Methods to measure the cutaneous transfer of radionuclides across the intact or lesioned epidermis,application to radiotoxicology

Although skin contamination by radionuclides is the most common cause of nuclear workers accidents, few studies dealing with the penetration of radioactive contamination through the skin are available. This work is a review of experimental methods that allow to assess transfer of radionuclides through the skin in occupational conditions, with or without skin trauma. The first section describes the different methods applied for skin transfer assessment of chemicals used in pharmacology. Major radionuclide contamination accidents can be associated with skin traumas. Thus, the second section describes the adaptation of these methods to radiotoxicology. Finally, the third section is an in vivo investigation of cobalt transfer (57CoCl2) through undamaged and damaged skin which simulates different industrial accident conditions (excoriation, acid or alcalin burn, scalding, branding).     

A local approach to reduce industrial uranium wound contamination in rats

The aim of this work is to develop a new approach to partially decontaminate wounds after industrial uranium contamination, during the interval of time between contamination and transfer of the patient to the infirmary. A wound dressing and a paste mixed or not with uranium-chelating ligands, ethane-1-hydroxy-1,1-bisphosphonate (EHBP) and carballylic amido bis phosphonic acid (CAPBP), were tested in vitro on muscles and in vivo on rats after deposit of uranium oxide compounds. The dressing and the paste, composed of carboxymethylcellulose-based hydrocolloids known to be highly absorbent, were applied on simulated wounds a few minutes after the contamination. The incorporation of chelating ligands did not improve the efficacy of the dressing or paste, and the best results were obtained with the dressing. In vivo, after 1 h of contact with the wound, the dressing absorbed about 30% and 60% of a UO4 compound deposited intra- and intermuscularly, respectively. After intramuscular deposit, the efficacy of the dressing was not reduced if the contact time decreased from 1 h to 15 min. Therefore, this wound dressing could be a practical option to treat uranium-contaminated wounds, but its efficacy depends on the localization of the uranium deposit.     

Rapidly Induced Wound Ethylene from Excised Segments of Etiolated Pisum sativum L., cv. Alaska: I. Characterization of the Response

A rapidly induced, transitory increase in the rate of ethylene synthesis occurred in wounded tissue excised from actively growing regions of etiolated barley, cucumber, maize, oat, pea, tomato, and wheat seedlings. Cutting intact stems or excising 9-mm segments of tissue from near the apex of 7-day-old etiolated Pisum sativum L., cv. Alaska seedlings induced a remarkably consistent pattern of ethylene production. At 25 C, wound-induced ethylene production by segments excised 9 mm below the apical hook increased linearly after a lag of 26 minutes from 2.7 nanoliters per g per hour to the first maxium of 11.3 nanoliters per g per hour at 56 minutes. The rate of production then decreased to a minimum at 90 minutes, increased to a lower second maximum at 131 minutes, and subsequently declined over a period of about 100 minutes to about 4 nanoliters per g per hour. Removal of endogenous ethylene, before the wound response commenced, had no effect on the kinetics of ethylene production. Tissue containing large amounts of dissolved ethylene released it as an exponential decay with no lag period. Rapidly induced wound ethylene is synthesized by the tissue and is not merely the result of facilitated diffusion of ethylene already present in the tissue through the newly exposed cut surfaces. Previously wounded apical sections did not exhibit a second response when rewounded. No significant correlation was found between wound-induced ethylene synthesis and either CO₂ or ethane production.     

Qualitative and quantitative study of wound healing processes in the coelenterate, Plexaurella fusifera: spatial, temporal, and environmental (light attenuation) influences

Following injury, the Caribbean soft coral, Plexaurella fusifera, forms an epithelial front containing amoebocytes and zooxanthellae, a photosynthetic endosymbiont. Amoebocytes may be responsible for extruding the connective mesogleal fibers necessary for regeneration of tissue and zooxanthellae may provide the energy for repair. This study examined the effects of time, space, and environment (light attenuation) on wound healing in this coral species and quantitatively confirmed the increase of amoebocyte concentrations in the injured area. A wound was made on coral branchlets by removing approximately 4.5 mm of coenenchyme. At assigned times after injury, samples were collected for gross morphological and histological evaluation, in which amoebocytes and zooxanthellae concentrations were quantified within 0.009 mm3 of tissue. Overall amoebocyte numbers within uninjured and wounded tissue were similar. However, when numbers of amoebocytes per area of injured tissue were calculated and compared to those of uninjured tissue, 82.4% more amoebocytes occurred at distances 0-0.5 mm from the wound edge, while areas of tissue >2 mm from the wound edge were occupied by fewer amoebocytes. Overall increases in concentrations of zooxanthellae also occurred within wounded coral, but no apparent temporal, spatial, or light-related pattern was detected. Therefore, this study supports the conjecture that amoebocyte accumulation at a wound site is an effect of cells migrating from uninjured tissue adjacent to the wounded edge. In addition, this movement occurs regardless of light attenuation. Light, which in this study was confined to ranges between 70 and 545 microE s-1 m-2, did not significantly affect the wound healing process in regard to either closure or cellular concentrations.     

Nuclear accumulation of exogenous DNA fragments in viable cells mediated by FGF-2 and DNA release upon cellular injury

We and others have previously shown that basic fibroblast growth factor (FGF-2 or bFGF) can be used as a targeting molecule to help carry plasmid DNA into cells when the growth factor molecule is physically coupled to the DNA molecule being delivered. Herein we report our observations on the FGF-mediated uptake of exogenous labeled DNA into cultured cells in a manner that is representative of that which may occur under physiological conditions at sites of wounded tissue. Cellular debris at such sites contains nucleic acid fragments released from dead cells, as well as growth factors such as FGF-2 that function early in the wound repair process. Using a cell culture model designed to mimic the local environment of a wound with respect to the presence of soluble FGF-2 and DNA fragments, we have shown that FGF-2 is able to direct the cellular uptake and nuclear localization of fragments of exogenous DNA via the FGF receptor into intact and healthy cells. Furthermore, we can monitor and quantitate this type of FGF-mediated DNA delivery by using indirect immunofluorescence of bromodeoxyuridine-labeled exogenous DNA. Our results suggest that this type of FGF-mediated DNA fragment uptake could allow for the transduction of viable nearest neighbor cells at sites of injury in vivo. Such a phenomenon may lead to mutational aberrations in the recipient cells and enhance the probability of wound carcinogenesis.     

Effects of compound 48/80 and exogenous histamine on wound healing in mice

Compound 48/80 has previously been shown to improve wound healing in rats, presumably through stimulation of histidine decarboxylase activity and mobilization of histamine from mast cells. In the present study, C57Bl/6 mice were wounded by dorsal skin incision followed by treatment with compound 48/80, exogenous histamine, or the combination of 48/80 plus histamine. Skin-breaking strength was significantly increased over saline-injected controls by the combined treatment with 48/80 and histamine. Neither 48/80 or histamine alone had any influence on wound healing. Histamine content of skin at the wound site was significantly reduced by 48/80 treatment, but was unaffected by 48/80 plus histamine or histamine given alone. In contrast, stomach and leg muscle histamine levels were significantly increased beyond those of unwounded, wounded saline- or 48/80-injected mice. These results were also confirmed in CD mice, and are in contrast to findings in rats in which treatment with 48/80 alone significantly improved wound healing of similarly injured animals.     

The effect of stretching on formation of myofibroblasts in mouse skin

Summary Wound contraction results from the contractile activity of modified fibroblasts, termed myofibroblasts, which are present in the granulation tissue of the healing wound. This study examines the relative role of mechanical tension (stretching) and wound healing as events capable of stimulating the formation of myofibroblasts in mouse skin. The skin of hairless mice was subjected to mechanical stretching and to a small incisional wound either separately or in combination. Animals were killed at intervals between 1 and 6 days and the dermis examined with the electron microscope. Stretching alone produced little evidence of inflammation at any time interval but cells with the ultrastructural characteristics of myofibroblasts were present at 4 days and abundant at 6 days. Skin that had been both stretched and wounded showed a marked inflammatory response and also contained myofibroblasts, but they were less frequent than in the skin subjected to stretching alone. Very few myofibroblasts were evident in skin that had only been wounded. It is suggested that the effect of mechanical tension alone may initiate formation of myofibroblasts in a tissue.     

Laminin 332 deposition is diminished in irradiated skin in an animal model of combined radiation and wound skin injury

Skin exposure to ionizing radiation affects the normal wound healing process and greatly impacts the prognosis of affected individuals. We investigated the effect of ionizing radiation on wound healing in a rat model of combined radiation and wound skin injury. Using a soft X-ray beam, a single dose of ionizing radiation (10-40 Gy) was delivered to the skin without significant exposure to internal organs. At 1 h postirradiation, two skin wounds were made on the back of each rat. Control and experimental animals were euthanized at 3, 7, 14, 21 and 30 days postirradiation. The wound areas were measured, and tissue samples were evaluated for laminin 332 and matrix metalloproteinase (MMP) 2 expression. Our results clearly demonstrate that radiation exposure significantly delayed wound healing in a dose-related manner. Evaluation of irradiated and wounded skin showed decreased deposition of laminin 332 protein in the epidermal basement membrane together with an elevated expression of all three laminin 332 genes within 3 days postirradiation. The elevated laminin 332 gene expression was paralleled by an elevated gene and protein expression of MMP2, suggesting that the reduced amount of laminin 332 in irradiated skin is due to an imbalance between laminin 332 secretion and its accelerated processing by elevated tissue metalloproteinases. Western blot analysis of cultured rat keratinocytes showed decreased laminin 332 deposition by irradiated cells, and incubation of irradiated keratinocytes with MMP inhibitor significantly increased the amount of deposited laminin 332. Furthermore, irradiated keratinocytes exhibited a longer time to close an artificial wound, and this delay was partially corrected by seeding keratinocytes on laminin 332-coated plates. These data strongly suggest that laminin 332 deposition is inhibited by ionizing radiation and, in combination with slower keratinocyte migration, can contribute to the delayed wound healing of irradiated skin.     

Ultrastructural analysis between fetal and adult wound healing process of marsupial opossum skin

The opossum delivers a newborn baby equivalent to tremature fetus state by postpregnancy. The peculiarity is advantageous for studies of fetus, because operations to take out fetus from the uterus of a mother are not necessary. When mammalian skin is wounded by full-thickness excision, fetal and adult wound healing processes differ. Fetal-type wound healing does not leave a scar. However, studies of how the fetal wound healing process differs in detail from the adult type are not advanced. We first observed the normal skin development of the gray short-tailed opossum (Monodelphis domestica) using an electron microscope. As for normal skin, an epidermis became multi-layered, and thickened from birth through to 7 days after birth. The quantity of extracellular matrix of the dermis increased thereafter, and several types of cells were found in the dermis. To examine the wound healing, we used material from a 1 day-old newborn baby, and from another 15 days after birth, and compared the wound healing style morphologically. Differences in the constitution of cells and fine structures of the skin were observed, it was obviously suggested that change in the wound healing style from fetal-type to adult-type occurred between 1 to 15 days after birth.     

Wound healing in X-irradiated mammalian skin.

Certain parameters of wound healing were investigated in mouse skin given 950 rads and 3325 rads of X-irradiation at various times relative to wounding. Increased inflammation, delayed dermal regeneration, and delayed contraction were noted in all irradiated groups. The activation of surrounding hair follicles, a process that usually accompanies wounding in mouse skin, occurred earlier, over a shorter elapsed time, and over a greater area of skin in animals irradiated prior to wounding than in the controls or in those irradiated and wounded simultaneously. Epidermal mitotic activity in wounds made at the time of irradiation was initially depressed but recovered by the second postoperative day. Wounds in pre-irradiated animals gave an unexpected result. They responded with an immediate burst of mitotic activity without the usual 24-hr lag that was seen in controls. In the pre-irradiated specimens a substantial number of cells appeared to die after dividing.     

Chaetognaths: a useful model for studying heat shock proteins. Effect of wound healing

The pattern of expression of Heat Shock Protein 70 (Hsp70), a highly conserved cellular protein chaperone, was investigated in Chaetognatha, a very important phylum of marine worms, which play a major role in marine food webs. The in toto distribution of Hsp70-like immunoreactivity was assessed in both intact and experimentally wounded specimens of Spadella cephaloptera Busch, 1851 that have been cut transversally just above the seminal vesicles. In intact animals, the ciliary organs, the corona ciliata, the coronal nerve and the spermatocytes express Hsp70 proteins. The kinetic of the expression pattern has been followed during the wound healing from the lesion to 5 days after. The Hsp70 immunoreactivity was observed according to time in the cerebral and suboesophageal ganglia and in the plexuses of the hood, the mouth, the neck and the tail region. At the wound level, the time-dependent Hsp70 expression was detected in the epidermal cells and along numerous muscle fibres of the tail region. Five days after the tail section, two Hsp70 immunoreactive areas were observed on both sides of the healed wound that correspond to the sites of formation of two new seminal vesicles. It is suggested that, in Chaetognaths, Hsp70 may be implicated in the regulation of several cellular processes especially at the level of the spermatocytes in intact and wounded specimens, and of the nervous system and muscular apparatus during the wound healing. It appears that chaetognaths are a good model as indicator of stress responses for experimental studies at the level of a whole organism.     

Co-operative Bmp- and Fgf-signaling inputs convert skin wound healing to limb formation in urodele amphibians

Urodele amphibians have remarkable organ regeneration capability, and their limb regeneration capability has been investigated as a representative phenomenon. In the early 19th century, nerves were reported to be an essential tissue for the successful induction of limb regeneration. Nerve substances that function in the induction of limb regeneration responses have long been sought. A new experimental system called the accessory limb model (ALM) has been established to identify the nerve factors. Skin wounding in urodele amphibians results in skin wound healing but never in limb induction. However, nerve deviation to the wounded skin induces limb formation in ALM. Thus, nerves can be considered to have the ability to transform skin wound healing to limb formation. In the present study, co-operative Bmp and Fgf application, instead of nerve deviation, to wounded skin transformed skin wound healing to limb formation in two urodele amphibians, axolotl (Ambystoma mexicanum) and newt (Pleurodeles waltl). Our findings demonstrate that defined factors can induce homeotic transformation in postembryonic bodies of urodele amphibians. The combination of Bmp and Fgf(s) may contribute to the development of novel treatments for organ regeneration.     

Modeling transmembrane transport through cell membrane wounds created by acoustic cavitation

Cells exposed to acoustic cavitation and other mechanical stresses can be transiently permeabilized to permit intracellular uptake of molecules, including drugs, proteins, and genes. Microscopic imaging and other studies suggest that intracellular loading occurs through plasma membrane wounds of submicrometer radius that reseal over time through the aggregation and fusion of lipid vesicles trafficked to the wound site. The goal of this study was to 1), determine the size of membrane wounds as a function of time after in vitro sonication of DU145 prostate cancer cells under conditions that caused extensive acoustic cavitation; and 2), theoretically model transport processes leading to intracellular loading. Our overall hypothesis was that intracellular loading is governed by passive diffusion through porous membrane wounds of up to 300-nm radius containing pores that permit entry of molecules up to at least 28-nm radius over a timescale of minutes. Experimental measurements showed intracellular loading of molecules with radii from 0.6 to 28 nm, where most loading occurred after sonication over a timescale up to minutes and where smaller molecules were taken up to a greater extent and over a longer timescale than larger molecules. Theoretical modeling predicted that membrane wounds would have a 300-nm radius initially and then would shrink, with a half life of 20 to 50 s. Uptake was shown to occur predominantly by diffusion and the increasing levels of uptake with decreasing molecular size was explained primarily by differences in molecular diffusivity and, for the largest molecule, geometrical hindrance within the wound. Mathematical modeling was simplified, because transport through porous wounds of possibly complex internal nanostructure was governed largely by transport at the edge of the wound, and depended only weakly on the size, number, and distribution of nanopores within the wound under the conditions relevant to this study. Overall, this study developed a theoretical framework for analysis of transmembrane transport through cell membrane wounds and thereby provided quantitative estimates of their size and lifetime.     

Mesoglycan induces keratinocyte activation by triggering syndecan-4 pathway and the formation of the annexin A1/S100A11 complex

Wound healing is a dynamic process comprising multiple events, such as inflammation, re-epithelialization, and tissue remodeling. Re-epithelialization phase is characterized by the engagement of several cell populations, mainly of keratinocytes that sequentially go through cycles of migration, proliferation, and differentiation to restore skin functions. Troubles can arise during the re-epithelialization phase of skin wound healing particularly in keratinocyte migration, resulting in chronic non-healing lesions, which represent a serious clinical problem. Over the last decades, the efforts aimed to find new pharmacological approaches for wound care were made, yet almost all current therapeutic strategies used remain inadequate or even ineffective. As such, it is crucial to identify new drugs that can enable a proper regeneration of the epithelium in wounded skin. Here, we have investigated the effects of the fibrinolytic drug mesoglycan, a glycosaminoglycans mixture derived from porcine intestinal mucosa on HaCaT human keratinocytes that were used as in vitro experimental model of skin re-epithelialization. We found that mesoglycan induces keratinocyte migration and early differentiation by triggering the syndecan-4/PKCα pathway and that these effects were at least in part, because of the formation of the annexin A1/S100A11 complex. Our data suggest that mesoglycan may be useful as a new pro-healing drug for skin wound care.     

PED/PEA-15 controls fibroblast motility and wound closure by ERK1/2-dependent mechanisms

Cell migration is dependent on the control of signaling events that play significant roles in creating contractile force and in contributing to wound closure. We evaluated wound closure in fibroblasts from mice overexpressing (TgPED) or lacking ped/pea-15 (KO), a gene overexpressed in patients with type 2 diabetes. Cultured skin fibroblasts isolated from TgPED mice showed a significant reduction in the ability to recolonize wounded area during scratch assay, compared to control fibroblasts. This difference was observed both in the absence and in the presence of mytomicin C, an inhibitor of mitosis. In time-lapse experiments, TgPED fibroblasts displayed about twofold lower velocity and diffusion coefficient, as compared to controls. These changes were accompanied by reduced spreading and decreased formation of stress fibers and focal adhesion plaques. At the molecular level, TgPED fibroblasts displayed decreased RhoA activation and increased abundance of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2). Inhibition of ERK1/2 activity by PD98059 restored RhoA activation, cytoskeleton organization and cell motility, and almost completely rescued wound closure of TgPED fibroblasts. Interestingly, skin fibroblasts isolated from KO mice displayed an increased wound closure ability. In vivo, healing of dorsal wounds was delayed in TgPED and accelerated in KO mice. Thus, PED/PEA-15 may affect fibroblast motility by a mechanism, at least in part, mediated by ERK1/2.     

An endogenous sodium current may mediate wound healing in Xenopus neurulae

We have studied healing of Xenopus neurulae in order to investigate the role of endogenous sodium-carried electric currents in wound closure. Transected embryos healed completely within 7 hr in an artificial pond water medium. Wound closure was biphasic, with a rapid purse string-like contraction that was independent of sodium concentration followed by a slower sodium-dependent phase. The initial contraction was reversibly inhibited by cytochalasin B. Healing was prevented when neurulae were wounded and left to heal in sodium-free medium. Healing was also prevented in the presence of amiloride, benzamil, or ouabain, drugs that inhibit sodium flux through the epithelium. The transepithelial potential measured in intact neurulae fell rapidly and reversibly by 70% in response to 10 microM amiloride. Currents measured leaving the wound also decreased by 70% following amiloride addition. Our results indicate that at least one phase of wound healing in Xenopus neurulae is dependent upon an endogenous sodium-carried electric current. We hypothesize that the current may act by guiding epithelial cells to the wound site.     

Fibrocytes contribute to the myofibroblast population in wounded skin and originate from the bone marrow

Myofibroblasts play a key role in wound closure but their origin is poorly understood. To investigate whether fibrocytes contribute to myofibroblast population, we examined the phenotype of fibrocytes and myofibroblasts present in the wounded skin of BALB/c mice. During wound healing, there was a marked increase in the number of cells expressing the myofibroblast marker alpha-smooth muscle actin in the granulation tissue. Between 4 and 7 days post-wounding, more than 50% of these cells also expressed the CD13 antigen. CD13(+)/collagen I+ fibrocytes could be isolated at an early stage of the healing process from digested fragments of wounded tissue by fluorescence-activated cell sorting. Like authentic fibrocytes, these cells were also CD45(+)/CD34(+)/CD14-. Between 4 and 7 days post-injury, 61.4% of the isolated fibrocytes were found to express alpha-smooth muscle actin gene and protein. We repeated similar experiments in female mice that had received a male whole bone marrow transplant after total body irradiation. By in situ hybridization, we identified the Y chromosome in the nuclei of the majority of fibrocytes isolated from the wounded tissue of these animals. Our data indicate that circulating fibrocytes contribute to the myofibroblast population in the wounded skin and that they originate from the bone marrow.     

Appearance of tenascin in healing skin of the mouse: possible involvement in seaming of wounded tissues

Distribution of the extracellular matrix glycoprotein tenascin during wound healing in mouse skin was studied immunohistochemically. Within 24 hours after wounding, and preceding the formation of granulation tissue, tenascin appeared in the basement membranes beneath epidermis and hair follicles adjacent to the wound edges and in the wounded edges of cutaneous muscle layer. Granulation tissue began to form in the wound space at about 1-2 days and was immediately covered by epidermis. Tenascin first appeared in the periphery of the granulation tissue beneath healing epidermis and around the wounded edges of cutaneous muscle layer. Then the tenascin-positive area extended into the inner region of granulation tissue. At about 5-7 days, all of the granulation tissue was intensely stained with anti-tenascin serum. Tenascin immunoreactivity decreased as granulation tissue was replaced with reconstructed dermal tissue at 7-14 days. In most cases, tenascin staining persisted longest in the dermis beneath the healing epidermis and at the juncture of healing edges of cutaneous muscle layer. It disappeared at about 10-14 days after wounding. These findings suggest that tenascin may play an important role in the seaming of wounded tissues.     

Maximal activities of glutaminase, citrate synthase, hexokinase, 6-phosphofructokinase and lactate dehydrogenase in skin of immune-competent Balb/c and immune-deficient Balb/c (nu/nu) mice during wound healing.

1. The maximal activities of hexokinase (HK), 6-phosphofructokinase (PFK), lactate dehydrogenase, citrate synthase (CS) and glutaminase (GLU) which provide quantitative and qualitative indices of flux through several important metabolic pathways have been examined in the wounded skin of haired immune competent Balb/c mice and hairless immune deficient Balb/c (nu/nu) mice of various ages during the first ten days of wound healing. 2. The potential for glucose utilization and for aerobic metabolism as suggested by the maximal activities of HK, PFK, CS, were raised in the skin of Balb/c mice of various ages on all post wounding days. Increases in the maximal activity of GLU was observed only in the skin of 6 and 10 weeks old Balb/c mice during wound healing. 3. There was no evidence of a contribution to the maximal activity of GLU by infiltrating cells of the immune system to the wound site in the skin of either haired or hairless mice.