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 C(T )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

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 60Co 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 (50×106 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

Persistent DNA Damage after High Dose In Vivo Gamma Exposure of Minipig Skin

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.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.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

Etude de propriétés antibactériennes d'hydrogels de chitosane à usage médical

LYON1-BU Santé (693882101) / SudocSudocFranceF

Intérêt des micronutriments pour lutter contre le stress oxydant chez le brûlé (pratique de supplémentation dans les Centres de Traitement des Brûlés)

LYON1-BU Santé (693882101) / SudocSudocFranceF

Involvement of the central nervous system in radiation-induced multi-organ dysfunction and/or failure.

International audienceThe presence of multiple organ dysfunction syndrome (MODS) in victims of the recent accidents in Nesvizh and Tokai-mura suggests that radiation-induced systemic inflammatory response syndrome (SIRS) occurs in acute radiation sickness (ARS). Multiple organ failure (MOF) refers to the gradual and sequential failure of organs occurring after a wide spectrum of insults. MOF is believed to be the consequence of the host's response to the insult and is strongly linked to SIRS. It is believed that SIRS is mediated by endogenous regulators that are released during the acute phase reaction. The resulting interplay of cytokines may compromise homeostasis of various organ systems, resulting in MODS. In the classical description of ARS, the role of the central nervous system (CNS) has been underestimated. Today, it is recognised that the CNS is a radiosensitive organ whose degree of dysfunction can be quantified by electrophysiological, biochemical and/or behavioural parameters. Abnormalities in CNS function defined by these parameters may occur at a low dose of whole body radiation. The evolving concept of radiation-induced MODS in ARS provides a framework for evaluating injury to the CNS. Ionising radiation also induces an inflammatory response that may be specific to the CNS. This response is observed after either local irradiation of the CNS or whole body irradiation. The relationship between inflammatory responses in the CNS and the peripheral nervous system is undefined. Whether or not the CNS inflammatory response syndrome is a consequence of SIRS or is an independent syndrome remains an open question. The answer to this question may have implications regarding therapy and medical management of irradiated victims

Development of a skin burn predictive model adapted to laser irradiation

International audienceLaser technology is increasingly used, and it is crucial for both safety and medical reasons that the impact of laser irradiation on human skin can be accurately predicted. This study is mainly focused on laser–skin interactions and potential lesions (burns). A mathematical model dedicated to heat transfers in skin exposed to infrared laser radiations has been developed. The model is validated by studying heat transfers in human skin and simultaneously performing experimentations an animal model (pig). For all experimental tests, pig’s skin surface temperature is recorded. Three laser wavelengths have been tested: 808&nbsp;nm, 1940&nbsp;nm and 10 600&nbsp;nm. The first is a diode laser producing radiation absorbed deep within the skin. The second wavelength has a more superficial effect. For the third wavelength, skin is an opaque material. The validity of the developed models is verified by comparison with experimental results (in vivo tests) and the results of previous studies reported in the literature. The comparison shows that the models accurately predict the burn degree caused by laser radiation over a wide range of conditions. The results show that the important parameter for burn prediction is the extinction coefficient. For the 1940&nbsp;nm wavelength especially, significant differences between modeling results and literature have been observed, mainly due to this coefficient’s value. This new model can be used as a predictive tool in order to estimate the amount of injury induced by several types (couple power-time) of laser aggressions on the arm, the face and on the palm of the hand

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

International audienceSkin 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

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

International audiencePrevious 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