Siegfried Schulz

Effects of Ozone Oxidative Preconditioning on TNF-α Release and Antioxidant-Prooxidant Intracellular Balance in Mice During Endotoxic Shock

Ozone oxidative preconditioning is a prophylactic approach, which favors the antioxidant-prooxidant balance for preservation of cell redox state by the increase of antioxidant endogenous systems in both in vivo and in vitro experimental models. Our aim is to analyze the effect of ozone oxidative preconditioning on serum TNF-α levels and as a modulator of oxidative stress on hepatic tissue in endotoxic shock model (mice treated with lipopolysaccharide (LPS)). Ozone/oxygen gaseous mixture which was administered intraperitoneally (0.2, 0.4, and 1.2 mg/kg) once daily for five days before LPS (0.1 mg/kg, intraperitoneal). TNF-α was measured by cytotoxicity on L-929 cells. Biochemical parameters such as thiobarbituric acid reactive substances (TBARS), enzymatic activity of catalase, glutathione peroxidase, and glutathione-S transferase were measured in hepatic tissue. One hour after LPS injection there was a significant increase in TNF-α levels in mouse serum. Ozone/oxygen gaseous mixture reduced serum TNF-α levels in a dose-dependent manner. Statistically significant decreases in TNF-α levels after LPS injection were observed in mice pretreated with ozone intraperitoneal applications at 0.2 (78%), 0.4 (98%), and 1.2 (99%). Also a significant increase in TBARS content was observed in the hepatic tissue of LPS-treated mice, whereas enzymatic activity of glutathion-S transferase and glutathione peroxidase was decreased. However in ozone-treated animals a significant decrease in TBARS content was appreciated as well as an increase in the activity of antioxidant enzymes. These results indicate that ozone oxidative preconditioning exerts inhibitory effects on TNF-α production and on the other hand it exerts influence on the antioxidant-prooxidant balance for preservation of cell redox state by the increase of endogenous antioxidant systems.

Preconditioning with ozone/oxygen mixture induces reversion of some indicators of oxidative stress and prevents organic damage in rats with fecal peritonitis

Objective and designReactive oxygen and nitrogen species are involved in the pathogenesis of sepsis syndrome with peritonitis and the septic shock. The aim of this study was to determine whether ozone oxidative preconditioning (OOP) may exert beneficial effects in the prevention and treatment of sepsis syndrome in rats inoculated by the intraperitoneal route (i.p.) with fecal material and also to determine if antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) may exert protective effects against this systemic inflammatory disorder.Materials and methodsMale Wistar rats were used. SOD and GPx activities were determined in erythrocytes. Thiobarbituric acid reactive substances (TBARS) content as biomarkers of oxidative stress, alanine amino transferase (ALT), aspartate amino transferase (AST) and creatinine (CRE) were measured in blood serum and myeloperoxidase (MPO) in lung tissue as markers of organs damage.ResultsIn rats submitted to OOP, SOD and GPx activities were significantly increased and it was accompanied by significant decrease of TBARS content in blood serum. OOP also significantly reduced levels of ALT, AST and CRE in blood serum as well as MPO in rat lung.ConclusionThe results support the important role of SOD and GPx in the protective effects of OOP against organ damage induced by fecal peritonitis in rats.

Intraglandular application of botulinum toxin leads to structural and functional changes in rat acinar cells.

Intraglandular injection of botulinum toxin (BoNT) leads to a transient denervation of the submandibular gland and this is associated with reduced salivary secretion. The purpose of the present study was to verify whether temporary acinar atrophy occurs simultaneously with chemical denervation of the glands.

Treatment with ozone/oxygen-pneumoperitoneum results in complete remission of rabbit squamous cell carcinomas

Head and neck squamous cell carcinomas (HNSCC) represent a group of metastasizing tumors with a high mortality rate in man and animals. Since the biomolecule ozone was found to inhibit growth of various carcinoma cells in vitro we here applied the highly aggressive and lethal VX2 carcinoma HNSCC tumor model of the New Zealand White rabbit to test whether ozone exerts antitumorous effects in vivo. Therapeutic insufflation of medical ozone/oxygen (O3/O2) gas mixture into the peritoneum (O3/O2‐pneumoperitoneum) at an advanced stage of tumor disease led to a survival rate of 7/14 rabbits. Six of the seven surviving rabbits presented full tumor regression and the absence of local or distant lung metastases. Insufflation of pure oxygen (O2) resulted in a survival rate of 3/13 animals accompanied by full tumor remission in 2 of the 3 surviving animals. Of the 14 sham‐treated animals only 1 had spontaneous tumor remission and survived. No adverse effects or changes in standard blood parameters were observed after repeated intraperitoneal insufflations of the O3/O2 or O2 gas. Animals with O3/O2‐induced tumor eradication developed tolerance against reimplantation of the VX2 tumor. This could be reversed by immune suppression with a combination of dexamethasone and cyclosporin A suggesting an antitumorous effect of O3/O2‐mediated activation of the bodys own immunosurveillance. Although the exact mechanisms of action are still unclear the present data point to O3/O2‐pneumoperitoneum as a promising new strategy in anticancer therapy.

Antioxidant mechanism is involved in the gastroprotective effects of ozonized sunflower oil in ethanol-induced ulcers in rats.

This research was performed in order to determine the potential protective effects of ozonized sunflower oil (OSO) in the injury of rat gastric mucosa induced by absolute ethanol and as well as to elucidate the role of reactive oxygen species (ROS), lipid peroxidation, and some important constituents of antioxidant defense such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in these effects. OSO was administered to rats intragastrically by a cannula and it was applied during four days to animals. The doses of OSO administered daily to each group of rats were 4, 12, and 24 mg/kg, respectively, and one hour after the last treatment, absolute ethanol (1 mL/200 mg body weight) was administered. Our results showed that gastric ulcer index was significantly reduced in rats pretreated with OSO as compared with ethanol-treated controls. However, in rats pretreated with OSO, no significant reduction of TBARS content in gastric mucosa was found as compared to those rats treated with ethanol alone. In contrast, SOD and GSH-Px activities were significantly increased in gastric mucosa of OSO-pretreated rats with respect to those treated with ethanol alone. In summary, our results demonstrate that OSO pretreatment exerts protective effects in ethanol-induced gastric ulcers in rats. Furthermore, these results provide evidence that these protective effects of OSO are mediated at least partially by stimulation of some important antioxidant enzymes such as SOD and GSH-Px, which are scavengers of ROS and therefore prevent gastric injury induced by them.

Evaluation of ozonated oxygen in an experimental animal model of osteomyelitis as a further treatment option for skull-base osteomyelitis.

Abstract The standard treatment of chronic skull-base osteomyelitis is antibiotics and surgical removal of sequestrums. Hyperbaric oxygen therapy has been found to be a useful method for managing refractory cases of chronic osteomyelitis. Since a minimal blood supply is needed for hyperbaric oxygen therapy, chronic osteomyelitis can produce necrotic infected areas that are not nutrified and therefore not assessible for hyperbaric oxygen therapy. Ozone is known to be an oxidizing medium with a strong bactericidal effect. We investigated the influence of locally applied ozonated oxygen on the development of chronic osteomyelitis in an experimental animal model using the femur of the rabbit. The proximal sides of the femurs of 40 rabbits were prepared and a needle inserted into the intramedullary cavity. Osteomyelitis was induced with an infusion of Staphyloccus aureus and sodium morrhuate into the bone. The needle was left in a intramedullar location. After a 4-week delay animals were randomly separated into treatment and control groups. The infected femur of treated animals was flushed three times a day with 20 ml of ozonated oxygen at an ozone concentration of 107 μg/ml O2 over periods of 2 or 4 weeks. Clinical, radiographic and microbiological findings were documented. Chronic osteomyelitis occurred in all animals. Ten rabbits were excluded from further study during the investigation because of excessive weight loss (>15% of the original weight). Bacterial cultures showed no significant reduction of S. aureus concentrations in the ozone-treated group, although comparison of radiographic results revealed less serious osteomyelitis-related bone damage in these animals (P < 0.01). These findings indicate that refractory osteomyelitis in the head and neck may benefit from locally applied “flush” therapy with ozonated oxygen in addtion to treatment with antibiotics, surgery and hyperbaric oxygen.

Repetitive pneumoperitoneum with ozonized oxygen as a preventive in lethal polymicrobial sepsis in rats.

The aim of this study was to test whether repetitive pretreatments of rats with ozonized oxygen at relatively low gas volumes into the abdomen (20 ml per rat per day) have any beneficial or detrimental effects on the course of a polymicrobial-induced lethal peritonitis. Peritonitis was induced in a surgical or a nonsurgical model by usage of fecal material from the cecum. As the biological read out we used the mortality analysis. To include possible mechanisms by which ozone might influence the septic outcome, we characterized the gene expression of the pro-inflammatory cytokines IL-1β, IL-2, and TNF-α mRNA in lymphoid organs. In both models, we found a significant beneficial influence of a dose-dependent O2/O3 pneumoperitoneum on the survival rate when compared to control animals or to room air. The ozone-enhanced survival seems to be independent from altered cytokine expression because there were no differences noticed in the levels of bacterial-induced gene expression of IL-1β and TNF-α in septic animals pretreated with ozonized oxygen when compared to control animals.

Influence of different anaesthetics on pro-inflammatory cytokine expression in rat spleen

We examined the effect of five anaesthetic drugs commonly used in laboratory animal research (tribromoethanol, ketamine/xylazine, chloral hydrate, pentobarbital, and urethane) on the expression of four pro-inflammatory cytokines. The anaesthetic agents were applied at dosages normally used for deep surgical anaesthesia. Semiquantitative image analysis of interleukin (IL)-1β, IL-2, IL-6, and tumour necrosis factor alpha (TNFα) mRNA expression in the spleen of male Wistar rats 4 h after application of the anaesthetic drugs showed that these had moderate immunomodulatory effects. Ketamine/xylazine, chloral hydrate, and pentobarbital enhanced the basal expression of IL-1β and IL-6 mRNA in rat spleen, while urethane reduced splenic IL-1β mRNA expression. Tribromoethanol, ketamine/ xylazine, and urethane reduced the basal TNFα mRNA levels, whereas TNFα mRNA expression was unaffected by chloral hydrate and by pentobarbital. The data demonstrate that these anaesthetics have slight, but significant, effects on the basal immune status of rats.

Lymph node topography of the head and neck in New Zealand White rabbits

Investigations of the lymphogenic metastatic spread of VX2 carcinomas in New Zealand White rabbits require an exact knowledge of the topography of cervical and facial lymph nodes. The topography of neck lymph nodes was evaluated from 16 rabbits macroscopically, histologically and by lymphographic investigations, and the possibility of their surgical removal (neck dissection) was examined. The upper aerodigestive tract and the ear of New Zealand White rabbits drain via four consistent groups of 12–18 lymph nodes. Except for the paratracheal lymph node, they are all easily accessible to surgery. The data presented in this study encourage the use of induced VX2 carcinomas in New Zealand White rabbits as an animal model to study the lymphogenic metastatic spread of squamous cell carcinomas of the head and neck. Such investigations could lead to an improvement of surgical and pharmaceutical treatment of this tumour entity.

Chronic Osteomyelitis in a New Rabbit Model

Dogs, rats, and rabbits are the most suitable species to induce chronic osteomyelitis and to study different methods of treatment. In rabbits, the incidence of and mortality from Staphylococcus aureus-induced osteomyelitis of the tibia depends on the method of prelesions and the amount and virulence of species-specific bacteria used. In this study two different lesions were combined simultaneously in the medullary canal of the femurs by aspiration of bone marrow, leaving the insertion needle in situ. A sclerosing agent was then inoculated followed by 300,000 bacteria of a rabbit-derived S. aureus strain to initiate infection. With this method, the incidence of chronic progressive osteomyelitis of the femur was increased to 100%. A relatively low mortality was observed, probably due to a lower number of inoculated bacteria as compared to other rabbit models described. The incidence of acute to chronic osteomyelitis was diagnosed by local signs, x-rays, microbiological recovery, and gross pathology of the femur. Initial fever, weight loss, abscess formation in soft tissues, and pain on palpation characterize the clinical features in the course of development of this chronic disease.