BABOON RADIATION QUALITY (MIXED-FIELD NEUTRON AND GAMMA, GAMMA ALONE) DOSE-RESPONSE MODEL SYSTEMS: ASSESSMENT OF H-ARS SEVERITY USING HAEMATOLOGIC BIOMARKERS.

Results from archived (1986 and 1996) experiments were used to establish a baboon radiation-quality dose-response database with haematology biomarker time-course data following exposure to mixed-fields (i.e. neutron to gamma ratio: 5.5; dose: 0-8 Gy) and 60Co gamma-ray exposures (0-15 Gy). Time-course (i.e. 0-40 d) haematology changes for relevant blood-cell types for both mixed-field (neutron to gamma ratio = 5.5) and gamma ray alone were compared and models developed that showed significant differences using the maximum likehood ratio test. A consensus METREPOL-like haematology ARS (H-ARS) severity scoring system for baboons was established using these results. The data for mixed-field and the gamma only cohorts appeared similar, and so the cohorts were pooled into a single consensus H-ARS severity scoring system. These findings provide proof-of-concept for the use of a METREPOL H-ARS severity scoring system following mixed-field and gamma exposures.

No evidence for toxicity after long-term photobiomodulation in normal non-human primates.

In this study, we explored the effects of a longer term application, up to 12 weeks, of photobiomodulation in normal, naïve macaque monkeys. Monkeys (n = 5) were implanted intracranially with an optical fibre device delivering photobiomodulation (red light, 670 nm) to a midline midbrain region. Animals were then aldehyde-fixed and their brains were processed for immunohistochemistry. In general, our results showed that longer term intracranial application of photobiomodulation had no adverse effects on the surrounding brain parenchyma or on the nearby dopaminergic cell system. We found no evidence for photobiomodulation generating an inflammatory glial response or neuronal degeneration near the implant site; further, photobiomodulation did not induce an abnormal activation or mitochondrial stress in nearby cells, nor did it cause an abnormal arrangement of the surrounding vasculature (endothelial basement membrane). Finally, because of our interest in Parkinson's disease, we noted that photobiomodulation had no impact on the number of midbrain dopaminergic cells and the density of their terminations in the striatum. In summary, we found no histological basis for any major biosafety concerns associated with photobiomodulation delivered by our intracranial approach and our findings set a key template for progress onto clinical trial on patients with Parkinson's disease.

Effects of a higher dose of near-infrared light on clinical signs and neuroprotection in a monkey model of Parkinson's disease.

We have reported previously that intracranial application of near-infrared light (NIr) - when delivered at the lower doses of 25J and 35J - reduces clinical signs and offers neuroprotection in a subacute MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) monkey model of Parkinson's disease. In this study, we explored whether a higher NIr dose (125J) generated beneficial effects in the same MPTP monkey model (n=15). We implanted an NIr (670nm) optical fibre device within a midline region of the midbrain in macaque monkeys, close to the substantia nigra of both sides. MPTP injections (1.8-2.1mg/kg) were made over a five day period, during which time the NIr device was turned on and left on continuously throughout the ensuing three week survival period. Monkeys were evaluated clinically and their brains processed for immunohistochemistry and stereology. Our results showed that the higher NIr dose did not have any toxic impact on cells at the midbrain implant site. Further, this NIr dose resulted in a higher number of nigral tyrosine hydroxylase immunoreactive cells when compared to the MPTP group. However, the higher NIr dose monkeys showed little evidence for an increase in mean clinical score, number of nigral Nissl-stained cells and density of striatal tyrosine hydroxylase terminations. In summary, the higher NIr dose of 125J was not as beneficial to MPTP-treated monkeys as compared to the lower doses of 25J and 35J, boding well for strategies of NIr dose delivery and device energy consumption in a future clinical trial.

Near-infrared light treatment reduces astrogliosis in MPTP-treated monkeys.

We have reported previously that intracranial application of near-infrared light (NIr) reduces clinical signs and offers neuroprotection in a subacute MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) monkey model of Parkinson's disease. In this study, we explored whether NIr reduces the gliosis in this animal model. Sections of midbrain (containing the substantia nigra pars compacta; SNc) and striatum were processed for glial fibrillary acidic protein (to label astrocytes; GFAP) and ionised calcium-binding adaptor molecule 1 (to label microglia; IBA1) immunohistochemistry. Cell counts were undertaken using stereology, and cell body sizes were measured using ImageJ. Our results showed that NIr treatment reduced dramatically (~75 %) MPTP-induced astrogliosis in both the SNc and striatum. Among microglia, however, NIr had a more limited impact in both nuclei; although there was a reduction in overall cell size, there were no changes in the number of microglia in the MPTP-treated monkeys after NIr treatment. In summary, we showed that NIr treatment influenced the glial response, particularly that of the astrocytes, in our monkey MPTP model of Parkinson's disease. Our findings raise the possibility of glial cells as a future therapeutic target using NIr.

Contribution of INTRAMUSCULAR Autologous Adipose Tissue-Derived Stem Cell Injections to Treat Cutaneous Radiation Syndrome: Preliminary Results.

Cutaneous radiation syndrome caused by high dose located irradiation is characterized by delayed symptoms, incomplete wound healing, and poor revascularization. Subcutaneous adipose tissue derived stromal/stem cells have been shown to improve skin repair in a minipig model of cutaneous radiation syndrome despite a subcutaneous defect being a consequence of radio-induced muscular fibrosis. Based on the pro-myogenic potential of stromal/stem cells, a new protocol combining subcutaneous and intramuscular injections was evaluated in a preliminary study. Six female minipigs were locally irradiated at the dose of 50 Gy using a Co source (0.6 Gy min) and randomly divided into two groups. Three animals received the vehicle (phosphate-buffer-saline solution) and three animals received three injections of 75 × 10 adipose tissue derived stromal/stem cells each time (day 25, 46, and 66 post-irradiation). Pigs were euthanized on day 76 post-irradiation before development of clinical skin symptoms. All minipigs exhibited a homogeneous skin evolution. Macroscopic observation of irradiated muscles showed prominent fibrosis and necrosis areas in controls as opposed to adipose tissue-derived stromal/stem cells injected animals. Moreover, muscle biopsy analysis highlighted a recruitment of myofibroblasts (Immune Reactive Score: p < 0.01), an interleukin 10 secretion and a muscle regeneration pathway activation after intramuscular injections of adipose tissue-derived stromal/stem cells (western-blot: respectively, 200-fold change difference and twofold higher in treated animals). Globally, these preliminary data suggest that intramuscular injections of adipose tissue-derived stromal/stem cells improve muscle regeneration in the cutaneous-radiation syndrome. Further work is ongoing to evaluate this therapeutic strategy on a larger animal number with a longer clinical follow-up.

Near-infrared light is neuroprotective in a monkey model of Parkinson disease.

OBJECTIVE: To examine whether near-infrared light (NIr) treatment reduces clinical signs and/or offers neuroprotection in a subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkey model of Parkinson disease. METHODS: We implanted an optical fiber device that delivered NIr (670 nm) to the midbrain of macaque monkeys, close to the substantia nigra of both sides. MPTP injections (1.5-2.1mg/kg) were made over a 5- to 7-day period, during which time the NIr device was turned on. This was then followed by a 3-week survival period. Monkeys were evaluated clinically (eg, posture, bradykinesia) and behaviorally (open field test), and their brains were processed for immunohistochemistry and stereology. RESULTS: All monkeys in the MPTP group developed severe clinical and behavioral impairment (mean clinical scores = 21-34; n = 11). By contrast, the MPTP-NIr group developed much less clinical and behavioral impairment (n = 9); some monkeys developed moderate clinical signs (mean scores = 11-15; n = 3), whereas the majority--quite remarkably--developed few clinical signs (mean scores = 1-6; n = 6). The monkeys that developed moderate clinical signs had hematic fluid in their optical fibers at postmortem, presumably limiting NIr exposure and overall clinical improvement. NIr was not toxic to brain tissue and offered neuroprotection to dopaminergic cells and their terminations against MPTP insult, particularly in animals that developed few clinical signs. INTERPRETATION: Our findings indicate NIr to be an effective therapeutic agent in a primate model of the disease and create the template for translation into clinical trials.

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.

Transient gene therapy to treat cutaneous radiation syndrome: development in a minipig model.

Cutaneous radiation syndrome is the delayed consequence of localized skin exposure to high doses of ionizing radiation. Adipocyte derived stem cells injection may improve tissue regeneration through secreted factors. Thus mesenchymal stem cells secretome optimization, using transient transfection, may represent a new strategy to treat this syndrome. Sonic hedgehog, a secreted protein involved in cell proliferation and angiogenesis, has been chosen as a first candidate. Here preliminary results are reported of the therapeutic potential of transient gene therapy to cure cutaneous radiation syndrome in a minipig model. Adipocyte derived stem cells were transiently transfected by electroporation with a plasmid coding for Sonic Hedgehog. Göttingen minipigs were locally irradiated using a (60)Co gamma source at the dose of 50 Gy and received Phosphate Buffer Salin (controls: n = 8), stem cells (50 × 106 each time, n = 5) or transfected stem cells (25±7 × 106 each time, n = 1). All controls exhibited a homogeneous clinical evolution of cutaneous radiation syndrome with final necrosis (day 91). In stem cell injected minipigs, an ultimate wound healing was observed in four out of five grafted animals (day 130 ± 28, complete in two of them) (historical results). The Sonic hedgehog animal, albeit injected with a lower number of transfected stem cells, presented a very similar evolution of skin healing without necrosis or uncontrolble pain. Globally this preliminary report suggests that local injection of Sonic Hedgehog transfected adipocyte derived stem cells may improve wound healing. Thus work is ongoing to evaluate this therapeutic strategy on a larger number of animals.

DNA damage focus analysis in blood samples of minipigs reveals acute partial body irradiation.

Radiation accidents frequently involve acute high dose partial body irradiation leading to victims with radiation sickness and cutaneous radiation syndrome that implements radiation-induced cell death. Cells that are not lethally hit seek to repair ionizing radiation (IR) induced damage, albeit at the expense of an increased risk of mutation and tumor formation due to misrepair of IR-induced DNA double strand breaks (DSBs). The response to DNA damage includes phosphorylation of histone H2AX in the vicinity of DSBs, creating foci in the nucleus whose enumeration can serve as a radiation biodosimeter. Here, we investigated γH2AX and DNA repair foci in peripheral blood lymphocytes of Göttingen minipigs that experienced acute partial body irradiation (PBI) with 49 Gy (± 6%) Co-60 γ-rays of the upper lumbar region. Blood samples taken 4, 24 and 168 hours post PBI were subjected to γ-H2AX, 53BP1 and MRE11 focus enumeration. Peripheral blood lymphocytes (PBL) of 49 Gy partial body irradiated minipigs were found to display 1-8 DNA damage foci/cell. These PBL values significantly deceed the high foci numbers observed in keratinocyte nuclei of the directly γ-irradiated minipig skin regions, indicating a limited resident time of PBL in the exposed tissue volume. Nonetheless, PBL samples obtained 4 h post IR in average contained 2.2% of cells displaying a pan-γH2AX signal, suggesting that these received a higher IR dose. Moreover, dispersion analysis indicated partial body irradiation for all 13 minipigs at 4 h post IR. While dose reconstruction using γH2AX DNA repair foci in lymphocytes after in vivo PBI represents a challenge, the DNA damage focus assay may serve as a rapid, first line indicator of radiation exposure. The occurrence of PBLs with pan-γH2AX staining and of cells with relatively high foci numbers that skew a Poisson distribution may be taken as indicator of acute high dose partial body irradiation, particularly when samples are available early after IR exposure.

Application of adipocyte-derived stem cells in treatment of cutaneous radiation syndrome.

Cutaneous radiation syndrome caused by local high dose irradiation is characterized by delayed outcome and incomplete healing. Recent therapeutic management of accidentally irradiated burn patients has suggested the benefit of local cellular therapy using mesenchymal stem cell grafting. According to the proposed strategy of early treatment, large amounts of stem cells would be necessary in the days following exposure and hospitalization, which would require allogeneic stem cells banking. In this context, the authors compared the benefit of local autologous and allogeneic adipocyte-derived stem cell injection in a large animal model. Minipigs were locally irradiated using a 60Co gamma source at a dose of 50 Gy and divided into three groups. Two groups were grafted with autologous (n = 5) or allogeneic (n = 5) adipocyte-derived stem cells four times after the radiation exposure, whereas the control group received the vehicle without cells (n = 8). A clinical score was elaborated to compare the efficiency of the three treatments. All controls exhibited local inflammatory injuries leading to a persistent painful necrosis, thus mimicking the clinical evolution in human victims. In the autologous adipocyte-derived stem cells group, skin healing without necrosis or uncontrollable pain was observed. In contrast, the clinical outcome was not significantly different in the adipocyte-derived stem cell allogeneic group when compared with controls. This study suggests that autologous adipocyte-derived stem cell grafting improves cutaneous radiation syndrome wound healing, whereas allogeneic adipocyte derived stem cells do not. Further studies will establish whether manipulation of allogeneic stem cells will improve their therapeutic potential.

Gene therapy to mitigate radiation-induced bone marrow aplasia: preliminary study in highly irradiated monkeys.

The hematopoietic syndrome represents the first therapeutic challenge following exposure to high doses of ionizing radiation. Today there is a crucial need to identify/develop new treatments in order to reach the transplantation threshold. The authors propose the concept of a global niche therapy strategy based on local and short-term secretion of selected morphogenes to favor a vascular niche in order to raise the transplantation threshold regeneration and to stimulate residual hematopoietic stem and progenitor cells. The present study was aimed at setting up a monkey model of gene therapy using Sonic hedgehog (Shh) as a first candidate. Multipotent mesenchymal stem cells from adipocyte tissues were nucleofected with mock and Sonic hedgehog pIRES2 plasmids using Amaxa technology. 8-Gy gamma irradiated monkeys were given a single intraosseous injection of manipulated or unmanipulated adipocyte stem cells 48 h following total body irradiation. Mock and Shh-grafts were well tolerated. This preliminary study establishes the feasibility of transient gene therapy in highly irradiated monkeys. Ongoing studies will determine the putative efficacy of this therapeutic strategy.

Assessment of total- and partial-body irradiation in a baboon model: preliminary results of a kinetic study including clinical, physical, and biological parameters.

This biodosimetry study used irradiated baboons to investigate the efficacy of a kinetic multiparameter (clinical, physical, and biological) approach for discriminating partial-body irradiation (PBI) and total-body irradiation (TBI). Animals were unilaterally (front) exposed to 60Co gamma rays (8 to 32 cGy min) using either TBI or vertical left hemi-body irradiation (HBI), as follows: 2.5 Gy TBI (n = 2), 5 Gy TBI (n = 2), 5 Gy HBI (n = 2), and 10 Gy HBI (n = 2). Midline tissue doses were measured at the anterior iliac crest level with an ionization chamber, and body dosimetry was performed using thermoluminescent dosimeters. Blood samples were collected before exposure and from 1 h until 200 d after irradiation. Clinical status, complete blood cell count, biochemical parameters, and cytogenetic analysis were evaluated. The partial least square discriminant analysis chosen for statistical analysis showed that the four groups of irradiated baboons were clearly separated. However, the dicentric chromosome assay may not distinguish HBI from TBI in confounding situations where equivalent whole-body doses are similar and the time of exposure is sufficient for peripheral blood lymphocyte homogenization. Interestingly, as bone marrow shielding in HBI animals prevented aplasia from happening, hematologic parameters such as the platelet count and Flt-3 ligand level helped to distinguish HBI and TBI. Moreover, the ratio of neutrophil to lymphocyte counts, creatine kinase, and citrulline levels may be discriminating biomarkers of dose or injury. Both early and delayed clinical signs and bioindicators appear to be useful for assessment of heterogeneous irradiation.

Persistent DNA damage after high dose in vivo gamma exposure of minipig skin.

BACKGROUND: Exposure to high doses of ionizing radiation (IR) can lead to localized radiation injury of the skin and exposed cells suffer dsDNA breaks that may elicit cell death or stochastic changes. Little is known about the DNA damage response after high-dose exposure of the skin. Here, we investigate the cellular and DNA damage response in acutely irradiated minipig skin. METHODS AND FINDINGS: IR-induced DNA damage, repair and cellular survival were studied in 15 cm(2) of minipig skin exposed in vivo to ~50 Co-60 γ rays. Skin biopsies of control and 4 h up to 96 days post exposure were investigated for radiation-induced foci (RIF) formation using γ-H2AX, 53BP1, and active ATM-p immunofluorescence. High-dose IR induced massive γ-H2AX phosphorylation and high 53BP1 RIF numbers 4 h, 20 h after IR. As time progressed RIF numbers dropped to a low of <1% of keratinocytes at 28-70 days. The latter contained large RIFs that included ATM-p, indicating the accumulation of complex DNA damage. At 96 days most of the cells with RIFs had disappeared. The frequency of active-caspase-3-positive apoptotic cells was 17-fold increased 3 days after IR and remained >3-fold elevated at all subsequent time points. Replicating basal cells (Ki67+) were reduced 3 days post IR followed by increased proliferation and recovery of epidermal cellularity after 28 days. CONCLUSIONS: Acute high dose irradiation of minipig epidermis impaired stem cell replication and induced elevated apoptosis from 3 days onward. DNA repair cleared the high numbers of DBSs in skin cells, while RIFs that persisted in <1% cells marked complex and potentially lethal DNA damage up to several weeks after exposure. An elevated frequency of keratinocytes with persistent RIFs may thus serve as indicator of previous acute radiation exposure, which may be useful in the follow up of nuclear or radiological accident scenarios.

Autologous adipocyte derived stem cells favour healing in a minipig model of cutaneous radiation syndrome.

Cutaneous radiation syndrome (CRS) is the delayed consequence of localized skin exposure to high doses of ionizing radiation. Here we examined for the first time in a large animal model the therapeutic potential of autologous adipose tissue-derived stroma cells (ASCs). For experiments, Göttingen minipigs were locally gamma irradiated using a (60)Co source at the dose of 50 Gy and grafted (n = 5) or not (n = 8). ASCs were cultured in MEM-alpha with 10% fetal calf serum and basic fibroblast growth factor (2 ng.mL(-1)) and post irradiation were intradermally injected on days 25, 46, 67 and finally between days 95 and 115 (50 × 10(6) ASCs each time) into the exposed area. All controls exhibited a clinical evolution with final necrosis (day 91). In grafted pigs an ultimate wound healing was observed in four out of five grafted animals (day 130 +/- 28). Immunohistological analysis of cytokeratin expression showed a complete epidermis recovery. Grafted ASCs accumulated at the dermis/subcutis barrier in which they attracted numerous immune cells, and even an increased vasculature in one pig. Globally this study suggests that local injection of ASCs may represent a useful strategy to mitigate CRS.

Skin burns after laser exposure: histological analysis and predictive simulation.

Thermal effects of laser irradiation on skin are investigated in this paper. The main purpose is to determine the damage level induced by a laser exposure. Potential burns induced by two lasers (wavelength 808nm and 1940nm) are studied and animal experimentations are performed. Several exposure durations and laser powers are tested. Based on previous works, a mathematical model dedicated to temperature prediction is proposed and finite-element method is implemented. This numerical predictive tool based on the bioheat equation takes into account heat losses due to the convection on skin surface, blood circulatory and also evaporation. Thermal behavior of each skin layer is also described considering distinct thermal and optical properties. Since the mathematical model is able to estimate damage levels, histological analyses were also carried through. It is confirmed that the mathematical model is an efficient predictive tool for estimation of damage caused by lasers and that thermal effects sharply depend on laser wavelength.

Multipotent mesenchymal stem cell grafting to treat cutaneous radiation syndrome: development of a new minipig model.

OBJECTIVE: Cutaneous radiation syndrome (CRS) is the delayed consequence of localized skin exposure to high doses of ionizing radiation. Recent grafting of three ionizing radiation-burned patients has suggested the benefit of local bone marrow mesenchymal stem cell (MSC) injection in favor of wound healing and pain control. Here, we have developed a new minipig model of severe CRS to study underlying mechanisms of this cell therapy approach. MATERIALS AND METHODS: Göttingen minipigs were locally irradiated using a (60)Co gamma source as follows: ungrafted 50 and 60 Gy (n = 4) and grafted 50 and 60 Gy (n = 3). Bone marrow MSCs were cultured in minimum essential medium with 10% fetal calf serum and basic fibroblast growth factor (2 ng.mL(-1)). Autologous MSCs were intradermally injected twice or three times from days 27 to 96 (range, 99-128.5 × 10(6) MSCs per injection). RESULTS: All animals exhibited a clinical evolution similar to humans after a latency phase of several weeks, including early erythema, hair loss, and dry/moist desquamation followed by necrosis during 81 to 222 days post-ionizing radiation. Skin damage in higher exposed animals appeared slightly earlier. Immunohistology revealed severe skin damage in all animals and rhabdomyolysis in the muscle tissue below the entry area, with the latter being more severe in controls. In grafted animals, MSCs led to local accumulation of lymphocytes at the dermis/subcutis border and improved vascularization. CONCLUSIONS: This study establishes a new minipig model that is close to human and allows development of stem cell therapy strategies that can be applied in treatment of human radiation burns.

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.