Alberto Alexandre Editor INTRODUCTION

THE USE OF OZONE IN SPINAL DEGENERATIVE PATHOLOGIES

Ozone is a very important gas in the stratosphere, reaching its maximum concentration (1000μg/m3above) at a height of 20-30km. Is an unstable gas. It is composed of three atoms of oxygen and has a high breakdown rate ranging in the order of 105-106mol/s. Ozone is 1.6 times denser and 10 times more soluble in water (49.0mLin 100mL of water at 0°C) than oxygen and even though ozone is not a radical molecule, it is the third most powerful oxidant after fluorine and perisulfate. Ozone is produced by three main sources of energy: chemical electrolysis, electrical shock, and UV radiation.

Ozone-theraphy has been used for therapeutic purposes since the late XVII century, in different therapeutic modalities with unexpected results in some pathologies.

In the scientific literature the first mention of ozone was by the Dutch physicist Van Marumom Mak in 1785. During experiments with a powerful facility for electrification he discovered that in the air around the “ Large lectic Machine” there was a gaseous substance with characteristic smell, which had strong oxidizing properties. In 1840 the professor of the University of Basel Christian Frederick Schonbein related the changes in the properties of oxygen with the formation of a particular gas which he called ozone (from the Greek word ozein “smelly”). Schonbein first detected ozone capacity to merge with biological substrates at positions corresponding to the double bonds. The German chemist Christian Friedrich Schonbein, is also known for the discovery of nitrocellulose.

In 1857 the first ozone-production apparatus was built with the help of “modern magnetic flux tube” created by Verner von Simens. It was used in an installation for the purification of drinking water. Since then industrial ozonation allows to obtain pure and hygienic drinking water safe for human consumption. One hundred years later Dr. Joachim Hansler built the first medical ozone generator which gave the possibility of accurately dosing the ozone-oxygen blend.

In 1885 the Medical Society of Florida (USA) published the book “Ozone”, written by Dr. Charles J. Kenworth, which gave details of the use of ozone for therapeutic purposes. In October 1893 in Holland (Ousbaden) the first water treatment system with ozone was installed currently extended to more than 3 000 water treatment plants with ozone. In September 1896, O3 generator system was patented by Nikola Tesla. In 1900 he formed the Tesla Ozone Company started to sell ozone-generators and ozonated olive oil for medical purposes.

In 1898 was founded in Berlin the Healing Institute with Ozone by Thauerkauf and Luth. Since that year they began experimenting with ozone administration by injection. In 1902 the dictionary Practical Medical Material by JH London Clarke describes the successful use of ozonated water which he called Oxygenium in the treatment of anemia, cancer, diabetes, influenza, morphine poisoning, canker, sores, and pertussis. This same year an article by Dr. Charles Linder describing the use of injections of O3 in his practice.appeared in a Washington newspaper.

Charles Marchand, a chemist from New York, published in 1904 the book “Medicinal Uses of hidrozon” (ozonated water) and glicozon (ozonated olive oil). In the book, which is preserved in the Library of United States Congress can be seen a stamp of the Association of General Surgeons.

Dr. Noble Eberhart of the Department of Physiology and Therapy of Loyola University in Chicago in 1911 published the Working Manual of High Frequency. In Chapter 9 he details the use of ozone in the treatment of tuberculosis, anemia, chlorosis, pertussis, tetanus, asthma, bronchitis, high fever, insomnia, pneumonia, diabetes, gout and syphilis. In 1913 he created the first German society for ozone under the direction of Dr. Eugene Blass which was named the Eastern Association for Oxygen Therapy.

3 During the First World War (1914-1918), Dr. Albert Wolff of Berlin promoted the use of ozone in the treatment of with HIV / AIDS study referred to comments made in 1991 on viral inactivation (HIV-1) dose dependent. wounds, trench foot (also known as trench foot or frozen foot), gangrene and in order of mitigating the effects of poison gas. Ozone down there was also indicated for colon cancer, cervical cancer and pressure ulcers. Professor Velio Bocci (University of Siena, Italy) spent his life studying and teaching the applications of ozone in humans. In 2002 he published the book “Ozone, a new drug” a landmark in the practice of ozone therapy. In 1926, Dr. Otto Warburg of the Kaiser Institute in Berlin reported that the cause of cancer was the lack of oxygen at cellular level. The researcher received the Nobel Prize in Medicine in 1931. The directors of the most Through the years 1992-2000 several Italian professionals developed the use of oxigen-ozone gas mixture in important hospitals in the U.S. in 1929 published the book “Ozone and Its Therapeutic Action”, which listed the treatment of spinal degenerative pathologies. 114 diseases and their treatment by ozone application. From them an enormous development came all over the world, with collection of very good clinical results, The Swiss dentist E.A. Fish (1899-1966) was the first to sense the enormous advantages of O3 in the local and with the progressive growth of analytical studies. The rapidly growing number of studies concerning the treatment. He worked with ozone and ozonated water even before 1932 when successfully treated medical use of ozone involve the explanation of the biochemical mechanisms by which ozone performs its gangrenous pulpitis with a gas injection. The patient treated was Dr. Edwin Payr (1871-1946) who immediately effects. understood the utility of ozone and was enthusiastic in its application in general surgery. He published in 1935 a 290-page report entitled “Treatment with ozone in surgery”, which was presented at the 59th congress of There are currently more than 47 national and international associations that bring together professionals the German Surgical Society. It was between 1934 and 1938 by Drs. Aubourg and Lacoste in France used who practice this therapy, indexed journals, continuing education courses and conferences on the subject. the ozone by rectal insufflation to treat fistula problems. In 1938 Paul Aubourg published an article on the Ozono-therapy is a safe procedure if well defined and precise doses, volumes and concentrations are used. successes achieved in the Beaujon Hospital (Clichy, Ile de France). The aim of this work, composed by articles of some of the most authoritative professionals involved in In 1933 the American Medical Association (AMA) directed by Dr. Simmons urged the U.S. government ozono-therapy for spinal degenerative pathologies, is to give an outlook on the specific knowledge: the status prohibiting all drug therapies that were not authorized and properly registered, and with this faded the use of of the art on the subject. ozone in this country. This gave the monopoly of therapies to the pharmaceutical companies, with all the obvious incomes. This book is presented in memory of our fellows: Dr Nivio Jucopilla, Dr Roberto Dall’Aglio, and Dr Nabil Mawsouf. Their contribution to the beginning of the application of ozone in Patients, and to the diffusion of The Simmons decision elicited unfavorable reactions within the AMA: Dr. Emanuel Josephson of New York ozono-therapy in the world thanks to their studies and publications is an example for all of us. who wrote describing the behavior of Dr. Simmons within the AMA as “conspiracy in restraint of trade, and extortion,” adding that “almost every branch of the Federal Government in the active field of medicine was Completely Dominated by the Association “.

In 1952, the National Cancer Institute verified the findings of Dr. Otto Warburg about the cause of cancer may originate from a deficit of tissue oxygen. Alberto Alexandre Since 1953 Dr. Hans Wolff (1924-1980) created the first school of ozone therapy training many doctors, and in 1961 introduced the techniques of self-hemotherapy major and minor. Created in 1972 with Dr. Joachim Haensler the German Society for Ozone Therapy. In 1979 he published his book “Das MedizinischeOzon” (Ozone in Medicine) (Heidelberg, VFM Publications, 1979).

In 1957, Dr. Joachim Haensler (1908-1981) patented his ozone generator which has been the basis for the expansion of ozone therapy in Germany. Today over 11 000 German health professionals use ozone in their daily work.In 1977, Dr. Renate Viebahn provided a technical description of the action of ozone in the body. Ten years later, in 1987, along with Dr. Siegfried Rilling published “The Use of Ozone in Medicine”, becoming one of the reference books.

In 1979, Dr. George Freibott began treating with ozone his first AIDS patient with encouraging results, followed by Dr. Horst Kieff who in 1980 reported its results. The journal Science published the article “Selective inhibition of ozone in human cancer growing cells. ”The first Ozone Research Center of the world was founded in Cuba. In 1990 success in the treatment of Retinitis Pigmentosa, Glaucoma, Retinopathy and conjunctivitis were posted there by a group of researchers led by Dr. Silvia Menendez, Dr. Frank Hernandez, Dr. Orfilio Pelaez and others. In 1992 a group of Russian researchers reported their experience treating large burns with saline baths to the saturation limit previously treated with ozone bubbling. The first uses of ozone were based on their bactericidal properties. In 1993 Carpendale and Freeberg found important applications of O3 in patients

4 5 MODALITIES OF OZONE TREATMENTS

V Bocci ¹, E Borrelli ² ¹Department of Biotechnologies ,Chemistry and Pharmacy , University of Siena, Italy ²Department of Medical Biotechnologies, University of Siena, Italy

INTRODUCTION

The use of ozone became a normal practice after the initial studies by Dr. H.H.Wolff (1979) in Germany. Today, in this Nation, is used by over 12.000 physician but only after Bocci’s study (1) it is used very proficiently either in chronic inflammatory diseases and in orthopedics. It has been shown to be very effective and absolutely atoxic when used in dosages which induce only a well tolerated oxidative stress either for autohaemotherapy or in orthopedics. Unfortunately in many countries such as Germany, Italy, Spain,China, Japan, United States and South America most of the ozonetherapists are not aware of this concept and therefore ozonetherapy may not be so effective. However by means of our papers and Congress’s reports it is hoped that slowly every ozonetherapist will become acquainted with our results. It will be described what ozone is, how it can be proficiently used, what are its messengers, what will be their effects and what are the therapeutic effects.

THE STATE OF THE ART IN SYSTEMIC AND ORTHOPAEDIC DISEASES a) basal concepts Ozone is made by three oxygen atoms (O3) and its molecular weight is of 48.000. Ozone has a cyclical structure with a distance among oxygen atoms of 1.26 A and exists in several mesomeric status in dynamic equilibrium. Its solubility (ml) in 100 ml water at 0°C of either ozone or oxygen is 49.0 ml or 4.89 (ten folds lower), respectively. Consequently the great solubility of ozone in water allows its immediate reaction with any soluble compounds and biomolecules present in biological fluids. Among oxidant agents, ozone is the third strongest, after fluorine and persulphate, a fact that explain its high reactivity.

Ozone is formed from pure oxygen via an endothermic process allowed by a very high voltage gradients set up between the electrodes of the Siemens’s tube 3O2 ----2O3 ---- 68.400 cal This reaction is reversible and consequently ozone is hardly storable. Indeed at 20 °C the ozone concentration is halved within 40 min at 30 °C within 25 min, while at -50 °C is halved only after 3 months. This is to say that ozone is a very unstable gas. In the tratosphere there is an ozone layer containing about 10 ppmv (part per million volume) equivalent to about 10 micrograms/ml. This layer is important because it absorbs most of the UV radiation (<290 nm) emitted by the sun. UV rays include band A (316-400 nm) responsible for suntan and bands B and C (from 100 up to 315 nm) which are far more mutagenic and able to induce skin ageing and carcinogenesis. However ozone able to control UV irradiation protects biological systems on earth. Unfortunately the increase of ozone at the ground level supports the dogma that ozone is always toxic particularly if breathed for many days. Indeed ozone should not ever breathed by humans or animals because it induces serious toxic effects in humans and animals. Indeed a terrestrial ozone concentration of 10,0 ppmv causes death within 4 hours. Thus ozone should never be breathed by humans: however if this happens in nature, it should never happen in clinics, where ozone is used daily. This is to say that a physician practicing ozonetherapy must have very safe ozone generator and a safe ventilator device. A normal medical ozone generator produce ozone concentrations equivalent from 1 up to 100 mcg/ml but for medical purposes ozone concentrations from 10 up to 40 micrograms/ml are used. Indeed ozone is carefully collected with a glass syringe in dosages ranging from 10 up to 40 μg/ml (2). b) how ozone is used in medicine? Ozone measured in terms of micrograms/ml is used with great care either for the autohaemotherapy or for direct use in case of orthopaedics. In all cases low dosages of ozone are used either because it is only necessary to induce the minimal oxidative stress which is sufficient to induce a medically useful response.

7 b1) The use of ozone with the patient’s blood (IV ozonetherapy) element (ARE) able to activate some 230 genes belonging to the phase II antioxidants and detoxification response. The induced genes include the most important synthesis of GSH, of various GSH enzymes, NADPH, NAD(p)-quinone- This is feasible because the therapeutic ozone dosage is mixed with the patient blood ex vivo. It is important to mention oxidoreductase 1 (NQO1), UDPsyaliltransferase and heme-oxygenase1 capable of increasing the level of CO and bilirubin. that human blood (both plasma and cells) contains a great number of antioxidants including hydrosoluble ones such as uric NrF2 also inhibits insulin and growth hormone pr duction,thus increasing stress resistance. The daily administration of acid, ascorbic acid, cysteine, glutathione, albumin, some chelating proteins such as albumin (CYS 34) and enzymes such as curcumin and L-sulforaphane may be useful but first of all their bioavailability must be improved. A daily food restriction in catalase, GSH redox system, NADPH and superoxide dismutases (SOD). The relevance of the antioxidants in plasma is conjunction with a daily exercise appears also useful but only a few subject can accept these approaches for a long time. enormous and it allows to perform ozonetherapy. In 100 ml of human blood it is possible to measure about 5 mg/dl of Ozonetherapy is very cheap and, if performed twice weekly, it is well accepted mostly because, within 3-4 weeks, the patient uric acid, 1,5 mg/dl ascorbic acid, albumin containing cysteine 34 and eleven nucleophilic groups. Thus the variety of reported to be rejuvenated (6). extracellular and intracellular antioxidants are able to explain how bland amounts of ozone can be perfectly tamed with the results of stimulating the biological system without deleterious effects. Thus, to a few chemists claiming that ozone is always c) the effect of ozone in orthopaedic diseases toxic, we can answer that they are wrong : the antioxidant reservoir decreases no more than 30% in relation to ozone doses between 10 and 40 mcg/ml of ozone per ml of blood. Moreover this partial depletion is corrected in about 20 minutes ex The pathophysiology of these diseases is complex, often characterized by the distruction of the articular cartilage with increased vivo thanks to the recycling of dehydroascorbic acid, GSSG,vitamin E radical, NAD(P) radical. Indeed the potent antioxidant matrix degradation due to the release of proteoglycanases. Moreover collagenase-activated chondrocytes and monocytes can reservoir of blood decreases no more than 35% after ozonation and fully recover in about 20 min ex vivo. Thus a first release IL-1,TNFalpha and prostaglandins which amplify the inflammation (7). conclusion is that we can perform safely ozonetherapy because a small ozone dose is fully controlled by the potent antioxidant It is surprising that after an initial pain, small doses of ozone induces great relief for a long time. system of blood. Consequently we measure a rapid antioxidant regeneration because we have demonstrated the manteinance Low back pain can affect up to about 80% of the world population. In 1988, Verga,a private ozonetherapist proposed an of a normal antioxidant system. Indeed if blood did not have a potent antioxidant system we could not perform ozonetherapy indirect approach by injecting small volumes of O2-O3 into the point localizable in the paravertebral muscle which is the locus (3). dolendi corresponding to a metamer of the herniated disc. Consequently two different approaches have been proposed and actuated: the simplest of the two is the indirect approach or chemical acupuncute because the 5-10 ml of oxygen ozone Which are the pathologies more suitable to be treated with a success? All chronic inflammations develop in conjunction (about 20%) injection is performed into the paravertebral muscle corresponding to the site of pain. This procedure has with a chronic oxidative stress. This means that protection of reactive oxygen and mitogen species (ROS/RNS) is very much become very popular in the world but the ozonetherapist must be warned to avoid an excessive dose of ozone, which may increased while the production of necrotizing antioxidants is very much depressed. Diseases with this problem are the induce a complex and dangerous neurovegetative over-reaction. The approach consists in one or two injections of 5-10 ml majority and include all vascular diseases such as those in the CNS either preceding a stroke or a chronic Limb Ischemia, gas to be done about 2 cm bilaterally to the spinosus process at the level of the pain. Results are: as about 40% optimal,35% Heart Insufficiency, Age - Related Macular Degeneration (dry form), Chronic Obstructive Pulmonary Disease, likely Multiple marked improvement and 15-20 % minimal or no results. The mechanism of action is due to the hig solubility of ozone in the Sclerosis, Parkinson’s and Alzheimer’s disease (4,5). On the whole they represent about 70% of chronic diseases and they muscle interstitial fluid. It is expected to suddently generate some H2O2 and lipoperoxides able to stimulate amyelinic fiber are very debilitating inducing loss of productivity and an earlier death. We tend to exclude viral diseases such as hepatitis B (nociceptors) able to elicit an antalgic response via the descending antinociceptive systems. The injection must be performed and C, HIV and cancer because they can be treated with orthodox medical approaches. Needless to say is that ozonetherapy very carefully and slowly for avoiding lipothymia. The mechanism of action is probably due to: integrates good medical drugs. Thus a mild ozonetherapy (twice weekly) performed with a progressive slow increase of ozone within the hormetic curve (first week:10 μg/ml ozone, ranging from 100 to 150 ml in relation to body weight; 2nd week:15 1) activation of the descending antinociceptive system. μg/ml of blood;3rd week: 20 μg/ml of blood;4th week: 25 μg/ml of blood;5th week:30 μg/ml of blood; 6th week: 35 μg ozone 2) elevation of the activation threshold linked to the oxidative degeneration of C-nociceptors. per ml of blood. With this scheme in most cases the patient reports a feeling of wellness but if he/she reports to be tired and sleepy, we must reduce the ozone dose. However about 85% of patients report a feeling of well being and increased energy. 3) release of endorphins able to block the transmission of the noxious signal to the thalamus and cortex. After six months therapy, one ozonetherapy is enough and, by the tenth month, the patient reduce the simptomatology and improve the performance status. 4) simultaneous psychogenic stimulation of the central analgesic system induced by the gas injection(s). The localized oxygenation and analgesia are important because they permit muscle relaxation and vasodilation, thus a reactivation of Why ozonetherapy is very effective in this particular set of diseases? muscle metabolism.

There are multiple reasons: When the paravertebral injection of ozone does not work ( in about 25-30 % of patients), the direct approach or intradiscal The first consists in having evaluated a weak oxidative stress. The very small amounts of ozone reacts immediately in the injection of oxygen-ozone may solve the problem. The typical orthopaedic operation destabilize the mechanical and functional glass bottle and, when the so-called ozonated blood is infused in the donor, ozone is no longer present. While oxygen slowly stability of the vertebral column and it is no longer used. It was substituted by the intradiscal injection of chymopapain. oxygenates all the erythrocytes, ozone reacts immediately with antioxidants present in the plasma and with polyunsaturated However also this chemonucleolysis has been abandoned in favour of the direct intradiscal injection of O2-O3, which must fatty acids (PUFA). Consequently the antioxidants decrease by 10-30% ( and they will be later rapidly reduced) while PUFA be carried out under radioscopic control: the needle is inserted in the center of the pathologic intersomatic space just undergo peroxidation. H2O2 and aldehydes are very important because H2O2 can penetrate all blood cells. The erythrocytes before the direct insufflations of 3-15 ml of the gas mixture: O2-O3. The ozone concentration ranges between 20-30 mcg/ will shift the hemoglobin curve to the right and this process will facilitate the oxygenation of ischemic tissues. The two final ml. Usually up to 70% of patients have shown a great outcome. The most likely mechanism follows: ozone dissolves in the alchenals, mostly represented by hydroxyl-nonenal (4-HNE) will be partly eliminated by the liver and kidneys but mostly will discal nucleus pulposus and generate both H2O2 and OH which are very reactive. Indeed they dissolve proteoglycans and bind to either the Cys 34 of albumin or to GSH or cysteine. These molecules, via the circulation, will easily transfer 4-HNE into collagen type I and II which are components of the degenerate nucleus pulposus leading to its breakdown. Thus the reabsorpion the cytoplasm of many cells. The cells contain an inactive transcription factor called NrF2 bound to a larger inactive factor with of hydrolytic products and water lead to progressive shrinkage and disappearance of the herniated material. The reduced several exposed-SH groups called Keap 1 rich in cysteins. Normally this complex: NrF2-Keap1 is destroyed in the proteasome mechanical irritation decreases the sensitivity of the nerve axons and also the inflammation disappears. This means that every about 20 minutes, but when 4-HNE binds to Cys 273 or Cys 288 of Keap1 causes the release of the important molecule ozone rapidly blocks inflammatory reactants and stimulates the restitutio ad integrum. Probably the release of TGFbeta1 and NrF2. This molecule (weight about 90.000) is now free to move into the cell nucleus and this step either prevents of replaces bFGF favour the reorganization of the residual nucleus pulposus with incipient fibrosis. Release of endorphins with activation the exacerbations of stress induced disease typical of cardiovascular diseases, COPD, age-related macular degeneration of the antinociceptive system and an elicitation of a placebo effect are very likely. A positive response may reach the level of and probably malaria as well. The NrF2 (90-11 m.w.) after binding to a small MAF protein, activates the antioxidant response 80% success. Side effect are very rare (8).

8 9 In conclusion today it appears that very small amounts of ozone surprisingly are able to solve a painful and invalidating THE MAJOR OZONATED AUTOHAEMOTHERAPY: problem. These results, due to a very small dosages of ozone, ought to stimulate an intelligent reflection of the most stubborn opponents of the use of the ozone in medicine. BIOCHEMICAL BACKGROUND AND A PROPOSED STUDY FOR AN INTEGRATED THERAPY IN NEUROSURGICAL DISEASES

Emma Borrelli, MD,PhD, Stephen John, MD* CONCLUSION Programme Director, Postgraduate Course of Oxygen Ozone Therapy, Department of Medical Biotechnologies, University of Siena,Italy Email [email protected] In this brief review we have analyzed the many “pros” and the very rare “con” capable of using O2-O3 either in parenteral or * EU.N.I. European Neurosurgical Institute, Treviso , Pordenone, Bologna, Roma localized therapies. It remains surprising that health authorities do not help to improve the situation and remain entangled in political problems. The so-called “major ozonated autohaemotherapy” (MOAT) was invented in Germany in the last Century and until now millions of treatments have been performed in patients all over the world without any acute or chronic toxicity. The correct method consists in collecting 100-200 ml of blood (plus an anticoagulant) in an ozone resistant glass bottle, adding an equivalent oxygen ozone gas volume containing ozone at a precise concentration ranged from 10 to 40 micrograms per milliliter of gas BIBLIOGRAPHY mixture, gently mixing for 2 min and returning the oxygenated-ozonated blood to the donor during the next 15 min, obviously without the gas. A complete description of the methodology was reported in previous articles (1-2). 1) Bocci V. Scientific and medical aspects of ozone therapy. State of the art. Arch. Med. Res. 2006, 37: 425-35 When human blood is exposed to a gas mixture composed of medical oxygen and ozone (about 96% and 4 % respectively), both gas present in the phase overlying a superficial layer of about 10μ of blood at first dissolve in the water of plasma. The 2) Bocci V. Borrelli E. Travagli V. Zanardi I. The ozone paradox. Ozone is a strong oxidant as well as a medical drug. Med Res gas solubilization goes on continuously when the blood is gently rotated in a glass bottle. Rev, 2009, 29: 646-82. Oxygen equilibrates with the extracellular and the intraerythrocytic water before becoming bound to hemoglobin until it is fully oxygenated, as shown by the rapid increase of the pO2 from about 40 up to 400 mmHg. On the contrary ozone, more soluble 3) Bocci V. Ozone: A new Medical drug. 2nd Edition, Springer Verlag, Dordrecht, The Netherland, 2011. than oxygen, readily dissolves in water and reacts instantaneously with several substrates, oxidizing ascorbic acid, urate, free cysteine, glutathione(GSH) molecules and albumin thiol groups (SH). In fact ozone doses within the therapeutical range (10-40 4) Borrelli E. Diadori A. Zalaffi A. Bocci V. Effects of major autohemotheraoy in the treatment of dry age related macular μg/ml of gas with a volumetric 1:1 gas-blood ratio) are mostly neutralized by the well known sacrificial reactions: for example degeneration: a randomized controlled clinical study. Int. J Ophthalmology 2012, 5: 708-13. albumin is considered the main sacrificial molecule due to large amount in plasma and surely it prevents lipoprotein damage.

5) Borrelli E. Bocci V. Oxygen ozone therapy in the treatment of chronic obstructive pulmonary disease: An integrative The ozonation process in blood is therefore characterized by the formation of only few molecules of reactive oxygen species approach. 2014, 2: 9-13 (ROS) and lipid oxidation products ( LOP) acting in two phases: while ROS ( mainly hydrogen peroxide) are acting immediately and disappear (early and short-acting messengers), LOPs (late and long-lasting messengers), via the circulation, distribute 6) Pecorelli A. Bocci V. Acquaviva A. Belmonte G. Gardi C: Virgili F. et al. Nrf2 activation is involved in ozonated human serum throughout the tissues and either bind to cell receptors or enter into the cell. upregulation of HO-1 in endothelial cells. Toxicol Appl Pharmacol 2013, 267: 30-40. The behavior and pharmacodynamic of hydrogen peroxide have been ascertained: the initial formation of a gradient between plasma and intracellular water allows its entrance into the erythrocytes and lymphocytes but its concentration remains around 7) Bocci V. Borrelli E, Zanardi I, Travagli V. The usefulness of ozone treatment in spinal pain. Drug Design, Development and a few micromoles because it is quickly reduced to water by free glutathione, catalase and glutathione-peroxidase. Its half-life Therapy 2015,9: 2677-2685. is less than one second and yet its intracellular concentration is critical because, in order to activate some biochemical pathways (formation of oxidized glutathione with consequent activation of the pentose cycle in pentose cycle in the red 8) Borrelli E. Mechanism of action of ozonetherapy in the treatment of disc herniation and low back pain. Acta Neurochir Suppl cell and activation of a tyrosine kinase in lymphocytes), it must reach a critical threshold that nonetheless, is not cytotoxic. 2011,108:123-125. Provided that the ozone dose is within a well defined, experimentally determined range (10-40 μg/ml or 0.21 and 0.84 microMoles per ml of blood) , there is only a transitory decrease (no more than 25 %) of the potent antioxidant capacity of plasma, fully reconstituted within 20 min owing to the efficiency of the redox system. There is neither damage to erythrocytes: haemolysis is negligible (from 0.4% up to 1.2 %) and methemoglobin remains normal, nor to other blood cells. It must be added that ozonated erythrocytes show an improved glycolysis with an increase of ATP and 2,3 DPG levels, which are able to shift the dissociation curve of oxyhemoglobin to the right, confirming the observation of an improved delivery of oxygen in peripheral obstructive arterial disease. The beneficial physiological cellular use of ROS is now being demonstrated in different field, including intracellular signals and redox regulation. It has been documented that low levels of ROS are signaling molecules, modulating cell proliferation and apoptosis, gene expression through activation of transcription factors like nuclearfactor kappa beta (Nfk) and hypoxia inducible factor (HIF).

What happens during the reinfusion of the hyperoxygenated-ozonated blood into the donor? The hyperoxygenation of blood (pO2 about 400 mmHg) is irrelevant because, during the 15 min infusion period, it mixes with about 6 liters of blood so that the final venous pO2 relative pressure is hardly modified. LOPs (mainly 4-hydroxynonhenale), as already mentioned, disappear from the circulation within a few minutes,and yet they can exert stimulatory effects throughout the body without toxicity because their concentration, at a submicromolar level, is transitory.

10 11 This is a crucial consideration to keep in mind and emphasizes how a small and precise ozone dose can act as a biological CONCLUSIONS response modifier. The atoxicity of blood ozonation is explained by the use of small and well calibrated doses of ozone that are tamed by the antioxidant system and the short span (only a few minutes) of ozone exposure. On the basis of biochemical and clinical studies performed in the last two decades, it has become clear that ozone, in very small dosages, behaves as a real drug and the biochemical and molecular mechanism of action are well within orthodox The repetition of ozonated autohemotherapies in patients upregulates the synthesis of several antioxidant enzymes medicine. The efficacy of the MOAT for restoring antioxidant balance is well ascertained. Patients with neurosurgical diseases (Superoxide dismutase, GSH-Peroxidase, GSH-Reductase, and G6PD) and heme-oxygenase-1 (HO-1) which is one of the most could benefit for a combined treatments of local and systemic ozone therapy with a better outcome and reduced expenses protective enzymes catalyzing the release of useful compounds such as bilirubin and CO from heme. The trace of hemolysis but unexplainably the World Health Authorities, always concerned with the increasing medical costs, still refuse to evaluate (0.4 -1,2 %), unavoidable when blood is ozonated in a glass bottle, is useful because it acts as an inducer of HO-1. Thus, a the ozone therapy and recognize its validity. small, acute stress on blood upregulates the antioxidant defences and the term “hormesis” describes these beneficial effects after an exposure to a low-intensity stressor. Whether ROS will act as damaging, protective or signaling factors depends on the delicate equilibrium between ROS production and scavenging at the proper time and side. On the basis of the ability of MOAT to restore the antioxidant potential in condition of chronic oxidative stress we integrated the BIBLIOGRAPHY ozone therapy in the treatment of patients affected by three serious and diffuse chronic oxidative stress diseases, the chronic obstructive pulmonary disease (COPD),the age related macular degeneration (ARMD), and the chronic peripheral ulcers. The 1) Bocci V, Borrelli E, Travagli V, Zanardi (2009) The ozone paradox. Ozone is a strong oxidant as well as a medical drug. Med results reported in published pilot studies suggested an efficacy of MOAT and a favourable cost benefit ratio (3-5). Res Rev;29:646-82.

Is MOAT useful in the integrated treatment of disc herniation and low back pain? 2) Bocci V(2006) Scientific and medical aspects of ozone therapy. State of the art. Arch. Med. Res ;37:425-35.

As previous reported, the use of MOAT in the treatment of the chronic oxidative stress diseases appears promising and 3) Borrelli E, Diadori A, Zalaffi A, Bocci V(2012) Effects of major autohemotherapy in the treatment of dry age related macular patients reported a subjective and objective benefit. In patients with neurosurgical disorders the pain has a multifactorial origin degeneration: a randomized controlled clinical study. Int. J. Ophthalmology;5:708-13. and the ozone can surprisingly displays a number of beneficial effects ranging from the inhibition of inflammation, correction of ischemia and venous stasis and finally an induction of a reflex therapy effect by stimulating anti-nociceptor analgesic 4) Borrelli E, Bocci V(2014) Oxygen ozone therapy in the treatment of chronic obstructive pulmonary disease: An integrative mechanisms (6). On this basis we wondered if in this patients the associated treatment with local ozone injection and major approach. Am. J. Clin. Exper. Med ;2(2) 9-13. ozonated autohaemotherapy could be more effective on the symptomatology respect to a simple local gas administration. At this purpose we recently performed a pilot clinical study on 40 male and homogeneous patients with disc herniation 5) Borrelli E, De Monte A, Bocci V(2015) Oxygen Ozone Therapy in the Integrated Treatment of Chronic ulcer: a case Series (unpublished data). All patients between 18 and 70 years of age with sciatica since 6-8 weeks are eligible for inclusion in Report. International Journal of Recent Scientific Research Vol. 6, Issue, 5, pp.4132-4136, May the study. A disc herniation at the appropriate level was assessed by Magnetic Resonance Imaging. Patients were enrolled in the study from February to November 2014. All subjects received one intradiscal injection of gas and eight major ozonated 6) Borrelli E (2011) Mechanism of action of oxygen ozone therapy in the treatment of disc herniation and low back pain. Acta autohaemotherapy two times a week ( 150 ml of blood + 150 ml of ozone at a increasing concentration of 20-30 micrograms/ Neurochir Suppl 108: 123-125. ml of gas according to Borrelli et al. ). Before and after the last treatment of MOAT we analyzed in all patients the plasma values of Reactive Oxygen Metabolites 7) Borrelli E, Alexandre A, Iliakis E, Alexandre A, Bocci V (2015) Disc Herniation and Knee Arthritis as Chronic Oxidative Stress (d-ROM test, Diacron International, Grosseto, Italy) and the SH proteins ( Diacron International, Grosseto, Italy) to better un- Diseases: The Therapeutic Role of Oxygen Ozone Therapy. J Arthritis 4: 161. doi:10.4172/2167-7921.1000161 derstand the time course of the oxidative stress and the antioxidant balance. The patients showed a marked increase in ROM value ( 480± 40 UCARR) and a decrease in protein SH content ( 300 ± 50 micromols/L) in plasma before the treatment. After the associated treatments intradiscal ozone + MOAT a decrease in d-ROM (320 ± 54 UCARR) and an increase of protein SH ( 480 ± 67 micromols/L) were observed. Moreover, we found a correlation between reduced plasma oxidation values and improvement of symptoms . A large study with the measures of more accurate markers of plasma lipid peroxidation and their correlation with the patient’s symptomatology is under way (7). Nevertheless these preliminary results suggest a beneficial action of the combined treatments ( intradiscal plus MOAT) in the reduction of low back pain and disability following disc herniation.

Other Applications of MOAT in neurosurgery

Surgical interventions (especially in neurological diseases) represent a well known situation of oxidative, immunological and inflammatory stress for the patients. In this case the use of MOAT should be effective for a reduction of the oxidative and inflammatory damage and for a more rapid recovery of the patients after surgery. We are planning a cycle of 4-6 MOAT at low concentrations (20 micrograms/ml) before and after neurosurgical interventions for the improvement of the outcome: the primary endpoints will be the postoperative reduced risk of infections and the reduction of the recovery time through the increase of blood antioxidant levels and the decrease of the surgical immunosuppression.

Moreover, a protocol about the use of intraoperative ozonated water and its wonderful antiseptic power is in progress.

12 13 DEGENERATIVE DISEASE OF THE SPINE: Growth factors, such as basic fibroblast growth factor (bFGF), transforming growth factor (TGF) and insulin-like growth factor (IGF), stimulate the chondrocyte or fibroblast to produce more matrix, and inhibit the production of MMPs.These growth factors DEGENERATIVE DISC DISEASE are normally bound by cartilage intermediate layer protein (CLIP)and are released if the matrix is degraded, in order to promote further synthesis. Tissue inhibitors of metalloproteinases (TIMP) suppress the activation of MMPs, thereby controlling Alexandre AM, Lozupone E, D’Argento F, Pedicelli A. degradation. A decrease in pH lessen the rate of synthesis of matrix proteoglycans. Institute of Radiological Sciences. Fondazione Policlinico A.Gemelli. Catholic University of Rome Largo F.Vito, 00168 Rome, Italy. On the othe hand, other cytokines, such as interleukin-1 (IL-1), interferon (IFN), and tumour necrosis factor-α (TNF-α) inhibit the synthesis of the matrix and promote the production of MMPs.These cytokines are produced by macrophages which enter INTRODUCTION the disc in response to injury.Macrophages also secrete superoxide (O2¯), which can degrade hyaluronic acid and proteoglycans, causing them to deaggregate, and can inhibit chondrocyte proliferation and synthesis. TNF-α and IL-1 Low Back Pain secondary to degenerative disk disease is a condition that affects men and women equally, in young to stimulate inducible nitric oxide synthetase to produce nitric oxide,which has a variety of degradative effects. It affects matrix middle-age with peak incidence at approximately 40 years. Disk degeneration increases with age, but degenerated disks are constituents directly, inhibits TIMPs, and thereby promotes matrix degradation and inhibits matrix synthesis. not necessarily painful. Probably bipedal ambulation this is the evolutionary event that made the lumbar spine susceptible to degenerative disease. The biochemical morphology of the end-plates is extremely similar to that of the disc: water, proteoglycans, collagen and Degeneration is a common event to structures that compone the functional spinal unit: two adjacent vertebral bodies and the chondrocytes. The concentration scheme of these components is also similar to that of the disc: the center of the end-plate is intervertebral disk. The disk and 2 zygapophyseal joints at the same level function as a trijoint complex. mostly rich in water and proteoglycan, while as we move outward toward the periphery, more and more collagen is present As humans age, they endure both macro and microtraumas and undergo changes in body habitus that alter and redistribute with less and less proteoglycans. This similar biochemical distribution scheme helps the diffusion of nutrients between the biomechanical forces unevenly on the lumbar spine. Natural progression of degeneration of the lumbar segment of motion subchondral bone of the vertebra and the depths of the disc. proceeds with characteristic anatomic, biomechanical, radiologic, and clinical findings in lumbar degenerative disk disease. The disc did not become the center of attention until 1933, when W. J. Mixter and J. S. Barr indicated the correct pathogenesis of lumbusciatic pain and nerve disfunction, and, hence, offered an appropriate surgical treatment. The neurogenic nature of PATHOPHYSIOLOGY OF DEGENERATIVE DISC DISEASE sciatica had first been described by Domenico Cotugno (1764). Following him, outstanding French neurologists such as Lasegue, Dejerine, and Sicard enriched our understanding of the basis of sciatica. The work of the German pathologists Nowadays the knowledge about degenerative disc disease is quite expanded, and even a classification scheme for lumbar Schmorl and Andrea (1927-1929) established the modern basis for understanding the intervertebral disc, by providing very intervertebral disc degeneration has been proposed. clear discussions of herniations as well as degenerations. Aging, genetic factors, nutrition, toxic factors, metabolic disorders, low-grade infections, neurogenic inflamation, mechanical ANATOMICAL CONSIDERATIONS: factors and even an autoimmune theory have colled the attention of reserchers as a key factor in the pathogenesis of THE NORMAL DISC AND THE DISCOVERTEBRAL COMPLEX degenerative disc disease.

The disc is made up of two fundamental structures: the nucleus pulposus and the anulus fibrosus. Although its composition Basically, degeneration is a process that begins early in life and is the consequence of a variety of genetic, physiologic, and percentage differs in cervical, dorsal and lumbar spine, it is made of three basic components: proteoglycan, collagen, and environmental (mechanical, traumatic, nutritional) factors, as well as normal aging. Disc degeneration per se is not painful and water. The nucleus pulposus is the water-rich, gelatinous center of the disc, and can tolerate very high pressure values in this is proven by the very high prevalence in the asymptomatic population. However, all the consequences that we mentioned standing and especially in the seated position. The anulus fibrosus has a much more fibrous structure, containing a much act in combination and the inflammatory response causes the complained low back pain. higher collagen percentage and lower water content. It serves as a limiting capsule of the nucleus because of the high pressure that is upon it. It is made of 15 25 concentric sheets of collagen (lamellae). The very outer fibers of the disc, Sharpey’s A very interesting flowchart of a hypothetical inflammatory cascade for degenerative disc disease has been proposed by fibers, anchor themselves into the vertebral endplates and into the anterior and posterior longitudinal ligament.The disc is Modic and Ross. A variety of mechanisms and mediators are involved, such as TNF, IL-1, macrophage recruitment, monocyte avascular, and disc cells depend on molecular diffusion from blood vessels at the disc’s margins to supply the nutrients es- chemoattractant protein-1 (MCP), granulocyte macrophage colony-stimulating factor (GM- CSF), vascular endothelial growth sential for cellular activity and to remove metabolic wastes. This is a key concept in the anatomy and pathophysiology of the factor (VEGF), fibroblast growth factor (FGF), IL-8, IL-6, matrix metalloproteinases (MMPs), nerve growth factor, calcitonin discovertebral complex, as it has been proved that a fall in nutrient supply can reduce the number of viable cells in the disc, gene-related peptide (CGRP), nitric oxide (NO), prostaglandin E2 (PGE2), phospholipase A2 (PLA2), substance P and Leukotrienes, leading to degeneration. all together resulting in pain.

At a microscopic level the lumbar intervertebral disc is similar to articular cartilage. In fact chondrocyte-like cells synthesise In fact disc degeneration will occur if the matrix is not normal: this can arise if the components synthesised are themselves type II collagen, proteoglycans, and non-collagenous proteins that collectively form the matrix of the nucleus pulposus and the abnormal, or if the balance between synthesis and degradation of normal components is disturbed in favour of degradation. cartilaginous vertebral endplate. Fibroblast-like cells synthesise type I and type II collagen for the annulus fibrosus. At a molecular level, degeneration will be expressed by the production of abnormal components of the matrix or by an increase Proteoglycans consist of a core protein from which radiate chains of glycosaminoglycans containing keratan sulphate and in the mediators of matrix degradation (IL-1, TNF-α, superoxide and nitric oxide)and of MMPs and a reduction in the levels chondroitin sulphate. Multiple proteoglycans, joined to a hyaluronic acid chain, form aggregates. Aggregates are held together of tissue inhibitor of matrix metalloproteinases (TIMPs). Meanwhile, increased concentrations of growth factors (bFGF, TGF) by type II collagen, which is cross-linked by type IX collagen. will reflect attempts to repair the degraded matrix.

The hydroscopic properties of the proteoglycan matrix giveto the nucleus hydrostatic properties,allowing it to accommodate Many studies have examined different noxious stimuli including increased load, osmotic pressure and reduced glucose, and compression loads and to brace the annulus. The constituents of the matrix are not static, in fact they are continually have shown they can lead to altered cell function, not only directly but also via stress-induced premature senescence.These degraded by enzymes, called matrix metalloproteinases (MMPs), which are secreted by the chondrocytes.Degradation of the senescent cells not only stop dividing and replicating, but can also produce increased levels of cytokines and matrix- matrix allows it to be refreshed by newly-synthesised components. degrading enzymes.

14 15 Once the disc becomes degenerate, it can cause LBP in different ways. One effect of disc degeneration is a loss of disc NOMENCLATURE heightwith subsequent altered stress on the facet joints and other spinal tissues, such as ligaments and muscles that can potentially lead to LBP. Loss of disc height can contribute further to compression of the exiting nerve root, resulting in pain As we mentioned before, from the radiological point of view a disc is considered “normal” when it is fully and normally in the buttock and lower limb, particularly if the nerve is sensitised in response to molecules like TNFα, which is produced in developed and free of any changes of disease, trauma, or aging. On the other hand clinically “normal” means asymptomatic the degenerate IVD. patient, even if in this case a variety of harmless imaging findings can be present (congenital or developmental variations of Olmarker and Rydevikdemonstrated that long-term compression of the exiting nerve root has been found to lead to changes discs, minor bulging of the annuli, age-related desiccation, osteophytes and so on). within the spinothalamic tract.In addition, neurohumeral markers, such as substance P, nerve growth factor (NGF), brain- Starting from the Recommendations of the combined task forces of the North American Spine Society, the American Society derived neurotrophic factor (BDNF) and platelet endothelial cell adhesion molecule (PECAM), which are not found in normal of Spine Radiology and the American Society of Neuroradiology, recently published by Fardon et al. in the version 2.0, we have IVD, have been seen in discs excised from patients with LBP. Other structures, incl ding the ligamentum flavum, also undergo to clarify the terminology about degenerative disc disease and it classification: changes in patients with LBP.In addition the growth of nociceptive nerves into the usually aneural IVD is a well-recognised feature of painful degenerative IVD. - Aging disc: disc demonstrating any of the various effects of aging on the disc. Loss of water content from the nucleus occurs before MRI changes, followed by the progression of MRI manifested changes consistent with the progressive loss RADIOLOGICAL CONSIDERATIONS: of water content and increase in collagen and aggregat-ing proteoglycans. See Pfirrmann classification.

MRI should be performed as the first examination. Sagittal image scanning must include both neural foramina, and the choice - Degenerated disc, degeneration (n), degenerate (v) between T1 or T2-weighted axial images should be based on what kind of equipment the (neuro)radiologist can rely on. The value of T2 relaxation times, especially, has been proved to characterize the structure of lumbar intervertebral discs. 1. Changes in a disc characterized to varying degrees by one or more of the following: desiccation, cleft formation, fibrosis, and gaseous degradation of the nucleus; mucinous degradation, fissuring, and loss of integrity of the annulus; defects in Whenever bone changes are present it is mandatory to apply adipose tissue suppression techniques, or complete the study and/or sclerosis of the end plates; and osteophytes at the vertebral apophyses. with a CT. The use of contrast agent should be reserved to well-selected patients, even if it can be very useful to highlight bone, intra-articular and muscular findings. Even though minimal signs of spondylolisthesis are noted in a standard supine 2. Imaging manifestation of such changes, including standard roentgenographic findings, such as disc space nar MRI, as for example exaggerated fluid in facet joints, bone instability must be suspected and a dynamic orthostatic X-ray or rowing and peridiscal osteophytes, MRI disc findings (Pfirrmann classification), CT disc findings (Dallas classification), an orthostatic MRI, if it is available, must be suggested to the patient. and/or MRI findings of vertebral end plate and marrow reactive changes adjacent to a disc (Modic classification).

Changes in discs are related to aging; in fact desiccation, fibrosis and cleft formation in the nucleus, fissuring and mucinous - Degenerative disc disease (nonstandard term when used as an imaging description): a condition characterized by degeneration of the anulus, defects and sclerosis of endplates, and/or osteophytes at the vertebral apophyses are frequently manifestations of disc degeneration and symptoms thought to be related to those of degenerative changes. Note: causal seen in the asymptomatic patient during radiological examinations. Clinical features must be considered to determine whether connections between degenerative changes and symptoms are often difficult clinical distinctions. The term ‘‘degenerative degenerative changes on imaging are pathologic and what may or may not have contributed to their development, even if disc disease’’ carries implications of illness that may not be appropriate if the only or primary indicators of illness are the distinction between aging changes and pathologic changes remains unclear. The role of imaging is to provide accurate from imaging studies, and thus this term should not be used when describing imaging findings. The preferred term morphologic information and influence therapeutic decision making. for description of imaging manifestations is ‘‘degenerated disc’’ or ‘‘disc degeneration,’’ rather than ‘‘degenerative disc disease.’’ Disc degenerations should be class fied with standard criteria using the Thompson Grading Scale, where, following a set of parameters, an X-ray radiographic inspection inspection of the disc is conducted and the gross morphology is used to - Dark disc (colloquial, nonstandard): disc with nucleus showing decreased signal intensity on T2-weighted images (dark), determine the extent of degeneration. usually because of desiccation of the nucleus secondary to degeneration. Also: black disc (colloquial, nonstandard). The term herniated disc means a focal lesion, and confusion with a bulging disc must be avoided. Unfortunately common classifications of herniated disc disease are not widely accepted. Radiologists are asked anyway, to specify the localization - Disc height: The distance between the planes of the end plates of the vertebral bodies craniad and caudad to the disc. Disc (median, paramedian, postero-lateral, lateral or foraminal and ‘‘far-lateral’), the fragment migration direction (cranial or height should be measured at the center of the disc, not at the periphery. If measured at the posterior or an-terior margin caudal) and the continuity with the original disc. Any signal intensity or signal density can be associated with a herniated disc, of the disc on a sagittal image of the spine, this should be clearly specified as such. even fluid or gas signal ones; these signal alterations can rapidly modify or even disappear. - Desiccated disc From the radiological point of view a disc is considered “normal” when it is fully and normally developed and free of any changes of disease, trauma, or aging. On the other hand clinically “normal” means asymptomatic patient, even if in this case 1. Disc with reduced water content, usually primarily of nuclear tissues. a variety of harmless imaging findings can be present (congenital or developmental variations of discs, minor bulging of the annuli, age-related desiccation, osteophytes and so on). 2. Imaging manifestations of reduced water content of the disc, such as decreased (dark) signal intensity on T2-weighted images, or of apparent reduced water content, as from alterations in the concentration of hydrophilic glycosaminoglycans. Lumbar discs alterations represent only a small part of degenerative spine pathology, in fact the same attention should be See also: dark disc (colloquial, nonstandard). given to modifications of other spine structures such as ligaments, zygapophyseal joints and vertebral body. These structures are involved by a mechanism cycle of degeneration that is instigated by small changes in the mechanical integrity of the - Dallas classification (of postdiscography imaging): commonly used grading system for the degree of annular fissur-ing intervertebral disc. In any case, compression effects on spinal canal, lateral recesses, neural foramina and their contents seen on CT imaging of discs after discography. Dallas Grade 0 is normal; Grade 1: leakage of contrast into the inner (thecal sac, nerve roots and ganglion) must be underlined if they are present. one-third of the annulus; Grade 2: leakage of contrast into the inner two-thirds of the annulus; Grade 3: leakage through Even dural venous plexus modifications must be searched for. If a central spinal canal stenosis is diagnosed, the extent of the the entire thickness of the annulus; Grade 4: contrast extends circumferentially; Grade 5: contrast extrava-sates into the stenosis can be confirmed by the presence of varicose and thicker nerve roots above the site of compression. epidural space.

16 17 - Modic classification (Type I, II, and III) : a classifiation of degenerative changes involving the vertebral end plates and adjacent generate a pressure by swelling from the attraction of water into the intervertebral disc from surrounding tissues - which vertebral bodies associated with disc inflammation and degenerative disc disease, as seen on MRIs. Type I refers to results in the vertebral bodies being pushed apart. This pressure is resisted by tension in the collagen fibres of the AF. The decreased signal intensity on T1-weighted spin echo images and increased signal intensity on T2-weighted images, balance between expansion of the NP and tension in the AF leads the intervertebral disc to resist compression. Consequently representing penetration of the end plate by fibrovascular tissue, inflammatory changes, and perhaps edema. Type I the whole composite disc can be weightbearing, while allowing flexion and torsion in an otherwise rigid structure. changes may be chronic or acute. Type II refers to increased signal intensity on T1-weighted images and isointense or increased signal intensity on T2-weighted images, indicatingreplacement of normal bone marrow by fat. Type III refers to Since the disc has a very limited blood supply, intervertebral disc cells, particularly those in the centre of the avascular NP, decreased signal intensity on both T1- and T2-weighted images, indicating reactive osteosclerosis. operate in an environment that would be unviable to most other cells. They appear to have adapted to this hypoxic, acidic environment. Accordingly, caution should be practised when studying their condition using a standard cell culture. This is an - Pfirrmann classification: a grading system for the severity of degenerative changes within the nucleus of the in- important factor, which influences the investigation of cell function and the understanding of the pathological processes that tervertebral disc. A Pfirrmann Grade I disc has a uniform high signal in the nucleus on T2-weighted MRI; Grade II shows occur within the intervertebral disc. a central horizontal line of low signal intensity on sagittal images; Grade III shows high intensity in the central part of the nucleus with lower intensity in the peripheral regions of the nucleus; Grade IV shows low signal intensity centrally and Posterior elements of the lumbar spinal functional unit bear less weight than anterior elements in all positions. Anterior blurring of the distinction between nucleus and annulus; and Grade V shows homogeneous low signal with no distinction elements bear over 90% of forces transmitted through the lumbar spine in sitting; during standing, this portion decreases to between nucleus and annulus. approximately 80%. The spine functions best within a realm of static and dynamic stability. Bony architecture and associated soft tissue structures, especially the intervertebral disk, provide static stability. Dynamic stability is accomplished through a - Thompson classification: a five-point grading scale of degenerative changes in the human intervertebral disc, from 0 system of muscular and ligamentous supports acting in concert during various activities. (normal) to 5 (severe degeneration), based on gross pathologic morphology of midsagittal sections of the lumbar spine. In conclusion the intervertebral disc can be thought as a soft pad that separates the vertebrae of the spine from one another. - Vacuum disc: a disc with imaging findings characteristic of gas (predominantly nitrogen) in the disc space, usually a ma Its functions are mainly three: it acts as a ligament by holding the vertebrae together; it acts as a shock absorber to carry axial nifestation of disc degeneration. load; and finally it acts as a pivot which allows spine bending, spine rotation and spine twisting.When functioning properly, the nucleus is a good example of what is called a closed hydraulic system, for the restrained water of the nucleus (by the MORPHOLOGICAL AND FUNCTIONAL CONSIDERATIONS annulus and endplates) becomes incompressible to the axial load and will in turn transfers these forces downward to the lower vertebra and set up the pivot-point. When this structure is damaged then the vicius circle starts. 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22 23 78) Altun I. Cytokine profile in degenerated painful intervertebral disc: variability with respect to duration of symptoms and MOLECULAR ASPECTS AND NEW BIOCHEMICAL PATHWAYS type of disease. Spine J. 2016 Jul;16(7):857-61. doi: 10.1016/j.spinee.2016.03.019. Epub 2016 Mar 11. UNDERLYING OZONE EFFECTS IN NEURO-ORTHOPAEDICAL 79) Fardon DF, Williams AL, Dohring EJ, Murtagh FR, Gabriel Rothman SL, Sze GK. Lumbar disc nomenclature: version 2.0: APPLICATIONS OF OZONE Recommendations of the combined task forces of the North American Spine Society, the American Society of Spine Radiology and the American Society of Neuroradiology. Spine J. 2014 Nov 1;14(11):2525-45. doi:10.1016/j.spinee.2014.04.022. Lamberto Re Epub 2014 Apr 24. MD, PhD in Pharmacological and Toxicological Sciences Former Professor of Pharmacology at the University of Ancona, Italy 80) Kozaci LD, Guner A, Oktay G, Guner G. Alterations in biochemical components of extracellular matrix in intervertebral disc Scientific Director of Medinat Clinic, Camerano, Italy herniation: role of MMP-2 and TIMP-2 in type II collagen loss. Cell Biochem Funct. 2006 Sep-Oct;24(5):431-6. In recent years emphasis and attention have been focused on the use of medical ozone. 81) Maataoui A, Vogl TJ, Khan MF. Magnetic resonance imaging-based interpretation of degenerative changes in the lower Despite ample clarification (3) confusion still persists concerning its potential toxicity as strong oxidant. lumbar segments and therapeutic consequences. World J Radiol. 2015 Aug 28;7(8):194-7. doi: 10.4329/wjr.v7.i8.194. This confusion is a major factor preventing a more widespread acceptance, despite its use in the medical field as a safety 82) Krock E, Currie JB, Weber MH, Ouellet JA, Stone LS, Rosenzweig DH, Haglund L. Nerve Growth Factor Is Regulated by Toll- remedy has been reported since the XIX century (9). Like Receptor 2 in Human Intervertebral Discs. J Biol Chem. 2016 Feb 12;291(7):3541-51. doi: 10.1074/jbc.M115.675900. Furthermore the use in specialties so different like neurology, orthopedics, internal medicine, sports medicine, endocrinology Epub 2015 Dec 14. and others makes difficult the placement of ozone as a single specialist branch. This fact may cause conflict between the different fields of application and the various medical areas. 83) Kim SM, Lee SH, Lee BR, Hwang JW. Analysis of the Correlation Among Age, Disc Morphology, Positive Discography and Prognosis in Patients With Chronic Low Back Pain. Ann Rehabil Med. 2015 Jun;39(3):340-6. doi: 10.5535/ Ozone is widely described as one of the best treatment for disc inter-vertebral pathologies (1). Regarding this application, we arm.2015.39.3.340. Epub 2015 Jun 30. again remember that the ozone effects are mainly due to its peculiar effects on the bio-humoral environment. Behind this effect, in recent years attention has been focused on the use of medical ozone and on its rationale in pain management (14; 84) Hadjipavlou AG1, Tzermiadianos MN, Bogduk N, Zindrick MR. The pathophysiology of disc degeneration: a critical review. 7; 16). Furthermore, it has been reported its action as an immune modulator and activator of cellular metabolism which shows J Bone Joint Surg Br. 2008 Oct;90(10):1261-70. doi: 10.1302/0301-620X.90B10.20910. long-term anti-inflammatory effects and reduces inflammation with an apparent low toxicity (6).

85) Hughes SP, Freemont AJ, Hukins DW, McGregor AH, Roberts S. The pathogenesis of degeneration of the intervertebral disc Ozone therapy increases the endogenous antioxidant system activities in endotoxic and septic shock models (23). However, and emerging therapies in the management of back pain. J Bone Joint Surg Br. 2012 Oct;94(10):1298-304. so far there is only one study aimed at identifying the possible molecular mechanisms of the anti-allodynic/hyperalgesic effect of ozone and the possible involvement of some inflammatory and apoptotic pathways (8). 86) Alexandre A, Alexandre AM. Minimally invasive treatments for spinal degenerative pathologies. Essential Practice of Neu rosurgery. Kalangu KN, Kato Y and Dechambenoit G. Nagoya, Japan, Access Publishing Company. 2009. ISBN 978-4- Moreover, evidence that antioxidant enzymes, nitric oxide pathways and other sub-cellular activities could be modulated by low 904992-01-2. Section VI, Chapter 13: 903-38 ozone doses is now proven and could support the surprising effects of ozone in many pathological conditions (2; 11; 4)

Regarding the pharmacological characterization of the ozone molecule, at the light of the more recent knowledge we can consider ozone as a pro-drug which, at certain non-toxic doses, can induce a rearrangement of the biochemical pathways with the activation of a second messenger in a cascade with a multiple system action. Ischemic preconditioning represents the best similarity in this context. Noteworthy, the report of Wentworth et al. (22). Indeed, their scientific data demonstrate the physiological presence of an ozone-similar mediator during inflammation, indicating ozone as a new bio-molecule with striking effects which must be considered and studied following new strategies with newly constructed randomized-standardized clinical studies.

In addition, a large number of inflammatory and signaling substances, such as tumor necrosis factor and interleukins (interleukin-1beta, interleukin-6, and interleukin-8) (5; 13) could be involved explaining the ozone action.

Looking at the more recent scientific discoveries, allow us to introduce a new terminology regarding the pharmacological mechanism of action characterizing the treatment with ozone. Indeed, being quite different from a drug, its action can’t be explained as a simple interaction between the molecule (drug) and the receptor (cellular membrane protein) - according to classical schemes of pharmacology currently spread in the faculties of medicine - but rather as “Hormetic Stress”.

Our scientific conviction that a molecule like ozone, i.e. a strong oxidant, could induce benefits in various ailments, when used at low doses was believed to be an unconventional theory. We are now very happy to note how this concept could finally gain a certain scientific credibility. Indeed, in a recent paper the Nobelist Dr. James Watson proposed an unconventional view on oxidative stress and diabetes. The conventional view is that oxidative stress causes insulin resistance.

24 25 In March 2014, Dr. James Watson, who co-discovered the double helix structure of DNA, proposed an unconventional view There is no doubt that this complexity makes us understand why it is not easy set up clinical trials demonstrating definitively on the cause of diabetes. (20). “The fundamental cause, I suggest, is a lack of biological oxidants, not an excess,” he says. the therapeutic power of these agents. On the other hand, any drug interaction, due to its direct bind with a receptor and “Physical exercise prompts the body to make large numbers of oxidants - molecules called reactive oxygen species, or ROS,” consequent biological function activated, is easily measurable and statistically standardized. In short, the stimulus of he continues, and that’s why exercise is beneficial to our bodies. This hypothesis certainly needs to be tested. It could be that Oxidative Stress, in the case of ozone, is able to activate Nrf2 protein (15) which, moving in the nucleus, starts production from it’s the balance that matters, and that disease results due to an imbalance on either side; i.e. both oxidative stress or oxidative part of Target Genes of proteins that promote cell functions (Fig. 1), strengthening the defenses, and optimizing the underlying deficiency (such as hypoxia), could lead to insulin resistance and other diseases. The idea that similar to brief exercise, the specific function. conditioning effects induced by small ozone doses, could be helpful for many of the biological functions of all the cells that burn oxygen to produce energy seems to be worthy of new and strong scientific studies dedicated to the matter.

Anyway, one of the issues raised by the scientific community is: how Ozone really acts on Humans? As already stated, ozone is quite different from a drug and its action is not a consequence of a binding reaction between one molecule (drug) and one receptor (cellular membrane protein). The fact makes more difficult the efforts of scientists in the evaluation of the molecular events underlying its clinical activity. For the above reasons the ozone action can’t be considered according to classical schemes of pharmacology and new concepts must be defined.

To our opinion, reactions induced by stress mechanisms require the introduction of a third parameter in addition to the DOSE and the EFFECT: i.e. the TIME. In fact, differently from what happens for a conventional drug that acts on a specific target with an immediate action, stressing agents promote several biological effects through a myriad of interactions that involve many cellular processes and metabolic pathways which in turn produce a stable clinical effect only after a certain time.

Like other Xenobiotics, agents not recognized in the metabolism of the body (from the Greek Xenos = Foreign and Bios = Life), such as heat, mechanical trauma, ionizing radiation or the same foods that we eat daily, even “ozone” molecule is able to influence the cellular functions. Indeed, following the stress, cells promote protection mechanisms that defend from the specific damage induced by the same stressing agent. The term xenobiotic has been introduced only recently (12), and the In our study, executed with Major Auto-Hemotherapy on healthy volunteers we demonstrated the involvement of Nrf2 details are still lacking of adequate scientific support regarding the involved mechanisms. As is obvious, this is reflected protein in vivo. Indeed, the levels of Nrf2 in peripheral blood mononuclear cells were found to increase immediately after ozone negatively in regards to ozone therapy too, and makes understandable, but not reasonable, the lack of attention of Health exposure (P<0.01). This effect was still detected (P<0.05) in total circulating PBMC when measured 30 min following Authorities devoted to the control of human health. reinfusion demonstrating that the oxidative stress was able to activate all the blood components. After a series of 3 MAH, Nrf2 returned back to the basal level. At the end of the experiment the activities of superoxide dismutase and catalase were So the studies to date are mostly directed to the characterization of the Mode of Action of the Stress Response (MOA), increased (P<0.05). These data demonstrate for the first time in vivo the activation of the Nrf2 pathway by a low dose of ozone understood as the definition of individual metabolic pathways activated at the cellular level by the various Xenobiotics. Ozone, followed by the promotion of the feedback mechanism that induces the synthesis of proteins which collectively favors cell like other agents, and unlike the common drugs that act on a specific receptor, induces small stress to the whole cell when survival (18). used at low doses. This, in turn, triggers a series of intracellular metabolic processes and promotes a myriad of intracellular activities. As a consequence of these reactions, the defense mechanisms of the cell are alarmed and enhanced to improve We can now better understand why we usually observe so different beneficial effects after ozone therapy. Indeed, this functionality, explaining in part the surprising therapeutic actions of this gas. A recent study published in the prestigious metabolic pathway is common to all the cell lines. So, we will see an aesthetic effect if the conditioned cell is part of the skin Toxicological Sciences (19) has fully explained the biochemical mechanism with its intracellular mediators (Transducers, tissue or a modulation action on inflammation if the cell is part of neuromuscular or bone tissues (Fig. 2). Sensors and Transfer Factor), where for each type of xenobiotic is possible to monitor the intracellular metabolic pathways (Table).

26 27 Taking into account these last papers, we can conclude that ozone could be very helpful as integrative and complementary 8. Fuccio C, Luongo C, Capodanno P, Giordano C, Scafuro MA, Siniscalco D, Lettieri B, Rossi F, Maione S, Berrino L (2009) A support for pharmacological therapy modulating the oxidative stress component in many illnesses. single subcutaneous injection of ozone prevents allodynia and decreases the over-expression of pro-inflammatory caspases in the orbitofrontal cortex of neuropathic mice. Eur J Pharmacol 603 (:42-49; Furthermore it could be emphasized its use in the elderly, where side effects and therapeutic costs are going to be even often a serious problem for the Health Authorities and for the medical care personnel (10). 9. Jacobs M (1986) Th-Untersusuchung uber zwischerfdlle und typische komplikationen in der Ozone Sauerstoff therapie Considering all the above, we can propose ozone therapy as a useful resource to complement and integrate the Ozonachrichten 5: 1-5; pharmacological approach actually utilized both for the most common symptoms and for rare diseases, still orphan of proper medical treatment (17). 10. Laroche ML, Charmes JP, Nouaille Y, Picard N, Merle L. (2007) Is inappropriate medication use a major cause of adverse drug reactions in the elderly? Br J Clin Pharmacol 63(2): 177-186; We believe that it is time for the scientific community to launch a serious assessment of ozone therapy that can rightfully be considered a strategic allied of the orthodox medicine, especially in the case of rare diseases still orphan of adequate drug 11. Martinez-Sanchez G, Al-Dalain SM, Menendez S, Re L, Giuliani A, Candelario-Jalil E, Álvarez H, Fernández-Montequín JI, treatment. A key factor of no small importance is also represented by the absence of important side effects, the incidence León OS. (2005) Therapeutic efficacy of ozone in patients with diabetic foot. Eur J Pharmacol 523(1-3):151-61; of which, considering the millions of patients submitted to this therapy all around the world over the last 40 years, it is represented by a number after the decimal point preceded by at least 5 zeros!!! 12. Mason HS, North JC, Vanneste M. Fed Proc. (1965) Sep-Oct, 24 (5): 1172-1180; Moreover, in the aim to defense this medical therapy that can play a primary role in the prevention and treatment of the elderly population, a careful assessment of a legal regulation could no further be delayed. Indeed, the proper education and training of 13. Miyamoto H, Saura S, T TH. (2000) The role of cyclooxygenase-2 and inflammatory cytokines in pain induction of herniated the medical and paramedical staff who will administer the therapy according to the most recent clinical indications approved lumbar intervertebral disc. Kobe J Med Sci 46 (1-2):13-28; by the scientific societies must be fairly ruled by the Health Authorities worldwide. 14. Paoloni M, Di Sante L, Cacchio A, Apuzzo D, Marotta S, Razzano M, Franzini M, Santilli V. (2009) Intramuscular oxygen- Ozone therapy, like other similar holistic approaches, could be included in the newly branch of Human Enhancement. This is ozone therapy in the treatment of acute back pain with lumbar disc herniation: a multicenter, randomized, double-blind, one of the reasons that prompted some experts to establish a non-profit medical society for “scientific research, bioethical clinical trial of active and simulated lumbar paravertebral injection. Spine 34(13):1337-44.; analysis and the enhancement of human health”. Due to the increasingly long-lived population, the aim will be also to broaden the knowledge in the field of integrated and natural therapies in the aim of preventing aging and most of the pain pathologies 15. Pecorelli A, Bocci V, Acquaviva A, Belmonte G, Gardi C, Virgili F, Ciccoli L, Valacchi G (2013) Toxicol Appl Pharmacol. 267(1): of the elderly. 30-40;

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28 29 THE LOW-DOSE OZONE CONCEPT: Chronic oxidative stress (pathologically increased values for malone dialdehyde MDA, hydrogen peroxide H2O2, total hydroperoxide TH etc.) and an antioxidant deficit (disbalanced superoxide dismutase and subgroups of SOD as well as GUIDELINES AND CLASSIFICATION ACCORDING TO catalase CAT and others) are phenomena common to all the mentioned diseases. It is here in particular that low-dose ozone EVIDENCE- BASED MEDICINE EBM. as a hormetic substance, shows a regulatory effect on pathological processes through regulation of antioxidants (upregulation in chronic, downregulation in acute inflammation) and downregulation of oxidative stress as a consequence. R. Vieban, Medical Society for the Use of Ozone in Prevention and Therapy, Iffezheim/Baden-Baden, D-76473, Germany Recommended concentration- and dosage ranges The Low-Dose Ozone-Concept with its moderate oxidative stress represents an ideal hormesis strategy (Rattan 2008). Dose SUMMARY response and concentration-effect relationship in the context of specific applications allow the setting of concentration ranges Background with therapeutical benefit, see table 1. On the well known basis of the mechanisms of action and pharmacology of medical ozone and the mostly standardized applications, the low-dose ozone concept is well established as a complementary medical method in the treatment of chronic Mechanism of action, biochemical and pharmacological effects of ozone underlying an effective ozone therapy inflammations or diseases associated with chronic inflammatory conditions. International guidelines have been defined in The formation of „ozone peroxides“ assume the role of physiologically active ozone metabolites: Due to the selective reaction the context of physiological and ozone resistant materials, indications, applications and the effective concentration and behavior of ozone, the 1,3-dipolar (electrophilic) addition to the isolated C=C double bonds of essential fatty acids according dosage range depending on the specific indications, reference substances to follow the treatment success are recommended. to classical ozonolysis as described by Crigée (1953) is the dominant reaction under physiological conditions with pH values Method and Findings With these prerequirements a literature search in international, data base listed journals and a ≤ 7,4 (5). classification of the clinical studies concerning the criteria of evidence-baced medicine have been performed for major autohemotherapy MAH and rectal ozone insufflation RI. When summarizing the evidence classification under RCT + CT Ozonolysis only takes fractions of a second, preferably forming short-chain hydroxy-hydroperoxides in whole blood; here (Randomized Controlled Trials + Controlled Trials), i.e. levels Ib and IIa, 12 studies with 657 ozone-treated patients were simply designated as „ozone peroxides“, less active than ozone itself but still with a short half-ife (contrary to the long chain obtained for MAH and 6 studies with 227 patients for RI. peroxides, formed from polyunsaturated fatty acids and oxygen or oxygen radicals, with a remarkable half life and responsible for the oxidative stress e.g. in chronic inflammatory conditions). Conclusion With its low tendency to radical reactions, the „ozone peroxide“ is able to initiate the regulation of antioxidant protective The evidence here assessed, justifies the two systemic ozone applications, MAH and RI beeing a constituent part of mechanisms as redox signal e.g. via the nuclear factors NFkB, in stress and inflammation processes via Nrf2 (8,9,13,14,16,18). evidence-based medicine. Both applications are effective, safe and economic, are in compliance with hygiene regulations and Figure 1 gives a schematic survey. meet the requirements for components: ozone resistent and biocompatible materials. H²O² formed from „ozone peroxides“, as proposed by other authors (2), will probably not fulfil this function, as the H²O² ontent is as a rule pathologically increased in the patients concerned, the corresponding signal transduction is insufficient and INTRODUCTION there is a deficit in antioxidant enzymes.

When used in specific diseases and conditions, medical ozone produces the same or similar therapy results worldwide. Reference substances Improper application in the form of erratic methods and doses is the most frequent cause of ineffectiveness and adverse A large number of cell and animal models as well as the clinical studies contributing to the pharmacological understanding of effects - and always the cause of violent controversies. For this reason, guidelines have been set up and published as a result medical ozone, show ozone-specific, reliable and reproducible parameters as suitable reference substances in a day-to-day of the most recent research and 30 years of experience (1,19). On this basis a remarkable number of clinical trials have been clinic: published and can now get evaluated and classified according to an EBM clasification system. Parameters representing the oxidative stress: TH total hydroperoxides, MDA malondialdehyde, NO nitrogen oxide... PREREQUIREMENTS FOR THE EBM CLASSIFICATION. Parameters representing the antioxidant status: Total SOD superoxidedismutase, GSH reduced Glutathion Indications and applications Principally, ozone is applied complementary to a corresponding basic therapy in chronic inflammatory conditions or Critical substances demonstrating an effective immunomodulation: Interleukine IL-1, IL-6, TNFα Tumornecrosisfactor-α diseases associated with inflammation: One or the other reference substance is to be given preference as indication demands; an example for this is found with NO where inflammatory vascular disease is concerned. In animal studies, the bioregulative effect of low-dosed ozone in the Major Autohaemotherapy and Rectal insufflation organs can be directly monitored on site, particularly in liver, kidney and spleen (15). Diabetes, Type 2 diabetes, chronic inflammatory diseases such as inflammatory vascular disease, in particular diabetic angiopathia, chronic hepatitis forms and chronic intestinal conditions belong to the classic indications within the low-dose METHOD AND RESULTS OF OZONE THERAPY CLASSIFICATION ACCORDINGTO EB ozone concept. Rheumatoid arthritis has proven to be a typical indication for a complementary ozone treatment system (6,10). Topical ozone treatment in herniated discs, a metaanalysis Topical: transcutanous ozone immersion „ozone gasbath“ and ozonized water 12 studies with 8,000 patients had been included and evaluated acc. to the classification of the Oxford Institute for External ulcers, infected burns, local infections (herpes, mycosis), eye injuries and infections Topical injections Pain Evidence-Based Medicine by Steppan et al. 2010 with the conclusion: the ozone treatment of herniated discs is an effective management, trigger points, acupuncture points, intraarticular, intradiscal, periarticular applications and related conditions. and extremly safe procedure (17).

Contraindications Major Autohaemotherapy MAH and Rectal Ozone Insufflation RI MAH: G-6-PDH deficiency (favism, acute hemolytic anemia), hyperthyroidism if not controlled, the first 3 months of pregnancy. As accepted by the criteria for EBM according to Oxford 2009 (11) and Cochrane 2015 (4) a comprehensive literature search MAH is not indicated in leucemia. and evidence assessment of the systemic ozone applications, Major Autohaemotherapy MAH and Rectal Ozone Insufflation

30 31 RI, has been carried out (20), see Figure 2. According to strict exclusion and inclusion criteria, quality assessment has been 14. Re L, Martínez-Sanchez G, Bordicchia M, Malcangi G, Pocognoli A, Morales-Segura MA, Rothchild J, Rojas A (2014) Is performed in 21 data sets for MAH and in 13 for RI and assigned according to the EBM classification. ozone preconditioning effect linked to Nrf2/EpRE activation pathway in vivo? A preliminary result. Eur J Pharm 742: 158-162

In the case of MAH, the majority of the clinical studies refer to evidence level Ib and IIa, 12 controlled clinical studies (CT), 15. Safwat M, El-Sawalhi, M, Mawsouf, Nabil Shaheen A (2015) The role of ozone in ameliorating oxidative stress associated 2 of them randomized (RCT), comprising 357 ozone patients; for rectal administration, 6 controlled (Ib and IIa) studies, 2 with ageing in rat liver, kidney and blood Paper presented at the 22nd Ozone World Congress of the International Ozone randomized, with 227 ozone patients, 6 studies without a control group comprising 484 ozone patients and evidence level IIb. All studies show statistically significant clinical and/or pharmacological improvements, without adverse effects or adverse Association, Barcelona, Spain, June 28 – July 3. reactions. and both therapy forms were assessed as being effective and safe in application: MAH in 577 patients of every age at ≥ 11,207 applications; RI in 716 patients and ≥ 46,984 applications. 16. Sagai M. Bocci V (2011) Mechanisms of action involved in ozone therapy: is healing induced via a mild oxidative stress? Hygiene standards and regulations when working with blood, are strictly adhered to, all used materials must be ozone- Med Gas Res 1: 1-29 resistant and biocompatible and follow the Medical Device Directives MDD 93/42 EEC (in Europe); studies cited in two references clearly point this out (3,7). When summarizing the evidence classification under RCT and CT, i.e. levels Ib and IIa, 12 studies with 357 patients are 17. Steppan J, Meaders T, Muto M, Murphy K (2010) A metaanalysis of the effectiveness and safety of ozone treatments for obtained for MAH, 6 studies with 227 patients for RI. herniated lumbar discs. J Vasc Interv Radiol 21: 534-548

CONCLUSION 18. Viebahn-Hänsler R (2006) Ozon-Peroxide” als Second-Messenger Moleküle. Zellprotektiver Mechanismus durch Regulation der zellulären Antioxidantien und Radikalfänger”. In: Viebahn-Hänsler R, Knoch HG (eds) Ozon-Handbuch, As a result of this evidence assessment, the systemic ozone applications, Major Autohaemotherapy and Rectal Insufflation are Ecomed-Publisher Landsberg, Germany evidence-based and thus part of Evidence-Based Medicine. 19. Viebahn-Hänsler R, León Fernández OS, Fahmy Z (2012) Ozone in medicine: The low-dose ozone concept. Guidelines and REFERENCES treatment strategies. Ozone: Sci. Eng. 34: 408-424 1. Beck EG, Wasser G.H, Viebahn-Hänsler R (1998) Ozontherapie in Wissenschaft und Empirie Z.Komplement Med 5: 61-75 20. Viebahn-Hänsler R, León Fernández OS, Fahmy Z (2016) Ozone in Medicine: Clinical evaluation and evidence 2. Bocci V, Zanardi I, Travagli V (2011) Oxygen/Ozone as a Medical gas mixture. A Critical evaluation. Med Gas Res 1: 6 classification of the systemic ozone applications, Major Autohemotherapy and Rectal Insufflation, according to the Requirements for Evidence-Based Medicine. Currently being printed in Ozone: Sci. Eng. 38 3. Ciborowski M, Lipska A, Godzien J, Ferrarini A, Korsak J, Radziwon P, Tomasiak M, Barbas C (2012) Combination of LC-MS- and GC-MS-based metabolomics to study the effect of ozonated autohemotherapy on human blood. J Proteome Renate Viebahn-Hänsler* , Olga Sonia León Fernández**, Ziad Fahmy* Res 11:6231-41! * Medical Society for the Use of Ozone in Prevention and Therapy, D-76473 Iffezheim, Germany 4. Cochrane Database and EBM classification 2009 http://www.cochrane.de ** Pharmacy and Food Institute, University of Havana. San Lázaro y L, Havana, Cuba 5. Criegee R (1953) Die Ozonolyse. Liebigs Annalen der Chemie 538:9 Correspondence should be addressed to: Dr. Renate Viebahn-Haensler, Nordring 8, 6. Dranguet Vaillant J, Fraga A, Diaz MT, Mallok A, Viebahn-Hänsler R, Fahmy Z, Barberá A, Delgado L, Menéndez S, Leon D-6473 Iffezheim/Baden-Baden E-mail: [email protected] Fernandez OS (2013) Ozone oxidative postconditioning ameliorates joint damage and decreases pro-inflammatory cytokine levels and oxidative stress in PG/PS-induced arthritis in rats. Eur J Pharm 714: 318-324

7. Faustini A, Capobianchi MR, Martinelli M, Abbate I, Cappiello G, Perucci CA (2005) A cluster of hepatitis C virus infections associated with ozone-enriched transfusion of autologous blood in Rome, Italy. Infection Control and Hospital Epidemiology APPLICATION OZONE CONCENTRATION OZONE VOLUME DOSAGE / OZONE 26: 762-767 RANGE AMOUNT PER TREATMENT SYSTEMIC TREATMENT 8. Gough, NR (2009) The Long and Short of Redox Signaling. Sci Signal 2:12 Major 10 - 30 μg/mL 50 mL 500 – 1,500 μg Autohemotherapy MAH max. 40 μg/mL) max. 2,000 μg 9. Léon OS, Menendéz S, Merino N, Castillo R, Sam S, Pérez L, Cruz, E, Bocci V (1998) Ozone Oxidative Preconditioning: a Rectal Insufflation 10 – 25 μg/mL max. 300 mL 3,000 – 7,500 μg protection against Cellular Damage by Free Radicals. Med Inflamm 7: 289-294 Minor Autohemotherapy 10 – 20 μg/mL 10 mL / 2-3 mL blood 100 – 200 μg (intramuscular) 10. León Fernández OS (2015) Targets of medical ozone in rheumatoid arthritis. How do they impact in the clinical scenery? Paper presented at the 22nd Ozone World Congress of the International Ozone Association, Barcelona, Spain, June 28 - TOPICAL TREATMENT July 3. Wound cleansing 80 – 100 μg/mL Wound healing 10 – 25 μg/mL 11. Oxford 2009. Oxford Center for Evidence Based Medicine: http://www.cebm.net Injections in pain syndrome 1 – 10 μg/mL 1 mL – 20 mL 1 mL – 20 mL In combination with local ane- 10 – 20 μg/mL 1 – 200 μg 10 - 400 μg 12. Rattan S et al (2008) Hormesis in Aging. Ageing Res Rev 7: 63-78 sthetics 13. Pecorelli A, Bocci V, Acquaviva A, Belmonte G, Gardi C, Virgili F, Ciccoli L, Valacchi G (2014) Nrf2 activation is involved in ozonated human serum upregulation of HO-1 in endothelial cells. Toxic Appl Pharm 267: 30-40 Table 1. Application-relevant concentrations and dosage ranges in ozone therapy

32 33 LUMBAR DISC HERNIATION: OUTCOMES OF TREATEMENT EITHER BY OPEN MICRODISCECTOMY, OR BY INTRADISCAL OZONE INJECTION

A. Alexandre, L.Corò, H. Baddredine, S. John, M. De Pretto, M. Brunori, A. Albarreal* *Clinica de Fatima, Sevilla , Spain

EU.N.I. European Neurosurgical Institute, Treviso, Bologna, Roma, Italy *

SUMMARY

Disc herniation with radiculopathy and chronic discogenic pain are the result of degenerative processes. Treatment approach in face of this problem has largely been debated in the last years. A number of reviews on surgical treatments in the ’80s and ’90s have been published and various new techniques have been introduced among which ozone discolysis is one non- invasive intradiscal treatment method. In a 3-year follow-up period we have investigated the different outcomes of 150 patients who received microdiscectomy and 150 patients who received intradiscal ozone injection. In this series results are in favour of discolysis for contained disc herniations and of microdiscectomy for large migrated fragments with pain so severe that open surgery was mandatory. Apart from this, our results with the two techniques are equivalent also in face of mild neurological motor deficits.

INTRODUCTION Figure 1. Pharmacological effects of ozone: signal tranduction via glutathione or cysteine oxidation by „ozone peroxides“, information of nuclear factors and finally regulation of the response proteins such as antioxidants or cytokines. Lumbar disc degeneration occurs because of a variety of factors and results in a multitude of conditions and of clinical alterations. Structural modifications in the vertebral endplate cause derangement of nucleus nutrition and disc degeneration. Aging, apoptosis, abnormalities in collagen, vascular ingrowths, loads placed on the disc, and abnormal proteoglycan all contribute to disc degeneration. Disc herniation with radiculopathy and chronic discogenic pain are the result of this degenerative process. The treatment approach in face of lumbar disc herniations has largely been debated in the last years. A number of reviews on surgical treatments in the ’80s and ’90s have been published and various new techniques have been introduced. Criticism has grown regarding long term results of discectomy and a large number of neurosurgeons focused their attention on minimally invasive percutaneous intradiscal procedures. Ozone discolysis is one non invasive intradiscal treatment method which has shown to be effective and safe for these problems [1–3]. On the basis of these considerations we investigated in a 6 year follow-up period the different outcomes of 550 patients who received microdiscectomy and 550 patients who received intradiscal ozone injection. The aim was to find out whether injection of this substance in the disc is effective and useful.

PATIENTS AND METHODS

This analysis is based on the series patients affected by lumbar disc pathology coming to EUNI in Italy, and patients treated by the spanish group leaded by Dr A. Albarreal, in Spain.

Patients have been treated either by microdiscectomy or by percutaneous O2-O3 discolysis. Figure 2. Results of data search and evaluatioMAH: Major Auto Hemotherapy, RI: Rectal Insufflation RCT: randomized, controlled trial, CT: controlled trial We selected 550 patients in each group with most similar characteristics. Only patients affected by lumbar disc herniation at just one level were included in this study. Patient distribution according to the level of pathology is shown on Table 1.

Kind and site of herniation in both groups are found in Tables 2 and 3.

All patients were examined by plain lumbosacral X-rays, EMG, lumbo-sacral CT/MRI before surgery, and 6 months, 1 year and 3 years after surgery. Clinical data were collected preoperatively and postoperatively at the above time intervals and were lassified according to the Roland Morris Scale; evaluation of pain was done according to the VAS Scale.

34 35 Table 1. Patient distribution based on level Table 7. Regression of sensory dysfunction at 3 years Microdiscectomy cases Discolysis cases Microdiscectomy Discolysis L1-L2 0.6% 1.3% Complete 82.5% 80.4% L2-L3 2% 1.3% Partial 12.4% 11.9% L3-L4 10% 12% Insignificant 5.15% 7.6% L4-L5 52.6% 53.3% L5-S1 34.6% 30% Table 8. Regression of motor deficit after 3 years RESULTS Microdiscectomy Discolysis Complete 86.6% 85.7% Results obtained for pain are presented in Table 4. Pain regression in respect to the kind of the herniation is analyzed in Table Partial 9.8% 7.4% 6. Regression of sensory dysfunction at 3 years is shown in Table 7. As shown in Table 8, 9 and 10 motor deficit improvement Insignificant 3.6% 7.1% is similar in the two groups among which we didn’t find significant differences, not even in EMG recovery. Morphological results are obviously different in the two series. After discolysis total elimination of herniation was observed in a low percentage in the first months (44.6%) and grew relevant Table 9. Regression of an initially severe motor deficit after 3 years after one year (57.3%) or after two years (68%). Microdiscectomy Discolysis Complete 44.4% 40% Table 2. Kind and type of herniation in Patients treated by microdiscectomy Partial 22.2% 20% Insignificant 33.3% (40%) Total 9 5 Kind of herniation Site of herniation Contained 65.3% posterior median 10.6% Extruded 31.3% posterior paramedian 32.6% In this series results were in favour of discolysis for contained disc herniations and of microdiscectomy for large migrated posterior median-param. 21.3% ragments, for which pain was so severe that open surgery had become a must. Apart from that, the results we obtained with Migrated 3.3% posterolateral 27.3% the two techniques were equivalent also with regard to mild neurological motor deficit. intraforaminal 8% COMMENT AND CONCLUSIONS Table 3. Kind and type of herniation in Patients treated by 0203 discolysis Kind of herniation Site of herniation Greenfield and coauthors [4] reported on the 1-year outcome for 80 patients who were randomly assigned to surgical or non-surgical groups for treatment of single-level lumbar disc herniations. All patients complained of back and radiating leg Contained 68% posterior median 9.3% pain. Patients with major neurologic deficits and incapacitating pain were excluded from his study. Extruded 29.3% posterior paramedian 35.3% posterior median-param. 24% Patients in the surgical group had a microdiscectomy, and the non-surgical group was treated conservatively with Migrated 26% posterolateral 22.6% intraforaminal 8.6% aerobic exercise and education. Measurement tools to evaluate outcomes included visual analogic scales for pain and Oswestry Disability Index for disability. Patients treated with microdiscectomy had a greater reduction in both back and leg pain and had greater improvements in their Oswestry Disability Index scores at all study intervals. This prospective study Table 4. Pain regression at controls established the efficacy of microdiscectomy in the first 12 months for treatment of patients with small-to-moderate lumbar disc Controls Microdiscectomy Discolysis herniations and moderate complaints of back and leg pain. Other non-randomized studies demonstrated the benefits of 92.6% non-surgical treatment for lumbar disc herniations at a 10-year follow-up: long-term outcomes approach those of surgery, 4–6 months 98% and some authors stress that the early benefits of surgery in the first 12 months may outweigh the risks of surgery. Our 1 year 91.3% 87.3% 3 year 85.3% 79.3% results show that, after time, discolysis lead to an evolving situation in respect of morphology, since total elimination of herniation was observed modest in percentage in the first months (44.6%) and grew relevant after one year (57.3%) or after two years (68%), but this doesn’t correlate with the clinical course. This is probably due to the fact that progress of disc Table 6. Pain regression related to kind of herniation degeneration entails a loss in volume within the nucleus pulposus due to a decrease in proteoglycan and water concentration [5]. Kind of herniation Microdiscectomy Discolysis Percutaneous intradiscal entry is a technique which gives minimal disruption of the anular structure, and will thereforeproduce minimal epidural scarring [6]. This is confirmed by the images we obtained one year after treatment in the two Contained 82.6% 68% groups of patients. It is too early yet to comment on recurrences within this series, but we think that the entity of fibrotic tissue Extruded 89.3% 77.2% will play a role in clinical significance. Regression of pain and of motor dysfunction three years after surgery is quite similar in Migrated 100% 0% Intraforaminal 9.6% 84.6% the two groups. In severe pain cases who presented with a large disc herniation open surgery o¤ered an advantage in the short term, but after time there is no significant di¤erence between the techniques employed for treating the problem. Discolysis is

36 37 a very simple method to practice and is safe. It can be employed also in elderly patients without danger. In consideration of “NOT JUST HERNIATED DISC” BACK PAIN: the long term results, the technique is a practical alternative for those cases in which surgery can be avoided and a solution can be o¤ered without fear of future complications such as scarring and peridural fibrosis. OUTCOME OF OXYGEN-OZONE TREATMENT IN SELECTED APPLICATIONS

REFERENCES Matteo Bonetti¹, Alessio Zambello², Marco Leonardi³,4, Ciro Princiotta³ ¹Dept of Neuroradiology – Istituto Clinico Città di Brescia 1. Alexandre A, Fumo G (1998) Discolisi percutanea mediante 0203 nell’ernia discale lombare. In: Ceccherelli F, Ricciardi A ²Dept of Anesthesiology - “Fondazione Borghi” Hospital Brebbia (Varese) (eds) Lombalgie e lombosciatalgie. Criteri di diagnosi e cura. Edizioni Libreria Cortina, Torino, pp 367-377 ³Dept of Neuroradiology, IRCCS Institute of Neurological Sciences of Bologna, Bologna, Italy 4DIMES Dept University of Bologna 2. Andreula CF, Simonetti L, De Santis F, Agati R, Ricci R, Leonardi M (2003) Minimally invasive oxygen-ozone therapy for lumbar disk herniation. AJNR Am J Neuroradiol 24(5): 996-1000 SUMMARY 3. D’Erme M, Scarchilli A, Artale AM, Pasquali Lasagni M (1998) L’ozonoterapia nel dolore lombosciatalgico. Radiol Med (Torino) 95(1-2): 21-24 Low back pain and sciatica arehighly debilitating conditions affecting all socioeconomic groups at an increasingly early age. They are caused by different often concomitant spinal disorders:disc or facet joint disease, spondylolisthesis (with or 4. Greenfield K, Egger M, Nelson RJ, Findlay GF, Sanford E (2002) Microdiscectomy vs. conservative treatment for lumbar disc withoutlisthesis), vertebral body and interapophyseal arthrosis, spinal stenosis, radicular and synovial cysts and,more rarely, herniation: a randomised clinical trial. Program and abstracts of the 17th Annual Meeting of the North American Spine infections and primary or metastatic cancer. Society; October 29–November 2, Montreal, Quebec, Canada. Abstract 1 Treatment of low back pain and/or sciatica requires an accurate diagnosis based on thorough history-taking and physical examination followed by appropriate imaging tests, namely computed tomography and/or magnetic resonance scans in 5. Guiot B, Fessler R (2000) Molecular biology of degenerative disc disease. Neuro- surgery 47: 1034-1040 addition to standard X-rays of the spine. In recent years, several reports have demonstrated the utility of oxygen-ozone therapy in reducing the size of 6. Mochida J, Tos F, Nomura T et al (2001) The risks and benefits of percutaneous nucleotomy for lumbar disc herniations: a herniated discs. The present study reports on the outcome of oxygen-ozone treatment in 416 patients with non-discogenic 10 year longitudinal study. Bone Joint Surg Br 1: 501-505 low back pain caused by degenerative disease of the posterior vertebral compartment(facet synovitis, Baastrup syndrome, spondylolysisandspondylolisthesis, facet degeneration). 7. Paradiso R., Alexandre A.: The different outcomes of patients with disc herniation treated either by microdiscectomy, or by intradiscal ozone injection Acta Neurochir (2005) [Suppl] 92: 139-142 INTRODUCTION

Oxygen-ozone therapy was first used to treat herniated discs in 1985 (1,3-5,7,8,12-26, 28,33). Since then, many literature studies have reported positive outcomesranging from 75% to almost 90% in patient cohorts treated for low back pain with or without sciatica due tonerve root compression caused by a herniated intervertebral disc (1,3-5,7,8,12-26,28-30,32,33). Low back pain and sciatica are highly debilitating conditions affecting all socioeconomic groups at an increasingly early age. Onset may be acute following injury or an unusual movement, or slowly progressive with gradually worsening pain. They are caused by different often concomitant spinal disorders: disc or facet joint disease, spondylolysis (with or without listhesis), vertebral body and interapophyseal arthrosis, spinal stenosis, radicular and synovial cysts, and, more rarely, infections and primary or metastic cancer. (31). Treatment of low back pain and/or sciatica requires an accurate diagnosis based on thorough history-taking and physical examination followed by appropriate imaging tests, namely computed tomography (CT) and/or magnetic resonance (MR) scans in addition to standard X-rays of the spine). The present study reports on the outcome of oxygen-ozone treatment in 416 selected patients with low back pain and/or sciatica due to causes other than disc herniation or protrusion. We focused ondegenerative disease of the posterior vertebral compartmentas a possible source of pain.

1) FACET SYNOVITIS Facet synovitis is an inflammatory disease of the synovial membrane lining the spinal interapophyseal joints, usually caused by injury or recurrent microtraumas over time. The disease may also arise in young adults following excessive stress on the spine, typically induced by extreme sports. In most cases, onset consists of acute lumbar pain with monolateral symptoms if only one joint is affected or bilateral low back pain in a band-like distribution if both facet joints are involved (6).Our cohort included only patients with post-traumatic conditions, excluding infectious synovitis and possible SAPHO syndrome (synovitis, acne, pustulosis, hyperostosis, osteitis) (11,27).Facet synovitis diagnosis is often challenging and spine MR scans with contrast administration are helpful to confirm the diagnosis (6,9,10). 38 39 2) BAASTRUP SYNDROME Gand 25 G. CT guidance also served to check the correct position of the needle in the joint in cases of facet synovitis and Baastrup syndromeor “kissing spines” wasfirst described by the Danish radiologist Christian Baastrupin 1933. It is degeneration, in the interspinous space in patients with Baastrup syndrome and in the root canal and isthmic lysis points to characterized by degenerative changes to the spinous processes of adjoining lumbar vertebraethat form new joints between treat spondylolysis. them, often causing low back pain refractory to conservative anti-inflammatory and analgesictreatment.Women are mostly An aseptic technique was used to fill a 10 ml polyethylenesyringe with a gas mixture of oxygen-ozone at a concentration of affected with anF:M ratio of 4:1 and the syndrome is usually diagnosed in the third decade of life. 25 μg/mland between 3 ml and 5 ml was injected depending on the condition to treat, using a microporous filter to minimize Diagnosis is radiological: spinal X-ray will depict extreme hyperlordosis of the lumbar spine with degenerative changes and the risk of contamination. Further CT scans were done after infiltration to confirm the correct distribution of the gas mixture in contact between adjacent spinous processes. the treatment site. Patients were then kept under observation for around 30 min and subsequently discharged. The disease has a progressive courses and worsening low back pain is an indication for MR study of the lumber spine. Scans should include fat saturation (Fat/Sat) sequences and possibly i.v. gadolinium administration to disclose any signs of acute FACET SYNOVITIS interspinous inflammation (9,10). Most of the 12 patients treated with oxygen-ozone therapy for facet synovitis were relatively young (75% were under 40) 3) SPONDYLOLYSIS AND SPONDYLOLISTHESIS aged between17 and 56 years. In all cases, the onset of acute lumbar pain was linked to injuryand the side of facet synovitis Spondylolysis is a bony defect of the neural arch. If the defect results in a forward shift of one vertebral body on another, this correlated with the side of the patient’s clinical symptoms. No signs of ongoing infection were present. MR scans withFat/Sat iscalled spondylolisthesis (a term coined by Kilian in 1854). The severity of spondylolys the sisisdetermined by the Meyerding sequences after paramagnetic contrast (gadolinium) administration confirmed the diagnosis (Fig.1). classification in four grades depending on the degree of vertebral body slippage over the vertebra beneath it (Grade I 0-33%, Grade II 34-66%, Grade III 67-99%, Grade IV 100%). BAASTRUP SYNDROME Grade I spondylolysthesisis often an occasional finding and most patients are asymptomatic. However a small percentage present low back pain with or without sciatica. These cases do not require surgery and treatment mainly involves physiotherapy Sixteen patients with Baastrup syndrome (11F-4M, aged range 51/78 years) were treated. In all cases, MR scans depicted and exercise. However, symptoms refractory to conservative therapy may warrant spinal stabilization surgery. inflammation in the interspinous ligament and perispinal soft tissues (Fig. 2A). Patients presented pain in the medio-inferior dorsal and lumbosacral spinewhere degenerative changeswere most evident, 4) FACET DEGENERATION and all had an associated antalgic contracture of the paraspinal muscles. Four cc of the oxygen-ozone gas mixture were Between 25 and 45% of cases presenting chronic low back pain are due to facet joint syndrome that may be flanked by other injected into the inflamed interspinous ligaments depicted on the MR scan using 23G needles followed by infiltrations into the conditions, thereby exacerbating the patient’s pain and disability. paraspinal muscles affected by antalgic contracture. Facet joint syndrome is commonly encountered in elderly patients. It has multiple causes, the most frequent being articular degenerative changes. The condition leads to a reduction or loss of joint cartilage, erosion of the adjacent bone margin, SPONDYLOLYSIS AND SPONDYLOLISTHESIS abnormal bone growth of the facet joints and articular processes, and lastly joint instability that may result in degenerative spinal subluxation (pseudospondylolisthesis).Back pain is caused by irritation to the sensory nerve endingsin the facet joints Given the widely acknowledged success of oxygen-ozone therapy for nerveroot compression caused by herniated disc,we and surrounding tissues innervated by the medial ramus of the posterior branch of the spinal nerve. administered oxygen-ozone to62 patients (19F-43M, aged range 21/58years) with Grade I spondylolysthesis (44 with L5 Candidates for interventional treatment are selected on the basis of history-taking and clinical findings supported by over S1 and 18 with L4 over L5) with bilateral isthmic lysis and associated disc disease (45 bilateral median-paramedian diagnostic imaging.Standard X-rays are accompanied by CT scans to disclose the joint relations, growth and deformation protrusions and 17 contained lateral disc herniations)and low back pain with or without sciatica. Seventeen patients also of the articular bones and narrowed joint spaces, an indirect index of cartilage rearrangement. MR scans, namely T1 and presented associated disc herniation. Diagnosis was confirmed in all cases by standard X-raywith morphodynamic (flexion- T2-weighted Fat/Sat sequenceswith paramagnetic contrast administration, are used to depict the active inflammatory process extension) tests and CT scan of the lumbosacral spine. Treatment was administered by CT-guided bilateral periganglionic involving the facet joint and surrounding tissues (6,9,10). ozone infiltration and injection of the gas mixture in close proximity to the lysis points in the neural arch (Figs 3,4). The different treatments proposed to relieve pain caused by facet joint syndrome includeintra-articular drug injections, nerve block with anesthetic and radiofrequency ablation. FACET DEGENERATION

MATERIALS AND METHODS We selected 326 patients with facet joint syndrome(185M-141F,aged between 58 and 91 years, average age 73.2years) for oxygen-ozone therapy. The clinical diagnosis was based on patient reports of pain during daily activities and following certain We selected 416 patients with different conditions involving the posterior vertebral compartment resulting in low back pain passive and activemovements designed to mobilize the facet joints. The main symptoms are listed in Table 2. All patients were with or without sciatica: 12 had facet synovitis, 16 had Baastrup syndrome, 62 had spondylolisthesis and 326 had facet treated by CT-guided injection of 2-3ccof the oxygen-ozone gas mixture into or around the facet jointusing a 22G needle. One degeneration.Diagnosis had been confirmed in all cases by MR scans on aSiemens Magnetom AERA 1.5 T system with level was treated in 184 patients (56.4%) of whom 71 (38.6%) only on the side of pain. Different levels were treated in the SYNGO MR D13 software using standard sequences followed by Fat/Sat sequences without and with contrast administration remaining142 patients. (Table 1). All patients underwent CT-guided targeted injection of an oxygen-ozone gas mixture using the deep paravertebral infiltration technique. RESULTS INFILTRATION TECHNIQUE FACET SYNOVITIS

After receiving written informed consent from the patients, the injection level was established on the basis of Eight (66.7%) of the 12 patients treated had an almost complete resolution of pain after just one oxygen-ozone infiltration so neuroradiological findings and clinical symptoms. The level was confirmed by preliminary CT scans with patients in a prone no further injections were administered. The remaining four patients (33.3%)underwent a second infiltration ten days after position to determine the point of needle entry.The skin was disinfected using a polyvinylpyrrolidone iodine solution after the first treatment. A multidisciplinary approach was adopted for this set of patients and a physiatrist devised a physiotherapy local anesthetic with ethyl chloride spray. CT-guided puncture was then performed using needles with a caliber between 22 program for each case based on postural re-education. Five patients (41.6%) also underwent associated Tecartherapy.

40 41 BAASTRUP SYNDROME CONCLUSIONS

All 16 patients treated reported a clear-cut improvement in pain one week after oxygen-ozone therapy, but all had a partial In recent years, the application of MR scans with fat saturation sequences and gadoli-niumadministration in routine return of pain six months later. A second infiltration was performed in seven cases (43.75%) again with a marked reduction diagnostic imaging has helped clinicians establish and confirm the diagnosisin patients with degenerative disease of the of pain and a decrease in perispinal contrast uptake at MR follow-up (Fig. 2B). lumbar spine and low back pain, leading to targeted treatment strategies. All patients underwenta physiotherapy and exerciseprogram to prevent relapse (in five cases the physiatrist prescribedcycles In patients with non-radicular low back pain, symptoms often stem from disease or degenerative changes in the posterior of mud balneotherapy every 6-12 months). elements of the lumbar spine (“theposterior vertebral compartment”).Appropriate patient selection for oxygen-ozone therapy and the choice of the best targeted treatment yield good clinical results in most cases. This was demonstrated by the SPONDYLOLYSIS AND SPONDYLOLISTHESIS successful treatment outcomes in between 61% and 74.5% of our cohort. The rapid resolution of pain with no complications, the ease of performing the technique andCT-guided control of infiltration All62 patientstreated by oxygen-ozone therapy were followed up at one, three and six months after treatment using a suggest oxygen-ozone administrationis a viable alternative to conservative therapies and probably the first choice treatment modifiedMacNabscale. A complete resolution of pain was obtained in 38patients(61%)after the first infiltration and clinical for disorders of the posterior vertebral compartment. In addition, oxygen-ozone therapy does not preclude subsequent wellbeing persisted throughout follow-up. The remaining24 patients (38.7%)had only a partial remission of pain and the infiltrations or surgical treatment. treatment was repeated after the first follow-up. These patients had a partial benefit from the second infiltration at the three-month follow-up after which eight patients (33.3%) underwent a third oxygen-ozone injection. FIGURES The24 patients with partial pain remission at six-month follow-up were referred for neurosurgical assessment and five (8%) underwentspinal stabilization surgery, while the remaining 19 received only rehabilitation with a physiotherapy and exercise Fig. 1 Fig. 2 program. T1 Fat/Sataxial MR (A,B) T1 Fat/Sataxial MR scan after Among the 17 patients(27.4%) with herniated disc associated with spondylolisthesis follow-up CT scan demonstrated scan after gadolinium administration: complete dehydration of the herniated disc in eight cases (47%), obviously with no change in thelisthesis grade (Fig. 5). gadolinium administration: (A) Before treatment contrast FACET DEGENERATION bilateral uptake is visible in the interspinous post-traumatic ligament and paraspinal soft facet synovitis tissues. At clinical follow-up ten days after oxygen-ozone infiltrationin the 326 treated patients, 243 (74.5%) reported a major (arrows). reduction of pain.The remaining 83 patients (25.5%) had an unsatisfactory therapeutic outcome. They declined further (B) After treatment there is a oxygen-ozone therapy and were referred to a physiatrist. marked reduction of inflammatory Of the 243 with an excellent or good initial outcome, 116 underwent clinical follow-up after three months. Of these, 83 contrast uptake in the paraspinal soft tissues. (71.5%) reported a persistent good outcome, whereas the remaining 33 (28.5%)complained of symptom recurrence and underwent a second infiltration; 26 of these cases (78.8%) again had an excellent therapeutic outcome with an almost total Fig. 3 Fig.4 resolution of pain. Intraprocedural CT (A,B): Intraprocedural spinal CT scan: DISCUSSION scan with a bone algorithm A) Correct positioning of the needle confirms the in the periganglionic region. The well-known mechanisms of action of oxygen-ozone infiltration are responsible for the excellent therapeutic outcome correct positioning obtained in our cohort of patients with facet synovitis, Baastrupsyndrome or facet degeneration. of the needle tip in B) CT-guided control of the An oxygen-ozone gas mixture injected into the site of inflammation exerts a strong anti-inflammatory and analgesic effect. close proximity to oxygen-ozone gas mixture injected This is ascribed to the capacity ofozone to normalize the cytokine and prostanglandin levels, increase superoxide dismutase, the isthmic lysis. into the foraminal region and facet joints (arrowheads). minimize reactive oxidant species and improve local periganglionic circulation with a eutrophic effect. In patients with post-traumatic facet synovitis the effect of oxygen-ozone infiltration is almost immediate and a single injection Fig.5 Fig.6 may be sufficient to resolve the condition. Instead, in patients with Baastrup syndrome the treatment proved effective in resolving pain with a secondary effect on muscle tone, reducing the antalgic contracture of the paraspinal muscles. This group (A,B) T2 sagittal MR scan Intraprocedural CT of patients obtained a clinical benefit after just one targeted oxygen-ozone infiltration. In line with the scientific literature, facet before and after oxygen-ozone scan in a patient therapy in a patients with with facet joint syndrome was the commonest cause of non-discogenic back pain in our cohort of patients (78.4%). Facet degenera- Meyerding Grade I degeneration: tion is currently treated by infiltration of drugs or using highly effective radiofrequency techniques. We substituted targeted spondylolysis and infiltration of steroids and anesthetic with injection of an oxygen-ozone gas mixture (Fig.6)obtaining a positive outcome in over spondylolisthesis: control of the 70% of ases, reserving radiofrequency ablation for patients failing to respond to ozone therapy. correct needle Oxygen-ozone infiltration yielded pain relief in 38 of our patients (61.3%) with spondylolisthesis and spondylolysis. In the A) Herniated disc in L5-S position in the facet before treatment. joint interline and light of this, ozone therapy could be proposed to treat patients with Grade I listhesis, although a good outcome requires regular distribution a multidisciplinary approach and the support of a physiatrist for postural re-education after oxygen-ozone infiltration to B) Complete dehydration of of the gas oxygen- ensure long-term benefit. In addition to physiatry referral, patients failing to respond to ozone therapy should be assessed by a the herniated disc at ozone gas mixturein neurosurgeon for possible spinal stabilization surgery. long-term follow-up after and around the treatment. joint.

42 43 TABLE 1. 8) De Santis F, Leonardi M, Simonetti L, Dall’Olio M, Princiotta C, Menetti F.(2009) Ossigeno-Ozonoterapia: la tecnic intradiscale. International Journal of Ozone Therapy 8:138-46. MRI scan protocol 9) D’Aprile P, Tarantino A, Lorusso V, Brindicci D. (2006) Fat saturation technique and gadolinium in MRI of lumbar spinal T2 SAG (Thickness 3 mm, Gap 20%, TR 3500, TE 100, Fov 300 mm, Matrix 384 Pd HF) degenerative disease. Neuroradiol J. 30;19(5):654-71 T1 SAG (Thickness 3 mm, Gap 20%, TR 550, TE 9.7, Fov 300 mm, Matrix 384 Pd HF) T2 AX (Thickness 3 mm, Gap 10%, TR 4280, TE 100, Fov 220 mm, Matrix 384 Pd AP) 10) D’Aprile P, Tarantino A, Jinkins JR, Brindicci D. (2007) The value of fat saturation sequences and contrast medium T2 SAG pair (Thickness 3 mm, Gap 20%, TR 3900, TE 100, Fov 300 mm, Matrix 384 Pd HF) administration in MRI of degenerative disease of the posterior/perispinal elements of the lumbosacral spine. EurRadiol. T1 COR (Thickness 3 mm, Gap 15%, TR 420, TE 9.1, Fov 300 mm, Matrix 384 Pd RL) 17(2):523-31. T1 FS SAG (Thickness 3 mm, Gap 20%, TR 2500, TE 39, Fov 300 mm, Matrix 384 Pd HF, Fat/Sat) T1 FS AX (Thickness 3 mm, Gap 20%, TR 3500, TE 39, Fov 220 mm, Matrix 384 Pd AP, Fat/Sat) 11) Freira S, Fonseca H, Ferreira PD, Vasconcelos P, Fonseca JE. (2014) SAPHO syndrome in an adolescent: a clinical case with unusual severe systemic impact. J Adolesc Health.55(2):304-6. TABLE 2. 12) Gallucci M., Limbucci N., et al.(2007) Sciatica: Treatment with Intradiscal and Intraforaminal Injections of Steroid and Main symptoms in facet degeneration Oxygen-Ozone versus Steroid Only. Radiology 242(3):907-13

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44 45 25) Magalhaes FN, Dotta L, Sasse A, Teixera MJ, Fonoff ET. (2012) Ozone therapy as a treatment for low back pain secondary THE ROLE AND THE EFFECTS OF THE OZONE PARAVERTEBRAL to herniated disc: a systematic review and meta-analysis of randomized controlled trials. Pain Physician. Mar-Apr: 15(2) INJECTIONS, IN DISC HERNIATION PATIENTS 26) Muto M, Andreula C, Leonardi M. (2004) Treatment of herniated lumbar disc by intradiscal and intraforaminal oxygen- ozone (O2-O3) injection. Journal de Neuroradiologie 31:183-9. Gabriel Calle , Lucio Huyhua , Alexis Martinez

27) Parlier-Cuau C, Laredo J.(2010) Vertebral involvement in SAPHO syndrome. J Radiol. 91(9 Pt 2):1068-78. Key words: discal herniation, ozone therapy, ozone paravertebral intramuscular injections, minimal invasive spinal approach, low Back pain 28) Pellicanò F, Martinetti F, Tavanti V, Bonetti M, Leonardi M, Muto M, Andreula C, Villari N.(2007) The Italian Oxygen-Ozone Therapy Federation (FIO) study on oxygen-ozone treatment of herniated disc. International Journal of Ozone Therapy 6:7-15. SUMMARY

29) Perri M, Grattacaso G, Di Tunno V, Marsecano C, Di Cesare E, Splendiani A, Gallucci M.(2015) MRI DWI/ADC signal predicts The results obtained from a group of patients suffering from discoradicular conflict syndrome treated with paravertebral shrinkage of lumbar disc herniation after O2-O3 discolysis. Neuroradiol J. 28(2):198-204. oxygen-ozone injections were analyzed. From a total number of 8500 patients treated with ozone during the period 2002/2015, 880 patients underwent intradiscal treatment (nucleolysis) and 7620 patients were treated with paravertebral ozone injections. 30) Rahimi-Movaghar V, Eslami V. (2012) The major efficient mechanisms of ozone therapy are obtained in intradiscal This paper analyses a subgroup of 1850 patients (24.28% of the patients who were treated with paravertebral injections), procedures. Pain Physician.Nov-Dec:15(6) including those patients who underwent the total 10-session treatment, complied with a 5-year follow-up and with the sample homogeneity parameters following a predictability therapeutic effectiveness (PET) index devised for such purpose (PET 31) Sizer PS Jr, Phelps V, Matthijs O.(2001) Pain generators of the lumbar spine. Pain Pract. 1(3):255-73. index O3) by Dr Calle in 2009. The outcomes were assessed based on the VAS score and modified Mac Nab criteria. Definite results determined positive post-treatment outcomes considered excellent and good in 81% of the cases. Such effectiveness 32) Splendiani A, Perri M, Conchiglia A, Fasano F, Di Egidio G, Masciocchi C, GallucciM. (2013) MR assessment of lumbar disk percentage resulted lower than the one achieved with the intradiscal nucleolysis technique (89%), and higher than the herniation treated with oxygen-ozone diskolysis: the role of DWI and related ADC versus intervertebral disk volumetric percentage seen in papers on the selective nerve root block technique. analysis fordetecting treatment response. Neuroradiol J. 26(3):347-56. INTRODUCTION 33) Steppan J,Meaders T, Muto M, Murphy KJ. (2010) A metaanalysis of the effectivenessand safety of ozone treatments for herniated lumbar discs. J VascIntervRadiol. Apr;21(4):534-48. Ever since Christian Friedrich Schönbein’s (1799 /1868) unexpected discovery of ozone in his laboratory, which was published in his paper Erzeugung des Ozons auf chemischen wege (5) in 1832, several applications of this gas have taken 34) Zhang Y, Ma Y, Jiang J, Ding T, Wang J. (2013) Treatment of the lumbar disc herniation with intradiscal and intraforaminal place, going through both periods of popularity and long periods of obscurity. Indeed, ozone was used due to its properties, injection of oxygen-ozone. J Back MusculoskeletRehabil. 26(3):317-22. medical treatments and other applications which do not seize to amaze us- especially because of its properties manifested in medical applications, as well as its safety, effectiveness, low-risk handling, and minimal invasion in treatment techniques. It is known that the German Army used ozone during the First World War to treat soldiers’ wounds. This gas was used as a disinfectant to reduce the risk of gangrene in war wounds. Even though ozone fell into disuse in countries such as the USA (where it had reached popularity since the beginning of the XX century) after the 1930s, many other European countries, especially Germany continued using and working with the gas showing that its applications were quite safe. There were exceptionally severe complications reported inherent to its use-only some sporadic cases, generally associated with practice carelessness rather than accidents caused while using the gas appropriately. The application through which ozone gained international prominence came much later with its usage in the spinal pathology. The Italian School was of great importance since it fostered the knowledge of the results achieved with ozone in the spinal pathology, especially in the discoradicular conflict syndrome and the spinal disc herniation. The first reports about ozone applicability in the treatment of spinal disc herniation through paravertebral injections were done by Verga (38) in Italy in the year 1989; and subsequently , Pietrogrande (28) reported intradiscal usage; but it was A. Alexandre (1,10,27) who later popularized this technique training disciples worldwide.

THE DISCORADICULAR CONFLICT: ANATOMY AND PHYSIOPATHOLOGY

During decades, it has been considered that the main cause of disc/radicular pain was induced by the effect of mechanical compression on the nerve roots, which produced discal herniation. Unfortunately, many practitioners still believe so. Nevertheless, in the last few years- especially in the last two decades- a great number of inflammatory, biochemical, immunological, neurogenic, reflexive, and other factors can cause pain beyond the mechanical factor imposed by the sole presence of the herniated disk material.

Moreover, we cannot say that there is a permanent parallelism between the images observed in the discal pathology MRIs and the pain the patient complains of.

46 47 These facts are the first thing to take into account in order to be able to understand the effectiveness of ozone when add, increase and optimize the therapeutic effects of the chosen treatment. Thus we combine different therapeutic effects treating the discoradicular conflict pathology, since pain generally is of mixed origin it comes both from direct root (mechanical/chemical etc). Furthermore, we consider surgery the last option within our protocol, always prioritizing minimally compression caused by the elements forming the spinal articular complex (disc, facet joints, intervertebral disc, pedicles, invasive and minimally aggressive techniques going from endoscopic approaches to dynamic stabilization or arthrodesis, vascular complexes-especially veins-etc) and quite particularly from the concentration of metabolites and catabolites present depending on the case. in such discoradicular complex. Indeed, it has clearly been shown that the aforementioned metabolites and catabolites are found in more concentration within the conflict area as the result of the degradation of herniated tissue and as inflammatory To summarize: processes originated due to the herniation itself. Within these processes, the following products are found: prostaglandins a2, leukotrienes, bradykinins, tumor necrosis factors, etc. Reactive accumulation of leukocytes, eosinophils, basophils and • A total of 8500 patients were treated using ozone therapy techniques (paravertebral and intradiscal) macrophages can also be found as inflammatory/immunologic response and thus, they work as anti-inflammatory, stabilizers and immunomodulators. To these mechanical, inflammatory, biochemical and immunological aspects, other aspects • 880 patients were treated using the intradiscal nucleolysis technique plus 5 sessions of paravertebral ozone therapy (a total frequently present due to particularities specific to the innervations of the intervertebral and periradicular disc should be of 10.35%; not included in this study). added. • 7620 patients were treated using solely paravertebral injections (89.65%). The intervertebral disc itself does not cause pain. What actually cause discal pain are the pericapsular discal innervations fibers (in areas of lateral layers outside the annulus fibrosus) which receive afferent branches from the recurrent anterior For this study, patients with complete treatment of paravertebral infiltrations (10 sessions) and with a 5-year follow up sensitive branch that supplies the dura mater, the posterior longitudinal ligament, and the outer half of the annulus fibrosus compliance were analyzed. They were grouped according to sample homogeneity, following a proposed index for pre- (recurrent nerves of Luschka). Histochemical studies using different nervous tissue markers have confirmed exclusive assessment. innervations of the annulus fibrosus and the absence of nerve terminals in its innermost part in patients with macroscopically normal intervertebral discs. Palmgren’s (25) marker studies have determined the immunoreactivity of the human From the initial total number of patients treated for discoradicular conflict syndrome/disc herniation with ozone therapy (8500 intervertebral disc concluding that, with the exception of the periphery (a few millimeters), the normal intervertebral disc patients) the following were excluded: has no innervations. The same does not apply for discs defined as painful (discogenic pain) where internal disc disruption is confirmed and in which cases a deeper and more extensive nerve supply was determined as compared with normal di- Patients undergoing Nucleolysis treatment Protocol: n: 880 (10.35% of the total sample) scs. Moreover, these innervation changes are also accompanied by changes in vascularization. These discs are particularly sensitive to postural changes, compression/distraction maneuvers or increases in axial load; reacting to such changes which From the remaining population of 7620 patients, 2667 (35%) were excluded due to early treatment abandonment, irrespective are seen during discal hypertension situations, or due to structure disruption or its degeneration, all of which would basically of their outcome. From the remaining 4953 (65%) patients who underwent a complete 10-session treatment, the present explain the discogenic pain and in such cases, mechanical factors do not necessarily justify the origin of the pain. analysis only includes 1850 patients (24.28% of the patients undergoing paravertebral ozone treatment) who complied with the 5-year follow up and with certain degree of sample homogeneity using assessment parameters we have developed for All these factors should be taken into account in order to evaluate the likelihood of ozone treatment and also to obtain an this purpose and that we decided to denominate therapeutic effectiveness predictive index (PET O3). approximation rate to forecast therapeutic feasibility. Although our experience suggests it is impossible to determine which patient will have good outcomes and which patient will not, we do get oriented to see which cases will have better Outcome assessment was performed at 1 month, 6 months, 1 year, 3 and 5 years; concluding in the present study with the possibilities of therapeutic success and which will have less likelihood of success; or even which cases will have scarce outcomes at 5 years. likelihood of success or no success at all. Therefore, although it is true that therapeutic selection is very important in every medical entity, in the case of ozone therapy, this selection has more predictive value than that of therapeutic exclusion. This Ozone therapy pre- and post treatment clinical evolution was assessed based on the VAS score and assessment of functional is an important observation when considering ozone therapy in the discoradicular conflict syndrome since such methodology recovery both in working and sport environments ( modified Mac Nab criteria). constitutes part of the initial therapeutic base in a high percentage of the cases before considering other possibilities of greater complexity. Inclusion criteria:

METHODS AND MATERIALS • Clinical:

We started our experience at the beginning of 2002, after intensive training in European centres of excellence; and we VAS greater than 6. gradually included our practice as we increased our experience and confidence in the therapeutic results of ozone as part of a Evolution greater than 1 month with conventional treatments and even selective nerve root block (-). protocol of initial treatment of spinal pathology, especially in the treatment of discopathy and discoradicular conflict syndrome. Patients having a PET index of 4 to 8.

Over the last 13 years, we have treated 8500 patients using percutaneous ozone therapy techniques, with a wide age • Radiological: distribution raging from 14 to 90 years of age; 880 of which were treated using intradiscal nucleolysis and paravertebral injections and the remaining 7620 were treated using paravertebral infiltration as a sole treatment. Discopathies of various locations and extensions (from protrusions to extruded and migrated disc herniation) The reason for such difference is due to the fact that the majority of patients had decided to start the most conservative and Up to Grade III disc degeneration according to Pfirrmann classification. less invasive treatment, leaving as a second choice intradiscal ozone treatment (nucleolysis) or the multimodal treatment Exclusion criteria: combined with other techniques (radiofrequency nucleoplasty, percutaneous aspirative nucleotomy, etc). In the cases Severe neurological motor deficit where patients chose the multimodal combined treatment, as first or second choice, the percutaneous nucleotomy or the Patients having a PET index < 4 radiofrequency nucleoplasty, we always combine such treatments with the application of intradiscal ozone in order to Pfirrmann IV and V

48 49 Paravertebral ozone infiltration: Assessment, therapeutic strategy and analysis. homogeneity within the group of patients to be treated. For the assessment 5 items considered relevant were suggested: type of discopathy, age, Container-content relationship, background of selective nerve root block, background of previous In order to assess the best therapeutic strategy as well as its effectiveness, such strategy is chosen according to a series surgery. of parameters, which also enables us to reach an approximation regarding the likelihood of therapeutic success or even the cases with less chance of effectiveness under paravertebral ozone therapy. IPET O3 assessment and scores:

A) Clinical evaluation: Background: “bulging disc¨/ Discopathy Acute/Lateral/Single Chronic/Foraminal/Multiple Extruded herniation • Process: acute / chronic ( < or > 3 months ). 1 2 2.5 • Age: , < 40 , 40 / 60 , > 60 years old. • Profession/Occupation. Age < 30 years old 30 / < 40 > 40 / < 50 > 50 / < 60 > 60 / < 70 > 70 • Athlete: Yes/ No. • Manual laborer. 1 1.5 2 2.5 3 3.5 • Pain: scale: VAS, other. • Medical records and background of previous selective nerve root blocks. Container-Content Relationship Normal Narrow Spinal Canal Narrowness > 1 Level • Previous surgery (Recurrences, FBSS). 0 2 + 0.5 per level B) Radiologic anatomy (X-ray, MRI): Considerations. Background of Selective Positive Positive with relapse Negative > 1 negative block • Type of discopathy: Nerve Root Block 0 1 2 + 0.5 x block Acute/Chronic. Previous Surgery Recurrence or postoperative pain Failed back surgery Size: Bulging/Protrusion, herniation (grade), location/ displacement of herniatio (medial, lateral,foraminal, extraforaminal, extreme lateral). 2 3

Presence of other relevant intercurrent events such as container/content relationship: associated (or not) spinal canal How to get the therapeutic effectiveness predictive index (PET O3): narrowness, facet hypertrophy,etc. This index is quantified from 1 to 8. C) Patient expectations: These considerations enable us to guide the patient towards the type of therapeutic strategy and chances of therapeutic A summation of the partial indexes is done. The result of the summation is then subtracted from the number 10. success with ozone therapy, especially using paravertebral ozone infiltration. E.g. A: Young patient, 30 years of age (1 point), with single lateral acute herniation (1 point).Total: 2 points. PET O3 index: Chances of therapeutic success. 10-2= 8 (high PET index)

Advisability of starting a basic treatment with ozone (paravertebral injections) is one of the options. Physicians may also E.g. B: 65-year-old patient (3 points), with narrow canal in one segmental level (2 points) and background of 3 negative blocks suggest the intradiscal treatment (ozone nucleolysis) alone or combined with other intradiscal techniques such as (2+0.5+0.5) = 3 points. Total: 3 (age) +2 (narrow canal) + 3 (3 blocks) = 8 points. radiofrequency nucleoplasty, percutaneous aspirative nucleotomy and/or other combinations. Hence, PET O3 Index for this patient would be 10-8=2. Therefore, this patient will have a very low PET Index and therapeutic Patients included in the present study were assessed for the first time at the beginning of the treatment; then, they were effectiveness with ozone in this case is scarce; thus other therapeutic option should be assessed and the patient is excluded assessed a second time after the fourth session and they underwent assessment for the third time at the tenth session of from the treatment group. paravertebral ozone application. Consequently, maximum acceptable PET O3 Index is 8 and the chance of positive treatment outcomes is directly proportional Subsequently, patients paid regular follow-up visits following these schedule: a visit every three months after the first year of to the aforementioned potential predictive index of therapeutic effectiveness. Conversely, likelihood of failure would be treatment; a visit every six months during the second year; and an annual visit after the third year. inversely proportional to such index. Minimum IPET O3 is 1 even when its score might result in a negative number.

Assessment and inclusion criteria according to the therapeutic effectiveness predictive index (PET O3). This assessment is done in order to simply share a thought about predictability with the patients, since statistical results encompass all cases without distinction of effectiveness likelihood, providing a final average where all cases are included. A Before the treatment, a predictive assessment parameter was designed to study the likelihood of therapeutic effectiveness simple evaluation of some aspects that may affect the final outcome is hence considered useful. This evaluation provides the with ozone. This parameter consists of assigning a score to evaluate pre-treatment therapeutic effectiveness according to patients with either a better predictive approach regarding their chances of therapeutic effectiveness or information on other clinical and radiological parameters in order to achieve effectiveness predictive criteria as well as the best sample recommended therapeutic strategy in keeping with the patient´s needs to get successful results. Even though this evaluation sessions (therapeutic assessment stage) and every fortnight after the fourth session onwards. This schedule enabled us to

50 51 does not intend to be the best, it enabled us to get a better selection of the treatment group making it as homogenous as BIOLOGY AND HISTOCHEMISTRY OF OZONE possible, continuing with the treatment of those patients with actual likelihood of getting good outcomes when undergoing IN DISCORADICULAR CONFLICT SYNDROME. the paravertebral ozone therapy treatment. These assessment parameters were proposed based on many years of experience treating patients with discoradicular conflict syndrome, and we consider they can be useful when providing the patient with The mechanisms of action which explain ozone action and effectiveness in the treatment of discoradicular conflict syndrome pre-treatment information, since asking about therapeutic effectiveness is a common question in the practice. should be considered in various levels and accordin to different theoretical points of view.

It is important to emphasize that this evaluation is merely representative, since it is impossible to predict which patients will These can be explained taking into account the ozone general actions and those directly or indirectly generated by the gas get positive outcomes with ozone treatment and which patients will not. in the application area. Ozone stimulates the production of a transient and moderate oxidative stress which is controlled by All the patients were treated with highly reliable ozone generators with a built-in photometer for the exact ozone concentration the endogenous antioxidant system. dose applied in real time, that is to say, when extracting the gas for its immediate application (Generator Ozoneline 80, made in Italy; and Ozonotec CX, made in Argentina). These generators are accurately calibrated periodically. For this treatment group Ozone biological properties: ozone concentrations of 15 μg per ml of oxygen were used, administering average doses of 20 cc paravertebral to each side • Oxidative stress reduction. of the conflict space. Treatment protocol included 10 ozone sessions, once per week during the first 4 sessions (therapeutic • Induction of oxygen metabolism (increasing its uptake and release by hemoglobin). assessment stage) and every fortnight after the fourth session onwards. This schedule enabled us to evaluate more accurately • Modulation of immune system. any improvements in therapeutic effectiveness. • High germicidal, fungicidal and virostatic effect. • Atoxic in therapeutical doses, but highly toxic in large doses (especially in the respiratory airways where the antioxidant TECHNIQUE system is rather ineffective). • It works as an oxidant interacting with pain mediators generated in the area, and this is one of its main mechanisms of The same technique was used in all cases as well as the same spine neurosurgeons specialized in ozone therapy and the action with analgesic/anti-inflammatory effect. same assessment and operative protocol. • Highly reactive with biomolecules such as polyunsaturated fatty acids (double bonds). It also reacts with amino acids and nitrogenated bases. For the paravertebral puncture 27 G X 1-1/1/2 atraumatic needles were used ( 40 x 0.4 mm) as well as 27 G x 2”- (50 x 0.4 • Intra and trans tissue oxygenation in the disease site with reduced hypoxia and venous stasis. mm) needles, depending on patient’s body structure. • Reduction of the cell-mediated process inhibiting proteinases release and an increase of immunosuppressor cytokines.

A routine dose of 0.5 ml to 0,75 ml IM Xilocaine was applied before injecting ozone in the puncture area since ozone is Periradicular space. painful and highly irritating, especially in the acute stage when local inflammation is greater, thus we reduce pain caused • Regional analgesic/anti-inflammatory effect. when injecting the ozone. Regarding this procedure, avoiding larger doses should be taken into account since there is a high • Oxygen supply for aerobic glycolysis increasing ATP and decreasing lactic acid. risk of decrease in blood pressure and vasovagal syncope. Special care should be taken when treating elderly patients. • Oxidant action together with regional pain mediators stimulating the production of antioxidant enzymes. • Oxidant action together with local pain mediators (2,34). As regards ozone doses, we usually apply an ozone/oxygen mixture of 15/20 ml per puncture with a concentration of 15 mcg/ • Proinflammatory prostaglandins (PG2)(6,19) synthesis is inhibited, or bradykinins and algogenic compounds are released. ml. The dose will depend on various aspects such as body structure, weight, age, etc. It is important to take into account that • Increased release of antagonists or soluble receptors capable of neutralizing proinflammatory cytokines like interleukin such dose must not exceed antioxidant capacity limits of enzymes such as catalase and superoxide dismutase as well as (IL)-1 , IL2, IL-8, IL-12, IL-15, alpha interferon; and increased release of immunosuppressive cytokines such as transforming glutathione to prevent accumulation of the superoxide anion (O2-) and hydrogen peroxide ( H2O2) (26) which can cause cell growth factor-beta 1 and IL-10 (19,25). membrane degradation (22.36) . Free radicals are mainly formed by ozone in medium with a pH higher than 8, whereas at pH • Local microvascularization improvement with the reduction of algogenic factors, resolution of inflammation, reduction of less than 7.5 the ozonolysis mechanism prevails, mainly leading to the formation of peroxides (30). muscular contracture and oedema.

The election target was taken from the midline, from the inferior medial border of the spinous process, bilaterally and Paravertebral muscle mass. outwards of the level to be treated: Opening of ion channels and vasodilator action on muscle structure with regional oxygen increase. Aerobiosis increase and oxygen consumption. Reduction of lactic acid, increase in ATP and decrease in muscle contracture reflex produced by pain. For L1L2 and L2L3 : 2 cm Immunomodulatory action. For L3L4 and L4L5 : 3 cm For L5S1 : 3.5 cm. Activation of lymphocytes and monocytes facilitating lytic activation and increase in immunosuppressive cytokines synthesis such as transforming growth factor beta and IL10 (19,25). In this case, the superior medial border of the L5 spinous process was taken as reference and a moderately inclined line was set in cephalocaudal and medial direction due to space conformation. Paravertebral ozone application was performed slowly with 10 ml syringes, since the use of bigger syringes are often Intervertebral disc: associated with more pain and pressure in the treated area, as well as greater risk of vasovagal syncope. In aqueous medium, it triggers biochemical changes in the amorphous matrix of polysaccharides, especially due to the disruption of glycosaminoglycans hydrophilic nature, which causes discal tissue moderate dehydration and volumetric After ozone application, it is advisable to let the patient rest for at least 5 minutes. (Fig.1) reduction. There is also indirect lytic action in the herniated disc content because ozone facilitates the affluence of macrophages resulting in the formation of scar tissue (this fact was extensively proven in anatomopathological studies of Average treatment duration: 4 months. patients who required discectomy surgery after ozone treatment).

52 53 RESULTS The treatment with ozone paravertebral infiltrations has shown high therapeutic effectiveness when treating pain coming from the discoradicular conflict syndrome. From the 1850 patients,37.35% of the total population who underwent complete paravertebral infiltration treatment due to Moreover, it has not generated any restrictions or constraints to undergo any other treatment at the same time or in the future. disc herniation) who were treated in accordance with complete selection protocol and complied with the 5-year follow-up requirement, the following results were obtained: Patients who required surgery due to therapeutic ineffectiveness did not present any aggravation to undergo the procedure.

• Excellent: Asymptomatic patients, with no residual pain, who went back to work, to doing sports and to their habitual We believe that the treatment with oxygene-ozone paravertebral infiltrations in the discoradicular conflict syndrome should be lifestyle without any restrictions. VAS reduction > 80 % .981 patients (53%).(Fig.2;Fig.3) considered one of the options to have in mind as a sensible and initial option to treat this syndrome, especially in those cases • Good: Asymptomatic patients, with no residual pain; but some isolated episodes which require analgesic therapy or rest where there is high surgical risk. despite immediate response to them. Normal working life, sports and lifestyle, but in moderation. VAS reduction: 60/80 %. 518 patients (28%). • Regular: Permanent lumbalgia but moderate which improves with NSAIDs and rest. Normal working and sport activity with REFERENCES restrictions. Frequent use of NSAIDs. Regular ozone applications required. EVA reduction: 40 to 60%. 222 patients (12%). • Bad: Patient might have attained some recovery, but pain persists. Patient complains of radicular pain and is drug 1. Alexandre A , Fumo G :Discolisi percutánea mediante O2O3 nell´hernia discale lombare . In: Ceccherelli F. Ricciardi A. eds. dependent. Patient cannot do sport, physical activity is restrained and there is frequently work incapacity when it requires Lombailgie e lombociatalgie. Criteri di diagnosi e cura . Torino: Edizioni Librería Cortina,1998:367-377. physical effort; in some cases patient needs to change job. EVA reduction: < 40 %. Surgery requirement. 129 patients (7%). 2. Andreula CF, Simonetti L, de Santis F, Agati R,Ricci R, Leonardi M. Minimally invasive oxygen-ozone therapy for lumbar disk Complications/Adverse effects. herniation. Am J Neuroradiol 2003;24:996-1000. We have not had severe complications with the paravertebral ozone infiltration treatment. All of the complications encountered were either spontaneously resolved or treated. They were more frequent during the first stage of our experience (first 5 years). 3. Bellomo G, Mirabelli F, Richellmi P, et al .Glutathione-mediated mechanism of defence against oxigen free radical-induced The total average of complications or adverse effects was 3.67%. Even though this percentage might seem significant, we must hepatotoxicity.Hum Toxicol 1989;8:152 take into account that with the exception of one unusual case of infection which required antibiotic treatment, the remaining cases experienced total reversion within the first 24 hours, most of the complications resolving spontaneously. If we take the 4. Bellomo G, Mirabelli f, Richelmi P, et al. Oxidative stress-induced plasma membrane blebbing and cytoskeletal alterations percentage of complications as compared with the number of procedures (since these should be multiplied by 10 times the in normal and cancer cells . Ann NY Acad Sci 1989;551:128-130 number of patients), we notice a decrease of 0.367% as compared with the total number of procedures. 5. Bocci V.Oxigeno-ozonoterapia. Comprensione dei meccanismi di azione e possibilita terapeutiche. Prima edizione: marzo The following is a list of the complications encountered in this study: 2000

Vasovagal syncope: : 33 ( 1.78 %); local pain in the application area: 32 ( 1.73 % ) ;neck pain: 12 (0.65); headaches: 10 (0.54%); 6. Bocci V , Luzzi E , Corradeschi F, et al. Studies on the biological effects of ozone: III, an attempt to define conditions for transient precordialgia: ( 0.38 %); transient ischemic attack cerebrovascular accident: 5,4 resolved within the 1st hour; 1 optimal induction of cytokines.Lymphokine Cytokine res 1993;12:121-126 resolved within the first 24 hours (transient amaurosis) (0,27 %); infections: 1, (0.054% ). 7. Bocci V, Pogni R, Corradeschi F, Busi E, Cervelli C, Bocchi L, Basosi R. Oxygen-ozone in orthopaedics: EPR detection of Most of these complications were seen during the first years of this study. hydroxyl free radicals in ozone-treated “nucleus pulposus” material. Riv Neuroradiol 2001;14:55-9.

8. Bonetti M , Fontana A,Cotticelli B,Volta GD, Guindani M,Leonardi M . Intraforaminal O2-O3 versus periradicular steroidal DISCUSSION infiltrationsin lower back pain :randomized controlledstudy.Am J Neuroradiol.2005;26:996-1000.

The treatment with O2O3 paravertebral infiltrations was found effective in a significant percentage of the population. If we 9. Bonetti M, Fontana A, Cotticelli B, Dalla-Volta G, Guindani M, Leonardi M. Intraforaminal O2-O3 versus periradicular steroidal take into account the total number of excellent and good cases (81%), such effectiveness percentage was lower than the one infiltrations in lower back pain: Randomized controlled study. Am J Neurol 2005;26:996-1000. obtained using the intradiscal nucleolysis technique, with an average of 89% excellent and good cases. 10. Buric J, Alexandre A, Corò L, Azuelos A. Intradiscal ozone treatment of non-contained disc herniations. 18 months The overall effectiveness results within satisfactory outcomes using this technique were higher than those found in studies follow-up. Rivista Italiana di Ossigeno-Ozonoterapia 2003;2:153-60. where blocks with anaesthetics and steroids were used and which effectiveness does not usually surpass 70%. It should also be mentioned that within our group of patients a background of negative selective nerve root block was not 11. Buric J. Ozone chemyonucleolysis vs microdiscectoy. Prospective controlled study with 18 months follow-up. Rivista classified as exclusion criteria and in fact, positive results were achieved in many of these patients. Italiana di Ossigeno-Ozonoterapia 2005;4:49-54.

The treatment with O2O3 paravertebral infiltrations has shown a high level of effectiveness associated with a very low level of 12. Buric J, Molino Lova R. Ozone chemonucleolysis in non-contained lumbar disc herniations: a pilot study with 12 months side effects and complications. follow-up. Acta Neurochir Suppl.2005;92:93-7.

This is an outpatient treatment, which enables most of the patients to continue with their work activities, but with the eventual 13. Castro M, Cánovas L, Martínez J, Pastor A, Segado I, Rocha F, Izquierdo C. Discólisis percutánea con ozono: nuestra restrictions each case might meet. experiencia. Rev Soc Esp Dolor 2009;16:405-9.

54 55 14. Curatte s. epidural corticosteroid inyections for sciatic due to herniated nucleo pulpsus. N. Engl. J med. 1997; 336:1634-40 33. Siddal PJ, Cousins MJ, Spine update spinal pain mechanism. Spine 1997, 22:98-104

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16. De Oliveira Junior, Jose O. Veloso Ozonotherapy for lumbociatic pain. Revista Dol. Vol.13 N. 3 Sao paulo july/sept; 2012 35. Staal JB,De Vet HC,Hildebrandt J,Nelemans P. Inyection therapy for subacute and chronic low back pain: an updated Cochrane review. Spine.2009;43:39-59. 17. Gallucci M,Limbucci N,Zugaro L,et al. Sciatica:tretment with intradiscal and intraforaminalinyections of steroid and oxygen-ozone versus steroid only. Radiology.-2007;242:907-13. 36. Stephen J, Thomas-Meaders BS, Muto M, Murphy KJ. A metaanalysis of the effectiveness and safety of ozone treatments for herniated lumbar discs. Journal of vascular and interventional radiology 2010;21:534-48.64. 18. Iliakis E. Utilizzo dell’ossigeno-ozonoterapia nella pratica ortopedica. Acta Toxic. Ther 1996;vol XVII:249-53 37. Torres LM, Terrero MJ, Vidal M, Aragón F, Martínez J. Discólisis con ozono intradiscal en el tratamiento de la ciática por 19. Iliakis E, Valadakis V, Tisiganos C P,Agapitos E.Rationalization of the activity of medical ozone on intervertebral disc :and hernia discal. Rev Soc Esp Dolor 2009;16:147-52. histological and biochemicalstudy. Rev Neuroradiol.2001;14:23-30. 38. Verga C. Nuovo approccio terapeutico alle ernie protusioni discali lombari. Riv Neuroradiol 1989;2:1489. 20. Lehnert T,Mundackatharappel S , Schwartz W,et al. Nucleolysis in the herniated disk.Radiloge.2006;46:513-9. 39. Villa G. C6-C7 herniated disc treatment with paravertebral oxygen-ozone infiltration. Rivista Italiana di Ossigeno- 21. LeonardiM . Disc puncture under fluoroscopic guidance. Riv Ital Ossigeno-ozonoterapia 2002; 1:73-78 Ozonoterapia 2002;1:199-201.

23. Moretti B, Lanzisera R, Pesce V, Moretti L, Patella S, Patella V. Simone C. O2-O3 vs. anti-inflammatory drugs in the 40. Zambello A, Fara B, Tabaracci G, Bianchi M. Epidural steroid injection vs. paravertebral O2/O3 infiltration for treatment of neck pain. Rivista Italiana di Ossigeno-Ozonoterapia 2004;3:131-7. symptomatic herniated disc refractory to conventional treatment. A prospective randomized study. Rivista Italiana di Ossigeno-Ozonoterapia 2006;5:123-7. 24. Muto M, Ambrosanio G, Guarnieri G, Capobianco E, Piccolo G, Annunziata G, Rotondo A. Low back pain and sciatica: Treatment with intradiscal-intraforaminal O2-O3 infection. Our experience. Radiol Med 2008;113:695-706.

25. Palmgren T, Gronbland M,Virri J, Kaapa E, Karaharju E. An Inmunohistochemical study of nerve structures in anulus

fibrosus of human normal lumbar intervertebral disc. Spine 1999, 24:2075-79. Fig.1. Fig.2

Ozone difussion after Paravertebral ozone 26. Paoloni M, Di Sante L, Caccio A, Apuzzo D, Marotta S, Razzano M, Franzini M, Santilli V. Intramuscular oxygen-ozone bilateral paravertebral pre and post-treatment therapy in the treatment of acute back pain with lumbar disc herniation: A multicenter, randomized, double-blind, clinical trial infiltration (MRI). of active and simulated lumbar paravertebral injection. Spine 2009;34:1337-44.

27. Paradiso R, Alexandre A.The different outcomes of patiens with disc herniation either by microdisectomy,or by intradiscal ozone inyection. Acta Neurochir Suppl.2005;92:139-42.

28. Pietrogrande V. The therapy inviving the infiltration of oxigen-ozono intradisc and interfacet. I Congreso de la Sociedad Fig.3 Española de Abordajes Percutaneos Vertebrales. Barcelona; 29-30 / 6 de 1995 . Paravertebral ozone pre and post-treatment 29. Qing H, Feng D, Tao L, Hui L, Xiao Fang L, Dong L. Report on 602 cases of percutaneous ozone puncture chemonucleolysis treating lumbar disc protusion. Rivista Italiana di Ossigeno-Ozonoterapia 2005;4:145-8.

30. Richellmi P, Valdenassi L , Berte F . Basi farmacologiche dell´azione dell´ossigeno-ozono terapia. Riv Neuroradiol 2001;14 (suppl 1 ):17-22 AUTHORS: 31. Robaina FJ.Situación actual de la cirugía de la columna vertebral degenerativa aplicada al manejo del dolor lumbar crónico. Estemosis de canal. Discopatía degenerativa, resultados basados en la evidenciacientífica. Rev Soc Esp Dolor. Gabriel Calle ,MD, neurosurgeon, director Staff Espinal Argentina 2006;3:167-72 Lucio Huayhua ,MD,neurosurgeon, Staff Espinal Argentina Alexis Martinez , MD, neurosurgeon, Staff Espinal Argentina 32. Robaina FJ . Situación actual de la cirugía de la columna vertebral degenerativa aplicada al manejo del dolor lumbar crónico. Estenosis de canal. Discopatia degenerativa, resultados basados en la evidencia científica. Rev Soc Esp Dolor. Address reprint requests to Gabriel Calle, MD, Staff Espinal Argentina, office: 2006;3:167 -72. Talcahuano 638, CABA, Buenos Aires, Argentina email: [email protected] /[email protected]

56 57 PARADIGM CHANGE: “THE OZONE A THERAPEUTIC The C reactive protein normally is higher in patients with MODIC I (Ronnou & cols). MEDIATOR IN MODIC PROCESSES”. The ozone is a biomodulator fluid in reduction oxidation processes in oxidative stress, in their effects produces inhibition of C amielinic fibers, activates the antinociceptive system, and raises the endorphins by increasing the muscular relaxation with AUTHOR consequent analgesia It raises the superoxide dismutase, catalase and glutatuione proxide and de prostaglandines degradations. Pepa Osvaldo Alberto MD; Instituto Central de Medicina, La Plata, Buenos Aires, Argentina. Key words: vertebral endplate, ozone, MODIC, discography, chronic low lumbar pain. The radiofrequency and nucleoplasty decreases the intradiscal pressure by nociceptive ablation by heat modulating the collagen fiber in the annulus fibrosus. It modifies the tertiary structure of the triple spiral, producing their contraction making INTRODUCTION them more stable and getting them closer decreasing the space in discal ruptures’.

The MODIC changes are the result of a degenerative process of the endplate, which combines an evolutionary triad: clinically RESULTS the pain, radiological evidence in the MRI and histological governed by the biomechanics and biochemistry; we developed our therapeutic strategy based on our clinical experience, besides the bibliography correspondence. We use the follow up of 168 patients from the SPINE MEDICINE SERVICE of MD: Osvaldo Pepa with chronic lumbar pain with more than 6 month of evolutions, between 2004 and 2014 with intradiscal The signal change of the vertebral endplates and the sub cartilage bone gave to the MODIC the capability of summarize and rupture and bone edema. All of them were treated with intradiscal ozone as only treatment; we observed the remission of combine in his classification of the morphologic changes and the causative mechanisms, that until today is not clear enough MODIC process. With one year follow up highlighting an average of total symptoms remition in 100 days. To objectivate the and we should understand and use therapeutically models that change the paradigm of this phenomenon. improvement in the evolution we use the Mc NAB scale and OSWESTRY scale and their follow up was made at the first, third, sixth month and the first year. During this period we find an improvement of their disability and we reach in almost all of our MATERIAL AND METHODS patients the disappearance of pain.

Based on the definition of scientific paradigm a situation is law until a new one takes its place. It’s a set of scientific In 45 % of the patients we had excellent results, in 31% good results and moderate in 24% of the patients. realizations universally known that during certain time proportions a model of problems and solutions to a critical community. So we conclude that based on our results, minimal invasive non-surgical treatment with O2 - O3 mixture can be considered In my pose I believe necessary: an effective TREATMENT for back pain.

1: Determine an intradiscal ozontherapy plan with discography (diagnostic and therapeutic) Compared to other surgical approaches, the less procedural risk and the lower costs can be regarded as a clear advantage. 2: Change the natural history of the MODIC We know that degenerative lumbar disease of the endplate, MODIC; is a clinical, radiological and evolutive constant, that has 3: Determine the use of intern spinous device 2 hypothesis of its origin, the biochemical and the biomechanical. The ozone plays an important role in the paradigm change 4: Avoid demolitive surgery in the treatment improving the healing time, giving a permanent solution and decreasing the complications, we hope that new 5: Determine the alternative intradiscal corticosteroid treatment (short term) investigation models and image studies make stand longer the law. 6: Evaluate how the thermal annuloplasty by radiofrequency does not stop the MODIC REFERENCES MODIC I T1 hipointense T2 hiperintense MODIC II T1 hiperintense T2 hiperintense 1. Adams MA, Freeman BJ, Morrison HP, Nelson IW Dolan P Mechanical initiation of intervertebral disc degeneration. Spine MODIC III T1 hipointense T2 hipointense 2000; 25 1625-1636.

MODIC I: edema of the vertebral body and hipervascularisation 2. Albert HB, Kjaer P, Jensen TS, Bendix T, Manniche C,Modic changes: possible causes and relation to low back pain. Med MODIC II: fat replacement of the bone marrow Hipotheses 2008;70:361-368 MONE III: under cartilage bone sclerosis Millier (1990) reclassifies in 4 grades giving the 0 grade as normal 3. Buttermann GR The effect of spinalsteroid injections for degenerative disc disease Spine J 2004.2004;4:495-505

The biomechanics changes occur on the vertebral endplates during the discal degeneration; this is submitted to calcifica- 4. Carragee EJ ,Alamin TF, Miller JL, Carragee Jm Discographic, MRI and psychosocial determinants of low back pain tions in the aging and changes with micro fractures. Those changes determine an unequal distribution of the loads throw the disability and remission a prospective study in subjects with bening persistent back pain, Spine J 2005;5 24 -35. disc being the cause of the endplate fissure. The endplate and the bone are weak links where the discal biological structure changes and the reveal of this micro fractures are an important source of MODIC. Therefore this is reflected by edema and 5. Jones A, Clarke a, Freeman BJ, Lam KS, Grevitt MP; The Modic classification inter-and intraobserver error in clinical vascularization after an accumulative trauma and an inflammatory answer after these lesions. We observed that the exit of practice.Spine 2005;30 :1867-1869. the disc goes with the MODIC phenomenon by healin degeneration of the annulus fibrosus. The biochemical changes in the nucleous pulposus produced an inflammatory environment with pain and raise of the proinflamatory mediators as IL 6 and IL 6. Marshman LA, Trewhella M, Frisem T, Reverse transformation of Modic tipe 2 changes of modic type 1 changes during 8 and PGE2. The gen product of protein (PGP) and nervous fibers inmunorreactive and the tumorous necrosis factor (TNF) this sustained chronic low back pain severity Reportbof two cases and review of the literature, J Neurosurg Spine 2007;6:152- was observed in the inmunorreactive cells of in the endplate in patients with MODIC. 155.

58 59 ELECTROMIOGRAFIC ANALYSIS OF THE RESULTS GIVEN damaged macromolecules (18) and probably leading to reduced tissue strength. Matrix synthesis decreases steadily throughout life but sometimes increases again in old and severely disrupted discs (8). Reduced synthesis is partly attributable BY LUMBAR DISCAL HERNIATION TREATAMENT to decreased cell density, although proteoglycan synthesis rates per cell also decrease (26). WITH INTRADISCAL OXIGEN-OZONE GAS MIXTURE INJECTION. All these characteristic aspects of disc degeneration are to be kept in mind when considering the nerve root disesase which Alexandre A. , Zalaffi A.* comes form disc degeneration which provoques both fisical compression on the nerve root with consequent hyschemia,and EU.N.I. European Neurosurgical Institute, TREVISO methabolic intoxication . Ingrowth of nerves and blood vessels has been shown to be an important feature of structurally * Istituto di Neurochirurgia , Università di Siena, SIENA disrupted discs, and appears to be directly, though variably, associated with pain.(27) Ingrowth could be facilitated by the loss of hydrostatic pressure that characterizes internal regions of intact discs, and that would collapse hollow capillaries. Reduced proteoglycan content in old and degenerated discs may also facilitate the ingrowth of nerves and capillaries because INTRODUCTION aggrecan can inhibit their growth in vitro.(28,29). Ingrowth of nerves has been thought as a possible base for the evolution of acute pain in chronic persistent pain. In the last years several statistical analysis have shown that sixty-five percent to 80% of adults have an episode of low back pain at some time in their lives, and although most cases resolve quickly, 40% recur and 5% result in a residual disability after Radicular pain often is the result of nerve root inflammation and irritation. Clinical practice and research demonstrate that 1 year. The extremely high frequency of the problem, and the discrepancy between pathological findings in neuro-imaging and mechanical compression on the nerves may cause only motor deficits and altered sensation, but will not cause pain. clinical situation, both before and after open surgery, have brought recently to a very ample revision of the pathophysiology Inflammation in the epidural space and nerve roots provoked by a herniated disk is a significant factor in causing radicular and reconsideration of the possible treatments. pain. Chronic compression of a nerve root can induce axon ischemia, impede venous return, promote extravasation of the plasma protein, and cause local inflammation. If dorsal root ganglia are chronically compressed and irritated, this theoretically Mechanisms of disc degeneration and subsequent nerve dysfunction and pain. can lead to their sensitization and resultant radicular pain. Similar mechanisms of radicular pain are postulated to occur in the thoracic and cervical spine as well. In summary, clinical practice and animal research suggest that radicular pain is the result Disc cells synthesize their matrix and break down existing matrix by producing and activating degradative enzymes, such of inflammation of the nerve root in the epidural space provoked by leakage of disk material, compression of the nerve root as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase (ADAMS). (1-7) Molecular markers of matrix vasculature, or irritation of dorsal root ganglia from spinal stenosis. turnover are naturally most plentiful during growth but usually decline thereafter (8)The proteoglycan content of the disc is maximal in the young adult and declines thereafter (8) presumably because of proteolysis. Disc cells appear to adapt the ELECTROMYOGRAPHY properties of their matrix to suit prevailing mechanical demands, although the low cell density and lack of a blood supply ensure that changes are not as rapid or pronounced as in adjacent vertebrae (9). Adaptive remodeling probably contributes to The classical instrument for studying nerve functioning alteration is EMGraphy. Altered EMGraphic tracings indicate the the large variation in compressive strength of adult discs, which ranges from 2.8 to 13.0 kN when they are tested in a manner dysfunction of muscles, due to inadequate transmission of electrical impulses along a peripheral nerve. A compressed or that causes failure in the disc rather than the adjacent vertebra (10). methabolically altered nerve looses its capability of active progressive point by point production and transmission of eletctrical impulses towards muscles. The instrument will record the dysfunction in the muscels, allowing to understand if the alteration Injured discs show increased levels of catabolic cytokines, increased MMP activity, (8,11) and scar formation, (12) especially has a central or peripheral nervous system origin. in the vicinity of anular tears.(12,13) They also show evidence of renewed matrix turnover (8,14) and a more variable range EMGraphic recording is normally associated with Electo Neurographic Recording. This consists in the recording of the of collagen fibril diameters.(15) However, gross injuries to a disc never fully heal. Scalpel cuts in the outer anulus fill with conduction quality and velocity in a nerve, and allows to demonstrate the anatomical site where obstacle exists. granulation tissue, with only the outer few millimeters being bridged by scar tissue.(16,17) Anular tears are not remodeled as in bone, presumably because the sparse cell population is unable to break down the large collagen fibe bundles of the anulus PATIENTS AND METHODS and replace them with new(18). Collagen turnover time in articular cartilage is approximately 100 years (19) and could be even longer in the disc. Proteoglycan turnover is faster, possibly 20 years, (18)and some regeneration of nucleus pulposus Among patients treated by intradiscal oxigen-ozone administration, in the period from Genuary to June 06, because of appears to be possible in young animals.(20) Injuries that affect the inner anulus or endplate decompress the nucleus (21), discoradicular conflict, we randomly selected a group of 200 cases for this study. Among these, 125 patients showed at and healing processes are then overtaken by severe degenerative changes (15). the moment of enrollement for treatment EMGraphic signs of mono or pluriradicular nerve dysfunction (125 among 200, that is 62.5%). The details of the dysfunction are given in Table 2. Repeated EMGraphic control during the treatment gives a A series of biochemical and histologic changes have been described as tipical of disc aging process. The blood supply to the valid parameter in order to quantify nerve root dysfunction: this is an objective, repeateble and precise.data. The treatment vertebral endplate decreases during early hildhood, and microstructural clefts and tears become common by the age of 15 consisted in intradiscal 0203 gas mixture administration at the doses of 10 to 15 ml (this depends on the fissuration of the years, especially in the nucleus and endplate (22). Cell density decreases throughout growth (23), and the nucleus pulposus annulus and subsequent diffusion of the gas in the periradicular and peridural space) and was performed by the senior author. tends to condense into several fibrous lumps, separated from each other and from the cartilage endplate by softer material EMGraphic controls were performed on enrollement, and at 4 and 12 months postoperatively, by the electophysiologist (A.Z.) (34). Proteoglycan fragmentation starts during childhood (31), and with increasing age, the overall proteoglycan and water as an independent observer. content of the disc decreases, especially in the nucleus (17) and the collagen content increases. Fine type II collagen fibrils in RESULTS the inner anulus are replaced by type I fibers as the anulus encroaches on the nucleus, and type I fibers throughout the disc become coarser. As long as the proteoglycan fragments remain entrapped in the disc, they can fulfill a functional role similar The evolution of EMGraphic picture doesn’t correspond always to the clinical situation. In several cases the normalization to that of the intact proteoglycan. Reduced matrix turnover in older discs enables collagen molecules and fibrils to become of the last will coexist with residual signs of EMGraphic dysfunction. This is notorious in radicular pathology, as well as in increasingly cross-linked with each other, and existing cross-links become more stable (23). In addition, reactions between peripheral nervous system pathology in general , which ever kind of medical treatment was undertaken. EMGraphic data will collagen and glucose lead to nonenzymatic glycation (extra cross-links that give old discs their characteristic yellow-brown express the nerve structure dysfunction as a consquence o relative hyschemization given by mechanic compression and by appearance) (24). Increased cross-linking inhibits matrix turnover and repair in old discs, encouraging the retention of the mentioned biochemical factors which make the basis of the problem. Persistence through the time of signs of damage

60 61 will express the fact that some nerve fibers are definitively altered. This may exist also in the absence of any clinical Table 4 manifestation. EMGraphic improvement on the other hand will indicate the recovery of the nerve root function, thanks to the normalized nourishment. in the series of 125 patients: Number of patients showing EMGgraphic alterations before and after the treatment. TABLES LITERATURE NERVE ROOTS 1. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: Structure, function, and LA L3 EMERGES FROM L2L3 CONJUGATION FORAMEN biochemistry. Circ Res 2003;92:827-39. LA L4 EMERGES FROM L3L4 CONJUGATION FORAMEN LA L5 EMERGES FROM L4L5 CONJUGATION FORAMEN 2. Mott JD, Werb Z. Regulation of matrix biology by matrix metalloproteinases. Curr Opin Cell Biol 2004;16:558-64. LA S1 EMERGES FROM L5S1 CONJUGATION FORAMEN Table 1 3. Duffy MJ, Lynn DJ, Lloyd AT, et al. The ADAMs family of proteins: From basic studies to potential clinical applications. Thromb Haemost 2003;89:622-31. The anatomical location of nerve roots, with respect to the disc level. 4. Tang BL. ADAMTS: A novel family of extracellular matrix proteases. Int J Biochem Cell Biol ‘01;33:33-44. 125 patients with EMGraphic signs of dysfunction before treatment: 5. Porter S, Clark IM, Kevorkian L, et al. The ADAMTS metalloproteinases. Biochem J 2005;386:15-27. monoradicular L3 in 6 patients (4.8%) 6. Roberts S, Caterson B, Menage J, et al. Matrix metalloproteinases and aggrecanase: Their role in disorders of the human L4 16 (12.8%) intervertebral disc. Spine 2000;25:3005 L5 53 (42.4%) S1 42 (33.6%) 7. Goupille P, Jayson MI, Valat JP, et al. Matrix metalloproteinases: The clue to intervertebral disc degeneration? Spine 1998;23:1612 pluriradicular L4 and L5 4 (3.2%) L5 and S1 4 (3.2%) 8. Antoniou J, Steffen T, Nelson F, et al. The human lumbar intervertebral disc: Evidence for changes in the biosynthesis and Table 2 denaturation of the extracellular matrix with growth, maturation, ageing, and degeneration. J Clin Invest 1996;98:996 9. Adams MA, Dolan P. Could sudden increases in physical activity cause degeneration of intervertebral discs? Lancet The distribution of signs of mono or pluri-radicular dysfunction in the series of 125 patients among 200, before treatment. 1997;350:734-5 125 patients with EMGraphic signs of dysfunction after treatment: 10. Adams MA, Hutton WC. Prolapsed intervertebral disc. A hyperflexion injury 1981 Volvo Award in Basic Science. Spine 1982;7:184-91 monoradicular L3 in 3 patients (2.4 %) L4 7 (5.6 %) 11. Kang JD, Georgescu HI, McIntyre-Larkin L, et al. Herniated lumbar intervertebral discs spontaneously produce matrix L5 20 (16 %) metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2. Spine 1996;21:271-7. S1 19 (15.2 %) 12. Nerlich AG, Schleicher ED, Boos N. 1997 Volvo Award winner in basic science studies. Immunohistologic markers for pluriradicular L4 and L5 3 (2.4 %) age-related changes of human lumbar intervertebral discs. Spine 1997;22:2781-95. L5 and S1 1 (0.8 %) Table 3 13. Weiler C, Nerlich AG, Zipperer J, et al. 2002 SSE Award Competition in Basic Science: Expression of major matrix metalloproteinases is associated with intervertebral disc degradation and resorption. Eur Spine J 2002;11:308-20. The distribution of signs of mono or pluri-radicular dysfunction in the series of 125 patients among 200, after 14. Duance VC, Crean JK, Sims TJ, et al. Changes in collagen cross-linking in degenerative disc disease and scoliosis. Spine treatment. An important modification of the percentages is observed. 1998;23:2545-51. PRE-TREATMENT POST-TREATMENT 15. Gruber HE, Hanley EN Jr. Ultrastructure of the human intervertebral disc during aging and degeneration: Comparison of surgical and control specimens. Spine 2002;27:798-805. EMG alterations 4 months 12 months Discrete 82 49 32 16. Melrose J, Ghosh P, Taylor TK, et al. A longitudinal study of the matrix changes induced in the intervertebral disc by Minor 43 12 10 surgical damage to the annulus fibrosus. J Orthop Res 1992;10:665-76

62 63 17. Kaigle AM, Holm SH, Hansson TH. 1997 Volvo Award winner in biomechanical studies. Kinematic behavior of the porcine INTRADISCAL INJECTION OF OXYGEN-OZONE GAS MIXTURE lumbar spine: A chronic lesion model. Spine 1997;22:2796-806. FOR THE TREATMENT OF CERVICAL DISC HERNIATIONS 18. Roughley PJ. Biology of intervertebral disc aging and degeneration: Involvement of the extracellular matrix. Spine 2004;29:2691-9. A. Alexandre, L. Coro , M. De Pretto, H. Baddredine EU.N.I. European Neurosurgical Institute, Roma, Treviso, Pordenone, Bologna Italy 19. Verzijl N, DeGroot J, Thorpe SR, et al. Effect of collagen turnover on the accumulation of advanced glycation end products. J Biol Chem 2000;275:39027-31. ABSTRACT

20. Bradford DS, Oegema TR Jr, Cooper KM, et al. Chymopapain, chemonucleolysis, and nucleus pulposus regeneration. A A series of patients affected by cervical disc pathology was treated by intradiscal injection of oxygen-ozone gas mixture. The biochemical and biomechanical study. Spine 1984;9:135-47. effects both on pain and on radicular dysfunction are impressive. For disc herniations the use of open surgical approaches is reduced since new percutaneous methods allowing shrinkage of the disc and improvement of the radicular function are 21. Adams MA, Freeman BJ, Morrison HP, et al. Mechanical initiation of intervertebral disc degeneration. Spine 2000;25:1625-36. gaining interest. Studies on the spontaneous disappearance of disc fragments have demonstrated autoimmune responses with a chronic inflammatoryreaction. Also radicular pain has been shown to be mostly due to biochemical mechanisms [10]. 22. Boos N, Weissbach S, Rohrbach H, et al. Classification of age-related changes in lumbar intervertebral discs: 2002 Volvo Researchers in different fields surprisingly noticed that a brief, calculated, oxidative stress by ozone administration may Award in basic science. Spine 2002;27:2631-44. correct a persistent imbalance due to excessive, chronic oxidative injury [4]. Oxygen-ozone gas injection in painful patients has a dramatic effect on clinical symptoms. On these bases the intradiscal injection of oxygen-ozone gas has been 23. Nerlich AG, Weiler C, Weissbach S, et al. Age-associated changes in the cell density of the human lumbar intervertebral conceived [1, 7, 9]. The morphological effect of the treatment was evaluated by radiological examination. After nervous and disc. Presented at: The 51st Annual Meeting of the Orthopaedic Research Society; Feb 20-23, 2005; Washington, DC 34- microcirculation compression, the metabolocal impairment in this anatomical area is quite important. Restoring blood perfusion and improving heamoreology has sufficient functional results, avoiding disconfort and risks of open surgery. 24. Adams MA, Dolan P, Hutton WC. The stages of disc degeneration as revealed by discograms. J Bone Joint Surg Br 1986;68:36-41. Keywords: Intradiscal injection; cervical disc herniation; oxygen-ozone.

25. DeGroot J, Verzijl N, Wenting-Van Wijk MJ, et al. Accumulation of advanced glycation end products as a molecular INTRODUCTION mechanism for aging as a risk factor in osteoarthritis. Arthritis Rheum 2004;50:1207-15. In cases of radicular dysfunction due to discalradicular conflict, the classical surgical treatment by open surgery has shown 26. Maeda S, Kokubun S. Changes with age in proteoglycan synthesis in cells cultured in vitro from the inner and outer rabbit to entail a number of complications or of partial results. Neurosurgeons have always been searching for a method that allows annulus fibrosus. Responses to interleukin-1 and interleukin-1 receptor antagonist protein. Spi shrinkage of the herniated or protruded disc in order to solve the problem of severe pain and dysfunction found in an enormous amount of patients. Thus a number of percutaneous non-invasive techniques have been conceived with the aim to remove 27. Freemont AJ, Peacock TE, Goupille P, et al. Nerve ingrowth into diseased intervertebral disc in chronic back pain. Lancet or provoke shrinkage of the discal tissue. The common principle of these techniques is that of acting directly on the discal 1997;350:178-81. structure without access to the spinal canal. This drastically reduces the epidural formation of scar tissue, which may lead to compression of the nerve root and adherence to the moving bones. In the last years percutaneous techniques applied in the 28. Johnson WE, Caterson B, Eisenstein SM, et al. Human intervertebral disc aggrecan inhibits nerve growth in vitro. Arthritis lumbar area have become increasingly the subject of research with regard to the various aspects of disc pathology and the Rheum 2002;46:2658-64. possible solutions of the problem. Studies on pain originating from this pathology show that it may be due to biochemical mechanisms of acid intoxication of the nerve, which may be somehow independent from the mechanical problem but may 29. Johnson WE, Caterson B, Eisenstein SM, et al. Human intervertebral disc aggrecan inhibits endothelial cell adhesion and result either from an autoimmune reaction, producing a chronic inflammatory response engendering an acid environment, cell migration in vitro. Spine 2005;30:1139-47. or a situation of ischemia [10]. These problems may be solved by biochemical treatment, reducing the need for surgical intervention [1–3, 7]. On the other hand, the mechanism of disc shrinkage and elimination of herniated fragments have carefully been studied and the development of an autoimmune response against a ‘‘non-self’’ material, leading to a chronic inflammatory reaction has been demonstrated [6]. The mixture of oxygen and ozone gases has been employed in medicine since the 30s for the treatment of pain and dysfunction in patients a¤ected by thrombotic and ischemic diseases. After decades of experience in these fields, the empirical observations of powerful and long lasting e¤ects of this gas mixture injected in paravertebral muscles for the treatment of pain and radicular dysfunction due to a discalradicular conflict have led to detailed studies on the subject. Working in different fields, researchers surprisingly noticed that a brief, calculated, oxidative stress, achieved by ozone administration, may correct a permanent imbalance caused by excessive or chronic oxidative injury. It has evolved that modest, repeated ozone treatment increases the activity of superoxide dismutase, catalase, and glutathione peroxidase, inducing a state of oxidative stress adaptation with very important therapeutic implications [4]. The mixture is produced by an apparatus (ozone generator) which activates the molecules of diatomic oxygen in a voltaic arch. Ultraviolet spectrophotometry allows a precise quantification of ozone percentages in the obtained mixture. Jacobs in 1982 reported [8] the absence of side effects in over five million ozone therapy sessions for different pathologies. The paravertebral intramuscular treatment produces pain relief in the majority of patients, together with decongestion, reabsorption of oedema

64 65 and increased mobility. This has triggered the idea of injecting the oxygen-ozone mixture in the intervertebral disc and the intradiscal injection, ozone can accelerate the degradation of proteoglycans in the degenerated nucleus pulposus, leading to conjugation foramen in order to obtain a powerful e¤ect directly on the pathological mechanism [1, 7, 9]. Since 1995 the its reabsorption and dehydration with the consequent reduction of herniated material responsible for nerve root compression application of these gases has been safely used also in cervical disc pathology. [3, 4]. In our opinion, the most important aspect is the biochemical modification of the medium in the extradural space. Studies on pain, which often is disproportionate to the morphological evidence of discal-radicular conflict, have demonstrated that it PATIENTS AND METHODS is provoked by the presence of acid metabolites coming from the degenerative processes inside the disc, and from ischemia of the nerve root and of the ganglion. In the 90s attention was brought to A2 phospholipase. Saal demonstrated that A2 From 1995 to 2010, a total of 252x3 756 patients were treated by intradiscal oxygen-ozone (0203) injection for cervical disc phospholipase is the cause of radicular pain, independent of the immunological response or a direct inflammatory disease in the different centres participating to this study. Mean patient age was 38 years, 47% were males. Each patient process [10]. A2 phospholipase is responsible for the arachidonic acid liberation, and hence prostaglandines. High levels of A2 underwent clinical and electrophysiological and neuroradiological investigation in order to establish a precise diagnosis. phospholipase have been demonstrated in herniated discs. Ozone injected in the disc and in the peridural space of the In each case the presence of a disc herniation was demonstrated. In 67x3 201 (39.8% of cases) multiple level herniation conjugation foramen and along the posterior longitudinal ligament acts as a powerful stimulus to the activation of antioxidant was observed. Patients affected by cervical spinal canal stenosis, discarthrosic processes, osteophytes or concomitant CSN defence, favouring the normalisation of redox balance with neutralisation of acidosis, increased synthesis of ATP, Careuptake pathologies were not included in the series. Patients enrolled had received pharmacological and physical therapy without and resolution of oedema [4, 6, 10]. The complete biochemical mechanism is not yet understood, but there is strong clinical remedial of the clinical picture. The perspective of solving the problem reducing drug administration and without conventional evidence that the effect is dramatic, and long lasting. Benefit is rapidly obtained on pain, and on nerve dysfunction, with surgical treatment was o¤ered to the patients who consented after detailed explanation. Dexamethasone administration, if progressive reduction of tingling. EMG controls have confirmed the recuperation of nerve function. We presume that this is pre-existing, was interrupted when starting 0203 injection. It was never associated with 0203 treatment. Non steroid drugs achieved by amelioration of nerve ischemia. Injections even in cases of extruded cervical disc pathology were performed and were allowed, if occasionally needed. had good results. This is probably due to the fact that the isolated fragment is separated from normal tissues, has higher The treatment consisted of an intradiscal injection of 0203 preceded and followed by 6 paravertebral injections paravertebral tendency to dehydration, and a degeneration process is engendered in the course of time. Much remains to be done, but injection consisted of administration of 20 ml of 0203 at 10 micrograms/ml concentration, divided into 2 sites of injection: in the possibility of treating patients by an easy method which is rapidly e¤ective for solving clinical problems is at hand. This the paravertebral muscles bilateraly, in the methameric level of the pathology. treatment is useful in patients who did not respond to physical therapy and conventional pain therapy, as a last step in Intradiscal injection is performed via the classical anterior cervial approach. Introduction of the needle in the disc through the conservative treatment before taking the decision of open surgery. Most of these patients will not need more surgery anterior approach orresponds to the classical approach as for open surgery. Its execution requires operative room equipment, anymore, since ozone may act directly on the cause eliminating clinical symptoms. The target of the doctor must be to solve allowing safe asepsis and anaesthesiologic tools, a radiological apparatus for direct vision of the spine, and the source of the clinical problems, not to correct a morphological aspect of a radiological image. This technique is simple, has no risks, offers oxygen-ozone mixture. Two to three ml of gas are injected, at 20 micrograms/ml concentration. to the patient a solution without the discomfort of surgery and the possible risks it entails. RESULTS REFERENCES

1. Among the 252x 3 756 patients pain symptomatology was completely abolished in 79.3% (200x3 600 patients), 1. Alexandre A (1996) Protocollo al Ministero per l’iniezione intradiscale di Ozono Medicale. Roma, Ottobre 1996: 1Congresso amelioration was obtained in 9.9% (25x 75 patients) and the result was poor in 10.7% (27x 81 patients). Italiano sull’applicazione dell’Ozono nel trattamento delle ernie discali

2. Sensory dysfunction was abolished in 78.1% (197x 591 patients) and improved in 16.6% (42x 126 patients). This makes a 2. Alexandre A, Soattin GB, Fumo G (1997) Intradiscal ozone injection: a new solution for herniated disc problems. Miami total of 94.7%. Dysfunction remained unchanged in 5.1% (13x 39 patients). Cedars University Course for Neurosurgery, Miami July 27

3. Various degrees of motor dysfunction were present in 78.9% of our 252x 756 patients, i.e. 199x 597 cases. In the great 3. Alexandre A, Fumo G (1998) Discolisi percutanea mediante 0203 nell’ernia discale lombare. In: Ceccherelli F, Ricciardi A majority of cases it was the question of a mild strength defect and was particularly evident when compared to the (eds). Lombalgie e lombosciatalgie. Criteri di diagnosi e cura. Edizioni Libreria Cortina, Torino, pp 367-377 non-affected side. The motor defect had pre-existed with a mean pre-duration time of 14 days. Among the total group of 252x 756 patients we observed complete regression of motor deficit in 61.9% (156x 468 patients), partial in 21.4% 4. Bocci V (1999) Biological and clinical e¤ects of ozone. Has ozone therapya future in medicine? Br J Biomed Sci 56: 270-279 (54x 162 patients), and insuffcient in 13.4% (34x 102 patients). This means a total of positive results in 83.3% of cases. 5. Donato G, Amorosi A, Lavano A et al (2000) Pathologic examination of the lumbar intervertebral disc. An appraisal about 4. Multiple level disc pathology was present in 67x 201 patients. The treatment was performed simultaneously in all utility and limits. Pathologica 92: 327–330 pathological discs. The results obtained do not differ from those obtained for single level pathology. 6. Fandino Rivera J (2000) Desaparicion espontanea de la hernia discal. Neurocirugia 11: 419-424 5. Patients underwent CT/MRI control at approximately 12 months after treatment. In 39.6% (100x 300 cases) we observed a significant reduction in volume of the hernia, but correlation with clinical signs was not statistically significant. 7. Fabris G, Tommasini G, Lavaroni A et al (1997) Percutaneous treatment of lumbar herniated disk. Riv. Neuroradiol 10: 523-532 DISCUSSION 8. Jacobs MT (1982) Untersuchung u ¨ber Zwischenfalle und typische Komplikationen in der Ozon-Sauersto¤-Therapie. Experimental models suggest that material from the nucleus pulposus may act as a chemical or immunologic irritant to the Ozonachrichte: 1-5 nerve and that these mechanisms may produce inflammatory response [10]. Up to now, studies have hypothesized that injection of such a powerful oxidant such as ozone induces overexpression of injection of such a powerful oxidant such as 9. Jucopilla N (1995) Personal communication ozone induces overexpression of antioxidant enzymes, which neutralise excessive reactive oxygen species (ROS) formation [4]. Ozone seems to reactivate immune system response. Several investigations have demonstrated that modest, repeated ozone 10. Saal J, Saal JS, Herzog R (1990) The natural history of the lumbar intervertebral disc extrusion treated nonoperatively. treatment increases the activity of superoxide dismutase, catalase, and other enzymes for antioxidant defence. After Spine 15: 683-686

66 67 OZONE THERAPY AS COMPLEMENTARY TREATMENT Risk factors such as obesity, diabetes, age over 60, chronic infections, prolonged operating time, blood loss, post-operative pain, anxiety, depression,etc., has been recognized as significant risk factors increasing the incidence of infection after spinal FOR SURGICAL INFECTION: CASE REPORT surgery (9,10)Up to date, there is no univocal idea on mechanisms of infection development. One theory favorsthe infection as a direct mechanical effect of bacterial virulence.Others are more incline to indicate the type of bacteria as being responsible Josip Buric1, Marco Damilano2, Pedro Berjano2 for infection initiation. (11,12,13) 1 Villa Torri Hospital GVM - via Quirico Filopanti 12 - 40126 Bologna - Italy 2 IRCCS Istituto Ortopedico Galeazzi - via Riccardo Galeazzi 4 - 20161 Milan - Italy Ozone is a triatomic allotrope of oxygen with a high electro-voltaic potential. Its known bactericidal, fungicidal and viristatic action is largely used in food and drinking water industry (14,15)The mechanism through which it explicatesthe bactericidal Corresponding author: action is believed to lay in its capacity to destroy the protein and lipid reactive sites on cell membranes of bacteria. (16) Josip Buric Laboratory findings indicate ozone to have a high destructive potential on vegetative and planktonic forms of bacteria e-mail: [email protected] immersed in water and/or saline solution. (17) Results on its activity on biofilms are not so univocal however, ozone seems to phone: +393356838866 have better destruction capacity then common treatment with antibiotics (18, 19, 20, 21, 22, 23, 24)Toxicological tests found that zone, as applied to laboratory animals in acute and subacute doses, didn’t show any tissue damage when studied either ABSTRACT under electronic microscopy or histology. (25)

Background: Infection is a serious surgical complication that increases significantly morbidity and mortality rates as well We report a case of post-operative spine surgery infection treated successfully with intra-wound application of gaseous form as health care expenses. Bacterial ever-growing resistance to antibiotics makes the treatment of such events even more of ozone. troublesome. CASE REPORT OBJECTIVE Female, 65 years of age, presented to the outpatient clinic due to progressively worsening low back pain started three years Report on a surgical infection case treated with ozone as a complementary therapy before with recent radiation of pain to both her lower extremities mainly to the right side. Radiological examination showed a case of spinal stenosis at L3/L4 and L4/L5 levels with significant sagittal imbalance: Pelvic Index 56°; Pelvic Tilt 24°; Lumbar Methods and Materials: Female, 65 years old, submitted to a complex surgical procedure for adult kyphotic deformity Lordosis 25° and C7 plumb line + 115 millimeters. correction that presented with early post-surgical infection. The patient was treated with revision surgery and antibiotics that (Figure 1&2) The patient’s BMI was 33.06 and the bone densitometry showed a T score of -0.6. No significant comorbidities or improved the condition but were unable to delete the infection. Ozone, in its gaseous form, was injected subcutaneously and risk factors were reported. The patient was submitted to a complex spinal surgical procedure for sagittal imbalance correction paravertebraly twice weekly for three weeks. and decompression. An anterior approach at L5/S1 was donewith an ALIF (anterior lumbar interbody fusion) cage insertion RESULTS and upon, the patient was turned laterally and an ACR XLIF (anterior column release extreme lateral interbody fusion) cage was positioned in L3/L4 obtaining an approximately 30 degrees of sagittal correction. After 3 weeks of treatment the wound healed completely and repeated visits and blood exams up to one year after the surgery Moreover, from the same lateral approach another two lordotic XLIF cages were positioned at L4/L5 and L2/L3 levels for did not show recurrence of infection. further improvement of the balance and anterior support to the spine. Upon, the patient was positioned prone on the Risser frame, and a decompression of the lumbar spine with pedicle screw instrumentation from the ileum up to L1 was done. CONCLUSIONS (Figure 3) On 5th post-op day the patient presented with hyperthermia (39.1°C) and wound discharge. The day after, blood culture was Although not a definite indication on validity of ozone therapy for surgical infections, the results of this case report indicate a taken and the resultshowed positivity for Enterococusfaecalis. C-Protein reactivity (CPR) showed values of 16.59. The MRI new way that merits to be explored. (magnetic resonance imaging) showed diffuse inflammatory changes of the paravertebral muscles and localized collections. (Figure 4 & 5) Due to aforementioned findings, arevision surgery with debridement was done. During the surgery, loosening INTRODUCTION of the upper screws was found and an extension of the instrumentation up to D10 was performed. Multiple tissue swabs taken during the surgery confirmed the results of blood culture test. The infectious disease specialist advised the following Infection is one of the most fearsome complications in spinal surgery and has the ability to transform even a simple treatment: Ampicilin 3gr x 4 a day and Gentamicin 320 mg a day. After 15 days, and significant improvement in health procedure in a nightmare situation with high postoperative morbidity, mortality and important increase in health care conditions, the medication was changed to Augmentin 1 gr x 3 a day. On discharge from the hospital, the pa- expenses.(1) Literature reports this event to range between 0.5% and 18.8%. (2,3)This extremely wide range can bejustified tient’s blood values showed significant reduction of inflammatory markers: White Blood Cells 4.79 and CPR 3.28. by the incidence of infection occurring in different spinal surgery procedures. More complex procedures, such as deformity Upon discharge the patient continued with the antibiotic treatment and was followed closely with weekly out-patient visits. corrections and long posterior instrumentations, bear a higher risk of infection while low complexity procedures such as The surgical wound healing was not optimal with two wound fistulasstill discharging yellowish, dense, liquid. The wound microscopically assisted ones, will have low or very low infection rate.(4,5) Surgical infections have three possible sources. continued to show redness and tenderness. After almost a month from the discharge the CPR values were almost the External, environmental source, originating from surgical instruments orthe theater air,an external, biological source, taking same (3.5) as it was the wound drainage.The patient started showing severe tiredness, loss of appetite and general health place from the skin near the incision site and an internal source, arising from internal mucous membranes such as gastro- deterioration. Before proceeding with a new revision surgery, a decision was taken to try using ozone therapy for its known intestinal, genito-urinary or oro-pharingeal sites of endogenous,usually chronic, infections. In order to become clinically bactericidal effects.Oxygen-ozone mixture in gaseous form was injected inside the wound with the following modality: evident, the infection requires a sufficient bacterial load, a sufficient tissue damage and a significant lowering of immune response of the host. (6,7)The most frequent pathogens giving rise to infections are the aerobic ones, however, anaerobic The procedure was done in outpatient clinic with the patient laying in lateral decubitus. Needle cannula of 16 G was inserted microorganisms were found responsible for infections in approximately 38% of cases.The scarce information on these last inside the fistula and thesyringe suction was applied as to remove the subcutaneous liquid collection.Approximately 120 ones is a consequence of a more difficult and cost-consuming procedureto verify them. (4,6) Indeed, it seems that the milliliters (ml) o yellowish and dense liquid was removed. Then, using a 60 ml syringe, ozone-oxygen mixture in concentration combined action of aerobic and anaerobic pathogens is frequently involved in the occurrence of the surgical infection (8) of 20 micrograms of ozone / milliliter of oxy gen, was injected into the subcutaneous space. Eight syringes of gas mixture

68 69 were applied for a total of 480 ml. Then another two, 60 ml injections, were introduced deep inside the paravertebral muscles anaerobic bacteria in postoperative wound sepsis. Arch Surg 121:924-929 using a 20 G, 5 centimeter-long, needles. These procedure was repeated twice weekly for two weeks and then again once on the third week. Already from the second week (third treatment), the wound presented in significantly better conditions. The 8. Duerden BI (1994) Virulence factors in anaerobes. Clin Infect Dis 18:S253-S25 fistulas were still open but the secretion,still yellowish, was not anymore dense. There was no more redness of the wound and the backpain diminished significantly. On the last session the patient presented in much better general health conditions with 9. Fang A, Hu SS, Endres N, Bradford DS (2005) Risk Factors for Infection After Spinal Surgery. Spine 30(12):1460-1465 significantly less pain and tiredness as compared to the first session.The previously described fistulas were completely closed so we had to puncture the skin for the last session. No more discharge could be found even on suction. The skin around the 10. Olsen MA, Nepple JJ, Riew KD, Lenke L, Bridwell KH, Mayfield J, Fraser VJ (2008) Risk factors for surgical site infection wound was flat and no redness could be seen anymore. The patient repeated her blood exams for inflammatory markers a following orthopaedic spinal operations. The Journal of Bone and Joint Surgery 90(1):62-69 week after the last session that showed normal values (CPR 1, Pro-calcitonin < 0.5). During the follow up of up to a year after surgery, the patient was seen once a month and repeated blood exams, initially once a month for two months and then upon 11. Saleh K, Sonesson A, Persson B, Riesbeck K, Schmidtchen A (2011) A descriptive study of bacterial load of full-thick on 6 and 12 months were done. No clinical or laboratory signs of infection presented ever more. ness surgical wounds in dermatologic surgery. DermatolSurg37(7):1014-22

DISCUSSION 12. Turtiainen J, Hakala T, Hakkarainen T, Karhukorpi J (2014) The Impact of Surgical Wound Bacterial Colonization on the Incidence of Surgical Site Infection After Lower Limb Vascular Surgery: A Prospective Observational Study. European Journal Due to the rise of multi-resistant bacteria and a decrease of effectiveness of antibiotics, infections are becoming a serious of Vascular and Endovascular Surgery 47(4):411-417 concern in the health care profession. (26, 27) Alternative treatment methods using traditional or complementary medicine are brought back in use. Ozone makes part of the complementary medicine and is widely used for the treatment of different 13. Lineaweaver WC, Jacob S, Yan H, Zhang F (2011)Wound cultures as predictors of complications in reconstructive flap medical conditions among which the treatment of bacterial, viral and fungal infections. (28) This case illustrates a severe procedures. Ann PlastSurg 66(5):572-4 post-surgical infection treated initially with,evidence based, treatment modalities of revision surgery and prolonged regime of antibiotics.Nonetheless, only partial healing was obtained. Adding the ozone into the treatment took over the “status quo” 14. Bermond CV (1998) A Review paper: The use of ozone and associated oxidation processes in drinking water treatment. situation and switched the patient on the healing side of the process. Experimental work of different authors indicate that Wat Res 32(11):3208-3222 ozone is a highly active bactericidal substance in vitro both on vegetative as well as planktonic forms of bacteria. Seemingly, it also shows superior results in the treatment of biofilms as compared to antibiotics. (17,19,20,21,22,23,24)Ozone therapy 15. Guzel-seydim ZB, Greene AK, Seydim AC (2004) Use of ozone in the food industry. LWT-Food Science and Technology for infections seems to have similarities with hyperbaric oxygen treatment. Animal studies comparing these two modalities 34(49):453-460 in acute pancreas infectionshowed positive result for both treatments with clear prevalence for the ozone therapy. (29) Literature review found a few published articles on the use of ozone for the prevention and treatment of surgical infections 16. Shinriki N (1994) Mechanism with Ozone. REPORTS OF HOKKAIDO NATIONAL INDUSTRAL RESEARCH INSTITUTE. 61:15-36 in clinical settings. (30, 31) The authors state that ozone treatment of the infections showed promising results and indicated ozone having a possible role in prevention of surgical infections, too. Of course, a single case is not a proven demonstration 17. Hems RS, Gulabivala K, Ng Y-L, Ready D, Spratt DA (2005) An in vitro evaluation of the ability of ozone to kill a strain of of clinical benefit, however, it indicates a new complementary treatment modalityof the aforementioned conditionthat should Enterococcus faecalis. Int Endodontic Journal 38:22-29 be explored, particularly in the era of ever growing bacterial resistance to antibiotics. 18. Marrie TJ, Costerton JW (1985) Mode of growth of bacterial pathogens in chronic polymicrobial human osteomyelitis. J LITERATURE Clin Microbiology 22:924-933

1. Calderone RR, Garland DE, Capen DA, Oster H (1996) Cost of medical care for postoperative spinal infections. OrthopClin 19. Huth KC, Quirling M, Maier S, Kamereck K, Alkhayer M, Paschos E, Welsch U, Miethke T, Brand K, Hickel R(2009) North Am 27:171–182 Effectiveness of ozone against endodontopathogenic microorganisms in a root canal biofilm model. IntEndod J 42(1):3-13

2. Olsen MA, Mayfield J, Lauryssen C, Polish LB, Jones M, Vest J, Fraser VJ (2003) Risk factors for surgical site infection in 20. Al-Saadi H, Potapova I, Rochford ET, Moriarty TF, Messmer P (2015) Ozonated saline shows activity against planktonic and spinal surgery. J Neurosurg 98:149-55 biofilm growing Staphylococcus aureus in vitro: a potential irrigant for infected wounds.Int Wound JJan 14

3. Pull terGunne AF, van Laarhoven CJHM, Cohen DB (2010) Incidence of surgical site infection following adult spinal deformity 21. Bialoszewski D, Pietruczuk-Padzik A, Kalicinska A, Bocian E, Czajkowska M, Bukowska B, Tyski S (2011) Activity of surgery: an analysis of patient risk. Eur Spine J 19:982-988 ozonated water and ozone against Staphylococcus aureus and Pseudomonas aeruginosa biofilms. Medical Science Moni- tor: International Medical Journal of Experimental and Clinical Research 17(11):BR339-344 4. Picada R, Winter RB, Lonstein JE, Denis F, Pinto MR, Smith MD (2000) Postoperative deep wound infection in adults afterposterior lumbosacral spine fusion with instrumentation: incidence and management. J Spinal Disord13:42-5 22. Koyama R, Okuda K-I, Kazuhiko M, Monroe B, Yoshimitsu M (2015) Antimicrobial and Antibiofilm Effects of Ozonated Water for Prevention and Treatment of Bone and Joint Infections. Journal of St. Marianna University 6(1):1-7 5. Gaynes RP, Culver DH, Horan TC, Edwards JR, Richards C, Tolson JS (2001) Surgical site infection (SSI) rates in the United States, 1992–1998: the National Nosocomial Infections Surveillance System basic SSI risk index. Clin Infect Dis 33:S69-77 23. Tachikawa M, Tezuka M, Yamanaka K, Nakamuro K (2006) Studies on the evaluation of disinfections and removal of biofilms by ozone water using a microbial model system. Bull Med &Hyg Ozone Res, Japan 13:50–56 6. Bowler PG, Deurden BI, Armstrong DG (2001) Wound microbiology and associated approaches to wound management. CliMicrobiol Rev 14(2):244-269 24. Broadwater WT, Hoehn RC, King PH (1973) Sensitivity of three selected bacterial species to ozone. Applied Microbio logy 26:391-393 7. Raahave D, Friis-Moller A, Bjerre-Jespen K, Thiis-Knudsen J, Rasmussen LB (1986) The infective dose of aerobic and

70 71 25. Hoshi S, Sakura M, Kitagawa S, Mori K, Saitou A, Akabori Y, Murakami A (1995) Toxicity Study of Ozonized Water. Journal NEW BOUNDERIES AND APPLICATION of Shizuoka Saisei Medicine 12: 89-95 OF OZONE THERAPY 26. Ventola C (2015) The Antibiotic Resistance Crisis Part 1: Causes and Threats. P T. 40(4):77-283 Paolo Tordiglione M.D. Ph.D., Università La Sapienza Roma 27. Harold CN (1992) The Crisis in Antibiotic Resistance. Science 257:1064-1073 Ozone therapy could be considered a procedure between homeopathy and naturopathy, in fact it is characterized by the use 28. Elvis AM, Ekta JS (2011) Ozone therapy: A clinical review. J Nat SciBiol Med 2(1):66-70 of a natural molecule which stimulates and increases the natural mechanisms already present inside the organism. It also attacks and solves the noxa patogena. 29. Bulent U, Mehmet Y, Nail E, Omer C, Abdullah K, Tuncer C, Bulent K, Sukru O, Ahmet K, Ahmet G (2010) Efficacy of Hyperbaric Oxygen Therapy and Medical Ozone Therapy in Experimental Acute Necrotizing Pancreatitis. Pancreas The interaction between ozone and biological tissue activates different mechanisms: red blood cells stimulation, micro blood 39:9-15 flow and delivery stimulation, anti-inflammatory activity and immune modulation. All the capacities of this molecule make it really versatile. 30. Białoszewski D (2003) The use of the intraooperative ozone - theraphy as prophylaxis of infections in surgery of Each cell line will be stimulated by ozone producing a cytoplasmic regenerative activity as described by Prof. Bocci’s study. locomotor system with special regard to total hip plasty - a preliminary study. OrtopTraumatolRehabil 30;5(6):781-786 We believe that ozone and all the techniques which use energy, such as shock waves, transdermal electrical stimulation, ultrasound, laser therapy, hyperthermia, reach the same aim: to stimulate the cell to react. It is important for the cell to be 31. Białoszewski D, Kowalewski M (2003) Superficially, longer, intermittent ozone theraphy in the treatment of the chronic, stimulated adequately. Too much energy could cause the apoptosis of the cell. infected wounds. OrtopTraumatolRehabil 30;5(5):652-658 In this chapter I would like to underline the release of energy which happen when the triatomic bound of ozone is broken. The amount of energy released by ozone is always the same and it is always capable of stimulating the cell regeneration. We will not talk about the classical pathology cured by ozone, such as low back pain and radiculopathies.

Figure 1 Figure 2 Figure 3 We want to propose the systemic therapeutical effects of this molecule. Considering ozone as a molecule which provide energy led us to study some of its extraordinary features. The reader can understand that investigating on such a versatile gas is not easy. As medical doctors and researcher of Policlinico Umberto I, Rome, Italy, we decided to focus on the first ozone feature ever used: its microbicidal effect. Ozone is one of the most oxidizing molecule1, 10 times more powerful than chlorine. Its effects on infective agent has been known for more than a century: the first municipal water purification was made in 1906. Many theories try to explain its microbicidal effect: ozonolyisis of bacterial membrane fatty acids, oxidation of bacterial protoplasm and swab system alteration. Virus are inactivated thanks to the receptors oxidation. To understand how valiant can be its therapeuthical effect, in 2013 we decided to test the microbicidal effect of O3 on some of the most common bacteria in our intensive care units and stocked in professor Mancini’s laboratory. Results were published in the same year in the following article: “In vitro evaluation of ozone activity in recent clinical isolated bacterial strain” 2.

Ozone effect was studied on 4 different bacteria: Escherichia Coli, Pseudomonas Aeruginosa, Staphilococcus Auereus, Figure 4 Figure 5 Enerococcus Faecalis. The experiment was repeated for three times in different cultures (saline solution 0,9% NaCl, agar culture, whole blood) with the same bacterial strain (0,2 Mc Farland in 2,5 ml). We observed different results. Ozone completely removed bacterial strains in all bacterial lineages in saline solution (NaCl 0,9%).

Nothing happened to the bacterial strain in the agar culture. Results on whole blood were different: E. Coli was the most sensible to ozone effect, its bacterial strain was removed in this case too. Other bacterial strain lowered considerably. Data showed the bactericide and virus-static ozone effect in the whole blood. The different systemic and local applications of ozone showed different results compared to data obtained in vitro. This discrepancy is brilliantly explained by Sagai-Bocci’s study, which shows the ozone immunomodulating effect.

Encouraged by these results, we decided for further investigations.

We devised the following protocol “Ozone: an instrument to content and prevent intensive care unit infections”. It was approved by the Ethical Committee of Policlinico Umberto I, Rome, Italy on March 2014. The study has been led in the Neurosurgical Intensive Care Unit since May 2014 and it is still not concluded.

We recruited 70 patients randomly divided into two groups: O group and C group. The O group underwent to hygienical cure with 2 liters of ozonized water [O3 37 mcg/l], ozonized soap and ozonized oils.

72 73 Two different kind of oils were used: the low concentration one was applied on all the skin, the high concentration one was But the ozone therapeutical effects are not restricted to the ones aforementioned. In particular, we have to underline its role applied only on sore and wheals. Moreover patients underwent to 250 ml ozonized gas [O3 80 mcg/l] rectal insufflation and in all vascular, infective and regenerative pathologies. We suggest further studies particularly on interstitial cystitis, herpes, 50 ml ozonized gas [O3 40 mcg/l] bladder insufflation once every two days. herpeszoster, pre-eclampsia, and sine causa sterility. The C group underwent to the traditional hygienical cure with clorexidine and common soaps. A recent published article focused on ozone effects on bladder inflammation in animal models. It demonstrates how the O3 decreases the production of inflammatory cells and, at the same time, it doesn’t injury the mucous membrane 8-9. Ozonized water was made with bidistilled water: in fact ozone reacts with water components, the more the water is pure the Today ozone therapy is spread in many fields, the ones on which we focused are only some of its possible uses in the higher is the ozone efficacy. various branches of medicine. Definitely many other peculiarities haven’t been discovered yet. It is up to the medical doctor- Ozone insufflations were made in the rectal ampoule; the gas reacting with the rectal mucous membrane causes researcher to use in the best way a molecule that after so many years is still capable of surprising. immunomodulation, improves the aerobic flora and removes the anaerobic flora. But at the same its versatility makes necessary more studies in every field in which this molecule is used. We suggests more studies to reach a greater knowledge and confidence on ozone. Exclusion criteria were: politrauma patients, HBV, HCV, HIV, diabetes, age <18 or> 75, recovery <28 days. The following parameters were registered during all the recovery period: infective episodes (number, duration, localization, agents); therapy REFERENCES (antibiotics, duration, dosage); fever; wheals, bed sores (staging); VES; PCR; PCT; AST; ALT; blood exams. Moreover an evaluation test on patient condition was administered to the nurses. 1) Bocci, V., Valacchi, G., Corradeschi, F., and Fanetti, G. - Studies on the biological effects of ozone 8: Effects on the total antioxidant status and on interleukin - 8 production. 1998 Pubb. Mediators of Inflammation 7, 313 - 317(1998). Preliminary results based on 20 patients (10 from O group and 10 from C group) showed how ozone is efficacy in this field too. The number of fever peaks (temperature >38 °C) was less in O group than in C group.: 104 vs 154. The number of 2) P. Tordiglione, F. Morselli, I. Scarpa, G. Puggioni, C. Mancini, G. Rosa and A. Giordano, “In Vitro Evaluation of Ozone Activity ventilator associated pneumonia (VAP) and urinary tract infection (UTI) was less in O group than in C group: 4 vs 10 and 2 on Recent Clinically Isolated Bacterial Strains,” Advances in Microbiology, Vol. 4 No. 2, 2014, pp. 106-115. vs 4. Moreover in C group there was the need for a greater antibiotics administration, especially Piperacillina/Tazobactam. These data suggests that ozone could prevent from new multiresistant bacterial strain formation and could reduce the cure 3) Di Paolo N., Bocci V., Marosi G., Borrelli E., Bravi A., Bruci A., Aldinucci C., Capotondo L. (2000) Extracorporeal blood costs of intensive care patients. But the most amazing data were on bed-sores: none of group O patients was affected by oxygenation and ozonization in man. Preliminary Report. Int J Artif Organs.23: 131-41. bed-sores. Instead the incidence of bed-sores in C group was of 50%. Even if ozone rectal insufflation is a safe technique it’s efficacy depends on the individual assimilation capacity in the rectal 4) Buckley R. D., Hackney J. D., Clark K,. Posin C. (1975) Ozone and human blood. Arch. EnvironHealth. 30(1): 40-43. ampoule. The fringes imposed by the ethical committee obliged us to use this technique instead of the major-auto-hemotherapy (MAHT). 5) Bocci V., Valacchi G., Corradeschi F., Aldinucci C., Silvestri S., Paccagnini E., Gerli R. (1998) Studies on the biological effects of ozone: 7. Generation of reactive oxygen species (ROS) after exposure of human blood to ozone. J BiolRegulHomeost MAHT consists in taking a blood sample of 250-300 ml thanks to a cannula needle linked to a silicone sack. Agents.12(3): 67-75. After the blood sample is mixed in the same sack with a volume of 250-300 ml of ozone[40mcg] for 10 minutes. Ozone shows its properties reacting with plasma and extra cellular matrix (MEC) components thanks to the formation of 6) Sagai M1, Bocci V. (2011) Mechanisms of Action Involved in Ozone Therapy: Is healing induced via a mild oxidative stress? reactive oxygen species3-4-5. Med Gas Res. Dec 20;1:29. The effects on blood are achieved thanks to the vasoactive amine release, the greater flexibility of red blood cells and the improvement of blood rheological function6. 7) Clavo B, Catalá L, Pérez JL, Rodríguez V, Robaina F. (2004) Ozone therapy on cerebral blood flow: a preliminary report. Ecam Recent studies attribute to ozone many therapeutical effects: red blood cells metabolism activation, a greater release of Evidence Based Complementary and Alternative Medicine. 1:315-319. oxygen which reduces blood viscosity and increases capillary filtration; increase of LOS, ROS, IL, SOD, TNF, G6PD, GSH-Rxand catalasis; increase of INF, NFKB, NO and red blood cell ATP; eme oxygenasis activation. Moreover it 8) Emilia A. Ripoll. Treating Interstitial Cystitis & Chronic Pelvic Pain with Ozone. shows analgesic and antiinflammatory effects reducing prostaglandins and bradykinines. Last but not least ozone increases the cerebral and myocardial microcirculation. All these events caused by ozone (the ghost 9) Bayrak O1, Erturhan S, Seckiner I, Erbagci A, Ustun A, Karakok M. (2014) Chemical cystitis developed in experimental molecule) will lead the body to react to an inflammatory state. animals model: Topical effect of intravesical ozone application to bladder. Urol Ann;6(2):122-6. In our PhD we studied the effect of ozone in nervous central system focusing on blood flow and oxygen delivery because only a study in the modern literature confirmed the hypothesis of a vasodilator effect of ozone7. Data showed an increase of cerebral blood flow of 40 % which is associated to important therapeutical effects of ozone, such as: red blood cells activation, increase of oxygen, IFN and citokines, inhibition of prostaglandins and bradykinins. It changes positively the emoreological parameters in patients affected by chronic obstructive arterial disease increasing the oxygen transfer from hemoglobin; it has also an immunomodulator effect. Ozone showed its therapeuthical effect in many chronic degenerative disease (Parkinson, Alzheimer and senile dementia), on pharmacological resistant depression and on hypoacusia caused by virus or vascuolpathy. The O3, thanks to a greater release of the endothelial nitric oxide, causes an increase of cerebral blood flow in medial cerebral artery (MCA) and common carotid (CC) as described by Clavo et al.

Our scientific curiosity led us to create a new protocol currently under decision by the Ethical Committee of Policlinico Umberto I. The aim is to study ozone capacity to enhance the post-operative cognitive recovery in the big aged. Post-operative cognitive dysfunction in old patients represents a really common problem which causes a great loss of money to the national sanitary system.

74 75 76 77 78 E' VIETATA LA RIPRODUZIONE CON QUALSIASI MEZZO ANCHE PARZIALE DELLA SEGUENTE DISPENSA E PER TUTTI I PAESI.