Quercetin attenuates inflammatory processes after spinal cord injury in an animal model.

OBJECTIVES: We have shown earlier that administration of the flavonoid quercetin significantly contributed to recovery of motor function after spinal cord compression injury in the adult rat. Using the same animal model, we have now designed a set of experiments to test the hypothesis that quercetin attenuates oxidative stress-related inflammatory processes early after spinal cord trauma. METHODS: Mid-thoracic spinal cord compression injury in adult male Wistar rats was caused by extradural application and closure of a 50 g calibrated aneurysm clip for 5 s. Myeloperoxidase (MPO) levels were determined in spinal cord tissue and serum of quercetin-treated animals and controls at 6, 12, 24 and 72 h after injury. The white blood count was followed until 72 h after injury. RESULTS: In quercetin-treated animals, MPO activity was significantly decreased in tissue at 12 and 24 h and in serum at 6, 12 and 24 h after injury, compared with saline controls. In quercetin-treated animals, the prevalence of ED-1 and MPO positive cells was significantly lower than in saline controls. White blood count in venous blood was significantly decreased in quercetin-treated animals at 12 and 24 h after injury. CONCLUSION: Quercetin attenuated the recruitment of neutrophils to the site of injury. The resulting lower MPO release in the injured tissue is likely to decrease the extent of secondary injury and might at least partially explain the neuroprotective effect of the flavonoid quercetin.

Quercetin administration after spinal cord trauma changes S-100 levels.

BACKGROUND: It has been shown previously that S-100beta levels in serum correspond with the severity of central nervous system (CNS) trauma. It also has been suggested that S-100beta in CNS tissue is involved in neuroprotection and neuroregeneration. We have previously shown that administration of quercetin results in improved motor function in an animal model of spinal cord trauma. METHODS: Mid-thoracic spinal cord compression injury was produced in adult male Wistar rats. Serum and tissue samples were acquired from quercetin-treated animals (25 micromol/kg) and saline controls at 6, 12 and 24 hours after the trauma. S-100beta levels were measured using a luminometric assay in the damaged tissue and in the serum of the animals. RESULTS: The increase in serum S-100beta levels seen in saline controls after spinal cord trauma was ameliorated in the quercetin-treated animals at all time points, although the difference to saline controls became statistically significant only at 24 hrs after the trauma. Compared to tissue S-100beta levels in healthy animals, values were significantly decreased in saline controls at all three time points, while they were decreased at 6 hrs and increased at both 12 and 24 hrs in quercetin-treated animals. At all three time points tissue S-100beta levels were significantly higher in quercetin-treated animals than in saline controls. CONCLUSIONS: Administration of quercetin results in modification of S-100beta levels in the setting of experimental spinal cord trauma. The kinetic patterns of the S-100beta fluctuations in serum and tissue suggest that post-traumatic administration of quercetin decreases the extent of CNS injury.

Troglitazone inhibits histone deacetylase activity in breast cancer cells.

We previously demonstrated that the PPARgamma agonist Troglitazone (TRG), a potent antiproliferative agent, in combination with the anthracycline antibiotic Doxorubicin (DOX), is an effective killer of multiple drug resistant (MDR) human cancer cells. Cell killing was accompanied by increased global histone H3 acetylation. Presently, we investigated the epigenetic and cell killing effects of TRG in estrogen receptor (ER) positive MCF7 breast cancer cells. MCF7 cells were treated with the Thiazolidinediones (TZDs) TRG and Ciglitazone (CIG), the non-TZD PPARgamma agonist 15PGJ2, and the histone deacetylase inhibitors (HDACi's) Trichostatin A (TSA), sodium butyrate and PXD101. Using MTT cell viability assays, Western analyzes and mass spectrometry, we showed a dose-dependent increase in cell killing in TRG and HDACi treated cells, that was associated with increased H3 lysine 9 (H3K9) and H3K23 acetylation, H2AX and H3S10 phosphorylation, and H3K79 mono- and di-methylation. These effects were mediated through an ER independent pathway. Using HDAC activity assays, TRG inhibited HDAC activity in cells and in cell lysates, similar to that observed with TSA. Furthermore, TRG and TSA induced a slower migrating HDAC1 species that was refractory to HDAC2 associations. Lastly, TRG and the HDACi's decreased total and phosphorylated AKT levels. These findings suggest that TRG's mode of killing may involve downregulation of PI3K signaling through HDAC inhibition, leading to increased global histone post-translational modifications.

Can admission S-100beta predict the extent of brain damage in head trauma patients?

BACKGROUND: As has been shown previously, S-100beta levels in serum can be a useful predictor of brain damage after head trauma. This pilot study was designed to investigate whether urine samples, which are much easier to obtain, could be used for the same purpose instead of serum samples. METHODS: Ninety-six consecutive patients admitted with head trauma were recruited in the study. After exclusion of 54 patients, mostly because of significant additional trauma, S-100beta levels were analyzed in serum and urine of 42 patients using a luminometric assay. A range for normal values was established based on samples from ten healthy volunteers. RESULTS: S-100beta serum levels increased proportional to the severity of the head trauma, as had been previously shown by several other groups. In many patients, initial increases in urine S-100beta levels were seen later than in serum, after which the kinetics of S-100beta levels in urine seemed to follow that established for serum levels. S-100beta values in urine were on average about 54% lower in urine than in serum. CONCLUSIONS: S-100beta levels in urine obtained on admission to the hospital are not a good indicator for the extent of brain damage. However, urine S-100beta levels obtained at later time points might be a useful indicator for the development of secondary brain injury.

In vitro production of tumor necrosis factor-alpha by human monocytes stimulated with lipopolysaccharide is positively correlated with increased blood monocytes after exposure to a swine barn.

Recently there has been interest in the air quality in and around intensive livestock production facilities, such as modern swine production barns, where agricultural workers and surrounding residents may be exposed to elevated levels of organic dusts. The health effects of these exposures are not completely understood. The study that is reported here is a component of a larger investigation of the relationships among the acute effects of high-concentration endotoxin exposure (swine barn dust), polymorphisms in the TLR4 gene, and respiratory outcomes following exposure to swine confinement buildings. The relationships among a mediator of acute lung inflammation, tumor necrosis factor alpha (TNF-alpha), and clinical responses to acute swine barn exposure were characterized. Analysis of the results showed that in vitro stimulation of human monocytes with as little as 1 ng/ml of lipopolysaccharide (LPS) produced a significant increase in the monocytes that produced TNF-alpha. Although the proportion of TNF-alpha-positive monocytes after in vitro stimulation with 1 ng/ml of LPS was not associated with gender or TLR4 genotype, it was positively associated with the concentration of monocytes in blood after barn exposure. Thus, these two responses to different forms of LPS exposure are significantly correlated, and more responsive monocytes in vitro indicate a forthcoming relative monocytosis, post barn exposure, which may initiate a cascade of chronic inflammation.

Synchrotron-based in vivo tracking of implanted mammalian cells.

We have developed an X-ray imaging protocol that permits 3D visualisation of a small number of implanted cells within bulk tissue. The cells are marked using natural endocytosis of inert gold nano-particles. The resulting local increase in electron density allows high imaging contrast to be obtained from small clusters of these marked cells. Using this technique we have imaged C6 glioma cells within the brain of a model animal. The cells were marked by exposing them to colloidal gold incorporated in the growth media. Gold-loaded glioma cells were implanted into the brains of adult male Wistar rats. After tumours had been allowed to develop for up to 2 weeks, the animals were sacrificed and images of the intact cranium were acquired at the SYRMEP imaging station on the Elettra synchrotron in Italy. Computed tomography was performed using mixed absorption and phase contrast techniques at an X-ray energy of 24 keV. In the resulting volume datasets the tumour bulk is clearly visible and the infiltrating nature of the malignant growth is well demonstrated. Although the protocol was developed using this particular model of malignant brain tumour, it is believed that it will be possible to use it with other cell lines.

A Fourier transform infrared microspectroscopic imaging investigation into an animal model exhibiting glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a highly malignant human brain tumour for which no cure is available at present. Numerous clinical studies as well as animal experiments are under way with the goal being to understand tumour biology and develop potential therapeutic approaches. C6 cell glioma in the adult rat is a frequently used and well accepted animal model for the malignant human glial tumour. By combining standard analytical methods such as histology and immunohistochemistry with Fourier Transform Infrared (FTIR) microspectroscopic imaging and multivariate statistical approaches, we are developing a novel approach to tumour diagnosis which allows us to obtain information about the structure and composition of tumour tissues that could not be obtained easily with either method alone. We have used a "Stingray" FTIR imaging spectrometer to analyse and compare the compositions of coronal brain tissue sections of a tumour-bearing animal and those from a healthy animal. We have found that the tumour tissue has a characteristic chemical signature, which distinguishes it from tumour-free brain tissue. The physical-chemical differences, determined by image and spectral comparison are consistent with changes in total protein absorbance, phosphodiester absorbance and physical dispersive artefacts. The results indicate that FTIR imaging analysis could become a valuable analytic method in brain tumour research and possibly in the diagnosis of human brain tumours.

Gender-related differences in advanced glycation endproducts, oxidative stress markers and nitric oxide synthases in rats.

An age- and blood pressure-associated increase in methylglyoxal (MG) and MG-induced advanced glycation endproducts (AGEs), including N(epsilon)-carboxyethyl-lysine (CEL) and N(epsilon)-carboxymethyl-lysine (CML), in the kidney of spontaneously hypertensive rats (SHR) has been shown. In the present study, gender-related changes in AGEs and nitric oxide synthase were investigated in Sprague-Dawley (SD) and stroke-prone SHR (SHRsp) rats. Immunohistochemical analyses were conducted on kidneys from 24-week-old male and female SD rats as well as SHRsp. The systolic blood pressure of SHRsp was significantly higher than that of SD rats. Male SD rats had more intense kidney staining for CEL than female SD rats. Both male and female SHRsp had more marked CEL and CML staining localized to kidney tubules, as opposed to SD rats. Female rats showed more staining in glomerular vessels than male rats in both SD and SHRsp. Nuclei containing nuclear factor-kappaB (NF-kappaB) p65 and activated macrophages were seen in the kidney from SHRsp, not so much in SD rats, localized to renal tubules in male and glomerular vessels in female SHRsp. A higher protein level of NF-kappaB p65 was found in SHRsp than in SD rats. SD rats had more intense kidney neuronal nitric oxide synthase staining than SHRsp. The intensity of inducible nitric oxide synthase staining was significantly higher in SHRsp than in SD rats, with no gender differences in either strain. SHRsp and male rats exhibited higher AGEs and oxidative stress than SD and female rats, respectively. These differences might partly account for the development of hypertension in SHRsp and the higher vulnerability of male animals to renal pathology.

Dietary approach to decrease aging-related CNS inflammation.

We demonstrate that the spontaneously hypertensive rat stroke-prone rat (SHRsp) undergoes premature aging of the CNS compared to the related normotensive Wistar Kyoto rat (WKY) as demonstrated by presence of activated microglia/macrophages, increased expression of inducible nitric oxide synthase and increased astrogliosis. We tested the hypothesis that dietary intake of phase 2 protein inducers would decrease these aging-associated degenerative changes. The source of dietary phase 2 protein inducers was dried broccoli sprouts of a cultivar containing high amounts of glucoraphanin that gives rise to phase 2 protein-inducing isothiocyanate sulforaphane. This diet significantly decreased the aging-related degenerative changes in the SHRsp CNS. We conclude that modest changes in diet may have profound effects on the aging CNS.

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.

Regulation of the glyoxalase pathway in human brain microvascular endothelium: effects of troglitazone and tertiary butylhydroperoxide.

The glyoxalase system, comprised of glyoxalase-I and glyoxalase-II with glutathione as the cofactor, plays an important role in the detoxification of methylglyoxal and other alpha-oxo-aldehydes. Such aldehydes, which increase with hyperglycemia, give rise to advanced glycation end products. The objective of this research was to examine the glyoxalase system in human cerebromicrovascular cells. The hypothesis tested was that this pathway would be regulated by phase 2 enzyme inducers such as t-butylhydroquinone and modulated by the insulin-sensitizing drug troglitazone. Human cerebromicrovascular endothelial cells were cultured and exposed to varying concentrations of t-butylhydroquinone or troglitazone. The activity of glyoxylase-I in human endothelial cells was similar to the activity present in hepatocytes. The phase 2 enzyme inducer t-butylhydroquinone had no effect on the glyoxalase enzymes activities but significantly increased glutathione levels and glutathione reductase activity, indicating that phase 2 enzyme inducers might promote alpha-oxo-aldehyde scavenging. Troglitazone decreased the activities of glyoxalase-I and -II and decreased glyoxalase-I mRNA. Troglitazone had no effect on glutathione levels or on the activity of glutathione reductase or glutathione peroxidase. We conclude that phase 2 enzyme inducers may promote scavenging of alpha-oxoaldehydes in endothelial cells.