Effect of Propionyl-L-Carnitine On Rat Spinal Cord Ischaemia and Post-Ischaemic Reperfusion Injury

In this study we have examined the effect of propionyl-L-carnitine (PC) on rat spinal cord ischaemia and post-ischaemic reperfusion injury by evaluating two lipid peroxidation indices, thiobarbituric acid reactive substances (TBARS) and diene conjugation, before and after the addition of an ADP-Fe⁺² complex to spinal cord homogenates. Aerobic, ischaemic, and post ischaemic reperfusion rat spinal cord homogenates from PC treated and untreated animals did not show any statistically significant difference in their TBARS and conjugated diene content. The addition of the ADP-Fe⁺² complex to these homogenates resulted in an increased production of both the lipid peroxidation indices, though the magnitude of such formation was related to the type of experimental intervention. The post-ischaemic reperfusion samples of untreated rats showed the highest TBARS and conjugated diene content, while ischaemic samples in either treated and untreated rats did not show any statistically significant difference with respect to the aerobic samples. The post-ischaemic reperfusion samples of treated rats showed a statistically significant decrease of TBARS and conjugated diene production in comparison to the untreated samples. In addition, PC was also able to partially inhibit TBARS and conjugated diene formation in linoleic acid micelles exposed to hemoglobin, though it did not protect albumin fragmentation from the irradiation of water with an X-ray source.     

Protective effects of a plant histaminase in myocardial ischaemia and reperfusion injury in vivo

Grass pea seedling histaminase (a copper-diamine oxidase) was found to exert a significant cardioprotection against post-ischaemic reperfusion damage. Electrocardiogram (ECG) recordings from the rats subjected in vivo to ischaemia and reperfusion showed ventricular tachycardia (VT) and ventricular fibrillations (VF) occurring in 9 out of 12 untreated rats whereas no ventricular arrhythmias were found under histaminase (80U/kg body weight) treatment (n=16 rats). Computer-assisted morphometry of the ischaemic reperfused hearts stained with nitroblue tetrazolium showed the extension of damaged myocardium (area at risk and infarct size) significantly reduced in rats treated with histaminase, in comparison with the non-treated rats, whereas no protection was found with the semicarbazide inactivated histaminase. Biochemical markers of ischaemia-reperfusion myocardial tissue damage: malonyldialdehyde (MDA), tissue calcium concentration, myeloperoxidase (MPO), and apoptosis indicator caspase-3 were significantly elevated in untreated post-ischaemic reperfused rats, but significantly reduced under histaminase protection. In conclusion, plant histaminase appears to protect hearts from ischaemia-reperfusion injury by more than one mechanism, essentially involving histamine oxidation, and possibly as reactive oxygen species scavenger, presenting good perspectives for a novel therapeutic approach in treatment of ischaemic heart pathology.     

Effects of Chronic Restraint Stress and 17-β-Estradiol Replacement on Oxidative Stress in the Spinal Cord of Ovariectomized Female Rats

Previous studies have shown sex-specific oxidative changes in spinal cord of rats submitted to chronic stress, which may be due to gonadal hormones. Here, we assessed total radical-trapping potential (TRAP), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and lipid peroxidation (evaluated by the TBARS test) in the spinal cord of ovariectomized (OVX) female rats. Female rats were subjected to OVX, and half of the animals received estradiol replacement. Animals were subdivided into controls and chronically stressed (for 40 days). Our findings demonstrate that chronic stress decreased TRAP, and increased SOD activity in spinal cord homogenates from ovariectomized female rats and had no effect on GPx activity. On the other hand, groups receiving 17β-estradiol replacement presented a decreased GPx activity, but no alteration in TRAP and in SOD activity. No differences in the TBARS test were found in any of the groups analyzed. In conclusion, our results support the idea that chronic stress induces an imbalance between SOD and GPx activities, additionally decreasing TRAP. Estradiol replacement did not reverse the effects of chronic stress, but induced a decrease in GPx activity. Therefore, estradiol replacement in ovariectomized chronically stressed rats could make the spinal cord more susceptible to oxidative injury.     

Effect of partial ischemia on phospholipids and postischemic lipid peroxidation in rabbit spinal cord

Rabbit spinal cord, subjected to severe partial ischemia induced by abdominal aorta ligation tightly below the renal arteries, was analyzed for phospholipid composition and levels of lipid peroxidation products after 10, 20, and 40 min of the insult. Under conditions when spinal cord blood flow was decreased below 5% of control, concentrations of inositol and ethanolamine phospholipids were decreased by 30% and 10%, respectively. Phosphatidic acid concentration was also altered during ischemia. No accumulation of thiobarbituric acid reactive substances (TBA-RS), conjugated dienes and fluorescent lipid soluble material was found throughout the ischemic period. Pattern of TBA-RS, conjugated diene, and fluorophore formation during postischemic in vitro incubation without and with a peroxidation couple (Fe²⁺, ascorbic acid) showed increased susceptibility to postischemic lipid peroxidation in tissues after 20 and 40 min of ischemia.     

Ischemia/Reperfusion-induced oxidative stress causes structural changes of brain membrane proteins and lipids

Oxidative stress is a recognized factor of ischemia reperfusion injury. It shares damage of lipids (LPO) and proteins (PPO), and consequently might cause changes in activity of transport systems. Global 15 min ischemia followed by 2, 24 and 48 hour reperfusion was induced by four-vessel occlusion in Wistar rats of both sexes. Levels of TBARS and conjugated dienes as parameters of LPO were analyzed in forebrain homogenates. Concentrations of total free sulfhydryl (SH) groups and emission spectra of tryptophan were measured to quantify PPO. Our results indicate that lipid peroxidation and protein oxidation occurs mainly during the period of reperfusion. However, significant increase in the level of conjugated dienes can be detected already after 15 min ischemia. Attack of proteins by free radicals leads to modification in structure of proteins seen as a decrease of free SH groups and tryptophan fluorescence. Ischemia/reperfusion induces formation of lipid peroxidation products as well as protein modifications.     

Increased susceptibility to lipid peroxidation in rabbit kidneys: a consequence of warm ischaemia and subsequent reperfusion

Rabbit kidneys were clamped and rendered warm ischaemic (WI) in situ for 60 and 120 min. They were then either removed immediately after the ischaemic insult or after reperfusion with blood for 60 min or 24 hr. Homogenates were assayed for phospholipid-Schiff base fluorescence (Ex. 360 nm, Em. 435 nm) and for diene conjugate formation by u.v. spectrophotometry (240 nm) as indices of lipid peroxidation. No alteration in tissue levels of Schiff base was evident immediately after WI but when the homogenates were incubated at 37 degrees C for 90 min, the rate of peroxidation was significantly elevated compared to controls (P less than 0.02 after WI of 60 min and P less than 0.001 after 120 min of WI). These values were still further elevated after reperfusion with blood for 60 min and 24 hr (P less than 0.001). Diene conjugates were raised after WI alone and further still after reperfusion. Thus an early index of lipid peroxidation (diene conjugation) suggested peroxidative damage during the warm ischaemic period itself, whilst detection of Schiff bases was only possible after in vitro incubation of the tissue. Both indices of oxygen-derived free radical damage were increased after reperfusion in vivo with blood and may relate to the degree of tissue damage sustained during ischaemia and reflow.     

Relationships between lipid peroxidation and anoxia tolerance in a range of species during post-anoxic reaeration

Peroxidation was studied in anoxically treated plant tissues and quantified as conjugated dienes/trienes in the total lipid fraction and as the production of thiobarbituric acid reactive substances (TBARS). Oxidative stress caused by re-exposure of plants to oxygen led to an increase of conjugated diene/triene formation in rhizomes of Iris germanica and roots of wheat ( Triticum aestivum L.) and oats ( Avena sativa L.), and after a long anoxic exposure (45 days) in the rhizomes of the very anoxia tolerant Iris pseudacorus . Second derivative (SD) spectrophotometry of the UV spectrum of lipid extracts confirmed the formation of dienes. However, determination of TBARS in Iris spp. showed no lipid peroxidation in the anoxia tolerant I. pseudacorus . In the rhizomes of the anoxia intolerant I. germanica , elevated levels of TBARS correlated positively with conjugated diene/triene formation. The results suggest that anoxic stress may induce qualitative changes in membrane lipids, as indicated by lipid peroxidation after restoration of aerobic conditions. The rate of lipid peroxidation correlated negatively with anoxic stress tolerance.     

A kinetic assay of TPNH-dependent microsomal lipid peroxidation by changes in difference spectra

In the presence of TPNH, O₂ and ADP-Fe⁺³ rat liver microsomes yield difference spectral changes at 237 nm and 267–270 nm that correlate with the kinetics of lipid peroxidation as measured by the rate of malonaldehyde formation and O₂ and TPNH consumption. Mn⁺² EDTA, aniline, and reduced glutathione were inhibitory. It is suggested that the difference spectral changes at 237 nm and 267–270 nm are essentially due to conjugated diene and malonaldehyde formation, respectively.     

Lipid Peroxidation In Vivo Induced by Reversible Global Ischemia in Rat Brain

It has been hypothesized that ischemia, followed by reperfusion, facilitates peroxidative free-radical chain processes in brain. To resolve this question, rats were subjected to reversible global ischemia. From coronal sections of brains frozen in situ, small (ca. 2 mg) amounts of tissue were sampled from neocortex, hippocampus, and thalamus of both cerebral hemispheres of four groups of rats exposed to 30 min cerebral ischemia followed by 0, 30, 60, and 240 min of reperfusion, and from a control group subjected to the same operative procedures, except for the induction of ischemia. Heptane-solubilized total lipid extracts from these samples were analyzed spectroscopically in the 190-330 nm range for content of isolated (nonconjugated) double bonds and of conjugated diene structures; the latter are formed from isolated double bonds during peroxidation of unsaturated fatty acids. Spectra derived from tissue regions of rats subjected to ischemia, or ischemia followed by reperfusion, were compared to averaged, region-specific control spectra and were normalized to the original content of isolated double bonds in the peroxidized samples. The resultant difference spectra were analyzed in terms of ratios of conjugated diene concentration to the concentration of isolated double bonds originally at risk in the specific tissue zones considered. The peak representing conjugated diene formation was centered at 238 +/- 1 nm and was usually well resolved when the molar ratio [conjugated diene]/[isolated double bonds], expressed as a percentage [( CD]/[IDB]), was greater than 0.25%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitamin E Protects DNA from Oxidative Damage in Human Hepatocellular Carcinoma Cell Lines

Expression of multiple drug resistant (MDR) phenotype and over-expression of P-glycoprotein (P-gp) in the human hepatocellular carcinoma (HCC) cell clone P1(0.5), derived from the PLC/PRF/5 cell line (P5), are associated with strong resistance to oxidative stress and a significant (?p<0.01) increase in intracellular vitamin E content as compared with the parental cell line. This study evaluates the role of vitamin E in conferring resistance to drugs and oxidative stress in P1(0.5) cells. Parental drug-sensitive cells, P5, were incubated in α-tocopherol succinate (α-TS, 5?μM for 24?h) enriched medium to increase intracellular vitamin E content to levels comparable to those observed in P1(0.5) cells at basal conditions. Susceptibility to lipid peroxidation and oxidative DNA damage were assessed by measuring the concentration of thiobarbituric-reactive substances (TBARS) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) at basal and after experimental conditions. Cell capacity to form colonies and resistance to doxorubicin were also studied. P5 cells, treated with α-TS, became resistant to ADP-Fe³⁺ and to ionizing radiation-induced lipid peroxidation as P1(0.5) cells. Exposure to ADP-Fe³⁺ or ionizing radiation increased TBARS and the 8-OHdG content in the P5 cells, while vitamin E enrichment abolished these effects. Irradiation doses at 5?cGy increased TBARS and 8-OHdG. They also inhibited cell capacity to form colonies in the untreated P5 cells. Incubation with α-TS fully reverted this effect and significantly (p<0.01) reduced the inhibitory effect of cell proliferation induced by irradiation doses at >500?cGy. Resistance to doxorubicin was not affected by α-TS. These observations demonstrate the role of vitamin E in conferring protection from lipid peroxidation, ionizing radiation and oxidative DNA damage on the human HCC cell line. They also rule out any role of P-gp over-expression as being responsible for these observations in cells with MDR phenotype expression.     

Protective effect of a new anti-oxidant on the rat brain exposed to ischemia-reperfusion injury: inhibition of free radical formation and lipid peroxidation.

A new oligomeric derivative was synthesized from prostaglandin B2 and ascorbic acid, and its effect on rat brain ischemia-reperfusion injury was studied. Brain ischemia was produced in the rat by the combination of bilateral common carotid artery occlusion and hemorrhagic hypotension (30 mmHg, 20 min). The cerebral cortex was homogenized in the presence of the spin trap agent, N-tert-butyl-alpha-phenyl-nitrone (PBN). Spin-adducts were detected using an electron spin resonance spectrometer (EPR). Lipid peroxidation was estimated from the amounts of both thiobarbituric acid reactive substances (TBAR) and conjugated diene. In control experiments, reperfusion induced a burst of free radical formation which peaked at 5 min reperfusion time (238 +/- 41%). Lipid peroxidation increased significantly after 20 min of reperfusion (TBAR, 161 +/- 50%; conjugated diene, 160 +/- 29%). When the oligomeric derivative was administered (9 mg/kg i.p. 30 min before ischemic insult), it significantly reduced both spin adduct formation (103 +/- 13%) and lipid peroxidation (TBAR, 109 +/- 14%; conjugated diene, 97 +/- 33%).     

Study on lipid peroxidation potential in different tissues induced by ascorbate-Fe2+: Possible factors involved in their differential susceptibility

Susceptibility of four major rat tissues to oxidative damage in terms of lipid peroxidation induced by in vitro by ascorbate-Fe²⁺ in homogenates and mitochondria has been examined. Lipid peroxidation, as assessed by thiobarbituric acid reactive substances (TBARS) and conjugated dienes was maximum in brain followed by liver, kidney and heart. However, the time course of lipid peroxidation showed different patterns in tissues examined. The higher susceptibilities of brain and liver can be explained by substrate availability and to a lesser extent the level of antioxidants. The differences observed in the tissues studied may reflect their susceptibility to degenerative diseases and xenobiotic toxicity which are considered as a result of oxidative damage to membranes.     

Ischaemic preconditioning modulates the activity of Kupffer cells during in vivo reperfusion injury of rat liver

This work was performed to elucidate further the main cellular events underlying the protective effect of ischaemic preconditioning in an in vivo rat liver model of 90 min ischaemia followed by 30 min reperfusion. A significant attenuation of the various aspects of post-ischaemic injury, namely necrosis and the levels of hydrogen peroxide and 5- and 15-hydroperoxyeicosatetraenoic acids, was afforded by the prior application of a short cycle of ischaemia/reperfusion (10 + 10 min) or when rats were previously treated with gadolinium chloride. However, when preconditioning was applied on Kupffer cell-depleted livers, no additional level of ischaemic tolerance was obtained. In terms of cellular pathology, this result could be suggestive of Kupffer cells as the target of the preconditioning phenomenon during the warm ischaemia/reperfusion injury. Accordingly, modulation of Kupffer cell activity was associated with a well-preserved hepatocyte integrity, together with low levels of pro-oxidant generation during reperfusion. As activated Kupffer cells can generate and release potentially toxic substances, their modulation by ischaemic preconditioning could help to provide new surgical and/or pharmacological strategies to protect the liver against reperfusion damage.     

Exercise-induced oxidative stress affects erythrocytes in sedentary rats but not exercise-trained rats.

Oxidant stress is one of the factors proposed to be responsible for damaged erythrocytes observed during and after exercise. The impact of exertional oxidant stress after acute exhaustive treadmill running on erythrocyte damage was investigated in sedentary (Sed) and exercise-trained (ET) rats treated with or without antioxidant vitamins C and E. Exhaustive exercise led to statistically significant increments in the levels of thiobarbituric acid-reactive substance (TBARS) and H2O2-induced TBARS in Sed rats and resulted in functional and structural alterations in erythrocytes (plasma hemoglobin concentrations, methemoglobin levels, and rise in osmotic fragility of erythrocytes with decrease in erythrocyte deformability). Administration of antioxidant vitamin for 1 mo before exhaustive exercises prevented lipid peroxidation (TBARS, H2O2-induced TBARS) in Sed rats without any functional or structural alterations in erythrocytes. Parameters indicating erythrocyte lipid peroxidation and deterioration after exhaustive exercise in rats trained regularly with treadmill running for 1 mo were not different from those in Sed controls. Erythrocyte lipid peroxidation (TBARS) increased in exhausted-ET rats compared with ET controls; however, the plasma hemoglobin, methemoglobin levels, and erythrocyte osmotic fragility and deformability did not differ. Exhaustive exercise-induced lipid peroxidation in ET rats on antioxidant vitamin treatment was prevented, whereas functional and structural parameters of erythrocytes were not different from those of the ET controls. We conclude that exertional oxidant stress contributed to erythrocyte deterioration due to exercise in Sed but not in ET rats.     

Antioxidant effect of caeruloplasmin on microsomal lipid peroxidation

Microsomal NADPH-dependent lipid peroxidation catalyzed by ADP-Fe³⁺ was inhibited by the addition of caeruloplasmin. The antioxidant effect of caeruloplasmin was independent of the superoxide anion (O^?₂ scavenging activity. Since caeruloplasmin enhanced the function of ADP-Fe³⁺ acting as electron acceptor for microsomal electron transport system, the antioxidant effect of caeruloplasmin is considered to depend on the ferroxidase activity.     

Effect of lead with vitamin E, C, or Spirulina on malondialdehyde, conjugated dienes and hydroperoxides in rats

Lead (100 ppm) was given in doubly deionised water for 30 days to one group of rats. The other groups received lead along with exogenous antioxidants like vitamin E (50 IU/kg), vitamin C (800 mg/kg) or Spirulina (1500 mg/kg) in food for a similar period. Levels of lipid peroxidation products such as malondialdehyde, conjugated diene and hydroperoxide were measured in liver, lung and kidney of treated rats. In lead treated animals there was a significant increase in the levels of these lipid peroxidative products. Administration of exogenous antioxidants in the lead treated animals reduced the levels of malondialdehyde, conjugated diene and hydroperoxide. It indicated that vitamin E, vitamin C and Spirulina had significant (P < 0.001) antioxidant activity thereby protecting the animals from lead induced toxicity.     

Resveratrol,an antioxidant,protects spinal cord injury in rats by suppressing MAPK pathway

Resveratrol, a polyphenol found in various plants, including grapes, plums and peanuts has shown various medIRInal properties, including antioxidant, protection of cardiovascular disease and cancer risk. However, the effects of resveratrol on spinal cord reperfusion injury have not been investigated. Hence, the present study was designed to evaluate the effect of resveratrol on nitric oxide synthase (iNOS)/p38MAPK signaling pathway and to elucidate its regulating effect on the protection of spinal cord injury. Spinal cord ischemia–reperfusion injury (IRI) was performed by the infrarenal abdominal aorta with mini aneurysm clip model. The expressions of iNOS and p38MAPK and the levels of biochemical parameters, including nitrite/nitrate, malondialdehyde (MDA), advanced oxidation products (AOPP), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) were measured in control and experimental groups. IRI-induced rats treated with 10 mg/kg resveratrol protected spinal cord from ischemia injury as supported by improved biological parameters measured in spinal cord tissue homogenates. The resveratrol treatment significantly decreased the levels of plasma nitrite/nitrate, iNOS mRNA and protein expressions and phosphorylation of p38MAPK in IRI-induced rats. Further, IRI-produced free radicals were reduced by resveratrol treatment by increasing enzymatic and non-enzymatic antioxidant levels such as GSH, SOD and CAT. Taken together, administration of resveratrol protects the damage caused by spinal cord ischemia with potential mechanism of suppressing the activation of iNOS/p38MAPK pathway and subsequent reduction of oxidative stress due to IRI.     

Lipid Peroxidation and Antioxidant Systems in Rat Brain: Effect of Chronic Alcohol Consumption

The effect of chronic ethanol exposure, in a liquid diet, on lipid peroxidation and some antioxidant systems of rat brain was investigated. Chronic ethanol administration induced a greater susceptibility to iron/ascorbate-induced lipid peroxidation, estimated as thiobarbituric reactive substances (TBARS) production, in the microsomal fraction, but a lower lipid peroxidation in the total homogenate. Glutathione (GSH) levels as well as GSH peroxidase and GSH reductase were unaffected, while the activity of Cu-Zn superoxide dismutase was decreased and that of catalase increased. Lipid peroxidation experiments performed in the presence of some hydroxyl radical scavengers suggested that a greater OH^· generation may be responsible of the greater TBARS production in the microsomal fraction of ethanol treated rats; differently, in total homogenate of control and ethanol rats a relationship was found between the redox state of iron and TBARS production, suggesting that the lower lipid peroxidation in treated rats may depend on a different modulation of the iron redox state.     

Diabetic heart and kidney exhibit increased resistance to lipid peroxidation

Alloxan-diabetic rats and age-matched controls were killed after 6 weeks of diabetes; heart and kidneys were removed and assayed for thiobarbituric acid-reactive substances (TBARS), lipid hydroperoxides, lipid phosphorus, total fatty acid composition and glutathione. Tissue homogenates from a second group of diabetic and control rats were incubated in oxygen-saturated buffer with and without the free radical generating system Fe2+/ascorbate (0.1/1.0 mM) and were assayed for lipid peroxidation. Diabetic hearts contained markedly lower levels of TBARS and lipid hydroperoxides (40% and 18%, respectively) than control hearts, whereas differences in TBARS were less pronounced in kidneys (9%). Incubation of homogenates of both organs in the presence or absence of Fe2+/ascorbate for up to 2 h yielded significantly lower levels of TBARS and lipid hydroperoxides with diabetic tissue. Diabetic hearts and kidneys contained higher levels of glutathione (28% and 13% over controls) and both diabetic tissues showed much higher linoleate/arachidonate ratios than did the controls (9.86 vs. 2.56 for heart, 2.01 vs. 0.86 for kidney). We conclude that diabetic tissues develop enhanced defense systems against oxidative stress and we assume tha the lower levels of arachidonate contribute to their resistance to lipid peroxidation as well.     

The role of phospholipase A2 in microsomal lipid peroxidation induced with t-butyl hydroperoxide

The role of phospholipase A2 (PlA2) in lipid peroxidation induced with t-butyl hydroperoxide was examined in rat liver microsomes. Exposure of microsomes to t-butyl hydroperoxide was associated with activation of endogenous PlA2. When PlA2 was inhibited with chlorpromazine, mepacrine, or p-bromphenacyl bromide, the accumulation of thiobarbituric acid reactive substances (TBARS) was reduced in a dose dependent manner. In contrast, the accumulation of conjugated dienes was not affected by chlorpromazine, and was slightly increased by mepacrine. When endogenous PlA2 was activated with mellitin prior to induction of peroxidation, accumulation of both TBARS and dienes was reduced. Analogously, pretreatment with exogenous PlA2 reduced both dienes and TBARS. In contrast, addition of mellitin following the induction of peroxidation did not alter either TBARS or dienes.