Ozone therapy – A comprehensive study of the literature. M.Nordfors and PA Öckerman.

Summary. The gas ozone (O3) was discovered in the 19th century. It consists of three atoms of oxygene. Ozone therapy has been used and studied for more than one hundred years. The effects are proven, consistent, safe and with minimal and preventable side effects. Medical O3 is used to disinfect and to treat diseases. O3 kills bacteria, viruses, fungi, yeast and protozoa, stimulates oxygene metabolism and blood circulation, activates antioxidant mechanisms and the immune system. Ozone is toxic to lung epithelium and the eyes, due to low activity of antioxidants in these tissues. Therefore, it is mainly given as an intravenous infusion, either directly into the blood, as an ozonized saline solution. It can also be given as so-called major autohemotherapy, taking out 50-200 mL blood from the body, which will be ozonized extracorporeally, then infused back again. This can be done from one to 30 times in a row, depending on the treatment tradition and treatment goals. However, most therapists do it only once. There is also a version called EBOO, where you ozonize 3-5 Liters of blood continuosly extracorporeally. Yet another method is to take out a small amount of blood (5-10 mL) , which is ozonized, and then inject it intramuscularly. This is called minor autohemotherapy. A rectal application is easy to perform, has good clinical effects, and can also be performed by the patients themselves in their home. The gas can be injected intramuscularly in a case of muscle damage or muscle pain, paravertebrally when treating back pain, in joints and directly into disc herniation under X-ray supervision as a safe and inexpensive alternative to surgery. Ozone is also widely used as a topical beauty treatment. You can also treat wrinkles with subcutaneous injections and ozone baths. It is also used in dentistry, treating caries and combating dental infections. One can enclose affected body parts in ozone gas baths in order to combat infections and improve wound healing. Ozone cream and ozone oil can also be used for this purpose. In this literature review, 77 clinical studies are summarized, of which 55 are controlled, 11 double-blind, randomized studies. 30 are randomized, but not blinded, 14 not randomized but controlled and 22 non-controlled case series. 76 of the 77 studies show statistically significant results or positive results compared to the entry status and/or expected prognosis. Treated conditions are heart failure, coronary vascular disease, peripheral vascular disease, diabetes, diabetic complications, ebola, Burulli ulcers, hepatitis, atypical pneumonia, fungal infections, infections of the gums and the oral cavity, genital infections, fibromyalgia, reumatoid arthritis, multiple sclerosis, ulcerative colitis, peritonitis, pancreatitis, gastric ulcers, bacterial vaginosis, placental insufficiency, preventatively before prostate surgery, urinary tract infections, chronic ulcers, burns, venous leg ulcers, traumatic wounds, pulmonary emphysema, cancer, osteonecrosis of the jaw, herniated disc, spondylosis, spondylolisthesis, osteoarthritis, jaw pains, vestibulocochlear problems, autonomic dysregulation and headache. It has also been used as anti-aging and as a general performance-enhancing agent in sports, etc. Ozone is not on WADA ´s anti-doping list, but reinfusion of more than 100 mL of blood is not permitted. We also make a brief review of possible mechanisms of action and the history of ozone therapy.

Introduction. Who would say no to a treatment that will kill bacteria, viruses and fungi as well as improve blood circulation, the immune system and mitochondrial energy production? And with practically no side effects. If the therapy was based on a substance that could be patented, it would have been used widely. In fact, it is well established since very long time by thousands of therapists in many countries such as USA, China, Russia, Italy, Spain, Germany, Brasil, Switzerland, Ukraine, Cuba and Austria. Side effects are mild and rare at 7 in 100 000 treatments [1). The use of ozone in alternative medicine has evoked critic by many due to the the fact that ozone is dangerous to inhale. Extensive research has demonstrated that O3:s dynamic resonance structure effects physiological interactions that can be used to treat many patological conditions. History of ozone treatment. The gas ozone (O3) was described in the 1840-ies. O3 is a highly water soluble inorganic molecule of three oxygen atoms. It has an unstable molecular structure and reacts easily with itself or water. Therefore, it was initially difficult to reach a concentration necessary for therapeutic effect [2]. O3 functions mainly as a prodrug, i.e. it is not ozone itself that gives rise to the medical effects but other molecules arisen through reaction with ozone, stimulating a cascade of reactions. But, ozone can also react directly with phospholipids, lipoproteins and cell membranes in bacteria and viruses. In spite of its many advantages, toxicity and clinical effects of O3 is dependent on how and at what dose it is administered [1,3,5,6]. It is for instance contraindicated to let O3 reach the lungs or the eyes due to low antioxidant capacity in these organs [7]. Ozone is also used in dentistry to treat caries and diseases in the oral cavity and the jaws. It is also used to disinfect drinking water and medical instruments and is known as one of the most effective disinfectants that exists. Methodology In this litteratur review we have done a comprehensive search on PubMed on clinical trials involving ozone therapy. We have also found a few interesting publications through personal connections with professor Euhenij Nazarov from Odessa, Ukraine [44, 45, 46, 47, 58]. Regarding working mechanisms and history we have used material from other review articles. [1,2,3,12] How ozone can be administered. Ozone therapy combines a mixture of O2 and O3 in a varied therapeutic interval from 10 to 80 ug per mL gas per mL blood [6-8]. The mode of administration depends on indication and treatment aim. The most used way to administer ozone is autohemotransfusion (O3-AHT). The use of O3-AHT has grown, since it makes it possible to take out a decided amount of blood to be mixed with a fixed composition of O2-O3 to be infused back again. The Austrian physician Johan Lahodny has introduced a method involving repetition of this treatment 5-30 times in sequence, giving anecdotal improvement of the results. Extracorporeal oxygenation and ozonization (EBOO) is another similar technique. The purpose is to obtain treatment of a larger volume of blood than 200-300 mL of blood obtained using O3-AHT. Using EBOO four to five liters of blood can be treated [7]. Other administration methods involves intramuscular injection, intradiscal injection and paravertebral injection. Rectal insufflation is a further common technique. Ozone has also been given in the nose, mouth, vagina, bladder, pleura and peritoneum. Exposure to the skin has also been effective and can be obtained by putting the part of the body in a sealed chamber and insufflate the O2-O3 mixture. Intravenous infusion of saline saturated with O3 has been much used in Russia. In the US slow i.v. infusion of a O2-O3 mixture has also been used. Mode of Action

1. Compensatory increase of antioxidant capacity In treatment with O3, a multifaceted endogenous reaction is initiated which releases biologically active substances in response to the transient and moderate oxidative stress induced by O3. O3 can cause this mild oxidative stress due to its ability to dissolve in the water soluble component of the blood. [9] By reaction with polyunsaturated fatty acids [PUFA] and water, O3 forms hydrogen peroxide (H2O2), a reactive oxygene species [ROS]. At the same time, O3 forms a mixture of lipid-ozonation products [LOP]. [10] These LOP:s are to a great extent responsible for the physiological action of O3. Moderate oxidative stress caused by O3 and LOP also increases the activation of the transcription factor mediating Nuclear Factor-Erythroid-related factor 2 [Nrf2]. Nrf2's domain is responsible for activating the transcription of Antioxidant Response Elements [ARE]. When inducing ARE transcription, the formation of antioxidant enzymes like superoxide dismutase [SOD], glutathione peroxidase [GPx], glutathione S- transferase [GST], catalase [CAT] and Heat Shock Proteins [HSP] is increased[10]. It is a well documented fact that an increase in the activity of these enzymes can have a beneficial effect on a wide spectrum of diseases. It is clear that the Heat Shock Response [HSR] provides a cytoprotective condition during inflammation, cancer, aging and neurodegenerative disorders. [12,13] Given its extensive cytoprotective properties, HSR is now an attractive target for the development of new pharmacological substances. [2]. Hsp70 is involved in co- and post-translational folding, quality control of folded proteins, [13] folding and mounting of de novo proteins in macromolecular complexes, as well as anti-aggregation, protein recovery, and degradation. [15] Hormesis is a potent, endogenous defense mechanism against lethal ischemic and oxidative attacks on multiple organ systems. [13] O3 may have a hormonal role in regulating the anti-inflammatory and proinflammatory effects of Carbon Monoxide(CO), which besides being a poison in high dose also has important anti-inflammatory effects in physiological concentrations [14]. Animal models have shown beneficial effects of prophylactic O3 therapy in controlling age-related oxidative stress. [17, 18] Evidence was given to show that low O3 dose administration had positive effects on age-related heart and hippocampal changes in rats. Further research has been conducted that allows for speculation that O3 therapy may affect mechanisms involved in rebalancing the dysregulated redox condition that occurs with increasing age. [19] In a trial investigating possible toxic components associated with O3 therapy, a study was made to assess the extent of lesions on Human Hematic Mononuclear Cells [HHMC], human thymic epithelium, murine macrophages, mouse splenocytes and B16 melanoma murine cells. A significant finding was that Hsp70 exhibited an O3-induced increase in biosynthesis in HHMC. Hsp70s are formed in response to thermal shock and other stressors to cope with the damage that stimulates their biosynthesis. [20] In addition, they stimulate several immune responses in lymphocytes and macrophages. The study provided evidence that O3 is a stressor that can regulate the biosynthesis of Hsp70, without toxicity to cell membranes.[20] Cisplatin, a treatment used against a variety of cancers has been observed to have nephrotoxicity in 25% of patients as a side effect. Ozone therapy was used to increase the antioxidant capacity of rats exposed to Cisplatin compared to a control group. Serum creatinine levels were significantly reduced with O3 compared to the control group. Additionally, thiobarbituric acid reactants [TBARS] were reduced, which is a marker for lipid peroxidation in the kidney. [21,22] In another study, the effect of O3-AHT therapy in combination with oral administration of coenzyme Q10 was investigated [10]. The control group received only O3-AHT. A significant increase in SD and Catalase was found in the group receiving O3 + Q10 while noting a reduction in the level of Malondialdehyde, a product of lipid peroxidation, an indicator of oxidative membrane damage. Several studies have shown that O3 therapy increases activation of Nrf2 by induction of moderate oxidative stress [15.23] This can be used for a longer time frame to restore the balance of the redox system. [21,22] The overall antioxidant status and plasma protein thiol group levels in a blood sample might be indicators of the exact amount of O3 required to optimize the treatment. By developing more precise antioxidant status indicators, a more optimized individual treatment could be achieved. [8,22,24] Vascular and Hematological Effects O3 is a stimulator for the flow of O2 through the cell 5 membrane. This leads to an increase of the intracellular O2 level which makes the mitochondrial respiratory tract more effective. [25] In red blood cells, O3-AHT can increase the activity of the energy producing enzyme phosphofructokinase, increasing the degree of oxygen dependent glycolysis, thus increasing the level of energy rich ATP inside the cell. Subsequently, a right-handed shift occurs in the oxyhemoglobin dissociation curve, allowing the oxygen-bound hemoglobin to be more readily released into ischemic tissues. Combined with the increase in the blood vessel dilating NO synthase activity, there is a marked increase in peripheral blood perfusion after autohemotherapy . [26] With repeated treatment, sufficient amounts of Lipide Ozonation Products(LOP) can be generated to stimulate the bone marrow to produce new erythrocytes.[2.27]. O3 has also been shown to improve blood circulation and oxygen supply to ischemic tissues. [29] Several studies have shown that the correction of chronic oxidative stress through the increase of antioxidant enzymes due to O3 can increase erythroblast differentiation. This leads to a progressive increase in erythrocytes and also gives them increased resistance to oxidative stress. This is called "oxidative preconditioning". [1,29] O3 also increases the levels of prostacyclin, a known vasodilator. [2] Studies have shown an increase in NO in association with O3 therapy, which led to speculation about O3's potential ability to activate genes associated with NO synthase production to further promote higher levels of NO formation. In addition, it is speculated that O3 stimulation of antioxidant enzymes may increase NO levels. [28.31] The prophylactic role of O3 has been investigated with hepatic ischemia / reperfusion [I / R] injury, a phenomenon associated with liver transplantation. Hepatic I / R is a clinically unsolved problem mainly due to the lack of knowledge of the mechanisms underlying this condition. In conclusion, it was found that the ozone oxidative preconditioners [ozonOPs] protect against liver I / R damage by mechanisms that promote regulation of endogenous NO concentrations and the maintenance of a sufficient cellular redox balance. OzoneOPs are also postulated to regulate endogenous antioxidant systems and generate an increase in NO production, both of which are protective mechanisms against liver and pancreatic lesions. The results in this animal model provided evidence that ozonOPs protected against liver I / R via an increase in the concentrations of endogenous NO and stimulation cells to a more balanced cellular redox system. [32] The effect of O3 on kidney I / R in rats has also been studied. Renal I / R is a primary cause of acute renal failure after transplant surgery. The results of a study by Orakdogen et al. [33] showed an increase in endothelial NO synthase and inducible NO synthase expression of ozone treatment. ozonOPs were closely related to the increasing NO production while reducing renal damage by suppressing endothelin [2, 33]. Cerebral vasospasm following subarachnoid hemorrhage is a major problem for the rehabilitation of these patients. In an animal model, the effects of intravenous O3 therapy on vasospasm in the femoral artery were examined in rats. Histopathological and morphometric measurements provided evidence that O3 therapy reduced morphometric changes, endothelial cell disorders, and bleeding resulting from vasospasm. The study speculated that the antioxidant and anti-inflammatory effects of O3 may be a potential new treatment option in the treatment of post-hemorrhagic vasospasm. [34] Pathogen Inactivation When bacteria are exposed to O3 in vitro, the phospholipids and lipoproteins that are within the bacterial cell membrane are oxidized. Thus, the stability of the cell membrane is reduced. O3 also interacts with fungal cell membranes. This interferes with the integrity of the cytosolic membrane and helps to oxidize glycoproteins, glycolipids and block enzymatic function. The combination of these reactions causes inhibition of fungal growth and mortality in bacteria and fungi. [2, 4, 6] In vitro, O3 has been shown to interfere with the contact between virus and cells, thus interfering with viral reproductive cycles [2, 4, 35] Specifically, animal models have shown that O3 therapy in addition to vancomycin enhances the animal's ability to eliminate methicillin-resistant Staphylococcus aureus mediastinitis. [36] Another study has shown improved treatment effect when combining O3 treatment and antibiotics, compared to antibiotics alone in the treatment of long-term pneumonias. [37]. Immune System Activation In vivo, O3 treatment has been shown to have multifaceted effects in interaction with PolyUnsaturated Fatty Acids (PUFA). O3 reacts with PUFA, thereby forming H2O2 and various peroxidation compounds. H2O2 diffuses easily into immune cells and has been shown to act as a regulatory step in signal transduction, which has been shown to facilitate a variety of immune responses. [35,37] Specifically, increases in interferon, TNF (Tumor Nuclear Factor) and interleukin (IL) -2 are seen. The increases of IL-2 are known to initiate immune response mechanisms. [2] In addition, H2O2 activates nuclear factor kappa B (NF-κB) and transforming growth factor beta [TGF-β], which enhances immune-active cytokine release and tissue remodeling. The increase in H2O2 and NO levels also stimulates a remarkable increase in IL-8. IL-8 also activates NF-KB, which enables increased production of ROS scavengers. [8] Animal models using O3 have been shown to reduce and prevent inflammatory reactions arising from the presence of E. coli in the kidney system. [25,37] In another study[24] synovial fibroblast cells from human patients with rheumatoid arthritis were purified and injected into immunocompromised mouse joints. The authors discovered that a 3% and 5% O3 application to the blood vessels in the local area significantly reduced the proinflammatory cytokines IL-1β, IL-6 and TNF-α without any toxicity or serious side effects. [24] Studies have shown that human cancer cells from lung, breast and uterine tumors are inhibited in a dose-dependent manner by O3 therapy in vitro. O3 concentrations of 0.3 and 0.5 ppm inhibited cancer cell growth by 40% and 60%, respectively. Furthermore, the non-cancerous control cells were not affected by these levels of O3. At 0.8 ppm, cancer cell growth was inhibited by more than 90%. However, the inhibition of control cell growth was less than 50%. [38] Clinical Applications With its ever-growing diversity of uses, O3 therapy finds a place in many branches of medicine and medical specialties. In fact, its clinical use can be systematically arranged in the treatment of cardiovascular diseases, wound care, peripheral vascular disease, neurological disorders, head and neck, orthopedic diseases, gastrointestinal disorders, genitoureal diseases, autoimmune diseases, cancer and infections. These indications are based on several human clinical randomized studies. No serious side effects have been noted during the studies. Ozone is also used in dentistry and veterinary medicine.

Cardiovascular Diseases

Treatment of Heart Failure In the so-called The ACCLAIM study [40], a randomized placebo-controlled study published in Lancet 2008, Immunomodulatory Therapy (IMT) was tested. IMT consists of a combination of minor autohemotherapy with ozone and UvB treatment, where 10 ml of blood is exposed to an oxygen /ozone gas mixture (ozone concentration of 15-35 g / m 3) and ultraviolet light at a temperature of 42.5 ° C for about 20 minutes. The treated blood sample was then administered by intragluteal injection to the same individual from which the blood sample was obtained. 2426 chronic heart failure patients received IMT (n = 1213) or placebo (n = 1213) on day 1, 2, 14 and every 28 days thereafter. Endpoint events were deaths and hospitalizations in the two groups. During an average follow-up of 10.2 months, 399 primary endpoint events were found in the IMT group and 429 in the placebo group p = 0.22. In two predefined subgroups of patients, those lacking prior myocardial infarction (n = 919) and those with heart failure (n = 689), IMT was associated with a decrease of endpoints of 26% p = 0.02, and 39% p = 0.0003. The conclusion was that IMT can be a promising treatment for patients with heart failure without prior myocardial infarction and for patients with NYHA II heart failure. No side effects were noted except for mild local reactions at the injection site. The study has been criticized [41] by one of the leading ozone experts, Professor Velo Bocci of Italy for its protocol to depart from the generally recognized optimum principles of ozone treatment, and thus not including managed to utilize ozone's vasodilatory properties, and for using saline injections as placebo instead of autologous blood.

Treatment of coronary artery disease In a randomized controlled clinical trial in patients with coronary artery disease from Cuba, [42], 27 patients received antithrombotic treatment Aspirin and policosanol, a natural supplement from sugarcane commonly used in Cuba to lower cholesterol levels, and the other patient group (n = 26) antithrombotic treatment plus rectal insufflation of O3. A parallel age and sex matched group (n = 50) was used as a reference for the experiment. The effects of the treatments were evaluated by comparing hemostatic indices and biochemical markers of oxidative stress after 20 days of treatment. Ozone treatment significantly improved prothrombin time (P <0.001) and antioxidant status in the O3 group compared to the control group without modification of bleeding time. Sycheva with co-workers [43] published a study in 2013 on the effect of ozone in addition to standard treatment and spa treatment of 145 post-heart attack patients. 56 patients received standard treatment, and 89 standard treatment + ozone. The results of the study indicate that ozone therapy in combination with medical treatment reduces the rehabilitation time after myocardial infarction and reduces tissue hypoxia, improves microcirculation and general health status. In a study from Ukraine [44], 157 patients with stable angina pectoris II-III according to WHO, and heart failure grade 2-3 of the New York Heart Association, as well as indications of endothelial dysfunction regarding the were examined. The control group of 32 patients received standard treatment with digoxin, amlodipine, aspirin and, furosemide. The trial group consisted of 63 patients who received, in addition to the standard treatment, 10 infusions of ozonized saline, while the Placebo group of 62 subjects received corresponding infusions of physiological saline. 6 months after the completion of ozone treatment, the experimental group was divided into two subgroups, one of which received a new series of ozone treatments. All patients underwent clinical follow-up with standard lab tests, work samples, Holter monitoring ECG, doppler arteriography of the brachial artery, Doppler echocardiography, heart ultrasound examination, etc. In the experimental group, the functional Angina Pectoris level was reduced from 2.3 to 1.5. This effect lasted for 6 months but disappeared almost completely within 12 months.

However, the effect could be restored by repeated ozone treatments. It also found a decrease in the total number of ischemic attacks by 55.7% and the daily amount of ischemic episodes of 61.3% in the test group compared to the control group. Ozone therapy corrected the endothelial-dependent dysfunction, resulting in an increase in endothelial-dependent vasodilation, by 83%, that is, 2.1 times more than with medical therapy alone. The need for additional anti-anginal therapy was also reduced with 61.5% and the frequency of repeated hospitalizations with 58.2%. In Russia 30 patients in the rehabilitation stage after a coronary artery surgery and 30 patients with coronary artery disease were allowed to bathe for 20 minutes daily in warm water(36 degrees Celsius) with an ozone concentration of 8-10 mg / l [49]. a decrease in tissue hypoxemia in the experimental specimens. There is no data on any control group in this study In a study from Cuba [53], twenty-two patients who had had a heart attack between 3 months and 1 year prior to the study were examined. They were treated with ozone through autohemotherapy for 15 sessions. A statistically significant reduction in total plasma cholesterol and low density lipoprotein was observed. High, biologically significant increases in the levels of erythrocyte glutathione peroxidase and glucose 6-phosphate dehydrogenase activities were found. There was no change in the plasma lipid peroxidation level. It was concluded that endogenous ozone treatment has a beneficial effect on blood lipid metabolism in patients with myocardial infarction, and activates antioxidant protection systems. Peripheral artery disease Since 1990, a group of Italian researchers [45] has developed a type of ozone treatment called Extracorporeal Blood Oxygenation and Ozonization (EBOO), in order to enhance the results observed with ozone autohemotherapy. Twenty-eight patients with Peripheral Artery Disease (PAD) were randomized to receive EBOO or intravenous prostacyclin in a controlled clinical trial. The patients treated with EBOO showed a significant improvement over the control group on the occurrence of circulatory skin changes, pain, itching, heavy bones and well-being. No significant differences in the vascularization of the lower extremities before and after treatment were found in any group, suggesting that the positive effect of the treatment had a different cause. No side effects or complications were recorded. In a Russian study [46], the effect of Ozone therapy on the treatment of Claudicatio Intermittens was studied. In the control group, 35 patients received traditional russian treatment consisting IV infusion of rheopolyglucin 400 ml, 200 ml of 0.05% solution of pentoxifylin, thiamine hydrochloride, pyrodoxine hydrochloride IM 1 ml, xanthine nicotinate 2 ml IM, aspirin 0.325 mg daily and placebo therapy with 200 ml physiological saline every other day. In the experimental group, 54 patients received ozone treatment - IV infusion 200 ml ozone-saline solution with a concentration of ozone of 1.2 µg / ml every other day in addition to traditional therapy. The walking distance became more than doubled for the group receiving ozonated saline compared to the control group.

Professor Zazorin of the University of Ural showed in another study [47] that infusions of ozonized saline solution increased the capillary blood flow in the lower extremities. Makarov with co-workers [48] performed a prospective randomized study on three parallel groups (139 patients). The first group (n = 57) received standard medical therapy in combination with ozone treatment and was divided into two subgroups: The patients in subgroup 1a (n = 28) received intravenous ozonated saline solution, subgroup 1b (n = 29) got major autohemotherapy with ozone. The second group (n = 62) - received extensive treatment, including so-called gravitational treatment in addition to ozone, with the same type of ozone treatments as in group 1a + 1b. (N = 31 + 31) The control group (n = 20) received only standard medical therapy. Patients were monitored for the need for surgery and hospital admissions for up to 7 years. The best result was seen in group 2a, which received a combination of gravitational therapy and treatment with ozonized saline solution. In another study from Italy[50], patients with low blood circulation in the lower extremities with resting or ischemic ulcers and a transcutaneous oxygen tension <40 mmHg and / or toe pressure <50 mmHg received placebo (n = 74) or a non- specific immunomodulation therapy (IMT) (n = 77) where autologous blood was exposed to an oxygen/ozone mixture and then injected intragluteally on day 1, 2, 7 and then once a week for at least 22 weeks while simultaneously measuring a variety of inflammatory markers. The transcutaneous oxygen tension and the level of endothelial progenitor cells increased after 22 weeks in the IMT group (P <0.01), while no changes were observed in the placebo group. TNF-alpha ( a marker of inflammation) levels decreased at 6 months in the IMT group (P <0.001), while no changes were observed in the placebo group. There was a strong positive correlation between the level of endothelial progenitor cells and the transcutaneous oxygen tension (r = 0.56, P <0.01). In addition, there was an inverse correlation between endothelial progenitor cells and TNF alpha (r = -0.51, P <0.01). It was concluded that IMT can improve wound healing in patients with peripheral occlusive arterial disease. In a study from the University of Naples [51] on patients with claudicatio intermittens, 27 patients received ozone treatment with major autohemotherapy. Whole blood viscosity, erythrocyte filterability, hematocrit, and fibrinogen levels were assessed at basal time and 30 minutes after reinfusion of ozonized blood. At the same time, p50 standard values (p50std) (an indicator of hemoglobin oxygen affinity) and plasma values of malonyl dialdehyde (MDA, an indicator of lipid peroxidation) were measured. At baseline, patients had significantly higher (p <0.05-p <0.001) whole blood viscosity, MDA and p50std values, and significantly lower blood filtration (p <0.01) compared to matched healthy volunteers (controls). Thirty minutes after the end of ozone treatment, whole blood viscosity decreased significantly (p <0.01). This was accompanied by a significant decrease in plasma fibrinogen content (p <0.01) with no change in hematocrit. Blood filterability, MDA plasma levels and p50std values increased significantly (p <0.01-p <0.005). 2,3-DPG value did not change. No significant changes occurred when the same patients received a non-ozonized autohemotransfusion (control test). ).

In a study from Cuba [52], 72 non-diabetic patients with Claudicatio Intermittens, stage II, were randomized to four different groups. Three of the groups were treated with ozone: one of them intravenous, another intramuscular and the last rectal; The fourth group received conventional medical treatment (control group). There were no significant differences between the three ozone treatment groups, but all three differed significantly from the control group, with extended walking distance and walking speed as prime results. The rectal treatment was the simplest and cheapest to perform. In another study conducted by the same research group [53], fifteen patients with Claudicatio Obliterans in the lower limbs, who were not candidates for revascularizing surgery were treated. A statistically significant improvement was noted in the treatment group with a reduced amputation rate of 26.7% and a decreased need for pain surgery (13.3%) compared to the control group (46.7 and 26.7%, respectively). Diabetes and Diabetes Complications In a study from Israel [55] on 62 patients with diabetic foot ulcers, 32 patients were randomized to ozone treatment + standard therapy and 29 to placebo + standard treatment for 12 weeks. Wainstein and his colleagues did not show a statistically significant difference in overall wound healing. 41% compared to 33%, P = 0.34). However, among the 34 patients who completed the study per protocol (16 in the ozone group, 18 in the placebo group), a significantly higher rate of complete wound closure in the ozone group was noted (81% vs. 44%, P = 0.03). Among PP patients with wound size ≤5 cm, the proportion of total wound closure was 100% compared to 50% in the placebo group (P = 0.006). A 55.5% relative increase in the healed wound area was detected in the ozone group versus the placebo group (4.2 ± 4.9 cm vs. 2.7 ± 1.5 cm, P = 0.23). In a study from Ukraine [56], bacterial cultures were performed on 49 patients with diabetic foot ulcers before and after ozone treatment. Prior to ozone treatment, the wounds were populated by a high level of pathogenic microorganisms such as staphylococci. Following local treatment with ozone gas, a positive effect was noted on the wound healing process and a significant reduction in the incidence of pathological microorganisms. In a study from Cuba [57], the therapeutic effect of ozone on the treatment of patients with type 2 diabetes with foot complications was compared to antibiotic treatment. 101 patients were randomized to one group (n = 52) treated with ozone (local and rectal) and a control group treated with local and systemic antibiotics (n = 49). The ozone treatment improved glycemic control, prevented oxidative stress, normalized levels of organic peroxides, and was found to activate superoxide dismutase. Furthermore, the wound healing improved, resulting in fewer amputations in the test group compared to the control group. No side effects were noted. In a doctoral thesis from Russia [58], 84 patients with type 2 diabetes were treated, of whom 62 were women and 22 men aged 45-75. The duration of the disease was up to 10 years. 71% of patients had an average severity and 86% had complications. The most common complications were angiopathy (33%) and diabetic polyneuropathy (31%). 76% of the patients had various diseases of the cardiovascular system. All patients were treated with diet therapy, insulin therapy and oral hypoglycaemic drugs. In addition, the control group received an intravenous infusion of 200 ml of oxygenated saline solution daily for one week The test group group received, in addition to standard treatment, seven intravenous infusions of ozonized saline of the same volume. The dosage of ozone was 20 µg/kg, which corresponded to the ozone concentration in saline from 0.8 µg / ml to 2 µg / ml. The ozone treatment had a significant glucose-lowering effect (on average about 26%). In the control group, on the other hand, a non-statistically significant increase in sugar levels was found.

At the same time, a 39% decrease in HbA1C was found in the experimental group, which according to the article author is typical of all types of systemic ozone treatment.

Positive changes were also observed in the lipid spectrum with a statistically significant reduction in the atherogenic fraction of beta-lipoproteins and triglycerides (TG). In addition, an increase in the level of the anti-atherogenic alpha-lipoprotein fractions was observed.

In summary, 96% of patients experienced a significant improvement in their symptoms, while 4% in the experimental group did not experience any effect. In the control group, a clinical effect was obtained in only a few patients, not specified in the summary. Infection Treatment

Treatment of tropical infections Among the most spectacular treatment results for ozone treatment are several case studies on rapid healing of EBOLA infections in Sierra Leone, West Africa [59]. Ebola Virus Disease (EVD) has plagued three countries in West Africa. Mortality is extremely high, and it is not only perceived as a threat to Africa but to the whole world. There is no known treatment to date except administration of convalescent blood or experimental monoclonal antibodies, both of which often fail. Ozone therapy (OT) has been shown to have physiological effects, which directly inactivates the virus itself and modulates its harmful effects. Five patients with suspected EBOLA infection were treated by a combination of direct intravenous ozone administration, rectal ozone and ozonized water. Three of the patients had a virologically verified known acute EVD, one with apparent acute infection and one with extremely high risk due to a needle stick with ebola infected blood. Treatment was performed for up to ten days despite rapid total remission of symptoms. The patients also received oral vitamin C and glutathione supplements, All four symptomatic patients recovered within 2-4 days. The only case of non- symptomatic high-risk exposure treated preventively did not develop symptoms. Unfortunately, the trial was stopped by the authorities of Sierra Leone for unknown reasons, despite the extremely promising preliminary results and that the researchers were initially invited by the same authorities. Buruli Ulcer (wound) (BU) [60] is a dramatic disease caused by Mycobacterium

Ulcerans, and occurs as an epidemic in Central Africa. Here, in a case description published in BMJ, the symptoms have been successfully treated with local administration of ozone gas for twenty minutes four times a week. This simple and inexpensive treatment can be an effective alternative to managing BU as an alternative to antibiotics or surgical treatments. The pictures above show the wound before, and after two weeks of treatment. Before that, no effects had been achieved with eight weeks of intensive antibiotic treatment.

Treatment of Hepatitis In a study from China[61], 85 cases of chronic severe hepatitis were randomly divided into one trial group (43 cases) and one control group (42 cases). All patients were treated with standard care. Patients in the study group also received ozone therapy with autohemotherapy once every second day for 20 days. One was mainly interested in measuring the frequency of kidney complications, which is a serious mortality factor in severe hepatitis. In the experimental group, only two patients received kidney complications (5%), while nine patients received renal complications in the control group (21%) (χ2 = 5.295, P <0.05). Thirty-three patients (77%) survived of those who received ozone therapy while only 16 cases (38%) survived in the control group (χ2 = 12,993, P <0.01). Panel reactive antibodies (PRA), Angiotensin II and Aldosterone levels were normalized and renal blood flow increased among those receiving ozone therapy.

In a study from Egypt [62], 40 patients with chronic hepatitis C received ozone therapy with major autohemotherapy, minor autohemotherapy, and rectal ozone insufflation. The control group consisted of12 patients that were treated with Silymarine and/or multivitamins. ALT and ASAT levels were normalized in 57.5% and 60% in the ozone group, compared to 16.7% and 8% in the conventional group. Polymerase chain reaction (PCR) for HCV RNA was negative among 25% and 44.4% after 30 and 60 sessions of ozone therapy, compared to 8% in the conventionally treated group. In another study from China [63], 42 chronic hepatitis B patients were divided into two groups. 22 patients received standard therapy and 20 patients also received ozone therapy. Standard parameters such as liver enzyme and virus tires were measured. After 8 weeks of treatment, the treatment group showed 10% total healing and 35% partial healing. In the control group, the corresponding figures were 4.6% and 13.6% (P <0.05). Treatment of Chronic Atypical Pneumonia After three weeks or more of antibiotic treatment [64], 36 patients who were found to have X-ray signs of pneumonia fever, productive cough and weakness were randomized into two matched groups. The test group received, in addition to standard antibiotic therapy, intravenous infusions of 400 ml ozonized sodium chloride solution (pO3) containing 1.6 µg / ml O3 twice weekly for 21 days. The ozone treatment significantly accelerated the radiographic clearance time so that the radiographic infiltrations were undetectable in all study group patients at week 4 while this was the case for only 61.1% of the patients in the control group. Bloodozonization in combination with antibiotics also gave a negative sputum culture against Chlamydia Pneumoniae and Mycoplasma 2-3 weeks earlier compared to the control group. The patients in the experimental group also experienced a faster recovery, with less weakness, fever and cough compared to the control group. Our comment: It would have been interesting to have another experimental group with only ozone treatment without antibiotics ....

Treatment of fungal infections In India, 40 patients [65] with oral candidiasis were randomized to either mouthwash with ozone water or local treatment with clotrimazole. At the end of the treatment, Candida CFU numbers were reduced by 60.5% in the ozone group compared to 32.3% in the clotrimazole group. Candida infection completely disappeared in 14 patients (70%) in the ozone group while disappearing in only 8 patients (40%) in the clotrimazole group. Due to the limited number of patients in the study, the difference was not statistically significant. Ozone water appears to be at least as effective as local treatment with Clotrimazole in the treatment of oral candidiasis In China, 60 patients [66] were randomized to either naphthinfin hydrochloride and ketoconazole cream once a day or 65 ozonized water baths with subsequent ozone-treated oil once daily. for four weeks. After 4 weeks of treatment, 6 patients in the control group showed a positive mycological culture while only 1 patient was positive in the ozone group, with no significant difference between the two groups (P> 0.05). There was no statistically significant difference in clinical signs between the two groups. No side effects were noted. In conclusion, ozone bath + oil was at least as effective as standard treatment of Tinea Pedis. In an Iranian study [67], 100 women with vulvovaginal candidiasis were randomized to either treatment with ozonated olive oil or clotrimazole for 7 days. Ozone and clotrimazole significantly reduced both symptoms and led to a negative culture of vaginal candidiasis (P <0.05). There were no differences in symptoms between the two groups, except that clotrimazole reduced burning sensations faster. The article author therefore concludes that vulvovaginary candidiasis can be treated with ozonized olive oil. In a randomized phase III study from Cuba [68], 400 patients with nail fungus were randomly divided into two groups: one received ozonized sunflower seed oil OLEOZON (®), twice daily and the control group received ketoconazole cream 2% twice daily for 3 months. A patient was considered cured when the infected nails regained their normal color, growth and thickness and produced a negative fungal culture. In the test group, most were cured already after the first month of treatment, while it usually took three months in the control group. All patients treated with OLEOZON (®) had improvement in their condition (9.5%) or cure (90.5%). In the control group, only 13.5% of patients were cured, 27.5% improved and 59% remained unchanged, with statistically significant differences between the two groups. After 1 year of follow-up, 2.8% recurrence was seen in the study group and 44.4% in the control group. Ozonated sunflower seed oil was therefore clearly superior to ketoconazole cream in the treatment of nail fungus. No side effects were observed. In another study from the same research team [69], treatment with Oleozon was compared to treatment with ketoconazole cream 2% (Nizoral) in 200 patients (100 in each group) twice daily for a period of 6 weeks. Complete clinical and mycological healing was obtained in 75% and 60%, respectively. 81% for Oleozon and Ketoconazole, with no significant differences between both groups. No side effects or bacterial superinfection were observed. No relapses were noted within 6 months.

Infections of gums and oral cavity. There is a large number of randomized, double-blind studies in odontology that show good effect of ozone treatment in gum and oral infections, and as infectious prophylaxis after dental surgery [70-72] For time and space reasons, we do not go into more detail here.

Genitoural Infections 127 Russian men [73] with laboratory diagnosed chronic urogenital chlamydia infection, 72 of whom also had a chronic urethroprostatitis received treatment with fromilide (clarithromycin) as well as transurethral and transrectal ozone treatment. The symptoms were relieved after 4-6 weeks. Repeated enzyme immunoassays and polymerase chain reaction tests also revealed eradication of the infectious agent. In addition, an improvement in hemodynamics, urinary tract symptoms and prostatic microcirculation following ozone treatment was noted.

HIV Ozone has been shown to inactivate the HIV virus in serum in sub-cytotoxic concentrations. In an uncontrolled study [74] on 10 HIV positive patents, Bocci with coworkers found neither positive nor negative effects on lab samples or the disease course of treatment twice weekly with major autohemotherapy.

Autoimmune Diseases

Fibromyalgia 36 Spanish patients with fibromyalgia [75] received 24 sessions of rectal ozone therapy over a 12 week period. At each session, the dose of ozone administered was 8 mg (200 ml of gas at a concentration of 40 µg / ml). Ozone sessions were given 5 days a week during the first 2 weeks, twice a week from week 3-6 and weekly from weeks 7-12. Fibromyalgia Impact Questionnaire (FIQ) was the main evaluation tool and was administered at baseline and at weeks 4, 8 and 12. In addition, the Pittsburgh Sleep Quality Index, the Beck Depression Inventory, the State and Trait Anxiety Inventory, and the SF-12 were used (the shortened form of the Short Form Health Survey). The FIQ score decreased significantly during the study period, with the largest decrease in the first 4 weeks of the study. Significant improvement was seen both in the Beck Depression Inventory, and in the physical summary of the SF-12 rating scale. Transient meteorism was the most reported side effect. 65 patients with a diagnosis of fibromyalgia according to the definition of the American College of Rheumatology were treated by ozone autohemotransfusion or rectal insufflation twice a week for one month and then for twice a month as maintenance therapy [75A]. A significant improvement of symptoms (>50%) was seen in 45 of the patients.

Rheumatoid Arthritis A randomized clinical study [76] with 60 patients was performed divided into two groups: one (n = 30) treated with methotrexate (MTX), folic acid and Ibuprofen (MTX group), the other group (n = 30) receiving the same treatment as the MTX group + medical ozone with rectal insufflation (MTX + ozone group). The clinical response in patients was evaluated by comparing disease activity scores 28 (DAS28), the Health Assessment Questionnaire Disability Index (HAQ-DI), Anti-CCP levels, acute phase reactants, and oxidative stress biochemical markers before and after 20 days of treatment. MTX + ozone reduced the activity of the disease while MTX only showed a tendency to reduce the variables. MTX + ozone reduced anti-CCP levels and increased the level of antioxidants and showed signs of reduced oxidative stress, while MTX showed no such changes. MTX + ozone increased MTX clinical response in patients with rheumatoid arthritis. No side effects were observed. The conclusion was that medical ozone treatment can improve the effect of MTX therapy in the treatment of rheumatoid arthritis.

Multiple sclerosis Molinari with coworkers believes that autohemotherapy with ozone is effective in reducing the symptoms of multiple sclerosis (MS) in patients [77], but the effects on the brain have still not been clear. To clarify this, a study was performed on 10 MS patients with Remitting Relapsing MS, who in addition to standard treatment also received autohemotherapy with ozone. The cerebrovascular pattern in MS patients was compared to a control group of 10 healthy subjects using close infrared spectroscopy (NIRS) as functional and vascular technique. An improvement in the cerebrovascular pattern of all subjects who could be noted as an increase in entropy of the NIRS signals was shown. It was therefore concluded that ozone therapy increased brain metabolism and helped it recover from the lower levels of activity that are predominant in MS patients. In another similar study [78] performed of the same main author, 20 MS patients were investigated. In addition to changes in the blood circulation in the brain, increased levels of Cytochrome C oxidase and oxygenated hemoglobin could be noted, which in turn indicates a reduced level of the type of oxidative stress. which is typical for MS patients. Delgado-Roche with co-workers [79] believes that oxidative stress and inflammation play a key role in the pathogenesis of multiple sclerosis (MS). Various drugs have been used in clinical practice, but there is no fully effective treatment. Due to its potential therapeutic effect, medical ozone represents a promising approach to neurodegenerative disorders. The purpose of the study was to address the role of ozone therapy on cellular redox status in MS patients. Ozone (20 µg / ml) was administered three times a week for one month by rectal insufflation. The effect of ozone treatment on biomarkers of oxidative stress and inflammation was measured by the effect of ozone on CK2 expression and Nrf2 phosphorylation by western blot analysis. Medical ozone significantly improved (P <0.05) the activity of antioxidant enzymes and increased levels of cellular reduced glutathione. Accordingly, a significant reduction (P <0.05) of oxidative damage to lipids and proteins was observed in ozone-treated patients. The levels of pro-inflammatory cytokines TNFα and IL-1β were also lower after ozone treatment. Ozone therapy increased CK2 expression along with Nrf2 phosphorylation in mononuclear cells in MS patients. These results indicate that the ozone antioxidant and anti-inflammatory effects may be partially associated with an induction of Nrf2 phosphorylation and activation. The authors therefore see Ozone Treatment as a potential therapeutic alternative for MS patients. Gastrointestinal Diseases

Ulcerative Colitis In a prospective randomized controlled clinical study [80], 54 patients with mild to moderate active distal ulcerative colitis were randomized to 3 groups of 18 subjects in each group (n = 18). The first group received sulfasalazine as standard rectal application, the second group got sulfasalazine via a special colon therapy system, and the third group was sulfasalazine via a colon therapy system + ozone treatment. The treatment was performed daily for four weeks. Then, the colon was examined with coloscopy after 0, 2 and 4 weeks of treatment, respectively, and a histological analysis was performed at 0 + 4 weeks to evaluate the treatment outcome. Ozone treatment plus colon therapy resulted in the fastest relief of clinical symptoms and also resulted in better histological healing without any adverse side effects.

Peritonitis In a Russian study [81], 211 patients suffering from Extended(Protracted) Peritonitis (EP) of various etiology were analyzed. The degree of peritonitis was graded according to the Mannheim Index of Peritonitis (MIP). The patients were divided into two groups. 60 patients (comparator group) received standard treatment, without immune correction. The patients in the main group were divided into three subgroups. Patients in the first subgroup received 400 ml of ozonated isotonic saline (OIS) along with standard treatment, as well as peritoneal enteral detoxification / rinsing using OIS. In the second study group, a regional intra-abdominal endolymphatic ozone treatment (OTH) was performed on 57 patients with OIS and peritoneal-enteral detoxification / rinse with medical ozone. In the third subgroup, 51 patients received intravenous infusion of OIS and peritoneal enteral remediation with medical ozone. These patients also received a special type of cytokine therapy called splenopid intravenously, intraperitoneally and enterally simultaneously. A number of laboratory parameters (T and B immunity, phagocytic activity of neutrophils, TNF-alpha, IFN-gamma and a variety of Interleukins were then measured. The best result was obtained in test group III, combining systemic, intraperitoneal and enteral ozone therapy) with cytokine treatment. In another Russian study, the results of surgical treatment of 114 patients with spread peritonitis of different etiology were analyzed [82]. The control group received standard treatment. In the experimental group, intravenous infusion of ozonized perftoran (OP) was applied at a dose of 1 mg / kg with an ozone concentration of 5 mg / l, with or without the metabolic immune modulator and the detox substance glutoxime. A large amount of blood samples were taken on all patients. The article authors concluded that the combination treatment was medically substantiated. In a Russian study [83] on 40 experimental animals and 58 patients with diffuse purulent peritonitis [81], the experimental animals received either standard treatment or ozone + standard treatment. The mortality in the ozone group was 5.2% compared 16.6%, in the control group.

Pancreatitis In a Ukrainian study [84], 32 patients with acute necrotic pancreatitis were randomized to ozone treatment according to a schedule prepared in the clinic, along with conventional treatment or only conventional treatment. The application of ozone therapy resulted in a decrease in the need for operative interventions, cellular, humoral and local immunity improved and a normalization of biochemical indexes was noted. The need for hospitalization was 21.4 days on average in the test group compared to 34.5 days in the control group. In a study from Russia, 89 patients were analyzed [85] with sterile pancreatic necrosis. All patients received standard treatment while the experimental group also received intravenous ozone therapy and small doses (20-25mkA) of direct current treatment. A three fold decrease in the transition from sterile to infected pancreatic necrosis was recorded in the study group, while the hospital stay decreased 1.6 times and mortality 1.3 times.

Gastric ulcer In a randomized Russian study [86] on 152 patients with perforated gastroduodenal ulcers, the study group (n = 87) received ozone treatment in addition to standard treatment. The control group (n = 65) received standard treatment. Ozone treatment succeeded to make an earlier and more fully restored structure of the gastroduodenal mucosa, providing effective elimination of Helicobacter pylori, reducing the time of wound healing and reducing the risk of recurrence. Three methods of ozone treatment [87] (intravenous infusion of ozonized saline, oral intake of ozonized low-mineralized water and a combination of the two treatments) were compared with each other for efficacy on 215 patients with gastroduodenal pathology associated with Helicobacter pylori. All ozone therapy techniques proved to be effective over regression of clinical symptoms, regeneration processes, and eradication of Helicobacter pylori, but the combined regimen was best. Genito Urethral Diseases

Bacterial Vaginosis In a Russian study [88], 102 elderly patients with bacterial vaginosis were randomized to 3 groups, matched for age and disease severity Group III received only hybrid therapy, the patients in group I received local ozone therapy and the patients in group II a combination of local and generalized ozone therapy. The study showed the best effect in group II receiving combination therapy.

Placental Insufficiency A clinical study from Russia [89] was designed to evaluate the influence of an ozone-oxygen mixture on the properties of blood supply in pregnant women with chronic placental insufficiency. The study demonstrated how ozone modulates the oxygen-transporting function of the blood and promotes the adaptive ability of the organism by activating aerobic metabolism and cell membrane stabilization. The conclusion is that the stated type of ozone treatment can be recommended for the treatment of women with complicated pregnancy to improve tolerance to and minimize the detrimental effect of hypoxemia.

In connection with prostate surgery Prostate Adenoma patients [90] with cystostomic drainage who had undergone a Transnational Resection TUR were preoperatively prepared with Ozone Therapy (OT). The control group consisted of comparable patients but without ozone pretreatment. The OT effect was assessed by analyzing the frequency of Pyogenic Inflammatory Complications (PIC), results of immunological examination, positive changes in prostatic secretion, urine analysis, HB value, and the degree of bacterial infections. In the study group, PIC (acute urethritis) was developed in 1 patient. In the control group in 6 patients (3 cases of acute urethritis, 2 cases of acute prostatitis and 1 case of acute epididymitis). OT led to lowering of the mean leukocyturia from 18.1 to 14.3 on the day of surgery, to 10.9 after 4 days and to 8.7 on day 8 postoperatively, compared to 18.8 to 15.4, to 15 , 8 and 13.5 respectively. The study group showed an increase in absolute number of blood leukocytes, lymphocytes and decreases Erythrocyte Sedimentation Rate(ESR). OT significantly increased phagocytic counts and activity, the concentration of mature T lymphocytes (CD3), T helper (CD4), cytotoxic T lymphocytes (CD8), B lymphocytes (CD20), T-NK cells (CD16), T lymphocyte activation markers (CD3 +, CD16 +, CD56 +, CD3 +, CD25 +, HLADR +, CD3 +. The concentration of IgG, IgM, IgA remained high. The author therefore recommends performing ozone treatment prior to a TUR operation.

Treatment of urinary tract infections Multi-resistant drug bacteria are an emerging problem worldwide. A decreased resistance to infection have been seen in chronic kidney disease (CKD) and kidney transplant patients as well as some metabolic conditions such as hyperglycemia and glycosuria or clinical conditions such as neurogenic bladder. These conditions constitutes a major risk factor for recurrent urinary tract infections (UTIs). The usual and unreasonable use of antibiotics only gives patients a transient or partial improvement of urinary discomfort and increases the risk of multi-drug resistant bacteria. Thus, a major effort is being made to develop novel antibacterial approaches, particularly in the determination of multi-resistant pathogens. Bonforte et al [91] reports in a series of cases from Italy about some promising but still preliminary results regarding the use of ozone treatment for recurrent UTI. Wound Care

Burns Several studies have shown that ozonated oil is effective in cutaneous wound healing. In a clinical study from Italy [92], the clinical effect of topical application of ozonized oil for 12 weeks were evaluated on 30 patients suffering from second-degree skin burns in the re-epithelialization phase. Each burn was divided into two symmetrical parts. One part was treated with occlusive application of ozonized oil; The contralateral part of the lesion was treated with topical application of hyaluronic acid gel, once a day for 12 weeks. A clinical evaluation and a video capillaroscopy were performed on each patient at baseline, 6 and 12 weeks after. Results: All treated lesions improved regardless of the treatment used. Ozone oil was as effective as hyaluronic acid in enhancing erythema, stress, itching and burning sensation, but it was more effective than hyaluronic acid to reduce post-lesional hyperpigmentation.

Diabetes wounds Treatment of diabetic foot ulcers (DFU) is a major challenge for clinicians. Although the oxygen-ozone treatment improves the results, few clinical trials have verified the effect and elucidated the underlying mechanisms of oxygen- ozone treatment on DFU. In a study from China [93], 50 type 2 diabetic patients with DFU, Wagner Stage 2 ~ 4 were randomized to a control group treated with standard therapy only, and an ozone group treated with standard therapy plus oxygen-ozone treatment. The therapeutic effects were assessed according to 4 levels from grade 0 (no change) to grade 3 (wound healing). The lesions and sizes of the wounds were measured at baseline and day 20, respectively. Tissue biopsies were performed at baseline and day 11. Meanwhile, the levels of vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) were measured. The proportion of responders in the ozone group was significantly better than in the control group (92% versus 64%, P <0.05). The reduction in wound size was also significantly higher in the ozone group compared to the control group. (P <0.001). After treatment, the level of VEGF, TGF-β and PDGF proteins at day 11 was significantly higher in the ozone group than in the control group. Conclusion: Ozone therapy promotes wound healing of DFUs via potential induction of VEGF, TGF-β and PDGF.

Venous leg ulcers In a study from Romania [94], a total of 29 patients with chronic venous leg ulcers were randomized to either daily application of ozonated oil and α- bisabolol or control cream (vitamin A, vitamin E, talc and zinc oxide) for 30 days. The patients were evaluated at 4 different visits: at days 0, 7, 14 and 30. At each visit, the wound surfaces were measured and the rate of wound healing was calculated. At the end of treatment, the proportion of patients with complete wound healing was higher with ozonated oil and α-bisabolol formulation (25% vs 0%). Furthermore, the changes in the wound surface were significantly improved in the ozone group. Only in the ozone and the α-bisabolol group (P <. 05), a significant and progressive reduction of the wound area was noted by 34%, 59% and 73% after 7, 14 and 30 days, respectively. In a Chinese study [95], 92 patients with venous ulcers were randomized to receive ozone gas baths combined with Endovenous laser therapy (EVLT) (OEVLT group) or EVLT alone (EVLT group). In the OEVLT group, the venous wounds were treated with ozone gas baths before EVLT. The minimum follow- up time was 12 months. There was no significant difference in venous occlusion between the two groups. The proportion of patients with wound healing was significantly higher in the OEVLT group compared to the EVLT group at 12 months of follow-up. Patients in the OEVLT group showed better satisfaction and fewer relapses compared to the EVLT group. No serious complications or side effects occurred in any of the groups.

Wound injuries from war In a prospective study [96] of 35 war-injured people, the effects of ozone on how well skin grafts with split thickness were integrated were examined. Each of the injured persons had at least two similar bullet wounds, one on the lower leg or the forearm and the other on the thigh or upper arm. During the first 10 days, all wounds were treated with 10% NaCl aqueous solution until the moment when healthy granulations could be observed. Thereafter, the defects were covered with Thiersch layered slabs. For technical reasons, grafts were treated on the lower leg and forearm with ozone following the usual schedule. Graft on the thigh or upper arm was treated in a conventional manner and served as a control group. More than 74.3% of the skin plastics with split thickness treated with ozone had an adhesion of more than 75% compared to only 40% of the graphs treated by conventional methods. The difference between the groups was statistically significant at P <0.01. Lung Diseases

Emphysema In a study from Cuba [97], sixty-four patients with pulmonary emphysema were randomized to receive rectal ozone in 20 daily sessions, rectal oxygen, or no treatment. The treatments were repeated three months later in the first two groups. At the beginning and end of the study, spirometry and clinical assessment were performed. Fifty patients completed the protocol. Initially, patients who received ozone treatment had significantly worse values for forced expiratory volume over one second (fEV1) and fEV1 / forced vital capacity. At the end of the treatment period, these parameters were similar in the three treatment groups, thereafter, only the group receiving ozone treatment improved significantly. No differences were observed in other spirometric parameters. The conclusion was that rectal ozone therapy may be useful for patients with pulmonary emphysema. Cancer In a review article on ozone therapy and cancer [98], Clavo and his colleagues say that for several decades, in vitro studies have been published in prestigious journals on the ozone's ability to induce direct damage to tumor cells and also improve the effects of radiotherapy and chemotherapy. Indirect effects have been demonstrated in animal models: Immune modulation through ozone alone and sensitizing effect of radiation therapy through simultaneous ozone administration. The effects of ozone in the modification of the hemoglobin dissociation curve, 2,3-diphosphoglycerate levels, local regional blood flow and tumor hypoxia provide additional support for potential beneficial effects in cancer treatment. Unfortunately, only a few clinical studies are available. Finally, some work and the authors' experience supporting the ozone treatment in the treatment of delayed healing after tumor resection is described, as well as in reducing radiation side effects and chemotherapy. Head and neck pectoral cancer (HNSCC) represents a group of metastatic tumors with high mortality in humans and animals. Since the biomolecule ozone was found to inhibit the growth of various cancer cells in vitro, [99] the highly aggressive and lethal VX2 carcinoma HNSCC tumor model was applied to rabbits to test whether ozone exerts anti-tumor effects in vivo on rabbits. Therapeutic insufflation of medical ozone / oxygen (O (3) / O (2)) gas mixture in the peritoneum (O (3) / O (2) pneumoperitoneum) at an advanced stage of tumor disease led to a survival rate of 7/14 rabbits. Six of the seven surviving rabbits showed a full tumor regression with the absence of local or distal lung metastases. Insufflation of pure oxygen (O (2)) resulted in a survival rate of 3/13 animals accompanied by complete tumor regimen in 2 of the 3 surviving animals. Of the 14 placebo-treated animals, only one had spontaneous tumor regression and survived. Ingested side effects or changes in standard blood parameters were observed after repeated intraperitoneal insufflation of the O 3/ O2 or O2 gas. Animals with O3 / O2 -induced tumor eradication developed resistance to re- implantation of the VX2 tumor. This could be reversed by immunosuppression with a combination of dexamethasone and cyclosporin This suggested an antitumor effect of O3/ O2 by activation of the body's own immunosurveillance. Although the exact mechanisms of action are still unclear, the current data points to O3 / O2 pneumoperitoneum as a promising new strategy against cancer.

In an uncontrolled pilot study [100], Bocci with co-workers gave ozone intraperitoneally as a slow gas infusion and an ozone / saline infusion to five patients with ascites and carcinomatosis due to ovary, colon and pancreatic cancer. All patients responded to the treatment with a decrease in tumor markers, improved well-being, and prolonged survival compared to what could be expected.

Osteonecrosis of the jaw Osteonecrosis of the jaw (ONJ) is a side effect reported in patients receiving cancer treatment regimens, including bevacizumab, bisphosphonates and denosumab. In an open prospective Phase I-II study [101], 10 patients treated with bisphosphonate received 10 local ozone treatments in the form of an oil suspension on BONJ lesions ≤2.5 cm. The treatment lasted for 10 minutes. In all patients, mucosal lesions were healed with complete reconstitution of mouth and jaw tissue, with 3-10 applications. No toxicity was reported. Unexpectedly, total sequestration of the necrotic bone was seen, with spontaneous healing in eight patients and new bone formation around the necrotic area in two patients. No patient required surgical intervention. . These preliminary results show the effect and tolerance of O3 delivered in an oil suspension applied directly to BONJ lesions, indicating that BONJ may be manageable and potentially curable. Musculoskeletal Diseases

Treatment of Herniated Discs with Intra-discal Injections The ozone treatment that has been verified most of all is the treatment of herniated discs with intra-discal ozone gas injections during fluroscopy or CT control. At least 15 large randomized, double-blind studies have been published, all with positive results. This therefore appears to be an effective, relatively side- effect free and inexpensive treatment compared to surgery. Most of the studies have been done in China and Italy, but also India is on the map. In an Italian study [102], six hundred patients with disc hernia were randomized to either treatment with 4 ml of ozone gas at a concentration of 27 micrograms / mL intradiscally, and 8 mL periganglionic (n = 300), or the same treatment + periganglionic injection with corticoid and local anesthesia. In group A, the treatment was successful (excellent or good result) in 70.3% and was considered unsuccessful (poor result or use of surgery) in the remaining 29.7%. The corresponding figures in group B were 78.3% and 21.7%, respectively. The difference was statistically significant (P <0.05), showing that combined treatment was better. In China, 262 patients [103] with lumbar disc herniation were injected intradiscally with medical ozone at a concentration of 40-50 µg / mL into the disc hernia and 30 µg / ml around the nerve roots percutaneously during CT guidance According to modified MacNab criteria, the therapeutic effect was excellent in 165 cases, OK in 64 cases, acceptable in 20 cases and poor in 13 cases, with a total success rate of 87.4%. No patient showed serious complications after treatment. Zhang with co-workers [104] conducted a randomized study in 172 patients comparing the effect of treatment with intradiscal and intraforaminal ozone with treatment with intradiskal and intraforaminal ozone + betamethasone (a corticosteroid). Both groups achieved good results. After three weeks, the betamethasone group had significantly better results, an effect that disappeared after 6 and 12 months, respectively. In Italy, 154 patients with lumbar disc herniation [105] were randomized to either intraforaminal injection of steroid and local anesthesia (n = 77) or the same treatment + injection of ozone. In the beginning, there was no statistically significant difference between the groups, but after 6 months, in the ozone group, 58 out of 77 patients had a successful outcome (75.3 %), compared with 30 (responders: 38.9 %) of 77 control group patients. The difference was statistically significant (χ2 = 31.31, P < 0.001) The power estimation was 0.99 (very good) and the effect size was 0.42. In China, 80 patients [106] were randomized to a control group, a group injected intradiscally with low dose ozone (20 µg / ml), a group of medium dose ozone (40 µg / mL) and a group of high dose ozone (60 µg / ml ). CT scanning and enzyme-linked immunosorbent assay (ELISA) were used to detect IL-6 level, SOD(Super Oxide Dismutase) activity, IgM and IgG levels at baseline as well as at 6 and 12 months. All patients showed disc retraction during the 6 and 12 month follow-up; The patients in the medium dose group (40 µg / ml) group showed the best effect on MR. IL-6, IgM, IgG and VAS levels (Pain) decreased significantly while SOD activity increased among all groups over time (p <0.05). Low concentrations of medical ozone (20 µg / mL and 40 µg / mL) reduced serum IL-6, IgG and IgM expression, which is typically analgesic and anti-inflammatory, while high concentrations of medical ozone (60 µg / mL) increased serum IL-6, IgG, IgM expression, which can be seen as pro-inflammatory effects. The medical ozone concentration of 40 µg / mL showed the best treatment effect. Perri with co-workers [107] randomized 517 patients to either a control group (n = 260) who received intra-foraminal injection of steroids + local anesthesia and a test group (n = 257) who additionally received intra-discal injection with O3. After 6 months, the O2-O3 lysis was successful in 106 (41.24%) test group patients compared to 9 control group patients (3.5%) (P <0.001). A radiologically verified decrease in disk herniation was observed in 185 study group patients (68.4%) compared to 4 control group patients (1.5%). In an Indian study [108], 91 patients with lumbar disc herniation were randomized to intra-discal ozone therapy or combination ozone and PIRFT (radio frequency treatment at 80 ° C in 360 s). Primary outcome measures included a visual analogue scale (VAS) for pain and the Oswestry Disability Index (ODI). Secondary outcomes were measured by pain relief, reduction of analgesic consumption, and patient satisfaction. ODI (Oswestri Disability Index) decreased significantly with both ozone and ozone-PIRFT compared to baseline values at all follow-up points; Ozone PIRFT, however, produced a significant decrease in VAS score and ODI compared to ozone at 2 weeks, 1 month, 3 months, 6 months and 1 year follow-up. The same applies to the secondary efficiency measures. Ozone-Pirft + Ozone thus had better effect than Ozone alone in this study. Two groups of [109] patients (n = 108) were treated with either injection of oxygen-ozone combined with collagenase intradiscally and in the epidural space; the second group was treated with traditional surgery. After treatment, the patients were followed and the therapeutic effect was evaluated at 2 weeks, 3 months and 12 months with the modified so-called. Macnab criteria. The success rate was 86.11% and 88.89% in the ozone / collagenase group at 3 and 12 months, respectively, and 92.59% and 95.37% in the surgical group. There was no statistically significant difference between the groups at 3 and 12 months (P = 0.123, P = 0.08). However, the surgical group produced statistically significantly greater improvement for back pain and disability during the first few weeks. (P = 0.0001). The success rate was 51.86% and 85.18% after 2 weeks in the minimally invasive group and the surgical group, respectively. No serious complications occurred in the ozone group. The authors argued that although the minimally invasive procedure has several advantages over the surgical procedure, each method has its indications and contraindications. For patients with spinal cord stenosis, protruding calcification and lateral access stenosis, surgery is preferred. In conclusion, the combination of collagenase with ozone shows statistically significant reductions in pain and improvement in function and can be considered as an alternative to surgical treatment. Wu with co-workers [109] randomized 75 patients with lumbar disc hernia who would be treated with intradiskal ozone treatment to either antibiotic prophylaxis with cephalotin V (2.0 g) intravenously 30 min before surgery, or no antibiotics at all. No signs of infection were found in any of the groups and it was therefore concluded that prophylactic antibiotic treatment is not necessary for this type of procedure. Galucci with co-workers [110] randomized 159 patients to intra-discal steroid / local anesthetic treatment (n = 77) or the same treatment + Ozone injection (n = 82). After 6 months, treatment was successful in 36 (47%) patients in group A and 61 (74%) of group B patients. The difference was statistically significant (P <0.01). In another Italian study [111], 306 patients (166 with primary disorder, 140 with back disorder who did not involve the disks) with acute or chronic low back pain and sciatica received a CT-controlled intraforaminal infiltration of ozone or a periradicular infiltration with steroids. At 1 week follow-up, most patients had a complete remission of pain, regardless of treatment. At 6 months of follow-up, differences occurred in favor of ozone therapy, which was significant in patients with disk disease (P = .0021) but not in those without disk disease (P = .0992). Clinical results were poor in 13 (15.1%) of the 86 patients receiving ozone infiltration and in 18 (22.5%) of the 80 patients receiving steroid injection (P = . 2226). Among patients without disc disease, six (8.6%) of 70 patients who received ozone infiltration and 21.4% of patients receiving steroid injections had poor results (P = .0332). Paradiso et al [112] analyzed the long-term effect (3 years of follow-up) of treatment in 150 patients who received microdiscectomy and 150 patients who received intradiscal ozone injection. In this series, ozone treatment was more beneficial in smaller, cohesive disc hernia, while surgery was better in large migrated fragments with pain so severe that open surgery was mandatory. Apart from this, the results with the two techniques were equivalent. In a study conducted by Alexandre with co-workers[113], 108 patients with disc hernia received treatment with intradiscal injection of ozone in year 2002-2003. One hundred seven patients were available for phone follow-up after 5 years. Sixty patients were available for a similar phone follow-up after ten years. Patients were asked to describe their clinical outcome after the injection. Surgical events were documented. The MRI images were reviewed to assess the reduction of disc hernia after six months: MRI images showed a consistent reduction in the size of the disk hernia. Seventy-five percent of the patients had a decrease in the volume of herniation and the average decrease was 56%. There were 19 patients who eventually needed surgery and 12 of them occurred during the first six months after the injection. One of these 12 was due to surgery on another level. Two operations involved an intermediate spacer indicated by stenosis or DDD (Degenerative Disc Disease). All other operations were disectomy. Of the patients who avoided surgery, 82% improved after 5 years and 88% improved after 10 years. In addition to subsequent operations, no spine-related complications were experienced. The conclusion is that ozone is safe and effective in about 75% of patients with disc hernia, and the effect is maintained through ten years. Intra- discal ozone injection may allow patients to correct the pain without epidural injections and surgery. The advantage of ozone is sustainable and does not exclude future surgical alternatives. The risk profile for this treatment is favorable.

Treatment of Hernia with Paravertebral Intramuscular Injections 20 patients with lumbar disc herniation [114] were treated with lumbar paravertebral injections of oxygen and ozone and 18 patients treated pharmacologically with anti-inflammatory analgesics. A reduction in pain and discomfort was found already after one week in the ozone group compared to pharmacological treatment. This difference in response became statistically significant after two weeks (50 percent vs. 16.6 percent) and was confirmed after 3 and 6 months, when 80 percent of the patients treated with injections were found to be painless compared to half of the patients treated pharmacologically. No statistical difference was found in MRI and EMG studies. No side effects were noted in any patient, In another study, sixty patients [115] were randomized with acute back pain caused by lumbar disc herniation to intramuscular paravertebral ozone treatment or to a control group with placebo injections. A significant difference in pain clearance between the two groups was observed at week 6 (61% versus 33%, P <0.05). The ozone group also had a lower mean value in terms of pain level. A significant improvement was also observed with regard to spine-related disabilities and analgesic consumption in the study group compared to the control group. No side effects were reported.

Treatment of Spondylosis and Spondylolistesis 18 patients [116] with low back pain and sciatica resistant to physical and medical management with a radiological diagnosis of spondylolistesis and spondylolysis received CT-controlled bilateral periganglionic O2-O3 infiltration and injection at lysis points. 15 patients (83.3%) received a complete remission of pain. None of the patients reported any recurrence of pain during clinical follow-up visits one, three and six months after treatment, respectively. In Italy, 58 patients with [117] cervical spondylosis (CS) were treated with ozone and laser via puncture needle. The VAS score for neck and shoulder pain decreased from 6.57 to 1.80 after one week, and was completely gone after one month. 29 patients responded excellently to the treatment; 23 well; and 6 were OK, ie 89.6% were successful). CONCLUSION: Ozone and laser combined therapy via puncture needle for treatment of CS is both safe and effective. Treatment of knee arthritis In a randomized, double-blind, placebo-controlled clinical trial [118] from Brazil, 98 patients with symptomatic knee arthritis (OA) received either intraarticular injection of 20 µg / ml ozone (OZ) or placebo (PBO) once weekly for 8 weeks. After 8 weeks of treatment, ozone was more effective than placebo p <0.004 with respect to VAS and a variety of other parameters. Adverse events occurred in 3 patients (2 in the placebo group and 1 in the ozone group) and included only puncture accidents. The study confirmed the known effect of ozone on pain relief, functional improvement and quality of life in patients with knee osteoarthritis. In an Italian study [119], 70 patients with knee arthritis (OA) were randomized to intraarticular injections with hyaluronic acid HA (n = 23) or O3 (n = 23) or combined HA + O3 (n = 24) once per week for 5 consecutive weeks. All three treatment groups showed statistically significant improvements compared to baseline data, but the combined treatment was found to have statistically significant best effect, especially at two months of follow-up. 102 patients [120] with mild to moderate and moderate knee osteoarthritis were randomized to either intraarticular injection with PRP (Platelet Rich Plasma) × 2, a single dose of HA (Hyaluronic acid) or ozone × four doses. At the end of the 1st month after injection, significant improvements were seen in all groups. During the third month, the improvements in WOMAC and VAS scores were similar in groups 1 and 2 while those in group 3 were lower (P <0.001). After 6 months, the clinical effects of PRP and HA remained, while the clinical effect of ozone had disappeared (p <0.001). At the end of the 12th month, PRP was both statistically and clinically superior to HA (p <0.001). Conclusion: The effect of a PRP injection lasts longer than a hyaluronic acid injection, which in turn lasts longer than an ozone injection.

A double-blind randomized clinical trial [121] was performed on 62 patients with knee Osteo Arthritis. The patients were randomly divided into two groups. In the first group 40 mg triamcinolone (1 cc) and in the second group 10 cc (15 µg/mL) oxygen-ozone (O2-O3) were injected into the knee joint under ultrasound guidance. Outcome measures included the Western Ontario and McMaster Universities Osteoarthritis (WOMAC), knee flexion range of motion (ROM), effusion in ultrasound images of the suprapatellar recess, and visual analog scale (VAS), which were evaluated before injection, 1 week, 1 month, and 3 months after the treatment. Sixty-two patients (10 men and 52 women) were enrolled with mean age of 57.9 years. VAS improved in both groups (steroid P value = 0.001, oxygen-ozone P value < 0.001). The improvements seen in VAS and WOMAC scores 3 months after treatment were in favor of the oxygen-ozone group when compared to the steroid group (P = 0.041 vs P = 0.19). There was no significant difference between the two groups in ROM and joint effusion seen under ultrasound (ROM p = 0.880, effusion p = 0.362). However, in the oxygen-ozone-receiving group, joint effusion was decreased significantly (p < 0.001). Both steroid and oxygen-ozone injections are effective in patients with knee osteoarthritis. This study showed that the effects of oxygen-ozone injection last longer than those of steroid injection to the knee joint.

Effect on jaw pain In a Turkish study [122], 40 women diagnosed with myofacial pain dysfunction syndrome were randomized to either ozone injection at trigger points (n = 20); or placebo(n = 20). Ozone and placebo were applied three times a week, a total of six sessions. Mandibular movements were examined, masticator muscle soreness was assessed and Pressure Pain Threshold PPT values were obtained. Subjective pain levels were evaluated using the visual analogue pain scale (VAS). These assessments were performed at baseline, 1 month and after 3 months. Ozone therapy reduced pain intensity and significantly increased PPT values from baseline to 1 month and 3 months in the ozone group compared to the placebo group. Overall improvements in VAS scores from baseline to 3 months were in OG 67.7%; PG 48, 4%. While ozone therapy may be accepted as an alternative treatment modality in mastoid muscle pain management, placebo injections also showed significant improvements in the tested parameters. In another Turkish study [123] 33 patients with Tempero Mandibular Joint problems (TMJ) received ozone therapy or oral anti-inflammatory medication with ketoprofen and thiocolicoside 2 x 1 for 7 days. This study showed that ozone therapy was a more effective treatment than oral medication to relieve TMJ pain. In a study from Egypt [124], the effect of intra-articular ozone injections was compared with oral anti-inflammatory medication in 60 patients with TMJ, with ozone treatment again being superior with statistical significance. Nervous System Diseases

Vestibulocochlear problems In a study from Cuba [125], 50 patients with peripheral vestibulocochlear syndrome received ozone injections laterally on the neck at C2-C3, for 20 sessions. The results showed improvements in vertigo, hearing loss, tinnitus and nystagmus by 90, 80, 65 and 100%, respectively. These patients were initially in a state of systemic oxidative stress, but at the end of the study in redox balance. No side effects were observed. 68 patients with tinnitus were randomized [126] to ozone treatment (n = 27), betahistine treatment (n = 26) or control group (n = 15). Ozone group patients received 10 major autohemotherapy sessions. The Betahistin group received 48 mg / day beta-histine tablets per os for 3 months. The control group was followed up without any treatment being given.The comparison between the groups in improving tinnitus strength was not significant (p = 0.821). Comparison of the original mean Tinnitus level (THI) and 3 and 6 months after treatment revealed a significant difference in the ozone and beta histine groups, but not in the control group. The results of the study do not provide sufficient evidence to support ozone and betahistine as a treatment for tinnitus and further research on the subject is recommended. Forty-five adult patients [127] with sudden sensorineural hearing loss were randomized to placebo (15 patients) or ozone therapy (auto-haemotherapy, 30 patients, twice weekly for 10 sessions. Significant recovery of hearing was observed in 23 patients (77 percent) receiving ozone therapy Compared to six (40%) patients who received placebo (p <0.05), airborne tones, dummy tones, speech understanding, and subjective recovery rates were significantly better in ozone-treated patients compared to placebo-treated patients (p <0.05). is a promising treatment for sudden sensorineural hearing loss, with no complications observed.

Autonomic Dysregulation In a Russian study [128] in 48 patients with chronic cerebrovascular disease and autonomic dysfunction, patients in the experimental group received ozone therapy plus standard medications. The patients in the control group received only standard medication. Disorders of vascular-autonomic regulation with dominance of ergotropic sympathetic effects were identified in 69.7% of patients with chronic cerebrovascular disease. Ozone therapy caused a shift in the autonomic balance against parasympathetic activity, as well as a decrease in the activity of the vasomotor center and the central control circuit, indicating an increase in defense mechanisms indicating normalization of the autonomic balance.

Headache Headache affects about 10% -15% of the general population. Mixed results are obtained from various therapies, usually drugs, but also oxygen therapy, behavioral therapy, physiotherapy and peripheral or central neurostimulation can be used. In therapy resistance, it has a serious impact on the quality of life.Five (5) patients [129] who had suffered from severe / persistent headache resistant to standard management (including 5-HT1 agonistriptriptan) were treated with ozone therapy (major autohemotherapy). The number of headache episodes (n = 80, range 5-200) decreased significantly during the first 6 months after the treatments (n = 0, interval 0-1, p = 0.042) and for 6 months before the last follow-up visit (n = 1, interval 0 -2; p = 0.043). The corresponding VAS scores were 8.7 ± 0.8 pretreatment compared to 1.1 ± 2.5, 6 months after treatment (p = 0.003) and versus 3.1 ± 3.3 the 6 months before the last follow-up visit (p = 0.036). The conclusion was that ozone treatment reduced headache and pain severity over a prolonged period. This new method is effective and deserves further research. Anti-Aging, Achievement and Cosmetic Treatment My good friend and leading ozone scientist from Russia, Professor Jevgenij Nazarov says that ozone treatment makes you 20 year younger, but the effect does not last forever. It must be maintained at least a few months apart to be permanent. Ozone removes wrinkles and increases the skin's elasticity, improves hair growth and hair quality, which can be clearly noted on patients who regularly receive ozone. The improved cardiac mitochondrial and circulatory effects also help athletes perform more. Ozone gas is not a doping classed substance, but it is not allowed to remove and reintroduce larger blood volumes, which means that rectal treatment can be an attractive alternative for athletes who want to increase their performance without side effects. As far as I know, no study has yet been carried out that relates regular ozone use to longevity, but I would guess, that the result would be positive. There are a large number of beauty clinics that use ozone to remove cellulite, wrinkles and regain hair growth in baldness. It has also been used to enhance potency and orgasmic potential in both men and women. Discussion O3 therapy may alter the course of a variety of disease states [3] and disorders, with potentially many more indications that have not yet been tested. There are randomized clinical trials on the treatment of heart failure, coronary artery disease, peripheral arterial disease, diabetes, diabetic ulcer, chronic atypical pneumonia, fungal infections, gum and oral infections, genitouretal infections, fibromyalgia, rheumatoid arthritis, multiple sclerosis, ulcerative colitis, rheumatoid arthritis gastric ulcer, bacterial vaginosis , placental insufficiency, urinary tract infections, wound treatment, burns, diabetes ulcers, venous leg ulcers, shot injuries, pulmonary emphysema, jaw osteonecrosis, disc herniation, spondylosis, knee arthritis, jaw pain, vestibulocochlear problems, sudden hearing loss and autonomic dysregulation. In addition, there is clinical experience and case descriptions that show good effect on a variety of conditions such as cancer, Mb Parkinson, Alzheimer's, ALS, chronic infections, psoriasis, epilepsy, depression, anxiety and probably also psychotic diseases. (There are a large number of double-blind studies linking schizophrenia to infection with chlamydia pneumoniae, and ozone has been shown to improve the release of chlamydia pneumoniae in combination with antibiotics [130]). Future laboratory and translation research should begin to develop protocols for O3-AHT in attempting to establish a dose-response relationship, as it has been shown to be useful in a variety of pathologists at varying concentrations. Despite the current convincing evidence, future studies should include more double- blind, randomized clinical trials with larger patient materials, determination of effect duration, and methods of measurement and analysis. Just like in most other areas within the medical science, we can assume that there is publication bias, conscious or unconscious manipulation of research results also in this area. However, it must be pointed out that the financial incentives for ozone research are significantly less than for patentable drugs, which should be a significant advantage in terms of reliability. In 2012, the former head of cancer research at the biotech company Amgen revealed that the results of the company's efforts to replicate 53 basic studies in hematology and oncology [131]. Only six of these studies could be reproduced ..... There is therefore always reason to read medical research with many pinches of salt, but one thing that is sure is: We do not know of any other medical treatment that has shown to have so much effect on as widely different conditions as ozone treatment. It also has almost no side effects. in addition to excessive flatulence and mild laxative effect in rectal treatment and things that do not have to do with Moreover, the results of ozone treatment can probably be further improved if the research receives more money for further development of methods and comparative studies between the different types of ozone treatments. Can our healthcare system really afford to neglect of such an effective, well-documented and side-effect treatment, or will we accept that former communist countries like Russia, China and Cuba will run away from us in the west? If our authorities have a serious interest in improving public health, a development, education research and documentation program on ozone treatment should be a high priority.

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