In the last 50 years several radiation accidents occurred in which industrial radiographers and others suffered severe radiation injuries from inadvertent contact with radiation sources. Such accidents involving acute localized injuries are characterized by a severe initial reaction progressing through erythema to skin necrosis with a spontaneous resolution of the lesion over a 2-mo period for the lower doses. However, the early symptoms observed on the skin give no indication as to the in-depth pathology, and cutaneous and muscular radionecrosis started generally from early epithelial, microvascular, and vascular lesions and from delayed muscular and connective tissue lesions. In a case of acute localized irradiation, different biophysical techniques are able to give real responses in biological dosimetry. More numerous are the methods, especially imaging methods, that make it possible for the clinician to evaluate the extent of the early injuries and to manage the medical intervention. We have developed animal experimental models of acute localized irradiation: overexposure to the gamma rays of a 192Ir industrial radiographic collimated source (in the pig and the rabbit) and overexposure to the beta rays of a 90Sr-90Y collimated source (in the pig). In these experimental models, most of the imaging techniques used in clinical practice, as infra-red thermography, microwave thermography, cutaneous and tissular vascular scintigraphy (beta or gamma emitters), cutaneous blood flow measurements by cutaneous laser Doppler, x ray computed tomography, nuclear magnetic resonance imaging, and skin topography, were correlated with clinical evaluation and histopathological observations, after high doses of gamma or beta irradiations ranging from 4 to 340 Gy at the skin surface. All these techniques are not for isolated use and the present review indicates that their combination is necessary to give an improved diagnostic and prognostic picture of early and late delayed radiation damage to the skin and subcutaneous tissues.

Experiments demonstrate the stimulation of transforming growth factor-β expression in porcine skin soon after ψ-irradiation in a dose of 64 Gy and in mouse macrophages after whole body irradiation in a dose of 4 Gy, which suggests the involvement of macrophages that produce transforming growth factor-β during the early postirradiation period in the development of postirradiation fibrosis.

To characterize the differences between fibrotic myofibroblasts and normal fibroblasts, we studied two differentiation markers: α-smooth muscle (SM) actin, a specific marker of myofibroblast differentiation, and β-actin, which is overexpressed in the fibrotic tissue. Experiments were performed on fibroblasts isolated from normal pig skin and on subcutaneous myofibroblasts isolated from pig radiation-induced fibrosis. Three culture models were used: cells in monolayers, equivalent dermis, consisting of fibroblasts embedded into a matrix composed of type I collagen, and in vitro reconstituted skin, in which the matrix and containing life fibroblasts were overlaid with keratinocytes. Samples were studied using immunofluorescence and western-blotting. In monolayers cultures, both fibrosis and normal cells expressed α-SM actin. Furthermore, similar amounts of β-actin protein were found. In these conditions, the resulting alterations in the phenotypes of cells made comparison of cultured fibrotic and normal cells irrelevant. Under the two 3-D culture models, normal fibroblasts no longer expressed α-SM actin. They expressed β-actin at the basal level. Moreover, the fibrotic myofibroblasts in both 3-D models retained their differentiation features, expressing α-SM actin and overexpressing β-actin. We found that this normalization was mainly related to the genomic programmation acquired by the cells in the tissue. Cellular motility and microenvironment were also involved, whereas cellular proliferation was not a major factor. Consequently, both three-dimensional models allowed the study of radiation-induced fibrosis in vitro, provided good extrapolations to in vivo conditions and avoided certain of culture artefacts.

In eukaryotic cells, various proteins homologous to the E. coli RecA protein are involved in the elimination of DNA damage. These proteins contribute to the repair of double-strand breaks and to genetic recombination. The mouse Kin17 protein is recognised by antibodies directed against the RecA protein. Kin17 has a zinc-finger domain allowing binding to curved DNA stretching over illegitimate recombination junctions. In the present study, we identified the human counterpart of the mouse Kin17 protein (named Hs Kin17) in skin cells. We employed an in vitro reconstructed skin model composed of an epidermal sheath lying on a dermal matrix with human fibroblasts embedded in rat collagen type I. The maturation programme (proliferation versus differentiation) of keratinocytes was highly dependent on stromal cells. Immunohistochemical staining of frozen sections obtained from skin specimens was monitored by an interactive laser cytometer. In this way we analysed protein levels in both dermal and epidermal compartments. After having characterised the epithelium, we focused our attention on Hs Kin17 expression. We detected Hs Kin17 in human keratinocytes. Hs Kin17 protein levels increased in proliferating epithelial keratinocytes after 7 days of culture. After 2 weeks of culture, epidermal sheaths acquired most of the differentiated features of mature epithelium. At this time, Hs Kin17 protein dropped below measurable levels in the stratum corneum, and diminished in nucleated cells. This study showed that Hs Kin17 is expressed in human reconstructed epithelium under conditions of hyperproliferation.

Purpose: To establish how far liposomal copper/zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD), respectively, reduce radiation-induced fibrosis (RIF), using a well-characterized pig model of RIF permitting the design of a controlled laboratory experiment.Methods and Materials: In this model of acute localized gamma irradiation simulating accidental overexposure in humans, three groups of five large white pigs were irradiated using a collimated Ir-192 source to deliver a single dose of 160 Gy onto the skin surface (100%) of the outer side of the thigh. A well-defined block of subcutaneous fibrosis involving skin and skeletal muscle developed 6 months after irradiation. One experimental group of five pigs was then injected i.m. with 10 mg/10 kg b.wt. of Cu/Zn-SOD, twice a week for 3 weeks, and another experimental group of five was infected with 10 mg/10 kg b.wt. of Mn-SOD, three times a week for 3 weeks. Five irradiated control pigs were injected with physiological serum. Animals were assessed for changes in the density of the palpated fibrotic block and in the dimensions of the projected cutaneous surface. Block depth was determined by ultrasound. Physical and sonographic findings were confirmed by autopsy 12-14 weeks after completing SOD injections. The density, length, width, and depth of the fibrotic block, and the areas and volume of its projected cutaneous surface were compared before treatment, 1, 3, and 6 weeks thereafter, and at autopsy, 12-14 weeks after treatment ended.Results: The experimental animals exhibited no change in behavior and no abnormal clinical or anatomic signs. Whether they were given Cu/Zn- dr Mn-SOD, significant and roughly equivalent softening and shrinking of the fibrotic block were noted in all treated animals between the first week after treatment ended and autopsy, when mean regression was 45% for length and width, 30% for depth, and 70% for area and volume. Histologic examination showed completely normal muscle and subcutaneous tissue surrounding the residual scar. This replacement of scar tissue by normal tissue in experimental animals and the 50% decrease in the linear dimensions of the scar were comparable to the results obtained in previous clinical studies and highly significant compared to the clinical and autopsy results for the control animals.Conclusions: Our results are striking and comparable to the results obtained in our previous clinical study after liposomal Cu/Zn-SOD treatment. To our knowledge, this is the first time that two agents have been shown to reverse the radiation-induced fibrotic process in experimental animals and to permit the regeneration of normal tissue in a zone of well-established postirradiation fibrosis.

The therapeutic or surgical management of acute localized irradiation is very complicated due to the delayed occurrence of ionizing radiation effects. There is a great need for non-invasive imaging techniques to delineate healthy from exposed tissues. Such a technique should be of value considering that spread of damage can occur from apparently silent fibrotic foci. Of the scintigraphic procedures, 99Tcm first-pass scintigraphy has already been recommended by the International Atomic Energy Agency (IAEA) for the evaluation of irradiated tissues. In order to improve the scintigraphic surveillance of accidental over-exposures, the potentials of 201T1 and 99Tcm-J001X were evaluated on an experimental porcine model reproducing the late fibrotic effect encountered after localized irradiation. J001X, an acylated poly-(1,3)-galactoside of bacterial origin, was used for the functional imaging of the inflammatory reaction which took place after irradiation. This scintigraphy, based on targeting of activated macrophages recruited by lesions, was performed together with 201T1 scans for the assessment of blood perfusion and cellular metabolism. Our results demonstrate that these two radiopharmaceuticals provide reliable information for the management of accidental localized over-exposure, J001X allowing the assessment of the inflammatory reaction and 201T1-chloride being mainly useful for imaging the delayed onset of fibrosis.

J001X, an acylated poly-(1,3)-galactoside isolated from Klebsiella pneumoniae proteoglycan, has been developed to target cells from the monocyte-macrophage lineage. Recent experimental work and initial clinical trials have proved the potential of this molecule labeled with 99mTc for the scintigraphy of inflammatory foci. In a model of radiation-induced inflammation in pigs, the scintigraphic contrast was observed to be very sensitive to a single injection of methylprednisolone given 12 h before scintigraphy. The present study was undertaken to confirm this effect and to estimate the possible interference of various anti-inflammatory agents on the in vivo targeting of macrophages by J001X. Methylprednisolone, dexamethasone, indomethacin and methotrexate used at an immunosuppressive dose were tested to assess the possible risk of false-negative examinations in patients thus treated. Analysis of the results indicated that among the four drugs tested, only methylprednisolone at 0.5-1 mg/kg could interfere with J001X scintigraphy.

The involvement of the nuclear protooncogenes c-fos and c-jun in the immediate early response of pig dermis cells was studied after in vivo gamma-irradiation. Following high radiation doses (8 to 48 Gy), the two protooncogenes were concomitantly induced, although c-fos induction was preferential. Both inductions were time and dose dependent. Therefore, the early response of the skin to high doses of radiation might involve heterodimeric activator protein 1 composed of c-Fos and c-Jun proteins. Following low radiation doses (0.5 to 2 Gy), c-jun was not induced. By contrast, dramatic c-fos induction was observed after 0.5 Gy, suggesting a specific role for c-fos at low doses.

Localized irradiation of the skin and subcutaneous tissues with large single doses of gamma-rays can induce delayed effects characterized by fibrosis which invades the irradiated tissues. In this study the depth of penetration of muscle fibrosis was measured in the pig 30 weeks after irradiation of the skin surface with single doses of 192Ir gamma-rays of 16-256 Gy. Irradiation was directed either to the outer side of the thigh or to the back, close to the mid-dorsal line. Fibrosis only developed in irradiated muscle after doses that induced moist desquamation of the skin in the acute phase of the reaction, i.e. after skin surface doses of 48-64 Gy. In skeletal muscles, the limit of fibrotic expansion was reached at a depth dose of 14 +/- 4 Gy (+/- SD) for skin surface doses exceeding 48 Gy.

L'administration d'"entérobloquants" aux ruminants (ovins), après arrêt d'une contamination orale, ne modifie pas de façon notable les paramètres des courbes de décorporation du radiocésium ingéré. Par contre, on observe une diminution très significative - d'un facteur 2 - par rapport aux animaux témoins (n = 4) de la radioactivité spécifique du lait et des urines chez les sujets recevant soit un dérivé du bleu de Prusse (l'AFCF) soit de la vermiculite (respectivement 5 et 6 animaux). Ces observations permettent d'une part d'apporter des précisions sur le transit métabolique du radiocésium chez les ruminants et d'autre part de disposer d'un moyen d'accélérer la décontamination du lait et, ainsi, de le rendre plus tôt utilisable pour l'alimentation humaine et celle des jeunes animaux.

Although radiation-induced fibrosis has long been characterized by excess fibroblast proliferation and extracellular matrix deposition, the origin of cell activation in these complications of radiotherapy or radiation accidents is still controversial. The present work was designed to test the hypothesis that the abnormal production of TGF-beta 1 in irradiated tissues results in continuous signals for tissue repair and long-term cell activation. We examined gene expression of this growth factor in a well-characterized pig model of radiation-induced fibrosis, using Northern-blot and slot-blot hybridizations and indirect immunofluorescence. We found that the TGF-beta 1 mRNA level was increased 19-fold in the irradiated skin during the early erythematous phase, which started 3 weeks after irradiation. During the later phases of fibrosis, from 6 to 12 months after irradiation, the TGF-beta 1 gene was highly expressed in the repaired skin and the underlying muscular fibrotic tissue, with 10- and 8-fold maximal increases, respectively. In addition, we found that the beta-actin mRNA level was increased in the fibrotic tissue. Immunostaining for TGF-beta 1 revealed the presence of the protein in endothelial cells of capillaries, in myofibroblasts, and in the collagenous matrix of the fibrotic tissue. These results suggest that TGF-beta 1 may be one of the key cytokines involved in the cascade of events that leads to radiation-induced fibrosis, at both early and late stages.

Sub-cutaneous and muscular fibrosis are common and irreversible late effect of radiation on normal tissues. An experiment was designed to test the effectiveness of superoxide dismutase in reducing late radiation injury. This study was performed in an experimental porcine model of acute localized gamma irradiation simulating human accidental overexposure: 12 Large White pigs were irradiated on the thigh with a collimated gamma 192Ir source, so that the dose was 160 Gy/skin (100%) and 40 Gy/2 cm depth (25%). In this model, fibrosis appears in 4 to 5 months. The heterogeneous sclerotic tissue is composed of stable fibrotic areas poorly cellularized and active areas with a high density of myofibroblasts and inflammatory perifibrotic part. Lipsod administration modalities were six intramuscular injections during 3 weeks (twice weekly) either 10 mg/inj (five pigs) or of 100 mg/inj (five pigs). A methodic evaluation by two examiners consisted of measurements being taken before and after treatment: sum of the two largest perpendicular measurable dimensions, cutaneous projected surface of palpated fibrotic block, ultrasound fibrosis deepness and extrapolated volume. We conclude that Lipsod is the first drug ever described that reduces radiation-induced fibrosis. Its efficacy in this model was highly significant, with a regression higher than 40% in size and 70% in surface and volume, 12 weeks after the end of treatment. This response was rapid, reproducible without dose-effect or toxicity in the limits studied. This work confirms previously published results in humans.

Sub-cutaneous and muscular fibrosis are common and irreversible late effect of radiation on normal tissues. An experiment was designed to test the effectiveness of superoxide dismutase in reducing late radiation injury. This study was performed in an experimental porcine model of acute localized gamma irradiation simulating human accidental overexposure: 12 Large White pigs were irradiated on the thigh with a collimated gamma Ir-192 source, so that the dose was 160 Gy/skin (100%) and 40 Gy/2 cm depth (25%). In this model, fibrosis appears in 4 to 5 months. The heterogenous sclerotic tissue is composed of stable fibrotic areas poorly cellularized and active areas with a high density of myofibroblasts and inflammatory perifibrotic part. Lipsod administration modalities were six intramuscular injections during 3 weeks (twice weekly) either 10 mg/inj (five pigs) or of 100 mg/inj (five pigs). A methodic evaluation by two examiners consisted of measurements being taken before and after treatment: sum of the two largest perpendicular measurable dimensions, cutaneous projected surface of palpated fibrotic block, ultrasound fibrosis deepness and extrapolated volume. We conclude that Lipsod is the first drug ever described that reduces radiation-induced fibrosis. Its efficacy in this model was highly significant, with a regression higher than 40% in size and 70% in surface and volume, 12 weeks after the end of treatment. This response was rapid, reproducible without dose-effect or toxicity in the limits studied. This work confirms previously published results in humans.

Localized irradiation of the skin and subcutaneous tissues with large single doses of gamma rays can induce delayed effects characterized by fibrosis that invades the irradiated tissues. The skin surface dose and the depth of penetration of 192Ir gamma rays, measured in phantoms and in frozen thighs of sacrificed pigs after acute single doses of irradiation and different skin-to-source distances, are reported in this note. Correlations between the dosimetry and the evolution of the lesions are considered.

D a n s un m o d è l e e x p é r i m e n t a l c h e z le p o r c , u n e i r r ad ia t i on g a m m a d e la p e a u p r o d u i t u n e f i b rose c i ca t r i c i e l l e . N o u s a v o n s t e s t é d a n s c e m o d è l e l ' h y p o t h è s e s e l o n laque l l e u n e p r o d u c t i o n a n o r m a l e d u f a c t e u r d e c r o i s s a n c e T G F B d a n s les t i s sus i r r ad iés p r o v o q u e r a i t u n e ac t i va t i on c h r o n i q u e d e s c e l l u l e s r e s p o n s a b l e s d e la f i b r o s e .

Quatre brebis gestantes ont reçu du foin contaminé par les retombées de l'accident de Tchernobyl, pendant environ 1 mois avant et un mois après le part. Le niveau de contamination (134Cs et 137Cs) des mères et des agneaux a été déterminé par comptage in toto ; la quantité de lait ingérée par les agneaux a été estimée par leur gain de poids journalier. Entre J10 et J30 après le part, la valeur moyenne des transferts a été de 4,7 ± 1,4 % pour le lait et 7,9 ± 1,7 % pour la viande. A la naissance, la charge corporelle moyenne des agneaux était égale à 3,2 ± 0,8 % celle de mère ; le coefficient moyen de rétention du césium ingéré était au sevrage (J30) de 39 ± 6 % pour les agneaux et de 27,3 ± 3,2 % pour les brebis. La période de décontamination spontanée était pour les agneaux après le sevrage d'environ 14,5 jours (courbe monoexponentielle) et d'environ 26 jours pour la composante longue de la courbe bi-exponentielle chez les brebis. Les transferts à la viande, les paramètres de charge et de décontamination spontanée ont été également étudiés : 1) chez 4 agneaux de 1 à 3 mois après le sevrage, 2) chez 3 jeunes animaux âgés de 9 à 10 mois. Les valeurs des différents paramètres du transit du césium chez les nouveaux-nés et chez les jeunes animaux sont discutées en rapport avec la gestion d'un accident de contamination.

After radiotherapy or radiation accidents, large doses of gamma irradiation can induce necrosis and fibrosis, and secondary cancers are occasionally observed. The identify of the target cells in such irradiated tissues is still controversial. As the role of the fibroblast in wound repair after acute irradiation is not well documented, this role was investigated in an experimental model. Pigs were gamma irradiated with an Ir-192 source on the thigh, which resulted in necrosis and then fibrosis of the skin and muscular tissues. The dose delivered to the skin was 160 or 120 Gy. It was found that in primary cultures, fibroblasts isolated from the fibrotic tissue exhibited an activated phenotype for up to two years after irradiation. They had a myofibroblastic morphology and their proliferation was activated. The fibroblasts synthesised an extracellular matrix similar to that of immature scars or developing tissues. Most fibroblasts carried numerous chromosome anomalies. In long-term cultures, fibrotic fibroblasts gave rise to established cell lines, in which the morphology of the cells resembled that of transformed cells. Such highly modified cells observed in vitro might be related to the appearance of secondary sarcoma in vivo. We conclude from these results that the fibroblast is clearly an important target cell in irradiated skin. The difficult healing of late radiation damage could be related to chronic inflammation and to the long-term activation of abnormal fibroblasts in irradiated tissues.

Few technics have been able to give a real response in biological dosimetry in the cases of acute localized irradiations up to now. More numerous are the methods, specially by imaging, that make it possible for the clinician to evaluate the extent and the intensity of the injuries, to give a prognosis and to manage the surgical intervention: thermography, vascular and tissular scintigraphy, CT tomography and NMR imaging. Most of these technics are yet experimental and their possible use in acute localized irradiation accident assessment is discussed.

In an attempt to study muscle and skin physio-pathology, we have established a method which allow repeated local and regional blood flow measurements of the hindlimb of the pig. The renal artery is chronically catheterized under general anaesthesia to allow intra-aortic administration of Xe-133 in saline. Radioxenon distribution is followed up in the whole thigh with a gamma-camera. Local clairance curves are then calculated and transferred to a micro-computer. A program for poly-exponential curve fitting, written in Asyst language has been developed. An iterative curve stripping is performed conventionally and then the coefficients are more precisely determined using a Gauss-Newton method. Considering the results obtained on healthy animals and on an experimentally radiation induced inflammatory lesion, this procedure which make use of all the potentialities of the radioactive isotopes of rare gases, allow repetitive and reproductive determination of blood flow in the hind limb of the animals.