Quercetin in an animal model of spinal cord compression injury: correlation of treatment duration with recovery of motor function.

OBJECTIVES: We have shown previously that administration of quercetin after spinal cord injury in a rat model induced significant recovery of motor function. In the same model for spinal cord compression injury, we now have correlated the treatment duration with the extent to which motor function is recovered. METHODS: Seventy-four male Wistar rats were assigned to eight experimental groups. Mid-thoracic spinal cord injury was produced in the animals of seven groups. Quercetin was administered intraperitoneally in individual doses of 25 micromol kg(-1). Treatment onset was 1 h after the injury. The length of treatment ranged from one single injection to 10 days, with injection frequencies of two or three times daily. BBB (Basso, Beattie and Bresnahan) scores were obtained and tissue preservation at the site of injury was analyzed. RESULTS: None of the untreated control animals recovered motor function sufficient to walk. When quercetin was administered twice daily over a period of either 3 or 10 days, about 50% of the animals recovered sufficient motor function to walk. Stepping/walking (BBB > or =10) were seen in two of six animals receiving only a single injection and in one of the six animal receiving three injections. Surprisingly, none of the animals that received quercetin injections three times daily recovered the ability to walk (all BBB < or = 9). CONCLUSION: Quercetin administration results in preservation of tissue bridges at the site of injury. Treatment success depends on frequency of administration and overall dose.

Quercetin promotes functional recovery following acute spinal cord injury.

We tested the hypothesis that quercetin, a potent Fe(2+)-chelating flavonoid, would decrease secondary damage following spinal cord trauma. MRI studies using the relaxation of the T1 proton signal caused by Fe(2+) ions and the dose-dependent reversal of this effect by addition of quercetin in aqueous solution were used to guide us to the dosage of quercetin to be used in animal experimentations. Forty-four male Wistar rats were used in two experimental series to test the hypothesis that administration of quercetin improves recovery of motor function after acute traumatic spinal cord injury. Animals were subjected to laminectomy and subjected to an extradural 40-g force clip compression for 5 sec at T7. Quercetin or saline was administered intraperitoneally 1 h after injury and then every 12 hr thereafter. Recovery of motor function was assessed using BBB scores at weekly intervals for 4 weeks. A dose of 2.5 micromoles quercetin/kg body weight did not result in significantly better functional outcome, whereas doses ranging from 5 to 100 micromoles quercetin/kg body weight resulted in a significantly better functional outcome with half or more of the animals walking, although with deficit; in contrast, no animals walked in the group of saline-treated animals. No significant differences in behavioral outcome were seen amongst the doses ranging from 5 to 100 micromol/kg, nor was there a difference if animals were treated for 4 or 10 days. Therapeutic outcome was coincident with more efficient iron clearance, suggesting that one possible mechanism whereby quercetin decreases secondary damage is through iron chelation.

Amelioration of experimental allergic encephalitis (EAE) through phase 2 enzyme induction.

The multiple sclerosis (MS) lesion is characterized by an inflammatory cell mediated attack on white matter. Oxidative stress appears to play a role in the onset and progression of MS. We reasoned that decreasing oxidative stress might ameliorate MS. One way of decreasing oxidative stress is to induce phase 2 enzymes. The model chosen to test this hypothesis was experimental allergenic encephalomyelitis (EAE) induced in the Lewis rat. The 26 animals were placed into two groups: 1) those on normal rat chow, 2) those on rat chow containing 250 mumoles t-butylhydroxyanisole (BHA)/kg. After 2 weeks, animals were administered 100 micrograms guinea pig myelin basic protein and examined daily in a blinded fashion. Twenty-nine days later, animals were sacrificed, blood collected for glutathione (GSH) measurements and tissues collected for histology. Six of the 13 control chow animals developed hindlimb weakness or paralysis while 5 developed tail weakness only. Only 1 BHA fed animal exhibited symptoms--hindlimb weakness. Clinical symptoms correlated well with the extent of perivascular lymphocyte infiltration. Animals with BHA in the diet had 20% higher red cell GSH indicting induction of phase 2 enzymes. We conclude that dietary phase 2 enzyme inducers should be examined for their ability to ameliorate MS.

Glucose transport and apoptosis after gene therapy with HSV thymidine kinase.

The relation between tumour metabolism and induction of apoptosis by gene therapy was investigated in a rat Morris hepatoma (MH3924A) model expressing the HSV thymidine kinase (HSVtk) gene. In vivo the amount of glucose transporter (GLUT1 and GLUT3 isoforms) expressing cells was determined in tumours of untreated and treated animals using immunohistochemistry. In vitro uptake studies with 2-fluoro-2-deoxy-D-glucose (FDG), 3-O-methylglucose and thymidine (TdR) and a TUNEL (TdT-mediated dUTP nick end labelling) assay for the assessment of apoptosis were done immediately and 24 h after treatment of the recombinant cells with different doses of ganciclovir (GCV). Immunohistochemistry revealed a significant increase in GLUT1 in treated tumours which showed enhanced transport activity for FDG. In vitro the FDG and 3-O-methylglucose uptake increased to 186% when compared with that of the non-treated cells immediately after incubation with GCV. However, 24 h later the FDG uptake had declined to its normal level, whereas the accumulation of 3-O-methylglucose remained elevated. The uptake of TdR, which was determined simultaneously, decreased in the acid-insoluble fraction of the cells to 27% and 11%, respectively, immediately and 24 h after therapy, while in the acid-soluble fraction it increased to 229% and to 167%, respectively. Employing the TUNEL technique, 25% of cells were found to be apoptotic 24 h after the termination of GCV treatment. Inhibition of glucose transport by cytochalasin B or competition with deoxyglucose resulted in a 78% (cytochalasin B) and 88% (deoxyglucose) decrease in FDG uptake in the recombinant hepatoma cells and in an increase in the apoptotic cell fraction. It is concluded that inhibition of enhanced glucose transport in GCV-treated cells increased apoptosis. Therefore, enhanced glucose transport seems to represent a stress reaction of tumour cells dedicated for the prevention of cell death.

Pre-column derivatization high-performance liquid chromatographic method for determination of cysteine, cysteinyl-glycine, homocysteine and glutathione in plasma and cell extracts.

A sensitive high-performance liquid chromatographic method for quantification of sulphydryl and disulfide amino acids in human plasma using ultra violet spectrophotometric detection was developed. Precolumn derivatization with 5,5'-dithio-bis-nitrobenzoic acid (DTNB) and an optional pre-derivatization reaction with dithiothreitol allowed both quantitative reduction of disulfides for measurement of total amino acid levels and the measurement of the reduced forms. A dynamic range of 500 nmol/l-750 micromol/l allowed the major analytes of interest to be quantified in plasma without sample dilution. The assay is a sensitive and precise method for the determination of sulphydryl and disulfide amino acids in plasma and cell extracts.

Apoptosis and changes in glucose transport early after treatment of Morris hepatoma with gemcitabine.

Apoptosis has been described as an energy-consuming process. This combined in vivo/in vitro study investigated the effects of the antineoplastic agent gemcitabine on tumour metabolism and on the induction of apoptosis. Dynamic positron emission tomography (PET) measurements of fluorine-18 fluorodeoxyglucose (FDG) uptake were done in rats bearing Morris hepatoma prior to and after therapy with 90 mg gemcitabine/kg b.w. Furthermore, thymidine (TdR) incorporation into the DNA of these tumours was determined. In vitro measurements of FDG and TdR uptake were performed immediately and 24 h after the end of gemcitabine treatment, and the amount of apoptotic cells was determined using the TUNEL reaction. In vivo an increase in FDG transport and phosphorylation occurred early after gemcitabine treatment, although TdR incorporation into the DNA of the tumours declined. In vitro, an enhanced glucose transport, an increase in TdR uptake in the cytoplasm and a decrease in TdR incorporation in the nucleic acid fraction early after treatment occurred. Inhibition of glucose transport caused an increase in the amount of apoptotic cells. The increase in glucose uptake and TdR metabolism early after therapy is interpreted as a stress reaction of the tumour cells, protecting the cells from apoptosis during this early period after exposure to cytotoxic drugs like gemcitabine.

Promoting glutathione synthesis after spinal cord trauma decreases secondary damage and promotes retention of function.

The study aimed to 1) quantify oxidative stress in spinal cord after crush injury at T6, 2) determine whether the administration of the procysteine compound L-2-oxothiazolidine-4-carboxylate (OTC) would up-regulate glutathione (GSH) synthesis and decrease oxidative stress, and 3) determine whether decreased oxidative stress results in better tissue and function retention. We demonstrate that spinal cord compression (5 s with a 50 g aneurysm clip) at T6 in rats results in oxidative stress that is extensive (significant increases in oxidative stress seen at C3 and L4) and rapid in onset. Indices of oxidative stress used were GSH content, protein carbonyl content, and inactivation of glutathione reductase. Administration of OTC resulted in a marked decrease in oxidative stress associated with a sparing of white matter at T6 (16+/-1.9% retained in OTC-treated animals vs. less than 1% in saline-treated). Behavioral indices in control, saline-treated, and OTC-treated animals after 6 wk were respectively: angle board scores (59 degrees, 32 degrees, and 42 degrees ), modified Tarlov score (7, 2.4, and 4.1), and Basso-Beattie-Bresnahan score (21, 5.3, and 12.9). We conclude that administration of OTC after spinal cord trauma greatly decreases oxidative stress and allows tissue preservation, thereby enabling otherwise paraplegic animals to locomote.

DNA array and biological characterization of the impact of the maturation status of mouse dendritic cells on their phenotype and antitumor vaccination efficacy.

We systematically investigated the impact of the relative maturation levels of dendritic cells (DCs) on their cell surface phenotype, expression of cytokines and chemokines/chemokine receptors (by DNA array and RNase protection analyses), biological activities, and abilities to induce tumor immunity. Mature DCs expressed significantly heightened levels of their antigen-presenting machinery (e.g., CD54, CD80, CD86) and numerous cytokines and chemokines/chemokine receptors (i.e., Flt-3L, G-CSF, IL-1alpha and -1beta, IL-6, IL-12, CCL-2, -3, -4, -5, -17, and -22, MIP-2, and CCR7) and were significantly better at inducing effector T cell responses in vitro. Furthermore, mice vaccinated with tumor peptide-pulsed mature DCs better survived challenge with a weakly immunogenic tumor (8 of 8 survivors) than did mice vaccinated with less mature (3 of 8 survived) or immature (0 of 8 survivors) DCs. Nevertheless, intermediate-maturity DCs expressed substantial levels of Flt-3L, IGF-1, IL-1alpha and -1beta, IL-6, CCL-2, -3, -4, -9/10, -17, and -22, MIP-2, osteopontin, CCR-1, -2, -5, and -7, and CXCR-4. Taken together, our data clearly underscore the critical nature of employing DCs of full maturity for DC-based antitumor vaccination strategies.

Sulfur amino acid deficiency depresses brain glutathione concentration.

Dietary sulfur amino acid content is a major determinant of glutathione concentration in some tissues. We examined whether brain glutathione (GSH), a key component of antioxidant defense important for minimizing ischemic injury, was also responsive to short-term sulfur amino acid deficiency. Female Long-Evans adult rats were fed a sulfur-deficient L-amino acid defined diet for five days; the control diet was supplemented with L-cystine and L-methionine (n = 6). Sulfur amino acid deficiency was confirmed by a reduction in liver cysteine and GSH concentrations, marked decreases in food intake, and weight loss. GSH concentration analyzed by reverse-phase high performance liquid chromatography was significantly depressed in the neocortex and thalamus of deficient rats. Brain cysteine was not decreased in a parallel manner. Classical glutathione peroxidase activity was increased in the liver and brain of sulfur amino acid deficient rats. This suggests an upregulation of antioxidant defense but these findings may be complicated by alterations in tissue composition. The depletion of brain GSH by a reduced supply of dietary precursors may be important during brain ischemia when the rate of GSH utilization and the need for synthesis are increased.

Monochlorobimane fluorometric method to measure tissue glutathione.

Glutathione (GSH) is the principal intracellular low-molecular-weight thiol and plays a critical role in the cellular defense against agents that impose oxidative stress. A common technique to measure GSH uses reversed-phase high-performance liquid chromatography (HPLC) following derivatization with 5, 5'-dithiobis(2-nitrobenzoic acid), a technique, although reliable and sensitive, that is time consuming and laborious. A common technique to measure GSH in cultured cells is to add monochlorobimane to the culture medium where it readily enters cells to form a fluorescent GSH-monochlorobimane adduct that can be measured fluorometrically. This reaction is catalyzed by glutathione S-transferase. We reasoned that adding glutathione S-transferase and monochlorobimane to tissue homogenates would allow a rapid reliable method to measure GSH. The accuracy of the new test was assessed in homogenates of rat livers. One-half of each homogenate was assayed for GSH using a HPLC approach while the other half was assayed using the monochlorobimane approach. The two methods were found to give identical results. We conclude that the monochlorobimane fluorescent method is sufficiently specific to reliably measure tissue GSH.

Characterization of apoptotic macrophages in atheromatous tissue of humans and heritable hyperlipidemic rabbits.

Apoptotic macrophages are regularly found in atherosclerotic plaques indicating programmed cell death as one of their regulatory controls. The objective of this study was to characterize in more detail apoptotic macrophages in atherosclerotic lesions of humans and heritable hyperlipidemic (HHL) rabbits. Macrophages were immunohistochemically analyzed using antibodies directed against alphaMbeta2-integrins (CD11b/CD18), CD44, major histocompatibility complex (MHC) class I and II, inducible nitric oxide synthase (iNOS), manganese superoxide dismutase (MnSOD), tumor necrosis factor alpha (TNFalpha), p53, c-jun/AP-1 and rabbit macrophages (RAM-11) and the TUNEL (TdT-mediated dUTP nick end labeling) technique. Colocalization studies of human atherosclerotic carotid and aortic tissue showed apoptotic plaque macrophages also being MnSOD-, alphaMbeta2-integrin-, CD44-, MHC class I- and II-, iNOS-, TNFalpha- and p53-immunoreactive. Similar results occurred in atherosclerotic aortas of HHL rabbits. Computer-assisted morphometric analyses revealed a positive correlation of the area density of MnSOD-immunoreactive macrophages with those of alphaMbeta2-integrin- and CD44-immunoreactive ones, but not with those of MHC class I- and II- as well as of RAM-11-immunoreactive macrophages. We conclude that apoptotic macrophages located in atherosclerotic vessel wall are activated, antigen-presenting, integrin-expressing and oxidatively stressed cells. Since all these processes have been demonstrated to cause apoptosis of macrophages in vitro, we propose their potency accelerates the susceptibility of the macrophages to programmed cell death in atherosclerotic lesions.

Complement C6 deficiency protects against diet-induced atherosclerosis in rabbits.

Low-density lipoprotein (LDL) can be transformed to an atherogenic moiety by nonoxidative, enzymatic degradation. Enzymatically degraded LDL induces macrophage foam cell formation, provokes release of cytokines, and also activates complement. To determine whether complement activation may contribute to atherogenesis, 6 pairs of homozygous C6-deficient rabbits and their non-C6-deficient heterozygous siblings were fed a cholesterol-rich diet for 14 weeks. Cholesterol levels and plasma lipoprotein profiles of the animals in the C6-competent and C6-deficient groups did not significantly differ, and the high density lipoprotein and LDL cholesterol ratios at the end of the experiment were 0.07+/-0.01 and 0.08+/-0.01 (SEM), respectively. However, differences in atherosclerotic plaque formation were discernible macroscopically, with extensive aortic lesions being visible in all C6-competent animals and absent in all C6-deficient animals. Aortas were sectioned from thorax to abdomen, and 10 sections were stained from each aorta. Quantification of atherosclerotic lesions and lumen stenosis with the use of computer-based morphometry documented a dramatic protective effect of C6 deficiency on the development of diet-induced atherosclerosis. We conclude that the terminal complement sequence is centrally involved in atherosclerotic lesion progression.

Apoptosis caused by oxidized LDL is manganese superoxide dismutase and p53 dependent.

Oxidized low density lipoprotein (oxLDL) induces apoptosis in human macrophages (Mphi), a significant feature in atherogenesis. We found that induction of apoptosis in Mphi by oxLDL, C2-ceramide, tumor necrosis factor alpha (TNF-alpha), and hydrogen peroxide (H2O2) was associated with enhanced expression of manganese superoxide dismutase (MnSOD) and p53. Treatment of cells with p53 or MnSOD antisense oligonucleotides prior to stimulation with oxLDL, C2-ceramide, TNF-alpha, or H2O2 caused an inhibition of the expression of the respective protein together with a marked reduction of apoptosis. Exposure to N-acetylcysteine before treatment with oxLDL, C2-ceramide, TNF-alpha, or H2O2 reversed a decrease in cellular glutathione concentrations as well as the enhanced production of p53 and MnSOD mRNA and protein. In apoptotic macrophages of human atherosclerotic plaques, colocalization of MnSOD and p53 immunoreactivity was found. These results indicate that in oxLDL-induced apoptosis, a concomitant induction of p53 and MnSOD is critical, and suggest that it is at least in part due to an enhancement of the sphingomyelin/ceramide pathway.

Effect of alterations of blood cholesterol levels on macrophages in the myocardium of New Zealand White rabbits.

We investigated the effect of alterations of blood cholesterol levels on macrophages (mphi) in the myocardium of New Zealand White (NZW) rabbits. Three groups of NZW rabbits were used: controls, rabbits fed a 0.5% cholesterol-enriched diet (CH-D) for 96 days, and rabbits fed a 0.5% CH-D for 96 days followed by normal chow for 4 months. Immunohistochemical analysis by mAbs directed against mphi (RAM-11) and Mn superoxide dismutase (MnSOD) were quantified by computer-assisted morphometry. Using cultured human and rabbit mphi, a cross-reaction of the human MnSOD mAbs was found as well as the predominant localization of MnSOD-immunoreactivity (IR) in mitochondria. In group 1, only a very few RAM-11-immunoreactive (ir) mphi occurred in the interstitial space of the myocardium. In group II blood cholesterol levels significantly increased in parallel with the numbers of mphi, which often contained lipid droplets (foam cells). Although blood cholesterol concentrations regressed about 10-fold in group III, mphi in the myocardium were found to be reduced only about 20%. Most mphi were also MnSOD-ir. In atherosclerotic coronary arteries RAM-11-IR was located in mphi and also extracellularly, whereas MnSOD-IR was found only in mphi. Drastically induced MnSOD in the mitochondria of mphi is suggested as an indicator of increased oxidative stress caused by in vitro conditions or by phagocytosis of low-density lipoprotein in vivo. Elevation of the cholesterol level leads to a long-term increase and its regression results in a delayed reduction of such mphi, which seem to play a key role in the atherogenesis of the coronary arteries as well.

Induction of mitochondrial manganese superoxide dismutase in macrophages by oxidized LDL: its relevance in atherosclerosis of humans and heritable hyperlipidemic rabbits.

The objective of the study was to analyze the intracellular antioxidative response of macrophages (Mphi) exposed to increased levels of low density lipoprotein (LDL). We studied manganese superoxide dismutase (MnSOD) and, in part, GSH in cultured human and rabbit Mphi, and in atheromatous arterial tissue of humans and heritable hyperlipidemic (HHL) rabbits. Incubation of human Mphi with oxidized-LDL (ox-LDL) resulted in an induction of MnSOD mRNA production as shown by RT-PCR. MnSOD immunoreactivity (IR) was found to be located in the mitochondria of Mphi. In HHL rabbits, MnSOD activity and GSH concentration were significantly increased in atherosclerotic intima compared to the media of the aorta, but significantly decreased (P<0.01) in larger plaques compared with smaller ones, resulting in a significant inverse correlation of MnSOD activity (r=-0.67, P<0.001) and GSH concentration (r=-0.57, P<0.01) with plaque size. Immunohistology of the atherosclerotic intima revealed MnSOD-IR in Mac-1 (CD 11b/CD 18)-immunoreactive (ir) Mphi of human arteries and, similarly, in RAM-11-ir Mphi of rabbit ones. The relation of MnSOD-ir Mphi decreased with plaque advancement, which is consistent with biochemical findings. Most MnSOD-ir Mphi in atherosclerotic plaques revealed TUNEL-positive nuclei, indicating DNA strand breaks, and p53-IR. We conclude that mitochondrial antioxidants such as MnSOD are induced in Mphi in vitro and in atherosclerotic arteries as a reply to increased mitochondrial oxidation. As normal consequences of an increased oxidative stress due to the exposure to ox-LDL nuclear DNA strand breaks occur, which are suggested to be a signal to increase p53 protein levels. Reactive oxygen species-mediated mitochondrial-dependent pathways are suggested as major contributing pathomechanisms to nuclear damage, which eventually may result in apoptosis. A common response to increased oxidative stress due to modified LDL is presumed in rabbit and human atherosclerotic plaques.