• Biological models and preclinical studies:

Protective effects of ozone oxidative preconditioning against liver ischemia/reperfusion injury in rats.
León O.S. and Ajamiech H. (Cuba)

Liver transplantation is a recommended therapy for end-stage liver disease and the demand for donor organs has surpassed the supply resulting in the death of thousands of patients. The damage by hepatic ischemia/reperfusion (I/R) is considered as the main responsible of organic failure after transplantation. On the other hand, 30% of transplants still fail by acute or chronic rejection within five years. The mechanisms of acute liver damage following I/R are thought to involve a complex interaction of immediate cellular damage caused by different mediators. In the process of prolonged ischemia, a major caused of primary liver dysfunction in donor grafts results from the generation of reactive oxygen species (ROS) which are associated to pathogenesis of inflammatory disorders including I/R injury. From the mechanism of Ozone Oxidative Preconditioning (OzoneOP) invoked by our research group, it is shown the protective effects of ozone on important enzymes as Xanthine Deshidrogenase-Xanthine Oxidase and Superoxide Dismutase: CuZn/SOD and Mn/SOD, among others). It is demonstrated that OzoneOP regulate the Nitric Oxide levels and other mediators which take part in the cellular redox balance. OzoneOP participation in the novo protein synthesis as protector mechanism for morphological integrity of the liver in the preconditionated groups is shown. The protection conferred by ozone treatment indicates that this therapeutic alternative may be successful against hepatic damage mediated by I/R associated to liver transplantation.

Protection by ozone preconditioning is mediated by antioxidant system in cisplatin-induced nephrotoxicity in rats.
González R., Borrego A., Zamora Z., Romay Ch., Menéndez S., Hernández F., Montero T. and Rojas E. (Cuba)

Ozone oxidative preconditioning is a prophylactic approach, which favors the antioxidant-prooxidant balance for preservation of cell redox state by increasing antioxidant endogenous systems in various in vivo and in vitro experimental models. The antitumor drug cisplatin is used for the treatment of a wide variety of cancers. The major side effect, nephrotoxicity, is dose-limiting and occurs either acutely or after repeated treatments. Taking into account that several lines of evidence suggest that reactive oxygen species or free radicals are involved in cisplatin nephrotoxicity; we decided to analyze the protective role of ozone oxidative preconditioning against cisplatin-induced nephrotoxicity. Male Sprague Dawley rats were pretreated with fifteen intra-rectal applications of ozone/oxygen mixture at 0.36, 0.72, 1,1, 1.8 and 2.5 mg/kg before cisplatin intraperitoneal injection (6 mg/kg). Serum and kidneys were collected and analyzed five days after cisplatin treatment. Creatinine was measured in serum and activity of antioxidant enzymes and thiobarbituric acid reactive substances (TBARS) and glutathione content were analyzed in renal homogenate. Ozone pretreatment prevented the increase in serum creatinine levels, the glutathione depletion and the inhibition of superoxide dismutase, catalase and glutathione peroxidase activities induced by cisplatin in rat kidney. Also, the renal content of thiobarbituric reactive substances was decreased by ozone therapy. The protective effects of ozone were dose dependent. Intra-rectal ozone therapy effectively prevented the renal antioxidant unbalance induced by cisplatin treatment.

Intrarectal applications of ozone/oxygen mixture after cisplatin treatment attenuate its nephrotoxicity.
Romay Ch., Borrego A., Zamora Z., González R., Menéndez S., Montero T., Hernández F.,and Rojas E. (Cuba)

Ozone therapy has become an useful treatment for many pathological processes, especially those in which the damage mediated by reactive oxygen species or free radicals is involved, because it favors the antioxidant-prooxidant balance for preservation of cell redox state by increasing antioxidant endogenous systems. Cisplatin is a highly effective anticancer drug against a wide variety of cancers, but its major side effect, nephrotoxicity, limits its clinical use. Taking into account that several lines of evidence suggest that reactive oxygen species or free radicals are involved in the nephrotoxicity of cisplatin; we decided to analyze the effect of the ozone/oxygen mixture after cisplatin application. Male Sprague Dawley rats were treated with five intra-rectal applications of ozone/oxygen mixture at 0.36, 1.1 and 1.8 mg/kg after cisplatin intraperitoneal injection (6 mg/kg). Serum and kidneys were collected and analyzed five days after cisplatin treatment. Creatinine was measured in serum and activity of antioxidant enzymes and thiobarbituric acid reactive substances (TBARS) and glutathione content were analyzed in renal homogenate. Ozone treatment diminished the increase in serum creatinine levels, the glutathione depletion and the inhibition of superoxide dismutase, catalase and glutathione peroxidase activities induced by cisplatin in the rat kidney. Also, the renal content of thiobarbituric reactive substances was decreased by ozone/oxygen mixture applied after cisplatin. The protective effects of ozone were dose dependent. Intra-rectal applications of ozone/oxygen mixture attenuated the renal antioxidant unbalance induced by cisplatin treatment by stimulating the antioxidant endogenous system.

Molecular bases associated to the protective effect of ozone on calcium homeostasis in a model of hepatotoxicity induced by carbon tetrachloride.
López R. and León O.S. (Cuba)

Ozone therapy has been used with therapeutic effectiveness, in the treatment of diverse pathologies of different origins. In spite of the effectiveness of this treatment, its clinical use remains controversial due to the scarce knowledge of its pharmacodynamic mechanism of action. The aim of this study is to evaluate the ozone (O₃/O₂) protective effects on calcium homeostasis, using a model of hepatotoxicity induced by carbon tetrachloride (CCl₄) in rats. Calcium homeostasis is an important event that is seriously affected by hepatotoxic mechanisms. Four groups of rats were classified as follows: 1-negative control, using intraperitoneal sunflower oil; 2-positive control, using CCl₄; 3-O₃/O₂ pretreatment, via rectal insufflation (15 sessions) and after it, CCl₄; 4-oxygen, as group 3 but using oxygen only. The results demonstrated that the O₃/O₂ is able to maintain the calcium homeostasis and to revert the hepatotoxic actions of CCl₄. A protective effect was obtained on calcium dependent ATPase, being one of the explanations that justified the maintenance of the basal concentrations of this cation. Oxygen group had a similar behaviour as positive control group. Ozone was able to maintain the intracelular calcium homeostasis and to preserve the calcium dependent ATPase in cytoplasm and in hepatic mitochondria. The results demonstrated that the treatment with O₃/O₂ regulated the lytic activity of phospholipase A2 and controlled the generation of reactive oxygen species produced by the CCl₄ oxidative challenger, decreasing lipid peroxidation and protease activation and increasing reduced glutathione. Ozone was able to counteract the deleterious effect induced by the hepatotoxin.

Ozone therapy in a cancer model (VX2 carcinoma: head and abdomen) in rabbits. A pilot study.
Schulz S., Sapundzhiev N., Dünne A., Ramaswamy A., Bette M., Nüsing R., Moll R. and Werner J.A. (Germany)

Recently, Babior et al 2003 detected that ozone is endogenously produced in the human body which opened many questions about the role of ozone in biological systems. Thus exogenously applied ozone probably exhibits beneficial, non-beneficial or detrimental effects under physiological or pathophysiological conditions. We here report for the first time that exogenously applied ozone (O₃/O₂-pneumoperitoneum) exhibits a strong anticarcinogenic and antimetastasing effect on primary tumor cell inoculations (VX2 tumor cells) VX2-tumor and on the lymphogenic metastatic spread in rabbits. This treatment was without any visible side effects such as diarrhoe etc. The VX2-carcinoma model is a highly malignant Shope virus-induced squamous cell carcinoma with a high mortality rate (approx. 100 %) within 100 days of observation. We used this established aggressive tumor model with lymphogenic metastatic spread (bi-auricular model) of the primary local tumor and a local spread of peritoneal disseminated tumor cells (tumor nodules). In the ear model (n=5 rabbits), from initiated 8 solid aggressive primary tumors, 4 tumors dissappeared after a combined ozone treatment (local and systemic application for 5 days). Fifty percent of the ozone treated animals survived and they were tumor-free for > 500 days. All results from the macroscopic primary tumor size development, the pathohistolgy of the head and neck lymph nodes and lung metastasis after the tumor cell inoculations are presented in details. Furthermore, we present the effect of ozone treatment on the dissemination of tumor cells in the abdomen of rabbits. We think that ozone might be benefical agent in the treatment of these malignant tumors.

Experimental research of biological effects of ozone, radiation and chitosan in cancer animals.
Scherbatyuk T., Moskovceva O., Frolov V. (Russia)

The experiment was done on 580 white male rats. The neoplasia was modelled through Lymphosarcoma clone inoculation. The animals were subject to following actions: mono-radial influence, intra-abdominally oxygen influence and gamma-irradiation; intra-abdominally, ozone influence and gamma-irradiation; chitosan per se influence. Ozonated physiological saline was used at an ozone concentration of 400 µg/L. State of free-radicals and antioxidant processes; glycometabolism; endotoxemia; phagocytosis; electrophoretic motility of erythrocytes and aggregation of erythrocytes were determined in blood and homogenates of tumour, lymphaden, and spleen tissues. Morphological and histological changes in tissues were observed. The new method (Shabalin, Shatichina, 1999) of morphological analysis of dehydrated blood plasma has been shown to be useful as a method of controlling efficacy of treatment in rats with limphosarcoma. The method being high sensitive, simple in performance. In the model of experimental oncology laboratory rats plus limphosarcoma it is shown, that ozone:

• In a complex of radial therapies increases antitumoral effect.
• In a complex of chitosan increases the antioxidant system of rats.

However in experiments concentration of ozone which causes a metastasis is revealed. Mechanisms of the effects are discussed.

Ozone therapy in the neuroprotection: new findings in experimental brain ischemia.
García-Salman J.D., García M., Cuba A., Coro R.M., Pérez-Saad H. and Menéndez S. (Cuba)

The brain can be considered as a main target organ for active oxygen species. It is the highest oxygen-demanding organ, has a high content of unsaturated fatty acids, a poor antioxidant defense system and a high ferrum ion content. During brain ischemia, free radical reactions increase. There are some evidences suggesting that SOD activity reduces edema, mortality and infarct volumes. Other findings found links between hydrogen peroxide and selective gene expression, differentiation and neuronal plasticity. On the other hand, ozone therapy is based in the generation of active metabolites as, ozonides, hydroperoxides, hydrogen peroxide and aldehydes. Then, these products could be carried by body fluids to the target tissue and exert therapeutic effects. It has been suggested that maximal effects be obtained after a period of preconditioning. In that sense, we treated Mongolian gerbils with intrarectal ozone (1 mg/Kg, 15 sessions) for three weeks before ligation of the right carotid artery. Ozone treatment prevented impairment of spontaneous exploratory activity, observed 7 days after ligation in oxygen-treated gerbils. In these animals a significant damage in CA1 pyramidal neurons of the right hippocampus was found, compared with the contralateral hemisphere. In another experiment, the spontaneous exploratory activity before and 7 days after 5 min bilateral carotid occlusion was tested. Ozone was administered in the six subsequent days of reperfusion. The results showed that ozone treatment also prevents impairments of the spontaneous exploratory activity. Also, the results showed morphological and functional evidences of brain protection using ozone. These findings suggest that ozone active metabolites - particularly hydrogen peroxide - are protecting memory and learning-related synapses through improving survival of brain cells. These findings could contribute to amplify the spectrum of ozone in the clinical therapeutics.

Multiple Organ Failure evolution in an experimental model of burnt mice treated with ozone therapy, Aloe B and Epidermal Growth Factor.
Montero T., Hurtado J., Menéndez S. and Berlanga J. (Cuba)

Burn disease is related with sepsis, generalized systemic inflammation syndrome, multiple organ dysfunction syndrome, shock, trauma, etc. Taking into account the therapeutical properties of ozone such as: germicide, immune modulator, antioxidant defense system activator, that of Aloe b as immune stimulator and that of Epidermal Growth Factor (EGF) as a cytoprotector preventing or ameliorating the systemic inflammatory response, a study of the multiple organ failure evolution, in an experimental model of critically burnt mice, using these medications was performed. A paravertebral dry burn model, with 11 % of burn area, in balb/c female mice of ± 20 g, previously anesthetized with ketamine intraperitoneal (ip) was performed. Animals were divided in 6 groups of 24 animals each: group 1, lesion and hydration with 1 mL of intraperitoneal (ip) saline solution 0.9 % after the burn; group 2, as group 1 but maintaining the hydration daily up to 14 days; group 3, hydration after the burn and then treated ip with 15 treatments of aloe b (0.15 mg/Kg in the first week and 0.10 mg/Kg in the second week); group 4, hydration after the burn and then treated with 15 treatments of ozone by rectal administration (ozone concentration - 37 mg/L, volume - 0.9 mL); group 5, hydration after the burn and treated with aloe b and ozone; and group 6, hydration after the burn and then treated ip with EGF (500 µg/kg, in one dose). Euthanasia, by immersion in liquid nitrogen, at 1, 3, 7 and 14 days (with n = 6), was done. The extracted organs (liver, lungs, heart, kidneys, brain) were studied by conventional morphometry. The results showed that in groups 1, 2, 3, 4, 5 and 6 died 75, 50, 54.2, 37.5, 100 and 16.7 % of the animals. Aloe b and ozone produced a lethal combination. Respect to vitality, groups 4 and 6 presented 75 and 79.6 % of the animals, respectively, with an active evolution in comparison with the other groups (0 %). The histological studies demonstrated a higher activity of the lymphatic tissue in the spleen and less liver and kidney damage in the ozone group, respect to the other groups. Groups 1 and 2 presented the higher manifestation of the multiple organ injury. The best results were obtained using Ozone and EGF, immediately after the burn. Both medications were capable to diminish mortality, to upregulate the body response and to decrease or eliminate the multiple organ failure.

Effects of different gases on bacterial growth. Experimental study in vitro.
Sad M.M., Molla O.L., Altenfelder R. and Pacheco A.M. (Brazil)

Insufflation of gases in the peritoneal cavity is the preferred technique to distend the abdominal wall during a video-surgery. This study aimed at creating an experimental model in vitro that could analyze the direct effect of some gases used in laparoscopy over bacterial growth, as well as comparing them with the effect of ozone. Thus, we performed some assays by displaying fifteen slide cultures inoculated with one of three different strains of bacteria: Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The concentration of bacteria used was of 10⁴ CFU/mL, the exposure time was of one hour, the flow of gases and the pressure were kept constant at 2L/mm and 11 mmHg, respectively. The gases used were carbon dioxide and helium, at the concentration of 99.99 % for both, the ozone at 0.4 % and the compressed air as control. Also a group of four plates of each bacterium was left without exposure to the gases as a control of the dilution technique. The examination was performed after incubation of twenty-four hours in incubator at 37 ^oC. The results separated the gases into two distinct groups: the first, containing only ozone, which promoted sterilization of the slide culture; and the second one, with the other gases, where no statistically meaningful variation between the slide cultures exposed to them and the unexposed control were recorded. Thus, we concluded that the exponent model devised is practical and efficient; that the gases currently used do not have direct effect upon the bacteria tested and that ozone has a much more effective bactericidal effect than the other gases studied.

Trial assessment of the use of ozonized oxygen in the peritoneal cavity of rats. Gasometric and histopathological analysis.
Molla O.L., Altenfelder R., Bonizzia A. and Pacheco A.M. (Brazil)

Ozone is a powerful oxidant with a microbicidal action when used in vitro, however, its usage in the treatment of peritonitis has not been studied as much as it should. The experiment at issue was carried out with the purpose of analyzing the effects of the use of ozone gas on blood gases and histopathological changes of the organs of the abdominal cavities with the performance of pneumoperitoneum in rats. The study focused 60 rats divided into three groups, in that group I (control), group II in which it was performed pneumoperitoneum with a 30 minute continuous ozonized oxygen infusion and immediate sacrifice immediately afterwards; and group III followed by sacrifice on the 21^st post-surgical day. The ozone concentration used was 8.55 mg of ozone per rat with a constant intra-abdominal pressure of 5 mmHg. The gasometric analysis was carried out by means of the collection of arterial blood from the carotid artery at the beginning and end of the experiment. Histopathological examinations of liver, spleen and intestine were performed by means of microscope examination. The results showed no meaningful differences in arterial gases and in the histopathological analysis of the tissues examined. No deaths were recorded among the animals. Thus, we can conclude that the use of ozonized oxygen the peritoneal cavity through pneumoperitoneum may be an alternative in the treatment of infections in the abdominal cavity without causing lesion on the abdominal organs by direct contact and for nor altering the pH, PaO₂, PaCO₂ for a 30 minute period.

Ozone treatment stimulates prostacyclin synthesis in rodents: a possible hint to the mechanism of ozone action?
Nüsing R.M. and Schulz S. (Germany)

Ozone is known as a powerful oxidizing agent which is produced mainly in chemical but also in biological systems. Under certain clinical conditions application of ozone exerts benefical effects described in empirical and/or complementary alternative medicine. However, mechanism of therapeutic ozone action is not understood. We studied the effect of bolus and repetitive intraperitoneal insufflation of ozonized oxygen in rodents, such as mouse, rat and rabbit. Specific focus was set on the endogenous prostanoid system in these animals. Prostanoids, such as prostaglandin (PG) E₂, prostacyclin or thromboxane (TX) are fatty acid mediators deriving from cyclooxygenase pathway of arachidonic acid which exert pleiotropic effects within the body. Upon ozone exposition at a concentration of 50 mg/mL we observed a 3-8 fold increase in renal excretion of 6-keto-PGF 1 a the measurable product of prostacyclin and a 5-10 fold increase of its metabolite 2,3-dinor-6-keto-PGF_1a. In contrast, excretion rates for other prostanoids such as PGE₂ or TXB₂ were unaffected by ozone application. Furthermore, isoprostanes which are regarded as endogenous biolological indicators of oxidative stress were unaffected. The stimulative effect of ozone on prostacyclin synthesis was observed in mice, rats and rabbits. Prostacyclin is an important vasodilator in the vessel system. We postulate that specific stimulation of endogenous systemic prostacyclin synthesis contributes to the beneficial effect of ozone treatment in certain pathological conditions.

Pre-conditioning of rats with intraperitoneally applied O₃/O₂ gas mixture enhances the efficacy of Piperacillin/Tazobactam in lethal peritonitis.
Bette M., Zamora Z., Mutters R., Schlimme S., Menéndez S. and Schulz S. (Germany)

Repeated insufflation of ozonized oxygen into the peritoneum (O₂/O₃-pneumoperitoneum, (O₂/O₃-PP)) of rats was found to reduce the lethality of a bacterial-induced severe peritonitis. Here we evaluate the prophylactic effect of O₂/O₃-PP combined with Piperacillin/Tazobactam (Pip/Tazo) in the septic shock model of Wistar rats. To analyse the effect of Pip/Tazo on the immune system the pro-inflammatory cytokine (IL-lb, IL-2, and TNFa) gene expression was also determined in different haematopoetic organs. Sepsis was induced by a single intraperitoneal injection of fecal material derived from donor Wistar rats. Application of Pip/Tazo alone at our dosage schedule of 65 mg/kg did not prevent mortality. To c/ndita. rats we pre-treated.

The O₃/O₂ pneumoperitoneum and the intraabdominal pressure in rats and rabbits.
Schulz S., Bette M. and Nüsing R. (Germany)

Insufflated and desufflated gases into the peritoneum, such as CO₂, helium or other inert gases are important for diagnostic laparoscopy, but they are still interesting for its possible unwanted time-dependent pressure-induced influences on abdominal infection/inflammation and cancer in patients. Therefore, some experimental research is still demanded to find out more about the role of different gases with various qualities. In rats and rabbits we used a gas mixture consisting of ozone and oxygen, which has not been used for laparoscopy because of its risk of combustion. We measured intraperitoneal pressures of the insufflated gas mixture of ozonized oxygen with different ozone concentrations and different gas volumes (mL/kg rat or rabbit) after single and/or repetitive insufflations (without desufflation), calculated amount of absorption and correlated these values with hematological data.

Protective effects of ozone oxidative preconditioning against liver ischemia/reperfusion injury in rats.
León O.S. and Ajamiech H. (Cuba)

Liver transplantation is a recommended therapy for end-stage liver disease and the demand for donor organs has surpassed the supply resulting in the death of thousands of patients. The damage by hepatic ischemia/reperfusion (I/R) is considered as the main responsible of organic failure after transplantation. On the other hand, 30 % of transplants still fail by acute or chronic rejection within five years. The mechanisms of acute liver damage following I/R are thought to involve a complex interaction of immediate cellular damage caused by different mediators. In the process of prolonged ischemia, a major caused of primary liver dysfunction in donor grafts results from the generation of reactive oxygen species (ROS) which are associated to pathogenesis of inflammatory disorders including I/R injury. From the mechanism of Ozone Oxidative Preconditioning (OzoneOP) invoked by our research group, it is shown the protective effects of ozone on important enzymes as Xanthine Deshidrogenase-Xanthine Oxidase and Superoxide Dismutase: CuZn/SOD and Mn/SOD, among others). It is demonstrated that OzoneOP regulate the Nitric Oxide levels and other mediators which take part in the cellular redox balance. OzoneOP participation in the novo protein synthesis as protector mechanism for morphological integrity of the liver in the preconditionated groups is shown. The protection conferred by ozone treatment indicates that this therapeutic alternative may be successful against hepatic damage mediated by I/R associated to liver transplantation.

Ozone oxidative preconditioning inhibits TNF-a release during endotoxic shock.
Zamora Z., Borrego A., López O.Y., Delgado R. Menéndez S., Schulz S. and Hernández F. (Cuba)

Ozone oxidative preconditioning is a prophylactic approach, which favors the antioxidant-prooxidant balance for preservation of cell redox state by increasing antioxidant endogenous systems in both in vivo and in vitro experimental models. The aim of this study was to analyze the effect of ozone oxidative preconditioning on the level of Tumor Necrosis Factor-a (TNF-a) in the serum of mice treated with lipopolysaccaride (LPS). Pretreatment with ozone/oxygen gaseous mixture was administered intraperitoneally (0.2, 0.4 and 1.2 mg/kg) or by rectal application (0.2 and 0.4 mg/kg) once daily, during five days before LPS (0.1 mg/kg, intraperitoneal). TNF-a was measured by cytotoxicity on L-929 cells. One hour after LPS injection there was a significantly mean increase of the TNF-a in mouse serum. Ozone/oxygen gaseous mixture reduced serum TNF-a levels in a dose-dependent manner. Statistically significant decreases in TNF-a levels after LPS injection were observed either with ozone intraperitoneal applications at 0.2 (78 %), 0.4 (98 %) and 1.2 (99 %) mg/kg or by rectal application at 0.2 (46 %) and 0.4 (87 %) mg/kg. These results showed that oxidative preconditioning with ozone has inhibitory effects on TNF-a production of mice.

Morphological study of different tissues of rats treated with intracardiac ozone therapy.
González L., Gómez H., Barber E., Del Río S., Banasco J., Ugarte C. and Alfonso J.P. (Cuba)

Ozone is used experimentally as a contrast media in Digital Subtraction Angiography, substituting the iodine in experimental models in dog, in order to visualize the abdominal aorta and its branches. For the introduction of this gas as a contrast media it is necessary to demonstrate that ozone is innocuous. A morphological study of different tissues of rats treated with intracardiac ozone therapy was done, in order to evaluate the presence or not of side effects. For this study, 18 males Wistar rats was used (300 - 350 g and of four to eight months age) and divided at random into 3 groups: negative control; positive control, rats treated with intracardic oxygen and ozone group, rats treated with intracardiac ozone at a concentration of 30 mg/L. Surgical procedure: an abdominal incision was done, previous anesthesia, and the aorta was catheterizated (the catheter was fixed 1 cm above the renal arteries). Ozone and oxygen (0.5 mL) were introduced slowly (during 15 minutes) through the catheter. After 1:30 h a cardiac puncture was done and animals were sacrificed. Liver, spleen and kidney samples were taken for functional studies. The samples were processed according to conventional techniques for light and electronic microscopy. By light microscopy, no significant alterations in none of the three organs studied were observed. By electronic microscopy, a slight fibrosis periportal (in 1 rat) and an increase of apoptosis (in 1 rat) were observed in the liver samples. The spleen and kidney samples presented normal structure.

Ozone therapy: a new option in Veterinary Medicine.
Camps A.M. and Elías-Calles B. (Cuba)

The effect of ozonized sunflower oil (OLEOZON^®) on tissue wounds performed by castration of Yorkshire race pigs was studied. The experiment was done after performing a wound in the scrotal region of fifteen pigs with 90 days old and sixty three pounds weight. They were divided into three groups (A, B and C) of five animals each. Group A - animals treated with placebo, once a day, topically, during five days. Group B - animals treated with OLEOZON^®, once a day, topically, during five days. Group C - animals treated with OLEOZON^®, twice a day, topically, during five days. The results showed an important positive effect of OLEOZON^® on wound healings, achieving the best results in group C in which the wound scarred more rapidly than in groups B and A, with significant differences (p < 0.05). Our data indicate that the topic application of OLEOZON^® has a cicatrization and regenerating effects on dermic wounds in pigs.

Ozone therapy effects by rectal insufflation in a model of renal failure.
Calunga J.L., Del Río S., Zamora Z., Barber E., Menéndez S., Borrego A., Concepción A. and Bacallao J. (Cuba)

Chronic renal failure (CRF) represents a world health problem. The aim of this paper is to evaluate the effect of ozone therapy, in the renal function and morphology, in an experimental model of CRF. Rats were divided into 3 groups: 1-negative control, rats without any treatment; 2-positive control, rats submitted to 5/6 reduction of the total renal mass (right kidney nephrectomy), with 2 branches of the renal arteria, of the left kidney, clamped); 3-ozone, as group 2, but receiving, rectally, 15 sessions of ozone (0.5 mg/kg), after the damage (partial nephrectomy). Plasmatic clearance of p-amino-hippurate and inulin, in order to know the renal plasma flow (RPF) and the glomerular filtration rate (GFR), respectively, were determinate. Also, plasmatic creatinine, protein excretion in 24 hours and systolic arterial pressure (SAP) were measured in all groups. The results demonstrated that RPF, GFR and SAP figures, in the animals treated with ozone, had similar behavior that group 1, however in group 2, RPF and GFR were decrease and SAP, increase. In relation to protein excretion and plasmatic creatinine, group 3 presented increased figures in comparison to negative control, but with significant decreased figures respect to positive control group. Histological study demonstrated that the animals treated with ozone presented less number of glomerular collapse and tubule degeneration, in comparison with positive control group. In this animal model of CRF, rectal administrations of ozone produced a delay in the advance of the disease, protecting the kidneys against the deleterious effects present in the CRF.

Ozone/oxygen mixture modifies the subcellular redistribution of Bax/Bcl-X_L proteins in renal tissue from rats treated with cisplatin.
Borrego A., Zamora Z., González R., Romay Ch., Menéndez S., Hernández F., Berlanga J. and Montero T. (Cuba)

Cisplatin is a potent antitumor compound that is widely used for the treatment of many malignancies. An important side effect of cisplatin is nephrotoxicity. Recently, it was suggested that the subcellular redistribution of Bax is a critical event in the apoptosis induced by cisplatin. We have demonstrated that protection and recovery with ozone/oxygen mixture from cisplatin induced renal damage is related with a significant increase in the antioxidant system in renal tissue. This study was undertaken to examine the subcellular expression pattern of Bax and Bcl-X_L proteins in renal tissue. A group of rats were pretreated with fifteen intra-rectal applications of ozone/oxygen (1.1 mg/kg) before intraperitoneal injection of cisplatin (6 mg/kg). Another group was treated with five intra-rectal applications of ozone/oxygen mixture after cisplatin. Serum and kidneys were extracted and analyzed five days after cisplatin treatment for creatinine in serum and subcellular distribution of Bax and Bcl-X_L in renal tissue by immunohistochemistry. Ozone pretreatment prevented the increase in serum creatinine levels and completely inhibited the acute tubular necrosis induced by cisplatin en renal tissue. This pretreatment diminished the cellular Bax/Bcl-X_L ratio in cisplatin treated kidneys and induced an increase in nuclear expression of Bcl-X_L. Ozone treatment after cisplatin diminished the increase in serum creatinine levels and the renal necrosis. Ozone treatment induced a lesser decrease of the Bax/Bcl-X_L ratio in cisplatin treated kidneys. In this model, intra-rectal ozone therapy induced a significant protection and recovery from cisplatin induced acute renal failure by improvement of the cytoprotective pathways mediated by Bcl-X_L.

Rectal applications of ozone/oxygen mixture increase the antibody response of Hepatitis B virus to greater surface antigen in mice.
Borrego A., Zamora Z., Menéndez S., López L. and Hernández F. (Cuba)

Ozone therapy has become an useful therapy for the treatment of many pathological processes such as hepatitis, probably due to the viral inactivation by generated endoperoxides, as well as stimulation of cytokines release. Taking into account the immunomodulator effects of ozone therapy and the previous reports of its beneficial properties, we decided to study the effects of the ozone/oxygen mixture on antibodies response to hepatitis B related antigen. Balb C mice were treated with fifteen intra-rectal applications of ozone/oxygen at 0.25, 0.8, 2.2 and 4.4 mg/kg during intraperitoneal management of the greater surface antigen hepatitis B virus by a 0-7-14 days scheme at a dose of 2 µg per animal. Blood extractions were performed at 0-7-21 days by retroorbital punction. A sandwich direct enzyme linked immune sorbent assay of double antigen was performed in order to measure the specific antibody response. Ozone/oxygen applications induce a significant increase (p< 0.05) in antibody response to the immunogen. The stimulant effect of ozone was dose-dependent, but 2,2 mg/kg of ozone provoked the greater stimulation of the antibody response on day 21. Nevertheless, we have not found out a significant decrease in antibody response of animals treated with 4.4 mg/kg of ozone. Stimulant effect of ozone/oxygen mixture on the antibody response to greater surface antigen of Hepatitis B virus was demonstrated. It was corroborated that ozone/oxygen mixture can be useful on the recovery of B Hepatitis infection by increasing the antibody response to the virus, which favors the inactivation and elimination of it.

Ozone therapy in an experimental model of adriamicyn induced toxic glomerulonephritis.
Calunga J.L., García M., Barber E., Menéndez S., Chaple M. and Merino N. (Cuba)

The experimental glomerulonephritis is cause of renal failure. It is characterized by a progressive damage of renal function leak to renal insufficiency. There are different schemes of treatment for this disease, nevertheless these are expensives and can produce adverse side effects, as Cushing's syndromes and inmunosupression, affecting the quality patient's life. That's why a research about new therapies is needed in order to stop the progressive kidney damage, and therefore increase the quality of life in these patients. Taking into account the ozone biological effects such as, increase in oxygen metabolism, inmuno system modulation and the increase of the antioxidant defense system, an evaluation of the ozone therapy efficacy to reduce kidney injuries in glomerulonephritis disease was performed. Forty female Wistar rats, of 200 g weight, were divided into four groups: 1-control; 2-positive control, which received iv adriamicine during an evolutive period of ten weeks, 3 and 4-experimental groups using an ozone dose of 0.3 mg per kg and 1.1 mg per kg, respectively. These two last groups received adiamicine as the positive control group, but they were treated daily with ozone by rectal insufflation at differents doses after the renal damage appeared. The renal function tests were determinated by proteinuria, systolic blood pressure, 24 hours diuresis and the weight evolution. The results showed that the ozone therapy effect was dose depending, being 0.3 mg per kg the best protective dose; 1.1 mg per kg hadn't a protected effect in this study.

Ozone oxidative precondicioning in the treatment of the hepatotoxicity induced by bacterial lipopolysaccharide.
Ferrer Y., López R. and Hernández I. (Cuba)

The bacterial lipopolysaccharide (LPS) or endotoxin iduces the oxidative damage in several organs due to the increment in the production of Reactive Oxygen Species (ROS) with the increase in lipid peroxidation. LPS also stimulates the production of nitric oxide, that can react with superoxide anion, with the formation of peroxinitrite, responsible of the oxidation of sulfhydryl groups and the generation of hydroxil anion. It has been proved that ozone, by means of a mechanism of oxidative preconditioning, stimulates the antioxidant defense system, protecting against the ROS and the damage in liver cells mediated by carbon tetrachloride, an agent whose toxic mechanism involves the formation of ROS. In this study we evaluate the ozone preconditioning effect in the hepatotoxicity model induced by LPS. Male Sprague Dawley rats (n = 24) were divided at random in three experimental groups: negative control, ozone plus LPS and LPS. The ozone dose was 1 mg/Kg by rectal insufflation (15 sessions) and 5 mg/Kg intraperitoneal of LPS. Determinations of malondialdehyde (MDA), reduced glutathione (GSH), catalase, superoxide dismutase, total proteins and calcium were carried out. The results demonstrated that ozone decreased the figures of free calcium and MDA and increases the antioxidant enzymes. Ozone oxidative preconditioning was able to protect the hepatic cells against the damage induced by the endotoxin.

The VX2 carcinoma in New Zealand white rabbits - an animal model to investigate lymphogenic metastatic spread in head and neck cancer.
Schulz S., Dünne A.A., Ramaswamy R. and Werner J.A.(Germany)

The aim of this study was to examine the auricular VX2 carcinoma of the New Zealand white rabbit as an animal model for human squamous cell carcinomas of the head and neck region (HNSCC), since both tumors tend to metastasize lymphatically, leading to early lymph node and subsequent distant metastasis. VX2 carcinomas were implanted into both ears of 22 New Zealand white rabbits. The animals were sacrificed at day 7, 14, 21, 28 or 32 after tumor implantation, followed by a detailed histopathological examination of their head and neck lymph nodes. At day 7 after tumor implantation 25 % of the animals had metastases in the parotid lymph node, which is the first draining lymph node of the tumor region. This number was rising to 87.5 % at day 28. At this time 12.5 % of all animals also had an additional metastasis in the second echelon node. 32 days after tumor implantation hemangiosis carcinomatosa was observed for the first time within the lung vessels. Frequent reproducible initial metastatic spread into the first and second draining lymph node could be demonstrated for the auricular VX2 carcinoma of the New Zealand white rabbit, followed by hematogenious spread after more than 4 weeks of tumor growth. The VX2 carcinoma therefore appears to be a highly suitable animal model for studying the sentinel node concept in the context of human HNSCC.