432 ZEMAN, CURTIS, SCARPELLI, AND KLEINFELD 



stained with PAS-gallocyanin. After the radiation-induced lesions has been 

 identified on these slides, the alternate sections were stained for cytoplasmic 

 proteins with Fast Green at pH2, for cytoplasmic RNA with Azure B at pH 

 4, and for nuclear DNA according to Feulgen's technique after digestion 

 with RN-ase. 



On these preparations, the following observations could be made. Depend- 

 ing on dose, the development of nerve cell necrosis begins at 2 to 5 days 

 after irradiation; higher doses usually resulting in a shorter latency. Using 

 beams of 75 /x and less, and doses of 100,000 rad and more, most nerve 

 cells within the beam path show a synchronous pattern of necrosis, while 

 wider beams and doses of less than 100,000 rad produce nerve cell necrosis 

 in a random distribution, i.e., some neurons are in a state of complete disinte- 

 gration, while others are still unaltered. The first stage of this radiation- 

 induced nerve cell necrosis consists of disintegration of the cytoplasm. About 

 the same time, the cytoplasmic DNA disintegrates. The nerve cell nuclei, 

 however, remain unaltered and appear naked. It is difficult to estimate how 

 long the nuclei rest in this state. They eventually become pyknotic, disinte- 

 grate, and disappear completely. It is not clear which cellular elements 

 finally phagocytize the debris from the necrotic neurons ; however, it appears 

 that the nerve cell necrosis takes place without any demonstrable evidence 

 of microgliocytic proliferation or activation. 



From these obseivations, it was assumed that radiation-induced nerve cell 

 necrosis might conceivably be an autolytic process generated by the activa- 

 tion of proteolytic enzymes within the irradiated ner\e cells. To test this 

 hypothesis, the following studies were undertaken. 



Fourteen mice received multiple beam irradiations over the visual cortex 

 and the cerebellum. Each animal of the 1st group received 4 irradiations 

 with 0.250 mm beams at 30,000, 60,000, 120,000, and 240,000 rad. Those 

 of the 2nd group were given 8 irradiations with 0.075 mm beams at 90,000, 

 180,000, 270,000. and 540,000 rad. These doses were measured in the ioniza- 

 tion chamber, thus indicating relative, but not necessarily absolute, tissue 

 dosages. The beams were directed to the visual cortex and the cerebellum by 

 means of telescopic focusing. They were arranged in a frontal plane, spaced 

 60 mils from center to center. The animals were sacrificed by neck disloca- 

 tion at 4, 7, 10, and 30 hours after irradiation. 



The brain was immediately removed and fixed in formol-calcium at 4° C. 

 After 24 hours fixation, the brains were cut in serial sections at 15 ^ on a 

 standard freezing microtome. The sections were incubated in a medium 

 prepared after Holt I 1958) at pH 5 for demonstration of esterases. To block 

 carboxylic esterases of the acetylcholinesterase, pseudocholinesterase, and 

 lipase type, the medium contained a 10"" M concentration of diisopropyl- 

 fluorophosphate. Thus, only A-type cathepsins were believed to be stained. 



