CENTRAL NERVOUS SYSTEM METABOLISM IN VITRO 



1829 



be quoted. The tissues may be prepared by extraction 

 with water (124, 221) and subsequent dialysis (226) 

 and by extraction with salts and buffers (105, 212). 

 Extraction has frequently been carried out in aqueous 

 solutions after an initial acetone-drying of the tissue 

 (71, 148, 187, 195); extraction fluids have in some 

 cases included cysteine (71, 78, 187) and the acetone 

 drying has been carried out with the frozen tissue 

 (152). In such extracts, metabolic potentialities may 

 for the purpose of study be limited to one or a few 

 enzyme reactions by supplying a specific substrate 

 (148, 149, 176), by the use of inhibitors (154) or 

 by the use of activators (176). The purification of 

 individual enzymes is beyond the scope of the 

 present review. 



Metabolii Conditions 



For preparations with intact cells, metabolites, 

 including oxygen which has already received com- 

 ment, are normally provided in an aqueous medium 

 which contains also salts, buffers and organic sub- 

 strates. The medium may be a modified or simplified 

 blood (60), physiological sail solution (35, 103, 177) 

 or various enriched salines (103, 146, 206, 207). 



There is no one metabolic criterion of the adequacy 

 of metabolic conditions; these musi be appraised in 

 relation to the object of the in vitro study. A minimum 

 requirement can usually be stated, namely, that the 

 main energ\ -yielding requirements of the tissue should 

 be met. This is best judged by the maintenance of 

 energy-rich phosphates in the tissue, or by the ability 

 of the tissue to show metabolic response i<> electrical 

 pulses (see below); respiration gives a much less 

 certain criterion. Parallel to such maintenance no 

 other activities of the tissue such as the incorporation 

 of isotopes into structural materials (47). This mini- 

 mum is provided by a salt mixture plus oxygen and 

 glucose. Glucose, with white matter (17) as well as 

 with gray, with subcortical ( 1 7 ) as well as with cortical 

 tissues, and with human (130) as well as with other 

 central nervous system tissues, is usually replaceable 

 by pyruvate but not by lactate, citrate, succinate or 

 fumarate. A high concentration of fructose may re- 

 place glucose, and glutamate is more effective with 

 human tissues than with tissues from other species. 



The minimal conditions give tissues known to be 

 depleted with respect to many of their organic consti- 

 tuents and of respiratory rate lower than in vivo. 

 Several specific additions to the minimal saline make 

 good some of the deficiencies; detailed study has been 

 made of glutamate and potassium salts (104, 191), 



chlorides (208), creatine (206), adenosine and guano- 

 sine derivatives (76, 207), glycogen (109, 147), 

 glutathione (144 1, glucose and lactate (147). When 

 any of these or related substances are added to salines 

 containing central nervous tissues, incubation for 

 appreciable times may be necessary for restoring 

 in urn composition or an approximation to it. Several 

 deficiencies are made good in a few minutes but 

 others such as glycogen and nucleotides may require 

 2 hr. at 37°C. 



The deficiencies already described arise spontane- 

 ously as a result of removing tissues from animals and 

 placing [hem in many times their volume of simple 

 salines. More m,i\ be learned about chemical require- 

 ments for tissue functioning b\ deliberately inducing 

 further depletion in the tissue by incubating it without 

 glucose (142) or without oxygen (135). Conversely, 

 the depletion may be minimized by incubatins; the 

 tissue with only very small volumes of salines (177) or 

 in oils (1771, experimental arrangements have been 

 described for supplying substrates and for electrically 

 stimulating cerebral tissues under these conditions. 



In the case of preparations not maintaining cell 

 structure, choice of metabolic conditions should 

 imitate intracellular rather than extracellular fluids. 

 Again, however, conditions must be appraised in rela- 

 tion to (he specific objective of the studv and ma\ 

 differ greatly. Regarding individual cerebral enzymes, 

 s\ stems .11c too diverse to receive general comment; 

 many individual examples have been quoted (136). 

 As many processes require maintenance of the main 

 energy-yielding reactions of the tissue, requirements 

 for these may be noted. For glycolysis in cell-free 

 sv stems conditions are well documented (43, 136); 

 they involve several intermediary metabolites and 

 coenzv mes and also some additions not native to the 

 tissue .is, lor example, high concentrations of nico- 

 tinamide to prevent loss of cozymase (145)- Require- 

 ments of oxidative phosphorylation (22, 25, 27) 

 include supplementing the tissue's store of phosphate 

 acceptors with added adenosine mono- or diphos- 

 phate, or glucose plus hexokinasc. When first studied 

 in mitochondrial preparations, oxidatively phos- 

 phorylating systems proved labile so that experiments 

 were often run below 37°C and for periods of onlv 

 5 to 30 min. More stable preparations have subse- 

 quently become available (27) which allow, with an 

 agent such as ethylencdiamine tetra-acetic acid, 

 much longer experimental periods. The tetra-acetic 

 acid may act in part by chelating traces of toxic 

 metals, to which cell-free systems are in general much 

 more susceptible (26, 134) than are cell-containing 



