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A two-stage fixation technique has been developed to obtain morphological preservation and retention of glucose 6-phosphatase (G6-Pase) activity for its demonstration in rat cerebral cortex. The technique was then employed to localize the enzyme in the cortex where it produced a dense reaction over the well developed granular endoplasmic reticulum cisternae in nerve cells and oligodendrocytes which contrasted with a thin reaction in astrocytes. Other membranous organelles showed no reaction.

A needle wound was made in the adult rat cerebral cortex. Responses of neurons and oligodendrocytes at the site of injury were followed over a period of 450 days and correlations made between morphological and enzyme cytochemical changes to clarify some phenomena previously unresolved. Evidence from acid phosphatase activity in degenerating neurons showed no increase in the number of cytochemically stained lysosomal profiles nor changes in the subcellular localization of the acid phosphatase reaction product. Our observations indicated that the majority of dying neurons were not digested by their own acid phosphatase 'autodigestion' but by the process of heterodigestion. The time-course study revealed that not all the traumatized neurons were eliminated but some persisted permanently in an attenuated 'atrophic' state. The atrophic neurons were small in size with low cytoplasmic-nuclear ratios and exhibited low levels of glucose-6-phosphatase and cytochrome oxidase activities. The acid phosphatase activity was slightly increased as evidenced by cytochemically stained hypertrophic Golgi cisternae and a slight increase in the number of lysosomes. The low level of enzyme activities concerned with carbohydrate metabolism reflected the low metabolic activity in atrophic neurons whilst an increase in Golgi-lysosomal enzyme activity suggested some anabolic process necessary for their survival. Oligodendrocytes displayed only minor changes in morphology, and their glucose-6-phosphatase and cytochrome oxidase activities were normal, suggesting that these cells have little or no involvement in the repair of a cerebral wound. The absence of significant changes in lysosomal acid phosphatase activity indicated a minimal role, if any, of oligodendrocytes in the process of phagocytosis.

Techniques for the ultrastructural demonstration of dehydrogenases in cerebral cortex are described. The best fixation for good fine structural preservation and retention of LDH and NADH-diphorase was obtained by perfusion with a misture of formaldehyde and glutaraldehyde and for SDH by perfusion with formaldehyde. Comparison of incubation conditions showed that consistent results were obtained using enzyme markers NBT and DS-NBT for LDH and NADH-diaphorase: DS-NBT was more satisfactory than NBT and BSPT for SDH. Penetration of incubation media was improved by Triton X-100: DMSO and ultrasonic treatment were less effective. The techniques enabled the first electron cytochemical demonstration of dehydrogenases in different elements of prefixed cerebral cortex. Ultrastructural sites of enzyme activities were localized within cristae and inter-membrane spaces of mitochondria in nerve cell cytoplasm and its processes, oligodendrocytes and astrocytes. Authenticity of the ultrastructural sites was confirmed by four different control experiments.

A procedure for the ultrastructural cytochemical localization of cytochrome oxidase via cytochrome c in the cerebral cortex is described. Vascular perfusion fixation by formaldehyde and glutaraldehyde of different concentrations and mixtures of the two gave varying results. A mixture of 4% formaldehyde and 0.5% glutaraldehyde gave the best combination of ultrastructural preservation and retention of enzyme activity. Histochemical methods were examined for optimum incubation conditions, based on the oxidative polymerization of 3,3'-diaminobenzidine (DAB) to an osmiophilic product. The reaction product was discretely localized within intercristate and the intermembrane space of mitochondria. The staining pattern was the same in nerve cells and in neuroglia and their processed. The DAB reaction product was also found in mitochondria of the endothelial cells.

A stab wound was produced in the adult rat cerebral cortex, and the progress of enzyme cytochemistry of phagocytosis was studied over 450 days. Light- and electron-microscopic observations were made to establish the origin of high acid phosphatase activity commonly seen at the site of brain lesions. Cells with phagocytic potential became differentiated and activated by the presence of degenerating neurons. The Golgi-lysosomal system of the phagocytes became elaborated, as evidenced by thiamine pyrophosphatase and acid phosphatase activities, the synthesis of acid phosphatase was increased, and the enzyme then secreted into the digestive vacuoles containing dead cells to be digested. Progress of the digestive process resulted in the accumulation of large amounts of acid phosphatase reaction product within the digestive vacuoles. The results showed that the phagocytes were the only detectable source of increased acid phosphatase activity at the site of injury in the cerebral cortex. In contrast to the phagocytes, newly formed multi-nucleated giant cells exhibited weak acid phosphatase, and intense cytochrome oxidase activities, the difference between the two cells reflecting the functional characteristics of each.