Triamcinolone acetonide protects the rat retina from STZ-induced acute inflammation and early vascular leakage

Streptozotocin (STZ) has been commonly used to induce in vivo and in vitro hyperglycemic diabetes and its toxicity leads to inflammation and vascular injury. Triamcinolone acetonide (TA), as an anti-angiogenic/anti-inflammatory drug, is clinically used to improve the visual acuity in neovascular and edematous ocular diseases. The aim of this study was to investigate the effect of TA on early inflammation and vascular leakage in the retina of STZ-induced hyperglycemic rats. Hyperglycemia was induced in 8-week-old male Sprague-Dawley (SD) rats by a single intraperitoneal injection of STZ (65 mg/kg); only rats with blood glucose levels >13.9 mmol/l 1 day after STZ injection were included in STZ-hyperglycemic group. Sex- and age-matched SD rats injected with buffer were used as the control group. One day before STZ and buffer injection, 2 microl TA (4 mg/ml in saline) and 2 microl saline were intravitreal-injected into the right and the left eyes of rats, respectively. Retinal vascular leakage was measured using the Evans-blue method. Changes in pro-inflammatory target genes, such as tumor necrotic factor (TNF)-alpha, intracellular adhesion molecule (ICAM)-1, and vascular endothelial growth factor (VEGF) were assessed by immunoblottings, immunostaining, and ELISA analyses. Vascular hyperleakage and up-regulation of most pro-inflammatory genes peaked within a few days after STZ injection and had recovered. However, these changes were blocked by TA pretreatment. Our data suggest that TA controls STZ-induced early vascular leakage and temporary pro-inflammatory signals in the rat retina.     

Adrenomedullin protects rat cerebral endothelial cells from oxidant damage in vitro

Increased permeability and reduced cerebral endothelial cell (CEC) viability induced by oxidative stress are the hallmarks of the blood-brain barrier disruption. In our experiments hydrogen peroxide (H2O2, 0.5 mM) induced a continuous decrease of the transendothelial electrical resistance (TEER) and resulted in intercellular gap formations in cultured rat CECs. Adrenomedullin (AM) increased TEER, enhanced peripheral localization of F-actin bands and attenuated the increased permeability induced by H2O2. Furthermore, AM treatment preserved mitochondrial membrane potential, attenuated cytochrome c release, and consequently improved CEC viability in H2O2 treated cultures. These results suggest that AM treatment protects CECs against oxidative injury.     

Dmp53 protects the Drosophila retina during a developmentally regulated DNA damage response

Ultraviolet (UV) light is absorbed by cellular proteins and DNA, promoting skin damage, aging and cancer. In this paper, we explore the UV response by cells of the Drosophila retina. We demonstrate that the retina enters a period of heightened UV sensitivity in the young developing pupa, a stage closely associated with its period of normal developmental programmed cell death. Injury to irradiated cells included morphology changes and apoptotic cell death; these defects could be completely accounted for by DNA damage. Cell death, but not morphological changes, was blocked by the caspase inhibitor P35. Utilizing genetic and microarray data, we provide evidence for the central role of Hid expression and for Diap1 protein stability in controlling the UV response. In contrast, we found that Reaper had no effect on UV sensitivity. Surprisingly, Dmp53 is required to protect cells from UV-mediated cell death, an effect attributed to its role in DNA repair. These in vivo results demonstrate that the cellular effects of DNA damage depend on the developmental status of the tissue.     

Ischemic preconditioning of the rat retina: protective role of ferritin

Ischemic preconditioning (IPC) of the retina, accomplished by ischemia of short duration, is highly effective in preventing subsequent severe injury caused by iron-dependent free radical burst after prolonged ischemia. To investigate the mechanistic basis for IPC rescue, we examined changes in the levels of the retinal redox-active and labile iron pool, ferritin, and ferritin-bound iron. Prolonged ischemia severely impaired retinal function, with total loss of the full-field electroretinographic response. IPC provided marked protection against such injury. Histological examination revealed that ischemia-associated structural damage and loss of cells in the outer and inner nuclear layers were largely prevented by IPC. Ferritin levels decreased after prolonged ischemia but remained close to normal when the ischemic episode was preceded by IPC. The protective effect of IPC on retinal function and ferritin was blocked by a zinc-desferrioxamine complex known to interfere with iron signaling. The results suggest a mechanism whereby IPC activates an iron signaling pathway leading to a marked increase in ferritin levels, which mediates resistance to prolonged ischemia.     

Ginkgo biloba extract protects rat kidney from diabetic and hypoxic damage

Ginkgo biloba extract EGb 761 was studied for its nephroprotective effects in experimentally diabetic and hypoxic rats. Duration of streptozotocin-induced diabetes was 4 months, that of respiratoric hypoxia of the diabetic group 20 min. The daily dose of 100 mg EGb/kg bodyweight started 1 month after induction of the diabetes. EGb reduced diabetes-induced morphological alterations of the kidney such as increase in volume of glomeruli, capillary tufts, urinary space, and thickening of Bowman's capsule basement membrane. Diabetically increased immunostaining of interstitial collagenes of types I, III, and VI was diminished by the EGb extract. EGb reduced the relative total SOD activity from 163% in diabetic kidney to 46%. Additional hypoxia-induced ultrastructural damage was also diminished.     

Ischemic tolerance in the brain

Endogenous tolerance to cerebral ischemia is nature's strategy for neuroprotection. Exploring the physiologic and molecular mechanism of this phenomenon may give us new means of protection against ischemia and other degenerative disorders. This article reviews the currently available experimental methods to induce ischemic tolerance in the brain and gives a brief summary of the potential mode of action.     

Leptin protects the cardiac myocyte cultures from hypoxic damage

Leptin, a circulating hormone mainly produced by adipose tissue, regulates fatty acid metabolism and causes multiple systemic biological actions even the regulation of cardiovascular function. It is previously known that leptin is a hypoxia-inducible hormone, that hypoxic conditions increase the expression of this peptide in various tissues such as placenta, pancreas and also in the heart. Since leptin receptors are present in the heart, we hypothesized that whether leptin was a protector response for tissues especially for the heart against the deleterious effects of hypoxia. Cultured cardiomyocytes from newborn rats were initially treated with 3000 ng/ml leptin incubation for 1, 5 and 20 h separately, then subjected to 120 min of hypoxia. Hypoxic damage of myocytes was assayed using the measurements of both lactate dehydrogenase and creatine kinase releases into the medium and performing morphological observations (ultrastructural and immunocytochemical) of plates. The obtained results from leptin treated and non-treated control groups were compared to each other, and these data have demonstrated that 5 h of leptin treatment before hypoxia provides a significant protection for cardiomyocytes against hypoxia. Neither 1- nor 20-h leptin treated groups exhibited sufficient protection against hypoxia. In conclusion, leptin protects the cardiomyocyte cultures from hypoxia, but this effect is selective and evident only in the 5-h treated myocytes.     

Caffeic acid protects rat heart mitochondria against isoproterenol-induced oxidative damage

Cardiac mitochondrial dysfunction plays an important role in the pathology of myocardial infarction. The protective effects of caffeic acid on mitochondrial dysfunction in isoproterenol-induced myocardial infarction were studied in Wistar rats. Rats were pretreated with caffeic acid (15 mg/kg) for 10 days. After the pretreatment period, isoproterenol (100 mg/kg) was subcutaneously injected to rats at an interval of 24 h for 2 days to induce myocardial infarction. Isoproterenol-induced rats showed considerable increased levels of serum troponins and heart mitochondrial lipid peroxidation products and considerable decreased glutathione peroxidase and reduced glutathione. Also, considerably decreased activities of isocitrate, succinate, malate, α-ketoglutarate, and NADH dehydrogenases and cytochrome-C-oxidase were observed in the mitochondria of myocardial-infarcted rats. The mitochondrial calcium, cholesterol, free fatty acids, and triglycerides were considerably increased and adenosine triphosphate and phospholipids were considerably decreased in isoproterenol-induced rats. Caffeic acid pretreatment showed considerable protective effects on all the biochemical parameters studied. Myocardial infarct size was much reduced in caffeic acid pretreated isoproterenol-induced rats. Transmission electron microscopic findings also confirmed the protective effects of caffeic acid. The possible mechanisms of caffeic acid on cardiac mitochondria protection might be due to decreasing free radicals, increasing multienzyme activities, reduced glutathione, and adenosine triphosphate levels and maintaining lipids and calcium. In vitro studies also confirmed the free-radical-scavenging activity of caffeic acid. Thus, caffeic acid protected rat’s heart mitochondria against isoproterenol-induced damage. This study may have a significant impact on myocardial-infarcted patients.     

UCP2 protects hypothalamic cells from TNF-alpha-induced damage

Uncoupling protein 2 (UCP2) is highly expressed in the hypothalamus; however, little is known about the functions it exerts in this part of the brain. Here, we hypothesized that UCP2 protects hypothalamic cells from oxidative and pro-apoptotic damage generated by inflammatory stimuli. Intracerebroventricular injection of tumor necrosis factor alpha (TNF-alpha)-induced an increase of UCP2 expression in the hypothalamus, which was accompanied by increased expression of markers of oxidative stress and pro-apoptotic proteins. The inhibition of UCP2 expression by an antisense oligonucleotide enhanced the damaging effects of TNF-alpha. Conversely, increasing the hypothalamic expression of UCP2 by cold exposure reversed most of the effects of the cytokine. Thus, UCP2 acts as a protective factor against cellular damage induced by an inflammatory stimulus in the hypothalamus.     

Estradiol protects cultured articular chondrocytes from oxygen-radical-induced damage

Osteoarthritis (OA) is aggravated in menopausal women possibly because of changed serum estrogen levels. Estradiol has been postulated to affect oxidative stress induced by reactive oxygen species (ROS) in articular chondrocytes. We generated ROS in cultured bovine articular chondrocytes by incubating them with combined Fe₂SO₄, vitamin C, and hydrogen peroxide. The release of thiobarbituric-acid-reactive substances (TBARS, lipid peroxidation) and lactate dehydrogenase (LDH, membrane damage) was measured photometrically. Various estradiol doses and vitamin E, serving as control with an established anti-oxidative capacity, were applied either upon each exchange of medium and during radical production (strategy 1) or only during radical production (strategy 2). In chondrocytes incubated according to strategy 1, the production of TBARS and LDH release were significantly suppressed by 10^–10–10^–4 M estradiol or by vitamin E. Under strategy 2, the production of TBARS was significantly suppressed at estradiol concentrations higher than 10^–6 M, whereas LDH release was inhibited at concentrations of 10^–6–10^–4 M. Vitamin E showed no significant effects. As repeated application of estradiol and vitamin E produced the best results, estradiol, like vitamin E, was speculated to accumulate in the plasma membrane and to decrease membrane fluidity resulting in protection against lipid peroxidation (non-genomic effect). Thus, in contrast to the neuroprotective effect of 17-estradiol in supraphysiological doses reported recently, the anti-oxidative potential of estradiol appears to protect articular chondrocytes from ROS-induced damage when the hormone is given repeatedly in a physiological range. Decreased estradiol levels may therefore contribute to menopausal OA in the long term.     

Hyperbaric oxygen preconditioning protects skin from UV-A damage

Hyperbaric oxygen therapy (HBOT) is used for a number of applications, including the treatment of diabetic foot ulcers and CO poisoning. However, we and others have shown that HBOT can mobilize cellular antioxidant defenses, suggesting that it may also be useful under circumstances in which tissue protection from oxidative damage is desired. To test the protective properties of hyperbaric oxygen (HBO) on a tissue level, we evaluated the ability of a preconditioning treatment regimen to protect cutaneous tissue from UV-A-induced oxidative damage. Three groups of hairless SKH1-E mice were exposed to UV-A 3 days per week for 22 weeks, with two of these groups receiving an HBO pretreatment either two or four times per week. UV-A exposure increased apoptosis and proliferation of the skin tissue, indicating elevated levels of epithelial damage and repair. Pretreatment with HBO significantly reduced UV-A-induced apoptosis and proliferation. A morphometric analysis of microscopic tissue folds also showed a significant increase in skin creasing following UV-A exposure, which was prevented by HBO pretreatment. Likewise, skin elasticity was found to be greatest in the group treated with HBO four times per week. The effects of HBO were also apparent systemically as reductions in caspase-3 activity and expression were observed in the liver. Our findings support a protective function of HBO pretreatment from a direct oxidative challenge of UV-A to skin tissue. Similar protection of other tissues may likewise be achievable.     

Thymodepressin protects murine bone marrow CFU-S from the hyperthermic damage

Was studied the influence of the Thymodepressin (dipeptide D-iEW--a new Russian immuno- and haemodepressant), on the hyperthermic sensitivity of haemopoietic precursors (CFU-S) and tumor model cells (EL-4 and Rauscher leukaemia). It was determined, that the injection of the Thymodepressin to donor mice, or the incubation with the preparation of the marrowy cells of normal mice provides the increasing of the CFU-S resistance to the following heating (43 degrees C). On the contrary, Thymodepressin-treated tumor cells became even more heat-sensitive. The data show that Thymodepressin can be useful for protection the haemopoietic precursors not only from radiation and chemotherapy, as it was shown earlier, but also from the hyperthermy.     

Involvement of calpain in hypoxia-induced damage in rat retina in vitro

Our previous study suggested that calpain isoforms played an important role in retinal ganglion cell death induced by ischemia-reperfusion in rats [Curr. Eye Res. 21 (2000) 571]. The purpose of the present study was to further establish the direct involvement of calpain in hypoxia-induced damage by administering calpain inhibitor SJA6017 to oxygen-starved, cultured retinas. Retinas were incubated in RPMI medium with glucose and 95% O2/5% CO2 to supply sufficient oxygen for retinal cell survival. To induce a hypoxic condition, retinas were incubated with 95% N2/5% CO2. Leakage of LDH in the medium was measured to assess retinal cell damage. Activation of calpain and proteolysis of calpain substrate alpha-spectrin were analyzed by casein zymography and immunoblotting. Large amounts of LDH leaked into the medium from retinas under hypoxic conditions for 12 h, and SJA6017 significantly reduced LDH leakage. Caseinolytic activity of mu- and m-calpains decreased with hypoxia for 5 and 12 h, suggesting calpain activation followed by autolytic degradation. SJA6017 partially inhibited decreased calpain activities. Proteolysis of 230 kDa alpha-spectrin to 150 and 145 kDa breakdown products was observed in retinas with hypoxia. SJA6017 completely inhibited production of the 145 kDa breakdown product and partially inhibited production of the 150 kDa breakdown product. These results confirm the direct involvement of calpains in retinal cell damage induced by hypoxia in vitro.     

Morin hydrate protects cultured rat glomerular mesangial cells against oxyradical damage

Cultured rat glomerular mesangial cells were damaged when exposed to oxyradicals generated either from xanthine oxidase plus hypoxanthine, or by superoxide radicals formed from menadione. Morin hydrate is an antioxidant extracted from yellow Brazil wood. When morin hydrate was added to cultured rat glomerular mesangial cells which were attacked by oxyradicals generated by xanthine oxidase plus hypoxanthine, the survival time of the cells was doubled. However, this protective effect of morin hydrate was less marked when the cells were attacked by menadione. Note that the protective effects of Trolox which is a polar analogue of vitamin E were miniscule relative to those of morin hydrate with both oxidants.     

Pretreatment with methylprednisolone protects the isolated rat heart against ischaemic and oxidative damage

Methylprednisolone (MP), a synthetic glucocorticoid, is widely used clinically and experimentally as acute antiinflammatory treatment. The molecular actions of MP indicate that pretreatment with this drug may be cardioprotective. We investigated if giving rats MP prior to excising their hearts for Langendorff-perfusion protected cardiac function against oxidative stress, and if this was mediated by increasing antioxidant defence or influencing myocardial nitric oxide synthase (NOS). Rats (n=6-11 in each group) were injected with MP (40 mg/kg i.m.) or vehicle 24 and 12 h before Langendorff-perfusion with 30 min global ischaemia and 60 min reperfusion, or 10 min perfusion with 180 μmol/L hydrogen peroxide. Other hearts were exposed to 30 min global ischaemia 5 days after MP-injection. Additional hearts were sampled before, during, and after ischaemia for analyzing tissue activity of antioxidant enzymes. Tissue endothelial and inducible NOS (eNOS and iNOS) were investigated by immunoblotting and semiquantitative RT-PCR in a time-course after MP injection. Pretreatment with MP improved left ventricular function and increased coronary flow during postischaemic reperfusion, and this effect was sustained 5 days afterwards. When exposing hearts to hydrogen peroxide, MP improved coronary flow. Catalase, glutathione peroxidase, and oxidized glutathione were increased during reperfusion of MP-treated hearts compared to vehicle only. MP did not influence eNOS at protein or mRNA level. iNOS could not be detected by immunoblotting, indicating low cardiac enzyme content. Its mRNA initially increased the first hour after injection, thereafter decreased. In conclusions, pretreating rats with MP protects the heart against ischaemia-reperfusion dysfunction. This effect could be due to increase of tissue antioxidant activity during reperfusion. MP did not influence cardiac eNOS. mRNA for iNOS was influenced by MP, but the corresponding protein could not be detected.     

Pretreatment with methylprednisolone protects the isolated rat heart against ischaemic and oxidative damage

In this study, we investigated whether a relationship exists between the levels of urate in vivo and lipid peroxidation during exercise. Seven healthy male subjects performed exhaustive cycling exercise under the following three conditions. The levels of urate, thiobarbituric acid reactive substances (TBARS) and allantoin in plasma and urine were examined before exercise and during a 3 h recovery period. (1) Benzbromarone administration experiment: benzbromarone (an uricosuric agent) was administered orally the day before exercise. (2) IMP administration experiment: inosine 5′-monophosphate disodium salt (a precursor of urate) was administered orally the day before exercise. (3) Control experiment: no test substance was administered. The main results obtained were as follows. Plasma urate levels and total peroxyl radical-trapping antioxidant parameter (TRAP) for deproteinized plasma in the resting period significantly decreased depending on the treatment, in the order of IMP > control > benzbromarone. A significant positive correlation was evident between plasma urate levels and TRAP values for deproteinized plasma. The increase in plasma levels of allantoin was observed only in the case of IMP treatment. A significant negative correlation between plasma levels of urate in the resting period and the amounts of urinary TBARS excreted during the recovery period was recognized. These results suggest that the urate level in vivo before exercise is a factor influencing lipid peroxidation during exhaustive exercise. Furthermore, these findings support the view that urate may serve as an important free-radical scavenger in vivo.     

Superoxide dismutase 1 protects retinal cells from oxidative damage

Bolstering the endogenous oxidative damage defense system is a good strategy for development of treatments to combat neurodegenerative diseases in which oxidative damage plays a role. A first step in such treatment development is to determine the role of various components of the defense system in cells that degenerate. In this study, we sought to determine the role of superoxide dismutase 1 (SOD1) in two models of oxidative damage-induced retinal degeneration. In one model, paraquat is injected into the vitreous cavity and then enters retinal cells and generates reactive oxygen species (ROS) that cause progressive retinal damage. Assessment of retinal function with serial electroretinograms (ERGs) showed that sod1 -/- mice were much more sensitive than sod1 +/+ mice to the damaging effects of paraquat, while sod1 +/- mice showed intermediate sensitivity. Compared to sod1 +/+ mice, sod1 -/- mice showed greater paraquat-induced oxidative damage and apoptosis. In the second model, mice were exposed to hyperoxia for several weeks, and sod1 -/- mice showed significantly greater reductions in ERG amplitudes than sod1 +/+ mice. In both of these models, transgenic mice carrying a sod1 transgene driven by a beta-actin promoter showed less oxidative stress-induced reduction in ERG amplitudes. These data demonstrate that SOD1 protects retinal cells against paraquat- and hyperoxia-induced oxidative damage and suggest that overexpression of SOD1 should be considered as one component of ocular gene therapy to prevent oxidative damage-induced retinal degeneration.     

Intracellular glutathione protects human monocyte-derived macrophages from hypochlorite damage

AimsMacrophages must function in an inflammatory environment of high oxidative stress due to the production of various oxidants. Hypochlorous acid (HOCl) is a potent cytotoxic agent generated by neutrophils and macrophages within inflammatory sites. This study determines whether glutathione is the key factors governing macrophage resistance to HOCl.Main methodsHuman monocyte derived macrophages (HMDM) were differentiated from human monocytes prepared from human blood. The HMDM cells were exposed to micromolar concentrations of HOCl and the timing of the cell viability loss was measured. Cellular oxidative damage was measured by loss of glutathione, cellular ATP, tyrosine oxidation, and inactivation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).Key findingsHOCl causes a rapid loss in HMDM cell viability above threshold concentrations. The cell death occurred within 10 min of treatment with the morphological characteristics of necrosis. The HOCl caused the extensive cellular protein oxidation with the loss of tyrosine residue and inactivation of GAPDH, which was accompanied with the loss of cellular ATP. This cellular damage was only observed after the loss of intracellular GSH from the cell. Removal of intracellular GSH with diethyl maleate (DEM) increased the cells' sensitivity to HOCl damage while protecting the intracellular GSH pool with the antioxidant 7,8-dihydroneopterin prevented the HOCl mediated viability loss. Variations in the HOCl LD₅₀ for inducing cell death were strongly correlated with initial intracellular GSH levels.SignificanceIn HMDM cells scavenging of HOCl by intracellular glutathione is sufficient to protect against oxidative loss of key metabolic functions within the cells.