162 



Appendix: analytical methods 



anaesthetization before freezing though this is not always used, 

 and may mask the effects being studied. Decapitation into hquid 

 air is a procedure which contains the risk of breakdown of labile 

 phosphates in the time intervening between severance of the head 

 and its immersion in the coolant, but has been used successfully by 

 various authors (cf. Kratzing and Narayanaswami, 1953; Gerlach 

 et al., 1958). Large animals such as the cat, dog or monkey are first 

 anaesthetized and the brain exposed by operative procedures 

 before pouring on liquid air or nitrogen (Kerr, 1935; Klein and 

 Olsen, 1947). The use of a carbon dioxide-ethyl chloride mixture 

 yielded variable and somewhat lower values for phosphocreatine in 

 cat brain than did the use of liquid air (Kerr, 1935). Generally, 

 freezing in situ is the technique to be preferred for an examination 

 of the levels of labile phosphates in the brain of rabbits determined 

 in this manner, and in the samples removed and transferred as 

 rapidly as possible to liquid air with a sharp, prechilled spoon, 

 revealed that even the briefest time lapse was sufficient to permit 

 considerable changes to have taken place (Table 24). 



The whole of the brain is not frozen immediately. Thus, it takes 

 about 16 sec to freeze completely the brain of a small animal and as 

 long as 30 sec to freeze the brain of larger animals to a depth of 

 1 cm (Richter, 1950). It seems probable that the blood supply to 

 the interior of the brain continues at the normal rate while the 

 wave of freezing is spreading from the cortex, thus preventing 

 anoxia. The intense cold is considered to block nervous transmis- 

 sion so that no stimulation occurs. Whatever the mechanisms 



Table 24. — The Effect upon Levels of Cerebral Phosphates of Two 

 Different Procedures for the Fixation of Brain Samples 



(Quantities are in /nmoles/g wet wt.) 



Data from Thorn et al. (1958), rabbit. 



