A MAPPING OF OXIDATIVE ENZYMES IN THE HUMAN BRAIN *

Histochemicul techiyues. Six normal adult human brains from average post mortem material were used. They were placed in neutral 10% formalin 3-9 hr after death and fixed at 5" for 2 days. After I-day fixation, the material was blocked into pieces 4-6 mm thick. Frozen sections were cut a t 30 p, rinsed in H,O and incubated for 2 hr at 38" with frequent agitation. The method of FARBER, STERNBERG and DUWAP (1956) was employed, using Nitro BT and tris buffer. Other techniques such as that of SCARPELLI, HESS and E (1958) and other tetrazolium salts had been used with identical resu1ts.t The pH of the incu medium was always adjusted to 7.35. Sections from each block were incubated in individual jars and the proportion of incubation media to tissue was approximately the same for each. The reaction was stopped by transferring the sections to 10% neutral formalin. Half of thc sections were mounted in glycerin gel; the others were dehydrated and mounted in Permount. Random material was used for the demonstration of succinic dehydrogenase (NACHLASS e f al., 1957) and cytochrome oxidasc (BURSTONE, 1Y58) in 60 11 unfixed sections. @rantifurire techniques. For quantitative DPN-diaphorase determinations large series were used from three normal human brains. They were fixed and sectioned i n the same way as sections used for histochemical studies. The incubation medium was the same except that the monotetrazolium salt INT was substituted for Nitro BT because Nitro BT formazan is very insoluble. The final concentration of tetrazolium salt in the incubation medium was 0.23 mgiml for both the histochemical and quantitative studies. The sections were stored in chilled distilled H,O until all the sections from one brain were cut. After all the sections were transferred to a beaker containing the buffer component of the incubation medium, they were gently poured into the rest of the incubation medium in a 2 I . Erlenmeyer flask which was shaken constantly in an Eberbach shaker-water bath a t 38" for 2 hr. The reaction was stopped by transferring the entire contents of the flask into a large quantity of lOu/d formalin. The constant shaking increased the rate of the reaction and prevented uneven staining due to folded tissue. To make an accurate comparison of the amount of INT reduced formazan in all areas it was necessary completely to eliminate even minute variations in the time and temperature of fixation and incubation, in the degree of shaking and, particularly, in the proportion of tissue to incubation medium. Thus, all the sections from one brain were incubated in a single large quantity (600ml) of incubation medium. When this is done, it is a reliable method for showing patterns within one specimen. It has been shown that the quantity of formazan formed is directly proportional to the enzyme activity (SHELTON and RICE, 1957). If the method was to be applied to experimental specimens, it was necessary to incubate sections from a control specimen in the same incubation bath each time. A 3 x 2 in. slide was covered with parafilm and each section was temporarily mounted on this to facilitate punching of small discs in the areas of interest. Sharpened stainless steel tubes, 1.1 mm and 2.4 mm in diameter were used to cut the discs. The INT formazan of each disc was extracted in 3 ml of 3: 1 (v/v) ethanol-tetrachloroethylene (Eastman, Rochester, N.Y.). One drop of I N-HCI was added to each tube of extracted forrnazan, to ensure that the colour would be stable for