SEQUENCE OF X-RAY DAMAGE IN MOUSE CEREBELLUM 205 



hydrolysis of chromatin decreases if pyknosis is induced by chemical asents. 

 It is conceivable that the increase in resistance of the pyknotic nuclei which 

 we found is attributable to greater density of the nuclear mass in that the 

 increased density reduces depolymerization and hydrolysis. 



We have emphasized that when RNA-containinsi material, e.g., the cyto- 

 plasm of the Purkinje cells, is stained with acridine oranse. pH 4.0 to 7.0, 

 it takes on a bright orange or red fluorescence. Since different types of 

 regressive changes can be observed in Purkinje cells during the course of 

 radiation damage, we have used the acridine orange method to study the 

 behavior of their cytoplasmic nucleic acids. Because of regressive changes 

 in these cells and because of the nuclear pyknosis in granule cells, the irradi- 

 ated area of the cerebellar tissue can easily be demonstrated by this method. 

 Since regressive changes in the Purkinje cells usually occur more strikingly 

 in the center of the irradiated area than along its margins, the red fluo- 

 rescence exhibited by the more peripheral cells gradually decreases in in- 

 tensity toward the center of the irradiated zone. The cytoplasm of unaltered 

 Purkinje cells fluoresces bright orange-red. Acute shrunken nei-ve cells show 

 the same fluorescence because their cytoplasm contains abimdant RNA. 

 Immediately following" irradiation, the swollen and \acuolated Purkinje cells 

 give off a slightly decreased orange fluorescence, but after sufficient time 

 has passed the cytoplasm exhibits only yellow or yellow-green fluorescence 

 because the RNA content of their altered cytoplasm is decreased. Purkinje 

 cells showing the homogenization type of necrosis have a green fluorescence 

 because they have lost all cytoplasmic RNA. Since color and intensity of 

 fluorescence in these Purkinje cells is similar to that of the neuroglia of the 

 molecular layer, it is somewhat difficult to recognize necrotic and homog- 

 enated Purkinje cells. It bears emphasis that the nuclear pyknosis in Purkinje 

 cells as well as in granule cells is not associated with depolvmerization of 

 DNA, since, when stained with acridine orange, the pyknotic nuclei still 

 fluoresce yellow-green. 



Discussion 



There ha\e been apparently conflicting reports in the literature as to the 

 primacy of irradiation damage in the central nervous system, whether in 

 vessels or in nerve cells. The cerebellum seems especially suitable for in\esti- 

 gation of this problem. .Some other workers already ha\e directed attention 

 to the radio\ulnerability of this part of the brain. In macaque monkeys, Hay- 

 maker et al. ( 1958) ha\-e studied the effect on the central nervous system of 

 whole-body BA'^"-LA"" i gamma i radiation. E\ idence of ner\e cell damage 

 in the cerebrum was scanty, but granule cells of the cerebellum were pyknotic 

 within a dose range of 5,000 to 30,000 r. The pyknosis occurred earlier and 



