7788) being considered a unifying factor in the current theories of Parkinson's disease (PD) pathogenesis. This is because of the links between genetic and potential environmental factors in the onset and progression of the disease. Those environmental toxins that have the strongest association with PD phenotypes either cause high amounts of oxidative stress, such as rotenone, or directly increase the rate of alpha-synuclein aggregation, as with copper and other heavy metals (J Biol Chem 2001;276:44284-96).
Slide 58 : Furthermore, the aggregation of alpha-synuclein itself can cause oxidative stress (Free Radic Biol Med 2001;30:1163-1170) and oxidative stress can in turn cause conformational changes in alpha-synuclein (Ann N Y Acad Sci 2003;991:93-100). Even if the factors initiating the pathogenesis of PD and related neurodegenerative synucleinopathies are still largely unclear, many studies indicate a multiple brain mitochondria dysfunction after systemic treatment with pesticides or rotenone (Am J Pathol 2007;170(2):658-666; J Biol Chem 2005;280(51):42026-42035; Nat Neurosci 2000;3(12):1301-1306).
Slide 59 : Rotenone is a classical, high affinity inhibitor of complex I, which has been widely used to understand the specific activity of the complex. Rotenone being extremely lipophilic, freely crosses the blood brain barrier and biological membranes, thus rapidly reaching the brain. Repeated systemic exposure of rotenone has been reported to cause nigrostriatal dopaminergic degeneration in rats, producing an in-vivo experimental model of PD (Antioxid Redox Signal 2005;7:1110-1116). The reductions in the activity of complex I of the mito-chondrial electron transfer chain (ETC) may play an im-portant role in rotenone-induced dopaminergic neuro-degeneration in PD (J Biol Chem 2004;279:51783-92).
Slide 60 : To evaluate the effect of OzoneOP on PD a study was conducted during a period of four weeks to evaluate the neurochemical effects of repeated exposure to rotenone in rats. The aim was to test the probable preventive effect of OzoneOP (as an indirect antioxidant) against rotenone-induced neurodegeneration in rats. It included sixty rats, Sprague-Daley strain, with an average weight of 175 g, divided into four groups:
Slide 61 : Group I: The control group. Group II: The ozone control group. The rats were given 5 ml of 25 µg/ml ozone in oxygen rectal insufflations (0.7 mg/kg). They received 20 sessions: 5 sessions per week for 4 weeks. Group III: Rotenone was injected subcutaneous in a dose of 2 mg/kg/day every other day for a total of six injections over 11 days. Group IV: The study group (OzoneOP). The rats were given 5 ml of 25 µg/ml ozone in oxygen rectal insufflations (0.7 mg/kg). They received 20 sessions 5 sessions per week for 4 weeks. After 10 sessions of ozone administration (two weeks) rotenone was injected s.c. at a dose of 2 mg/kg/day every other day for a total of six injections over 11 days.
Slide 62 : The rats were observed daily for the development of any signs of toxicity throughout the treatment period. Repeated rotenone treatment caused a marked decrease in both the food intake and the locomotor activity and induced muscle relaxation of both fore and hind limbs accompanied with high mortality rate in comparison to the other treatments. Ozone therapy remarkably increased food intake and rate of weight gain compared to the control group and prevented the mortality of the animals. Repeated treatment with rotenone significantly (p<0,05) decreased the levels of dopamine and norepinephrine in both the cortex and striatum. OzoneOP significantly (p<0,05) minimized the declining effect of rotenone on the levels of the two transmitters in the cortical and the striatal regions.
Slide 63 : In addition, rotenone treatment increased the level of NO, MDA, oxidized glutathione and protein carbonyls of brain cortex and striatum. Rotenone- treated animals exhibited a significant (p<0,05) decrease in the level of GSH, ATP and depressed enzymatic activity of SOD.   OzoneOP significantly (p<0,05) antagonized the disturbing effect of rotenone on the tested parameters in the cortical and the striatal regions.   Repeated rotenone treatment induced remarkable histopathological abnormalities in brain cortex which are manifested as inflammatory, hemorrhagic and neurodegenerative effects (Fig. 2) in comparison to normal control (Fig. 1).    
Slide 64 : OzoneOP remarkably attenuated the undesirable histopathological damage induced by rotenone (Fig. 3). A transverse section of cerebral cortex of control rat showing the normal structure of the meninges (M) and cerebral cortex (C). H& E, x : 40 A transverse section of cerebral cortex of rat injected with rotenone showing sever hemorrhages (arrow), edema (o) and hyperemic blood vessels in the meninges covering cerebral cortex. H& E, x: 40 A transverse section of the cerebral cortex of rat injected with rotenone and ozone showing normal histological structure in the cerebral cortex and surrounding meninges. H& E, x: 40
Slide 65 : The study (Arch Med Res. Jan;39(1):17-26. Epub 2007 Sep 29, 2008) suggests that oxidative stress plays an essential role in rotenone toxicity and that OzoneOP may offer a remarkable protective effect against rotenone induced brain toxicity. The data presented in this paper are indicative of potentially positive effects induced by treatment with low doses of Ozone. Particularly, an OzoneOP approach could be considered as a positive complement to the actual pharmacological therapies addressed to some pathologies such as diabetes and neurodegenerative disorders promoting the regulation of endogenous NO concentrations and the maintenance of an adequate cellular redox balance.
Slide 66 : Somebody still ask .. It will be or not?
Slide 67 : Major Auto-Haemo Therapy is applied for the various pathologies without any negative or collateral effect …
Slide 68 : Local Infiltrations for Achille’s Tendon and other injuries 30G 13mm – 27G 6mm 10 ?g/ml O3/O2 20 cc
Slide 69 : Plastic Bag Oxygen-Ozone 70 µg/ml Skin Ulcer Ankle Twist
Slide 70 : The Vaccination Theory a Surprising Similarity Edward Jenner and the Discovery of Vaccination The year 1996 marked the two hundredth anniversary of Edward Jenner's first experimental vaccination--that is, inoculation with the related cow-pox virus to build immunity against the deadly scourge of smallpox. Edward Jenner (1749-1823), after training in London and a period as an army surgeon, spent his whole career as a country doctor in his native county of Gloucestershire in the West of England. His research was based on careful case-studies and clinical observation more than a hundred years before scientists could explain the viruses themselves. So successful did his innovation prove that by 1840 the British government had banned alternative preventive treatments against smallpox. "Vaccination," the word Jenner invented for his treatment (from the Latin vacca, a cow), was adopted by Pasteur for immunization against any disease.
Slide 71 : John Baron, M.D., 1786-1822The Life of Edward Jenner, M.D., LL.D., F.R.S.London: Henry Colburn, 1838. 2 volumes. While still an apothecary's apprentice in the late 1760s, Jenner had been intrigued by possible relationships between smallpox, cowpox, and swinepox. At the time, he was ridiculed. By 1780, however, he returned to the idea, as evidenced in the conversation recorded here, and in 1789 he experimented by inoculating his own son, then aged one-and-a-half, with the swine pox, followed by conventional smallpox inoculation. Christian Charles Schieferdecker, M.D.Dr. C. G. G. Nittinger's Evils of Vaccination.Philadelphia: the editor, 1856. Because of the lack of clear scientific explanation of its effects, the frequent side-effects, and contaminated vaccines, vaccination itself remained controversial throughout the nineteenth century. It certainly carried risks for the infants being vaccinated, and this volume, playing on parental fears, argued, inter alia, that vaccination was nonsensical, unscientific, criminal, and even sinful.
Slide 72 : Every new theory passes through three phases: 1. It is attacked and declared absurd 2. Then it is admitted that it is true and obvious, but insignificant 3. To the end it is recognized the real importance and its detractors demand the honor to have discovered it 1842-1910 American psychologist and philosopher
Slide 73 : Thank you

 


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