The first results in cytogenetic studies suggested that fibrosis extracted fibroblasts (FEF) derived from irradiated cells. The high level of chromosome damages might be responsible for the pretransformed state of those fibroblasts and perhaps might lead to secondary radioinduced cancers. A consistent observation about this hypothesis was the spontaneous perennisation of FEF cell lines (at the present time more than 50 subcultures) when normal dermal fibroblasts cultures died after 10-15 subcultures.
In vitro modulation of the collagen phenotype by heparin fragments showed that the degradation products of the extracellular matrix might be regulatory factors of the fibrotic processes. As regards growth factors, their expression was studied as a function of time after irradiation, in order tounderstand the initial reactions in fibrosis genesis and the persistance of chronic inflammation in later stages; preliminary data showed that interleukin 2 and fibroblast growth factor could be involved in those processes.
This investigation concerns the following:
the relation between fibrosis extension in irradiated muscle and the dose according to the depth;
assessment of the functional ability of the skeletal muscle after irradiation (gamma scintigraphy, electromyography); and the possible role of transforming growth factor (TGF)b as an activation factor in the development of postirradiation fibrosis.
Experimental models of localized irradiation studies (all doses are given at 2 cm depth, ie 4 times higher at the skin basal layer level);
Large white pigs, 40 to 60 kg, were irradiated on the external side of the right thigh (biceps femoralis) with a collimated source of iridium-192 at 30, 40, 64 Gy (n=8 for each dose); and on the back (iliospinalis) with 6 irradiation areas per animal: 4, 8, 12, 16, 20 and 24 Gy (n=8).
The animals were sacrificed 30 weeks after irradiation and the depth of the muscular fibrosis measured.
It was noted that the irradiation procedure delivered very close to the skin surface (17 mm) produced radiolesions more similar to those observed after exposition to hot particles than those classically observed after overexposure to cobalt-60 gamma rays or X-rays with a SSD of 50 to 80 cm.
No microscopic modification of the muscular structures was observed after 4, 8 and 12 Gy; between 16 and 64 Gy fibrosis thickness, assessed 30 weeks after irradiation, increased with the dose. In pigs locally irradiated either on the thigh or on the back, fibrosis developed in irradiated muscle only when moist desquamation was observed on the skin lesion: the animal surface dose was comprised in these cases between 48 and 64 Gy (12 to 16 Gy at 2 cm depth); fibrosis spead in tissues irradiated at lower doses when applied doses were higher. In fact, the importance of the early inflammatory procesess after irradiation woudl control fibrosis extension in depth, although the early skin symptomatology gave no information on the pathology at depth and on the importance of late effects. Thus the limit of the deep extension of the fibrosis in the skeletal tissue corresponds to a deep dose of 14 Gy whatever was the applied dose (if higher than 12 Gy at 2 cm depth).
The characterization of tissues involved in necrotic and fibrotic process was performed with 201 Tl scintigraphy. The influence of blood flow changes during these explorations was shown using xenon-133 scintigraphy injected intraarterially. The accumulation phases of 201 Tl are, to a large extent, independent from blood flow changes. Specific antiinflammatory drugs were used concomitantly with a technetium-99 labelled lipoglycopeptide to make obvious its property of revealing activated macrophages.
To date, most of the biophysical methods used for the dosimetry of acute localized irradiation have a high threshold in the dose effect relationship: thermography, RMN imaging, scintigraphy. Attempts were made to develop a noninvasive method for assessing lower doses. Fatigue was induced by electrostimulation of thenervous branch of flexor carpi radialis which was easy to study individually in pigs. Performance change was estimated with implanted electrodes and strain gauge set on a restraint device.
Using periodic sequences working at 1 fixed frequency it was possible to characterize a decrease in excitibility due to fatigue induced by high frequency and a change in excitation-contraction coupling induced by low frequency. Postirradiation fibrosis exhibits, particularly in nonspontaneously healing lesions, a tendency to spread out in surrounding tissues. Fibroblasts isolated from radiation induced fibrotic tissues exhibit, in culture, an abnormal and activated phenotype. The role of TGFb which controls the extracellular matrix accumulation, was investigated.
Fibrotic tissues were removed from the normal dermis and the thigh lesion at 1, 4, 6 and 16 months after irradiation; ribonucleic acid (RNA) was hybridized to a molecular probe coding for exon 1 of TGFb. An intense hybridi zation could be seen in all fibroses, which was much higher than in normal dermis. RNA isolated from confluent primary cultures showed that the expression of TGFb was weak and variable in dermis and high and stable in fibrosis; in the latter no diminution of TGFb expression could be observed up to 2 years later.
TGFb stimulated collagen synthesis either in fibrosis or in normal dermis, and modified culture aspect (hills and vallyes) in monolayer fibrosis cultrues.
At low dose TGFb stimulated the synthesis of all proteins, especially the noncollagenic cones; at high dose collagen was still synthetized when noncollagen proteins were inhibited.
Thus an autocrine stimulating process does exist in postradiation fibrosis fibroblasts which could be a mechanism of cellular activty persistance in chronical fibrosis. On the other hand further mediators, especially the extracellular matrix and its degradation fragments, might interfere.
A. THERMOGRAPHIC TECHNIQUES BY MEANS OF MICROWAVES WILL BE IMPROVED BY FURTHER DEVELOPING THE INSTRUMENTATION TO MAKE IT POSSIBLE TO MEASURE AT 2 WAVE LENGHTS (3 AND 10 GHZ) AND TO 2 DIFFERENT DEPTHS. THE COMPUTER TREATMENT OF THE DATA WILL BE IMPROVED WITH RESPECT TO THE ANALYSIS OF THE IMAGE AND RECONSTITUTION OF THE HEAT PROFILES. OUR STUDIES DEALING WITH THE RELATION BETWEEN LOCAL THERMIC REACTION AND DOSE OR DOSE RATE WILL BE PURSUED.
B. TWO TYPES OF FUNCTIONAL INDICATORS WILL BE DEVELOPED WITH THE HELP OF A GAMMA CAMERA, DETERMINATION OF DYNAMIC FUNCTIONS BY MEANS OF XE-133 AND TISSUE ACCUMULATION OF RADIONUCLIDES MARKING MUSCULAR NECROSIS (T1-201, TC-99M COMPOUNDS) AND THROMBOSIS (PLATELETS LABELLED WITH IN-111).
C. FIBROBLAST CULTURES FROM BIOPSY SAMPLES WILL BE STUDIED TO EVALUATE LOCAL DOSES AND TO COMPLETE EARLIER IN VITRO INVESTIGATIONS.
THREE TYPES OF RESEARCH WILL BE EXPANDED TO BETTER DEFINE CERTAIN FUNDAMENTAL ASPECTS OF THE PATHOGENESIS OF THE LESIONS AND TO HELP, TO A CERTAIN DEGREE, THE PROGNOSTICS OF ACUTE LOCAL EXPOSURE.
A. THE LOCAL BIOCHEMICAL (TISSUE) AND GENERAL (HUMORAL) REACTIONS WILL BE CORRELATED WITH THE SEVERITY OF THE LESIONS (MARKER PROTEINS FOR INFLAMMATION,TISSUE ENZYMES...).
B. THE BEHAVIOUR OF THE EXTRACELLULAR MATRIX WILL BE COMPARED IN VIVO AFTER IRRADIATION AND IN VITRO IN FIBROBLAST CULTURES (LAMININE, FIBRONECTIN, DIFFERENT TYPES OF COLLAGEN...).
C. THE PATHOPHYSIOLOGY OF THE CELLS INVOLVED IN RADIATION-INDUCED FIBROSIS WILL BE INVESTIGATED ON THE BASIS OF BIOPSIES FROM SCAR CONNECTIVE TISSUE AFTER RADIATION AND SURGERY IN HEALTHY OR IRRADIATED TISSUES USING MORPHOLOGICAL, BIOCHEMICAL STUDIES AS WELL AS CULTURES OF FIBROBLASTS FROM BIOPSIES AND DETERMINING THE FACTORS RESPONSIBLE FOR THE EXTENSIVE CHARACTER OF RADIATION-INDUCED FIBROSIS.
THE STUDIES DEAL WITH THE ACTION OF ANTI-INFLAMMATORY AGENTS, ANTICOAGULANTS, AGENTS WHICH COUNTERACT PLATELET AGGREGATION, ANDROGENIC HORMONES AND VASODILATORS GIVEN ALONE AND TOGETHER. IT WILL BE ATTEMPTED TO MODIFY THE TREATMENT IN DEPENDENCE OF THE ANATOMO-CLINICAL DEVELOPMENT OF THE LESIONS. THE RESULTS WILL BE CONSIDERED IN THE LIGHT OF ANATOMO-PATHOLOGICAL CRITERIA, THERMOGRAPHY AND FUNCTIONAL TESTS FOR LOCAL CIRCULATION, BIOCHEMISTRY AND HISTOLOGY.