Elements of margin of safety, toxicity and action of sodium selenite in a lipopolysaccharide rat model.

PROJECT: Both septic shock and sodium selenite (Na2SeO3) lead to multiple organ failure through oxidation. Na2SeO3 has direct oxidant effects above the nutritional level and indirect anti-oxidant properties. In a lipopolysaccharide (LPS) rat model we assessed margin of safety, toxicity and beneficial effect of pentahydrate Na2SeO3 (5H2O·Na2SeO3) at oxidant doses. PROCEDURE: In a three-step study on 204 rats we: (i) observed toxic effects of Na2SeO3 injected intraperitoneously (IP) and determined its Minimum Dose Without Toxic effect (MDWT) 0.25-0.35 mg/kg selenium (Se) content; (ii) injected IP LPS at 70% lethal dose (LD) followed, or not, one hour later by IP Na2SeO3 at MDWT and (iii) by doses>MDWT. At 48 h, in survivors, we measured plasma creatinine, lactate, aspartate and alanine aminotransferase (AST, ALT), nitric oxide (NO) and Se concentrations. RESULTS: (i) Na2SeO3 alone did not increase NO and lactate. Encephalopathy appeared at 1mg Se/kg. Creatinine increased at 1-1.75 mg Se/kg, AST, ALT at 3-4.5 mg Se/kg, and the minimum LD was 3 mg Se/kg. (ii) Mortality after LPS was 37/50 (74%, [62-86%]) vs. 20/30 (67%, [50-84%]) when followed by Na2SeO3 at MDWT (p=0.483) with a decreased in NO (-31%, p=0.038) a trend for lactate decrease (-19%, p=0.068) and an increased Se in plasma of survivals. (iii) All rats died at doses ≥0.6 mg/kg (p<0.001). CONCLUSION: Mechanisms of LPS and Na2SeO3 toxicity differ (i.e. NO, lactate). In septic shock 5H2O·Na2SeO3 toxicity increased, margin of safety decrease, but IP administration of dose considered as oxidant of 5H2O·Na2SeO3 showed beneficial effects.

[What's new in biomedical applications for Terahertz (THz) technology]. / A la frontière onde-lumière : que peuvent apporter les techniques térahertz (THz) dans le domaine de la santé ?

Terahertz technologies have recently been applied to develop high resolution imaging. Since practical portable systems can be designed, the possibilty has emerged to easily screen for biohazards and concealed objects, a procedure which usually requires remote analysis. Applications of THz are also envisaged in the medical field, because this technology offers a degree of accuracy never reached before in molecule analysis. Skin abnormalities and dental health care are two promising targets of THz applications. Nevertheless, potential hazards and health effects of THz exposure should be monitored carefully, particularly since some data suggest induction of genomic instability.

Effect of sub-deficient zinc status on insulin sensitivity after burn injury in rats.

Although zinc status is an important parameter in insulin sensitivity, data concerning its implication in noxious burn-induced insulin resistance are scarce. The present study was designed to evaluate the impact of zinc status before burn on the recovery of injury with focus on plasma insulin and glucose levels. The experiment was performed in male adult Wistar rats fed from weaning with a zinc normal diet (80 ppm) or a depleted zinc diet (10 ppm) for 8 weeks and burned to third degree on 20% of their total body surface area. Blood and tissue samples were collected 3, 6, and 24 h after injury in order to study biochemical parameters and the glucose/insulin response in relation with the zinc status. After burn, zinc-depleted rats presented an exacerbated decrease in plasma zinc level. In addition, the burn-induced insulin resistance, leading to protein catabolism, was emphasized, with higher plasma insulin, glucose, and leptin levels in zinc-deficient animals versus normal-fed rats. Our experimental results underlined the interest to early control the zinc status in order to limit the deleterious effects of oxidative stress and insulin resistance in burned patients.

Burn-induced oxidative stress is altered by a low zinc status: kinetic study in burned rats fed a low zinc diet.

As an initial subdeficient status of zinc, considered as an essential antioxidant trace element, is frequent in burned patients, we aim to assess the effects of low zinc dietary intakes on burn-induced oxidative stress, in an animal model. After 8 weeks of conditioning diets containing 80 ppm (control group) or 10 ppm of zinc (depleted group), Wistar rats were 20% TBSA burned and sampled 1-10 days after injury. Kinetic evolutions of zinc status, plasma oxidative stress parameters, and antioxidant enzymes were also studied in blood and organs. The zinc-depleted diet induced, before injury, a significant decrease in zinc bone level and the increase of oxidative stress markers without stimulation of antioxidant enzyme activity. After burn, more markedly in zinc depleted animals than in controls, zinc levels decreased in plasma and bone, while increasing in liver. The decrease of thiol groups and GSH/GSSG ratio and the depression of GPx activity in liver are also moderately emphasized. Nevertheless, depleted zinc status could not be considered as determining for oxidative damages after burn injury. Further investigations must also be done to enlighten the mechanism of beneficial effects of zinc supplementation reported in burned patients.

Toxic effects of iterative intraperitoneal administration of zinc gluconate in rats.

Although zinc is an essential trace element involved in many physiological functions, toxicological data concerning acute exposure are scarce. The aim of our study was to determine the maximal iterative dose of zinc that can be administrated in rats without any adverse effect. Saline (control group) or zinc gluconate at 1, 2 or 4 mg/kg were intraperitoneally injected in animals daily during 7 days. The tolerance of zinc treatments was evaluated by the observation of clinical symptoms, haematological parameters and biochemistry, in relation to the zinc and copper levels in blood, liver, pancreas and faeces. We found no serious adverse effect within 1 week in rats injected intraperitoneally with 1 or 2 mg/kg/day of zinc gluconate, which tends to indicate that those doses could be useful in future therapeutic research. In contrast, the therapeutic treatment of adult rats with repeated intraperitoneal injections of a 4 mg/kg/day zinc dose should be cancelled, due to the occurrence of clinical adverse effects within a few days, as intraperitoneal local intolerance or major growth underdevelopment.

Interleukin-6, TNF-alpha and interleukin-1 beta levels in blood and tissue in severely burned rats.

Previous studies have demonstrated the early appearance of inflammatory cytokines in the systemic circulation after thermal injury both in humans and animals. The aim of this study was to evaluate the time course of several cytokines, IL-6, TNF-alpha and IL-1beta in serum, lung, liver and brain of severely burned rats during the first week after thermal injury. Cytokine measurements were performed by enzyme-linked immunosorbent assay (ELISA). The comparison between the sham-burned animals and animals with third-degree burns on 20% or 40% of their total body surface area allowed for the study of the inflammatory process relative to the size of the injury. Serum IL-6 levels, which were undetectable in sham-treated animals, peaked during the first hours after injury and were proportionate to the size of the area burned. After a few days, IL-6 increased once more, but only in the most severely burned rats. In lung, liver and brain, low but measurable basal levels of TNF-alpha and IL-1 were detected in sham-burned animals. Strikingly, IL-1beta levels remained significantly elevated in the lung after injury in animals having 20% and 40% burned skin area. Unexpectedly, both TNF-alpha and IL-1beta production decreased gradually in liver and brain after burn injury. Also, the inflammatory response after a burn injury appeared to be biphasic. The first period corresponded to the early release of IL-6 into the circulation, proportional to the severity of the injury. After a few days, a second period was marked by the extension of the inflammatory processes from the injured area to the rest of the body, particularly to lung, which could be considered as at potential risk of involvement in severely burned patients.

The use of physical hydrogels of chitosan for skin regeneration following third-degree burns.

Skin repair is an important field of the tissue engineering, especially in the case of extended third-degree burns, where the current treatments are still insufficient in promoting satisfying skin regeneration. Bio-inspired bi-layered physical hydrogels only constituted of chitosan and water were processed and applied to the treatment of full-thickness burn injuries. The aim of the study was at assessing whether this material was totally accepted by the host organism and allowed in vivo skin reconstruction of limited area third-degree burns. A first layer constituted of a rigid protective gel ensured good mechanical properties and gas exchanges. A second soft and flexible layer allowed the material to follow the geometry of the wound and ensured a good superficial contact. To compare, highly viscous solutions of chitosan were also considered. Veterinary experiments were performed on pig's skins and biopsies at days 9, 17, 22, 100 and 293, were analysed by histology and immuno-histochemistry. Only one chitosan material was used for each time. All the results showed that chitosan materials were well tolerated and promoted a good tissue regeneration. They induced inflammatory cells migration and angiogenetic activity favouring a high vascularisation of the neo-tissue. At day 22, type I and IV collagens were synthesised under the granulation tissue and the formation of the dermal-epidermal junction was observed. After 100 days, the new tissue was quite similar to a native skin, especially by its aesthetic aspect and its great flexibility.

Brain and cognitive impairments from burn injury in rats.

Severe burn injuries affect not only the release of stress hormones but also the metabolism of nitric oxide (NO), a substance playing a large role in cognition. We investigated the effect of third-degree burns both on central NO-levels and on short-term memory in rats. Burns were administered under halothane-anesthesia by dipping 20% of the skin area in hot water. In a first experiment, NO-changes were estimated over hours by differential normal pulse voltammetry (DPNV) with a sensor implanted chronically in the frontal cortex. In a second experiment, cognitive abilities were tested over days by comparing the spontaneous time used to explore objects that the animals had, either never- or already-encountered before. Cerebral NO appeared steadily depleted for at least 12h after the injury, not after control anesthesia. During nine days following the burn, discrimination performance was lower compared to controls. Putting together the results of the two experiments, especially on the day of burn, NO changes are likely to account for the behavioral effect. A choice of neuro-pharmacological agents involved in NO-metabolism, together with a choice of proper anesthetics, should now be tested as means to alleviate cognitive impairments following third-degree burns.

Kinetic changes of oxidative stress and selenium status in plasma and tissues following burn injury in selenium-deficient and selenium-supplemented rats.

BACKGROUND: This study investigates the relationship between the burn-induced oxidative stress and the selenium status. METHODS: The rats were fed with a selenium-adequate diet or a selenium-depleted diet for 5 weeks, before a third-degree thermal injury was applied to the animals. One group of selenium-depleted animals received injections of sodium selenite after the injury. The selenium status and the oxidative stress parameters were measured for 5 days. RESULTS: The selenium-deficient diet leads to oxidative stress with a high stimulation of the superoxide dismutase activity. After the burn injury, the oxidative stress appears important because the initial selenium status is already impaired and, in all animals, the selenium levels and the antioxidant seleno-dependent glutathione peroxidase (GPx) activity decrease in the plasma and the tissues. A treatment with daily selenium injections is efficient in normalizing selenium levels and restores the GPx activity, but fails to counteract the initial oxidative damages induced by the selenium-deficient diet. CONCLUSIONS: The selenium status before the burn injury is a modulating factor of the burn-induced oxidative stress. A single selenium supplement is not sufficient to counteract these oxidative damages and henceforth combined antioxidant supplementations should be investigated to improve the early treatment of the burn patients.

Burn-induced alterations of chromium and the glucose/insulin system in rats.

Our objective was to demonstrate a role of chromium (Cr) in response to severe burn. A third-degree burn involving 20% of total body surface was applied under anaesthesia in accord with ethical guidelines. Chromium concentrations in liver decreased progressively and were non-detectable on days 5 and 10 following injury. In quadriceps muscle, Cr concentrations increased 6h after injury and then declined significantly within the first day and remained at these levels the following 9 days. Urinary Cr losses were also increased. Changes in kidney, brain and serum Cr were not significant. Non-fasting glucose rose 6h after injury and then returned to levels measured before the burn. There was a significant rise in corticosterone reaching a maximum the first day after injury that was accompanied by significant increases in circulating insulin and glucagon that were maximal after 2 days. Changes in IGF-1 were not significant. In summary, changes in Cr concentrations were associated with an early hyperglycemia, hyperinsulinemia and increased secretion of stress hormones. These observations strongly suggest a mobilization and utilization of Cr following severe burn. Additional studies are needed to document that improved Cr status might lead to improved recovery following burn.

Optimization of selenium status by a single intraperitoneal injection of Se in Se-deficient rat: possible application to burned patient treatment.

In order to investigate the efficiency of a single selenium (Se) administration in restoring selenium status, Se and antioxidant enzymes were studied in an animal model of Se depletion. In Se-depleted animals receiving or not a single parenteral administration of Se, plasma, red blood cell (RBC), and tissue Se levels were measured concurrently with glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities. The oxidative stress was assessed by thiobarbituric acid-reactive species (TBARs), total thiol groups, glutathione, and tocopherol measurements. Our study showed that Se depletion with alterations in the antioxidant defense system (Se and GPx activity decreases) led to an increase of lipid peroxidation, a decrease of the plasma vitamin E level, and SOD activation. Sodium selenite injection resulted after 24 h in an optimal plasma Se level and a reactivation of GPx activity. In liver, brain, and kidney, Se levels in injected animals were higher than those in reference animals. However, this single administration of Se failed to decrease free radical damage induced by Se depletion. Therefore, in burned patients who exhibit an altered Se status despite a daily usually restricted Se supplementation, the early administration of a consistent Se amount to improve the GPx activity should be of great interest in preventing the impairment of the antioxidant status.

High levels of endogenous nitric oxide produced after burn injury in rats arrest activated T lymphocytes in the first G1 phase of the cell cycle and then induce their apoptosis.

Major physical traumas provoke a systemic inflammatory response and immune dysfunction. In a model of thermal injury in rats, we previously showed that an overproduction of nitric oxide (NO) was responsible for the collapse of lymphoproliferative responses. In the present work, we performed a time-course analysis of cell proliferation and cell death parameters in order to establish the sequence of events triggered by the high NO output in Wistar/Han rat splenocytes activated with Con A, 10 days after burn injury. We demonstrate that activated T cells from burned rats never divided whereas normal T cells underwent four division cycles. However, T cells from both burned and normal rat entered the G1 phase as shown by increase of cell size, mitochondria hyperpolarization, and expression of cyclin D1. Burned rat T cells progressed to the late G1 phase as shown by expression of the nuclear Ki-67 antigen, but they never entered the S phase. They underwent apoptosis as shown by morphological parameters, disruption of transmembrane mitochondrial potential, and DNA fragmentation. Persistent accumulation of the p53 protein accompanied these phenomena. NO synthase inhibitors antagonize alterations of cell proliferation and cell death parameters in burned rat T cells and accelerated p53 turnover.

Early evolution of selenium status and oxidative stress parameters in rat models of thermal injury.

The objective of the present study was to measure the relationship between selenium status and oxidative stress in two rat models of thermal injury. A non-lethal third-degree burn injury involving 20% (experiment 1) or 40% (experiment 2) of total body surface area (TBSA) was applied to male Wistar rats. Selenium level, glutathione peroxidase (GPx) activity in plasma, red blood cells (RBC) and tissues (liver, kidney, muscle, and brain), and plasma selenoalbumin (Se-alb) were measured in control rats and in burned rats respectively 6 hours after injury and daily from day 1 to day 5. In parallel, lipid and protein oxidative damages, monitored by plasma and tissue thiobarbituric acid reactive species (TBARs) levels and plasma total thiol groups were assessed. We observed a decrease of plasma Se and Se-albumin 6 hours after burn injury. In parallel, plasma GPx activity rapidly decreased and remained significantly lower than in control rats. These alterations were enhanced by the burn injury severity. Plasma TBARs followed the same pattern as that of plasma cholesterol, with an initial decrease and an increase at day 3 in 40% TBSA burned rats. Plasma thiol groups decreased in the two experiments indicating plasma protein oxidation. These results confirm an early oxidative stress in burn injury, and suggest an early selenium mobilization, which might counteract this oxidative stress. These data underline the crucial need of a restored selenium status in burned patients immediately after the burn injury.

Rat pro-inflammatory cytokine and cytokine related mRNA quantification by real-time polymerase chain reaction using SYBR green.

BACKGROUND: Cytokine mRNA quantification is widely used to investigate cytokine profiles, particularly in small samples. Real-time polymerase chain reaction is currently the most reliable method of quantifying low-level transcripts such as cytokine and cytokine receptor mRNAs. This accurate technique allows the quantification of a larger pattern of cytokines than quantification at the protein level, which is limited to a smaller number of proteins. RESULTS: Although fluorogenic probes are considered more sensitive than fluorescent dyes, we have developed SYBR Green real-time RT-PCR protocols to assay pro-inflammatory cytokines (IL1a, IL1b and IL6, TNFa), cytokine receptors (IL1-r1, IL1-r2, IL6-r, TNF-r2) and related molecules (IL1-RA, SOCS3) mRNA in rats. This method enables normalisation against several housekeeping genes (beta-actin, GAPDH, CypA, HPRT) dependent on the specific experimental treatments and tissues using either standard curve, or comparative CT quantification method. PCR efficiency and sensitivity allow the assessment of; i) basal mRNA levels in many tissues and even decreases in mRNA levels, ii) mRNA levels from very small samples. CONCLUSION: Real-time RT-PCR is currently the best way to investigate cytokine networks. The investigations should be completed by the analysis of genes regulated by cytokines or involved in cytokine signalling, providing indirect information on cytokine protein expression.

Effects of a thermal injury on brain and blood nitric oxide (NO) content in the rat.

In the present study, the effects of a thermal injury on the nitric oxide (NO)-ergic system was investigated in freely moving rats. Using a voltammetric method allowing direct and in situ NO measurements, a significant decrease in cortical NO concentration was observed during the 24h following burning procedure. Since in the burning procedure halothane was employed, it was verified that this anaesthetic did not induce significant effect on cortical NO level. Experiments conducted in ex vivo conditions showed that blood NO and nitrites (NO(2)(-)) + nitrates (NO(3)(-)) concentrations increased strongly after burn injury while hypothalamic inducible NO-synthase (NOS(2)) mRNA level decreased significantly. A thermal injury was thus accompanied by a rapid impairment of the NO-ergic pathways, which might partly have been responsible for numerous changes occurring after burn injury.

Nitric oxide inhibits spleen cell proliferative response after burn injury by inducing cytostasis, apoptosis, and necrosis of activated T lymphocytes: role of the guanylate cyclase.

We previously showed that an overproduction of nitric oxide (NO) by macrophages was responsible for the collapse of lymphoproliferative responses after burn injury in rats. First, we demonstrate here that 10 days post-burn, the inhibition of splenocyte response to concanavalin-A results from cytostatic, apoptotic, and necrotic effects of NO on activated T cells. This was evidenced by various criteria at the levels of DNA, mitochondria, and plasma membrane. Inhibition of NO synthase by S-methylisothiourea (10 microM) normalized all the parameters. Second, we show that two soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ, restored the proliferative response in a concentration-dependent manner. LY83583 (0.5 microM) rescued T cells from apoptosis. Similar results were obtained with KT5823 (5 microM) a specific inhibitor of protein kinase G (PKG). In contrast, neither LY83583 nor KT5823 inhibited NO-induced necrosis. These results suggest that NO blocked T cells in the G1 phase and induced apoptosis through a sGC-PKG-dependent pathway and necrosis through an independent one.