SEQUENCE OF X-RAY DAMAGE IN MOUSE CEREBELLUM 203 



contrast, the highly polymerized deoxyribonucleic acid (DNA) of the 

 nuclei shows an intense yellow or yellow-green fluorescence. Other elements 

 of the brain tissue present a weak green fluorescence. The diff"erence in the 

 staining raction of RNA and DNA is due to the high degree of polymeriza- 

 tion of the nuclear DNA in comparison with the cytoplasmic RNA. After 

 depolymerization, e.g., by placing the tissue slices in boiling water or 

 hydrochloric or perchloric acid, the DNA stained with acridine orange 

 shows a red fluorescence. The depolymerized DNA behaves, therefore, like 

 RNA, which is always more weakly polymerized. The acridine orange 

 method is more sensitive than the methyl green, which can, however, bring 

 out wide differences in the degree of polymerization in DNA. The occur- 

 I'ence of depolymerization in DNA thus can be proved by using the acridine 

 orange fluorescence technique. 



We tried to determine with this method whether alterations occur in the 

 structural organization of the DNA in the pyknotic nuclei of irradiated 

 granule cells. Many other cells besides those of the cerebellar granular layer 

 show clumping or other changes in the nuclear chromatin. In an histo- 

 chemical study of nuclear changes in the superficial epithelium of the 

 tongue of mice that had received radioactive chromic phosphate, Burstone 

 (1953) observed enlargement and hyperchromasia of the nuclei. In contrast 

 to nonirradiated controls, treatment with deoxyribonuclease (DNase) re- 

 sulted in decreased staining capacity of these nuclei to the Feulgen reaction 

 and to the methyl-green-pyronin method. Burstone (1953) believed that 

 this decreased resistance of the irradiated nuclei is due to a somewhat 

 decreased aggregation of the nuclear DNA. Based on such observations and 

 on radiation experiments on DNA solutions which show a splitting of DNA 

 molecules i Scholcs and Weiss, 1952) some still believe that the clumping 

 of chromatin after irradiation is a result of depolymerization. If such 

 depolymerization of DNA should occm- in the pyknotic nuclei of irradiated 

 cerebellar granule cells as a primary effect or a secondary reaction to ioniz- 

 ing radiation, then it should be demonstrable with acridine orange by the 

 nuclei exhibiting red fluorescence. We ha\e ne\er found this. Disregarding 

 the point that pyknosis causes a higher density which provokes a somewhat 

 more intense fluorescence, no difference in the fluorescent color of these 

 pyknotic nuclei compared to normal nuclei has been obser\ed. 



The fact that the DNA of these pyknotic nuclei is not significantly 

 depolymerized can also be shown by using methyl green. This dye stains 

 only highly polymerized DNA (Kurnik. 1950. 1952; Kurnik and Forster, 

 1950: Kurnik and Mirsky, 1950; Pollister and Leuchtenberger, 1949; Ver- 

 cauteren. 1950). The pyknotic nuclei should not be stainable with methyl 

 green if there had been depolymerization of nuclear DNA. But, as compared 

 with nonirradiated cells, no substantial diflference in the staining capacity 



