In order to elucidate the ultrastructural histochemical changes of tissues and cells in various organ systems in animals and men, tissue samples obtained from various aging stages starting from embryonic stage to newborn, suckling, weanling, juvenile and senescent stages of ddY mice bred in our laboratory, as well as biopsied samples of human tissues from juvenile, adult and senescent patients in clinical departments were collected and investigated by means of various histochemical procedures. The histochemical methods employed were fixations with routine chemical fixatives, such as buffered glutaraldehyde and osmium tetroxide followed by dehydration and plastic embedding for thick and thin sections, or rapidly frozen cryosections, or freezesubstituted or freeze-dried and embedded dry sections, which were routinely stained with toluidine blue for light microscopy or uranyl acetate and lead citrate for electron microscopy, or specific histochemical reactions such as enzyme histochemistr
y, radioautography after administration of various kinds of precursors for macromolecular compounds such as ^3H-thymidine (for DNA), ^3H-uridine (RNA), ^<3H>-leucine (proteins), ^3H-glucosamine or ^<35>SO_4 (glucides), ^3H-glycerol (lipids) either in vivo or in vitro, immunohistochemical reactions for proteins, and lectin histochemistry with biotinyl-lectins in combination with ABC and DAB methods to demonstrate glycoconjugates.
The results obtained are as follows.
1. Cell and Tissue Culture. Established cell line, HeLa cells, primary cultures from rat liver cells, human normal and cancer thyroid tissues were cultured in vitro and ultrastructural morphology, incorporation of ^3H-labeled thymidine and uridine were demonstrated. The labeling index of ^3H-thymidine incorporation increased from the initiation of cell culture, showed a peak on days 3 and 4 in culture and decreased.
2. Organs of Movement. The human lumber yellow ligaments from various kinds of diseased. patients in various ages were surgically taken out, chemically fixed or rapidly frozen, cryosectioned, freeze-dried, and both the morphology of elastic fibers and elementary analysis with an X-ray microanalyzer were performed. The results revealed that the fine structure of elastic fibers changed according to aging, classified into 4 types, and soluble calcium was found in the ligaments suggesting calcification and ossification.
3. Circulatory Organs. Mouse spleen cells from various stages of aging mice were examined. Acid phosphatase activity was demonstrated in the lysosomes of reticulocytes, macrophages and littoral cells and the intensity of the activity, which was estimated by X-ray microanalysis on Ce end-products, increased after birth reaching the maximum at 2 weeks and decreased to 10 months. The incorporations of ^3H-thymidine, -uridine and -leucine increased after birth, reaching the maxima at 2 weeks and then decreased.
4. Endocrine Organs. The thyroid tissues obtained surgically from human patients with goiters were examined with PCNA/cyclin immunohistochemical stainability equivalent to the incorporation of ^3H-thymidine. The results showed that the PCNA/cyclin positive cells were more than ^3H-thymidine. In primary cultured normal and tumor cells, intracellular localization of keratin and vimentin were demonstrated by immunohistochemical procedures and inhibitory effects of both TSH and anti-cancer drugs were observed. On the other hand, incorporation of ^3H-thymidine was observed in aging mouse adrenal gland by light microscopic radioautography which resulted in high labeling index in both adrenal cortex and medulla during the fetal stage, then decreasing after birth to senescence, showing different indices in superficial and deeper layers of the cortex and medulla.
5. Digestive Organs. The mucigen granules in the goblet cells of the colons of aging mice were examined by ^3H-glucosamine radioautography, X-ray microanalysis on S in the glycoproteins and lectin histochemistry. The results showed that the incorporation of ^3H-glucosamine and S contents increased after birth, reached the maxima at 2 weeks and decreased. The staining pattern with several lectins changed from fetal to senescent stages demonstrating the change of glycoconjugates composition. On the other hand in the liver, labeling indices of various kinds of celis composing the liver with ^3H-thymidine, ^3H-uridine, and ^3H-leucine were examined by both light and electron radioautography The results revealed that the labeling index of ^3H-thymidine in hepatocytes, sinusoidal endothelial cells and other cell types decreased from fetal stage and after birth reaching almost 0 in 2 years, demonstrating different pattern of frequencies. To the contrary, the incorporations of ^3H-uridine and ^3H-leucine increased after birth, reaching the maxima at 2 to 4 weeks and finally decreased. The pancreatic cells of aging mice also demonstrated similar pattern of ^3H-thymidine, ^3H-uridine and ^3H-leucine incorporations. In the pancreas, incorporations of ^3H-glucosamine, ^3Hglycerol revealed the similar pattern to uridine and leucine, reaching maxima at 2-4 weeks after birth.
6. Respiratory Organs. The tracheas of aging mice were examined after administration of ^<35>SO_4 by light microscopic radioautography, which demonstrated incorporation of glycoproteins into chondrocytes and cartilaginous matrix of the tracheal cartilages. The number of silver grains demonstrating glycoprotein synthesis increased after birth to 2 weeks, reaching a maximum and decreased. The incorporation of ^3Hthymidine is now being examined.
7. Uro-genital Organs. In the kidneys of mice at various ages, incorporation of ^3Hthymidine was examined by light microscopic radioautography and it was found that the labeling index of glomerular epithelial celli and uriniferous tubular cells increased during the fetal stage and decreased after birth showing different patterns in the superficial and deeper cortex. Lectin histochemistry also revealed changes of stainability to various kinds of lectins in respective ages, thus the stainability patters were categorized into 4 types. The testis, ovary, uterine tube and uterus of aging mice are now under investigation.
8. Nervous and Sensory Organs. Nucleic acid synthesis in mouse ocular tissues was studied by ^3H-thymidine and ^3H-uridine radioautography. The incorporation of ^3H-thymidine was demonstrated in the retina and retinal pigment epithelium, The labeling indices of these cells were high in the fetal stage and decreased after birth to the senescence. However, the indices in respective sites of the retina, i. e. from anterior to equatorial and posterior, as well as from external to internal layers, at each stage of development and aging, differed variably. The RNA contents as expressed by the number of silver grains in each cell at different sites, layers, and stages, varied, increasing after birth and showing maxima at 1 week and decreased. Lectin histochemistry revealed that several kinds of lectins showed characteristic changes in staining paftems in the retina due to aging.
From these results, histochemical changes in cells and tissues of many organs In various organ systems of experimental animals and men were demonstrated. It is expected that these results should be utilized in the fields of clinical medicine to elucidate the mechanism of aging and senescence in men in the future. Less