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Following axotomy, the regrowth of peripheral axons takes longer in older individuals than in young ones. The present study compares the crush-induced process of degeneration and regeneration in the buccal branch of the facial motor nerve in groups of rats aged 3 months and 15 months. Observations are based on qualitative and quantitative analyses of the nerve 20 mm from the site of injury in rats 1, 2, 4, 16, 21, 28, and 56 days after crush. The buccal branch is purely motor and contains a unimodal population of about 1,600 axons commonly in a single fascicle. During the first 28 days post crush (dpc) in the 3-month animals, the progression of myelin and axon degeneration, myelin clearance, regrowth of axon sprouts, and axon maturation are relatively synchronized and uniform. In the older rats, the degeneration of myelin and axons, myelin clearance, and the appearance of axon sprouts at the site of sample are all delayed. In the younger animals, axon sprouts increase in numbers from their first appearance at 4 dpc through the 2 weeks examined following the restoration of whisking behavior. The numbers of regenerating older axons increase at a rate comparable to that in the younger animals through the time that bilaterally symmetrical whisking behavior is evident, but afterwards the number of axon sprouts decreases. At 2 months after crush the young animals have 30% more fibers in the buccal branch than control nerves, while the older animals have fewer than control numbers. In the 3-month regenerated nerve, 2 months post crush, 30% of the regenerated fibers are of very small caliber, less than 3 microns2 in cross sectional area, and typically these small axons have unusually thick myelin sheaths; the older nerves do not have such a skewed distribution of axon areas. The older regenerated axons at 2 months post crush have an unusually high density of microtubules compared to the younger regenerated ones (and controls), and the ratio of neurofilaments to microtubules is very low. The conclusions are that motor neurons in older animals regenerate damaged axons after a delay not apparent in the young; the strong regenerative response apparent initially in animals of both age groups is not maintained in the older animals; and the relationship between the numerical density of cytoskeletal elements and the axon cross-sectional area deviates from normal in the regenerated axons of the older animals.

The basal dendritic trees of layer V pyramidal cells in the rat auditory cortex were examined quantitatively in a group of 3-month-old and a group of 34- and 36-month-old rats. Two forms of analysis were used on the Golgi preparations: (1) the number of intersections between the basal dendrites and a series of concentric circles whose common center lies over the perikaryon center, and (2) the number of dendritic branches, by order, per neuron. The data indicate that in the old animals the density of the dendritic tree has decreased significantly within a radius of about 150mu of the perikaryon, yet the extent of the dendritic domain has not changed appreciably. Analysis of the dendritic branching suggests that there has been a deterioration not only in the peripheral branches of the dendritic tree, but also that entire dendrites have been lost. This loss of primary branches was confirmed through the reconstruction of layer V neuronal perikarya and their proximal dendrites from 1-mu plastic serial sections of auditory cortex. Concomitant with the loss of dendrites which accompanies advancing age is a tendency for the perikaryon to be smaller, but not distorted, in the old animals.

The thiol ester N-t-Boc-L-alanine p-nitrothiophenyl ester (Boc-Ala-SNp) was synthesized and applied as an ultrastructural cytochemical substrate for intracellular elastase-like enzymes . Mature human neutrophils incubated with Boc-Ala-SNp and gold ions generate an electron-dense reaction product, gold p-nitrothiophenolate, which is found in the nuclear membrane, Golgi complex, endoplasmic reticulum, mitochondria, and granules of these cells . Enzyme activity against BocAla-SNp is also observed in developing monkey bone marrow neutrophils and in other blood cells. The intracellular neutrophil enzyme activity is elastase-like because it is characterized by a slightly alkaline pH optimum and is inactivated by exposure of the cells to general and specific active site inhibitors of neutrophil elastase . This substrate appears to have important potential for use in ultrastructural studies of intracellular elastase-like enzymes . KEY WORDS ultrastructural cytochemistry neutrophil -elastase-like enzymesElastolytic enzymes have been implicated in the pathogenesis of emphysema (20), glomerulonephritis (16), arthritis (8, 32), and atherosclerosis (30). A cytochemical substrate that can be used for the ultrastructural localization of elastase-like enzymes should aid considerably in further defining the role of these enzymes in normal and disease states. Although cytochemical substrates for elastase-like enzymes have been used for studies at the light microscope level (34), and elastase antibodies have been used for the extracellular localization of elastase in electron microscopy (29), elastase-like enzymes have not been described intracellularly in fine structural studies .This report describes the synthesis and use of the substrate N-t-Boc-L-alanine p-nitrothiophenyl ester (Boc-Ala-SNp) for the ultrastructural localization of intracellular elastase-like enzymes . The basic principle of this method has been applied to the detection of nonspecific esterases by Vatter et al . (43) . The intracellular hydrolysis of Boc-AlaSNp by enzymes of elastase-like specificity produces p-nitrothiophenolate (PNT) ion, which is immediately precipitated in the presence of gold ions at the enzyme site as an insoluble mercaptide . The heavy metal complex is readily identified in the electron microscope, thus localizing the subcellular sites of reactive enzyme . The present study reveals the association of elastase-like activity in human neutrophils not only with granules, as expected (28), but also with other membranous organelles within these cells. MATERIALS AND METHODSSynthesis of Boc-Ala-SNp t-Boc-L-Alanine (3 .70 g, 20 tnmol ; Schwartz/Mann Div ., Bec-J . CELL BIOLOGY

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