228 



G. LELLI, U. MAROTTA AND A. D AMORE 



In a second group of experiments in which we looked 

 for possible structural modifications of the collagen 

 fibrils after freezing we divided the tests, 20 in all, into 

 two subgroups. 



In the first of these we subjected samples of collagen 

 to temperatures ranging from - 50 C to - 70'C for 24 

 hours, they were then lacerated in double distilled 

 water and examined. Nothing particular regarding the 

 thickness, the periodic structure and the length of the 

 period was found in these fibrils. 



In the second subgroup, designed to obtain more exact 

 information, the collagen fibrils were lacerated in double 

 distilled water and then examined and repeatedly photo- 

 graphed. They were then subjected for 24 hours to a 

 temperature of - 70 C and then again photographed 

 electron microscopically in the same microscopic field. 



A comparative study of the electron micrographs 

 thus obtained and an acurate measurement of the 

 length of many periods showed that freezing produced 

 no substantial changes in the periodic structure of the 

 collagen. However it was evident from visual obser- 

 vation and even more so from checks of the electron 

 micrographs with the comparator that there is 

 a constant and uniform thinning of the fibrils after 



freezing. The degree of thinning is usually moderate 

 and at most one fifth of the original thickness. 



Conclusions. — (a) Extreme cold applied to collagen 

 produces in the fibrils an increased resistance to the 

 destructive action of acid solutions and to the action 

 of some proteolytic enzymes such as papain. 



The hardening which may occur in frozen meat 

 may be due to this increase in the resistance of the 

 collagen. 



(b) Cold does not produce appreciable structural 

 changes in the collagen fibrils when seen in the 

 electron microscope. This is in agreement with earlier 

 observations (I, 2). 



(c) Cold produces a greater or lesser degree of 

 thinning which is constant in collagen fibrils. 



References 



1. Lelli, G., Bonanome, A., and Sappa, M.,Z. wiss. Mikro- 



skop. 61, 298 (1953). 



2. Nemetschek, Th., Grassmann, W., and Hofmann, U., 



Z. Natiirforsch. 10 b, 61 (1955). 



Electron Microscopic Observations on Collagen Exposed to X-Rays 



G. Lelli, U. Marotta and A. D'Amore 



Istituto Superiore di Sanita, Roma 



In view of the ever-increasing application of radium 

 and x-ray therapy in neoplastic diseases, the study 

 of the effects of irradiation on the connective tissue 

 is of great practical interest. 



By means of the electron microscope we have 

 studied the efTects of x-rays on the collagen fibrils. 



We have irradiated at varying doses the abdominal 

 skin of 7 pigs aged 3 months, weighing about 15 kg, of 6 

 guinea pigs weighing about 700 g, and some bits of skin 

 taken from animals of the same species before irradiation. 



All treated animals showed rather early manifestations 

 of circumscribed radiodermatitis, apart from the general 

 toxic symptoms which in some cases killed the animal 

 in the course of a few days. 



Specimens for biopsy were taken regularly from the 

 irradiated area of the skin. 



In four cases the first biopsy was done immediately 

 after the irradiation, and successive ones after 4, 8, 16 

 and 24 days, provided the animal was still alive. 



In two cases 6 biopsies were done 24 hours after each 

 irradiation and after 8, 16 and 24 days if the animal was 

 then still alive. 



The dermis was isolated from the biopsy material 

 and from the pieces of skin removed prior to irradiation. 

 It was then lacerated in double distilled water and 

 examined under the electron microscope after previous 

 chromium shadowing. 



Careful examination of a very large number of 

 electron microscopic photographs showed that the 

 collagen fibrils had a normal transverse striation, 

 usually without changes of either shape or thickness. 

 We only occasionally encountered a fibril which was 



swollen, ruptured, flattened, winding and apparently 

 disintegrated in some parts. 



We attach little importance to such changes, as 

 identical pictures were seen in the controls. More- 

 over, these changes were not in any way related 

 to the x-ray dosage applied. 



Nemetschek et al. (2) using x-ray doses smaller 

 than ours, found under the electron microscope that 

 the normal transverse striation of the fibrils persisted 

 and that these showed a fine granular appearance. 

 This we could not see in our material which was 

 always examined after chromium shadowing. 



Also Marotta (I) excludes electron microscopi- 

 cally demonstrable changes in the collagen fibrils 

 after a single dose of 700 r. 



Our negative results are also in agreement with 

 those recently obtained by Nerli (3) using the method 

 of x-ray diffraction. Indeed this author found no 

 difference whatsoever in the diagrams he obtained 

 using dermis from the skin of rats irradiated with 

 a single dose of 1200 r without filter and non- 

 irradiated derma. According to Nerli this suggests 

 that "x-ray irradiation does not change the basic 

 structure of the polypeptide chains of the collagen". 



References 



1. Marotta, U., Reiul. ist. super. Sanita 18 (1955). 



2. Nemetschek, Th., Grassmann, W., and Hofmann, U., 



Naturwissenschaften 16. 371 (1954). 



3. Nerli, A., Rac/ioter. Radiobiol. e Fis. Med. 2, 166 (1956). 



