There is some anecdotal evidence that oxygen-ozone therapy may be beneficial in some human diseases. However so far only a few biochemical and pharmacodynamic mechanisms have been elucidated. On the basis of preliminary data we postulated that controlled ozone administration would promote an oxidative preconditioning preventing the hepatocellular damage mediated by free radicals. Six groups of rats were classified as follows: (1) negative control, using intraperitoneal sunflower oil; (2) positive control using carbon tetrachloride (CCl4) as an oxidative challenge; (3) oxygen-ozone, pretreatment via rectal insufflation (15 sessions) and after it, CCl4; (4) oxygen, as group 3 but using oxygen only; (5) control oxygen-ozone, as group 3, but without CCl4; group (6) control oxygen, as group 5, but using oxygen only. We have evaluated critical biochemical parameters such as levels of transaminase, cholinesterase, superoxide dismutase, catalase, phospholipase A, calcium dependent ATPase, reduced glutathione, glucose 6 phosphate dehydrogenase and lipid peroxidation. Interestingly, in spite of CCl4 administration, group 3 did not differ from group 1, while groups 2 and 4 showed significant differences from groups 1 and 3 and displayed hepatic damage. To our knowledge these are the first experimental results showing that repeated administration of ozone in atoxic doses is able to induce an adaptation to oxidative stress thus enabling the animals to maintain hepatocellular integrity after CCl4 poisoning.

Increased formation of MG (methylglyoxal) and related protein glycation in diabetes has been linked to the development of diabetic vascular complications. Diabetes is also associated with impaired wound healing. In the present study, we investigated if prolonged exposure of rats to MG (50-75 mg/kg of body weight) induced impairment of wound healing and diabetes-like vascular damage. MG treatment arrested growth, increased serum creatinine, induced hypercholesterolaemia (all P < 0.05) and impaired vasodilation (P < 0.01) compared with saline controls. Degenerative changes in cutaneous microvessels with loss of endothelial cells, basement membrane thickening and luminal occlusion were also detected. Acute granulation appeared immature (P< 0.01) and was associated with an impaired infiltration of regenerative cells with reduced proliferative rates (P < 0.01). Immunohistochemical staining indicated the presence of AGEs (advanced glycation end-products) in vascular structures, cutaneous tissue and peripheral nerve fibres. Expression of RAGE (receptor for AGEs) appeared to be increased in the cutaneous vasculature. There were also pro-inflammatory and profibrotic responses, including increased IL-1beta (interleukin-1beta) expression in intact epidermis, TNF-alpha (tumour necrosis factor-alpha) in regions of angiogenesis, CTGF (connective tissue growth factor) in medial layers of arteries, and TGF-beta (transforming growth factor-beta) in glomerular tufts, tubular epithelial cells and interstitial endothelial cells. We conclude that exposure to increased MG in vivo is associated with the onset of microvascular damage and other diabetes-like complications within a normoglycaemic context.

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On the basis that ozone (O3) can upregulate cellular antioxidant enzymes, a morphological, biochemical and functional renal study was performed in rats undergoing a prolonged treatment with O3 before renal ischaemia. Rats were divided into four groups: (1) control, a medial abdominal incision was performed to expose the kidneys; (2) ischaemia, in animals undergoing a bilateral renal ischaemia (30 min), with subsequent reperfusion (3 h); (3) O3 + ischaemia, as group 2, but with previous treatment with O3 (0.5 mg/kg per day given in 2.5 ml O2) via rectal administration for 15 treatments; (4) O2 + ischaemia, as group 3, but using oxygen (O2) alone. Biochemical parameters as fructosamine level, phospholipase A, and superoxide dismutases (SOD) activities, as well as renal plasma flow (RPF) and glomerular filtration rate (GFR), were measured by means of plasma clearance of p-amino-hippurate and inulin, respectively. In comparison with groups 1 and 3, the RPF and GFR were significantly decreased in groups 2 and 4. Interestingly, renal homogenates of the latter groups yielded significantly higher values of phospholipase A activity and fructosamine level in comparison with either the control (1) and the O3 (3) treated groups. Moreover renal SOD activity showed a significant increase in group 3 without significant differences among groups 1, 2 and 4. Morphological alterations of the kidney were present in 100%, 88% and 30% of the animals in groups 2, 4 and 3, respectively. It is proposed that the O3 protective effect can be ascribed to the substantial possibility of upregulating the antioxidant defence system capable of counteracting the damaging effect of ischaemia. These findings suggest that, whenever possible, ozone preconditioning may represent a prophylactic approach for minimizing renal damage before transplantation.

Metabolism of arachidonic acid by cyclooxygenase is one of the primary sources of reactive oxygen species in the ischemic brain. Neuronal overexpression of cyclooxygenase-2 has recently been shown to contribute to neurodegeneration following ischemic injury. In the present study, we examined the possibility that the neuroprotective effects of the cyclooxygenase-2 inhibitor nimesulide would depend upon reduction of oxidative stress following cerebral ischemia. Gerbils were subjected to 5 min of transient global cerebral ischemia followed by 48 h of reperfusion and markers of oxidative stress were measured in hippocampus of gerbils receiving vehicle or nimesulide treatment at three different clinically relevant doses (3, 6 or 12 mg/kg). Compared to vehicle, nimesulide significantly (P<0.05) reduced hippocampal glutathione depletion and lipid peroxidation, as assessed by the levels of malondialdehyde (MDA), 4-hydroxy-alkenals (4-HDA) and lipid hydroperoxides levels, even when the treatment was delayed until 6 h after ischemia. Biochemical evidences of nimesulide neuroprotection were supported by histofluorescence findings using the novel marker of neuronal degeneration Fluoro-Jade B. Few Fluoro-Jade B positive cells were seen in CA1 region of hippocampus in ischemic animals treated with nimesulide compared to vehicle. These results suggest that nimesulide may protect neurons by attenuating oxidative stress and reperfusion injury following the ischemic insult with a wide therapeutic window of protection.

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Summary The liver is damaged by sustained ischaemia during liver transplantation, and the reperfusion after ischaemia results in further functional impairment. Ozone oxidative preconditioning (OzoneOP) protected the liver against ischaemia/reperfusion (I/R) injury through different mechanisms. The aim of this study was to investigate the influence of the inhibition of protein synthesis on the protective actions conferred by OzoneOP in hepatic I/R. Rats were treated with cycloheximide (CHX) in order to promote protein synthesis inhibition after OzoneOP treatment. Plasma transaminases, malondialdehyde and 4‐hydroxyalkenals and morphological characteristics were measured as an index of hepatocellular damage; Cu/Zn‐superoxide dismutase (SOD), Mn‐SOD, catalase, total hydroperoxides and glutathione levels as markers of endogenous antioxidant system. OzoneOP increased Mn‐SOD isoform and ameliorated mitochondrial damage. CHX abrogated the protection conferred by OzonoOP and decreased Mn‐SOD activity. Cellular redox balance disappeared when CHX was introduced. Protein synthesis is involved in the protective mechanisms mediated by OzoneOP. Ozone treatment preserved mitochondrial functions and cellular redox balance.