Electron Microscopic Radioautograms Research Articles

Electron microscopic radioautographic study on mitochondrial RNA synthesis in adrenal cortical and medullary cells of aging mice

In order to study the aging changes of intramito-chondrial RNA synthesis of mouse adrenal cells, 10 groups of developing and aging mice, each consisting of 3 individuals, total 30, from fetal day 19 to postna-tal newborn at day 1, 3, 9, 14, adult at month 1, 2, 6 and senescent animals at month 12 (year 1) and 24 (year 2) were injected with 3H-uridine, an RNA pre-cursor, sacrificed 1 hr later and the adrenal tissues were fixed and processed for electron microscopic radioautography. On electron microscopic radio-autograms obtained from each animal, the number of mitochondria per cell, the number of labeled mito-chondria with 3H-uridine showing RNA synthesis per cell and the mitochondrial labeling index in each adreno-cortical cells, in 3 zones, as well as in each adreno-medullary cells, 2 types of cells in the medulla, the adrenalin cells and the noradrenalin cells, were calculated and the results in respective aging groups were compared with each others. The results demon-strated that the number of mitochondria in adreno-cortical cells in 3 zones, the zona glomerulosa, fasciculata and reticularis of respective mice at vari-ous ages increased from fetal day 19 to postnatal month 1 reaching the plateau from month 1 to 24 due to development and aging of animals, respectively, while the number of labeled mitochondria per cell and the labeling index of intramitochondrial RNA synthesis incorporating 3H-uridine increased from fetal day 19 to postnatal month 2, reaching the maxima and decreased slightly from month 6 to month 24. On the other hand, the number of mito-chondria per cell in the medulla increased from fetal day 19 to postnatal month 1 reaching the plateau from month 1 to 24, while the number of labeled mi-tochondria per cell and the labeling index of in-tramitochondrial RNA synthesis increased from fetal day 19 to postnatal day 14, reaching the maxima and decreased from month 1 to 24. From the results, it was demonstrated that the activity of intramitochno-drial RNA synthesis in both the cortical and me-dullary cells in developing and aging mice adrenals changed due to aging of individual animals.

Electron Microscopic Radioautographic Study on DNA Synthesis of Mitochondria in Adrenal Medullary Cells of Aging Mice~!2009-08-12~!2009-09-17~!2009-12-24~!

For the purpose of clarifying the aging changes of DNA synthesis of mouse mitochondria in adrenal medullary cells, 10 groups of aging mice, each consisting of 3 individuals, total 30, from fetal day 19 to postnatal newborn at day 1, 3, 9, 14, adult at month 1, 2, 6, 12 and 24, were injected with 3H-thymidine, sacrificed 1 hr later and the adrenal tissues were fixed and processed for electron microscopic radioautography. On electron microscopic radioautograms obtained from each animal, number of mitochondria and the number of labeled mitochondria labeled with silver grains due to 3H-thymidine showing DNA synthesis in each adrenalin cell and noradrenalin cell were counted and the results of labeling indices in respective developing groups were compared respectively.

Studies on mitochondrial macromolecular syntheses in various organs of aging animals labeled with 3H-precursors as revealed by electron microscopic radioautography

In order to study the aging changes of intramitochondrial macromolecular synthesis in various organs of aging animals during the development and aging, 10 groups of developing and aging mice, each consisting of 3 individuals, total 30, from fetal day 19 to postnatal newborn at day 1, 3, 9, 14 and adult at month 1, 2, 6, 12 to 24 were injected with 3H-thymidine a DNA precurson, another 10 groups consisting of 3 individuals, total 30, were injected with 3H-uridine a RNA precursor, and another 10 groups of 30 individuals were injected with 3H-leucine a protein precursor, total 90 individuals. Then, all the animals were sacrificed 1 hr after the injections and the liver tissues, the lung tissues, the kidney tissues, the testis and ovary tissues, the adrenal tissues were taken out, fixed and processed for electron microscopic radioautography. On electron microscopic radioautograms obtained from each animal, ten photographs in respective groups, numbers of mitochondria per cell profile area, numbers of labeled mitochondria per cell and the mitochondrial labeling index (LI) labeled with 3H-thymidine showing DNA synthesis, LI labeled with 3H-uridine showing RNA synthesis, and LI labeled with 3H-leucine showing protein synthesis, in various organs, were counted and the results in various organs in respective aging groups were compared, respectively. From the results, it was demonstrated that the numbers of mitochondria in hepatocytes as well as in 3 zones of the adrenal cortex, the zona glomerulosa, fasciculata and reticularis of mice at various ages increased from fetal day 19 to postnatal month 1 due to development and aging of animals. On the other hand, the numbers of labeled mitochondria and the labeling indices of intramitochondrial DNA syntheses incorporating 3H-thymidine in hepatocytes and 3 zones of cortical cells increased from fetal day 19 to postnatal month 1 and decreased to month 24. The numbers of labeled mitochondria and the labeling indices of intramitochondrial RNA syntheses incorporating 3H-uridine in hepatocytes and 3 zones of cortical cells increased from fetal day 19 to postnatal month 1 and decreased to month 24. The numbers of labeled mitochondria and the labeling indices of intramitochondrial RNA syntheses incorporating 3H-uridine in hepatocytes and 3 ones of cortical cells increased from fetal day 19 to postnatal month 1 and decreased to month 24. Moreover, some other organs such as the lung and the testis were also review and discussed. From the results, it was shown that the activity of intramitochnodrial DNA synthesis, RNA synthesis, and protein synthesis in hepatocytes, adrenal cortical cells, pulmonary cells and testicular cells in developing and aging mice as well as some other organs increased and decreased due to development and aging.

Recent Studies on Macromolecular Synthesis Labeled with 3H-Thymidine in Various Organs as Revealed by Electron Microscopic Radioautography

In order to study the aging changes of intramitochondrial DNA synthesis of mouse adrenal cells during the development, 10 groups of developing mice, each consisting of 3 individuals, total 30, from fetal day 19 to postnatal newborn at day 1, 3, 9, 14 and adult at month 1, 2, 6, 12 and 24 were injected with 3H-thymidine, sacrificed 1 hr later and the adrenal tissues were processed for electron microscopic radioautography. On electron microscopic radioautograms obtained from each animal, numbers of mitochondria per cell, numbers of labeled mitochondria and the mitochondrial labeling index labeled with 3H-thymidine showing DNA synthesis in each adrenal cortical cell, in 3 zones, as well as adrenal medullary cells were counted and the results in respective groups were compared, respectively. From the results, it was demonstrated that the numbers of mitochondria in 3 zones, the zona glomerulosa, fasciculata and reticularis of mice at various ages as well as the numbers of mitochondria in 2 types of medullary cells, adrenalin cells and noradrenalin cells, increased from fetal day 19 to postnatal month 1 due to development and aging of animals. On the other hand, the numbers of labeled mitochondria and the labeling indices of intramitochondrial DNA syntheses incorporating 3H-thymidine in 3 zones of cortical cells increased from fetal day 19 to postnatal month 1, while the numbers of labeled mitochondria and the labeling indices of intramitochondrial DNA syntheses in medullary cells, both the adrenalin and the noradrenalin cells incorporating 3H-thymidine increased from fetal day 19 to postnatal day 14 and decreased to month 1. From the results, it was shown that the activity of intramitochnodrial DNA synthesis in both the adrenal cortical and the medullary cells in developing mice changed due to development and aging but the cortical cells and the medullary cells were a little different from each other. The intramitochondrial DNA syntheses in some other organs were also reviewed and discussed. Keywords: Mouse adrenal cortex, medulla, electron microscopic radioautography, DNA synthesis, mitochondria, development, aging, hepatocyte

Electron Microscopic Radioautographic Study on Mitochondrial DNA Synthesis in Adrenal Cortical Cells of Developing and Aging Mice

In order to study the aging changes of intramitochondrial DNA synthesis of mouse adrenal cortical cells, eight groups of developing mice, each consisting of three individuals (total 24), from fetal day 19 to postnatal newborn at days 1, 3, 9, 14, to adult at months 1, 2, and 6, were injected with 3H-thymidine, sacrificed 1 h later, and the adrenal tissues were fixed and processed for electron microscopic (EM) radioautography. On EM radioautograms obtained from each animal, the number of mitochondria and the mitochondrial labeling index labeled with 3H-thymidine showing DNA synthesis in each adrenal cortical cell, in three zones, were counted and the results in respective developing groups were compared. From the results, it was demonstrated that the numbers of mitochondria in the three zones, the zona glomerulosa, fasciculata, and reticularis, of mice at various ages increased from fetal day 19 to postnatal month 6 due to development and aging of animals, respectively, while the number of labeled mitochondria and the labeling index of intramitochondrial DNA syntheses incorporating 3H-thymidine increased from fetal day 19 to postnatal month 2, reaching the maxima, and decreased to month 6. It was shown that the activity of intramitochondrial DNA synthesis in the adrenal cortical cells in developing and aging mice changed due to aging.

Electron Microscopic Radioautographic Study on Protein Synthesis in Mitochondria of Binucleate Hepatocytes of Aging Mice

In order to study the aging changes of intramitochondrial protein synthesis in mouse hepatocytes, 10 groups of aging mice, each consisting of three individuals, total 30, from fetal day 19 to postnatal year 2, were injected with 3H-leucine, a protein precursor, sacrificed 1 h later, and the liver tissues processed for electron microscopic radioautography. On electron microscopic radioautograms obtained from each animal, the numbers of mitochondria, the numbers of labeled mitochondria, and the mitochondrial labeling index labeled with 3H-leucine that showed protein synthesis in each hepatocyte, both mononucleate and binucleate cells, were counted and the averages in respective aging groups were compared. From the results, it was demonstrated that the numbers of mitochondria, the numbers of labeled mitochondria, and the labeling indices of intramitochondrial protein syntheses in both mononucleate and binucleate hepatocytes of mice at various ages increased due to development of animals. The numbers of mitochondria, the numbers of labeled mitochondria, and the labeling indices of intramitochondrial protein synthesis in binucleate hepatocytes were more than those of mononucleate hepatocytes at the same aging stages.

Electron Microscopic Radioautographic Study on Protein Synthesis in Hepatocyte Mitochondria of Aging Mice

For the purpose of studying the aging changes of intramitochondrial protein synthesis in mouse hepatocytes, 10 groups of aging mice, each consisting of 3 individuals (total 30), from fetal day 19 to postnatal month 24, were injected during development with 3H-leucine, a protein precursor, sacrificed 1 h later, and the liver tissues processed for electron microscopic (EM) radioautography. On EM radioautograms obtained from each animal, the number of mitochondria, the number of labeled mitochondria, and the mitochondrial labeling index labeled with silver grains due to H-leucine showing protein synthesis in each mononucleate hepatocytes were counted and the averages in respective aging groups were compared. From the results, it was demonstrated that the numbers of mitochondria, the numbers of labeled mitochondria, and the labeling indices of intramitochondrial protein syntheses in mononucleate hepatocytes of mice at various ages from embryonic day 19 to postnatal month 24 increased and decreased due to development and aging of animals.

Radioautographic Study on the Intracellular Localization of Bezafibrate in Cultured Rat Hepatocytes.

In order to demonstrate the intracellular localization of the peroxisome proliferator in the hepatocytes, we have examined the localization of silver grains due to 14C-labeled bezafibrate in cultured rat hepatocytes by means of light and electron microscopic radioautography. On light microscopic radioautograms of the epoxy resin sections at each incorporation period, about 90% of all the silver grains were localized over the cytoplasmi Then, statistical significance was evaluated on grain density in the cytoplasm. On electron microscopic radioautograms of whole mount cultured cells observed by high voltage electron microscopy, silver grains were localized on the cytoplasmic matrix, especially over the endoplasmic reticulum. From these results, it is concluded that bezafibrate was localized over the endoplasmic reticulum. This fact suggests than the receptor of peroxisome proliferator should be associated with the endoplasmic reticulum or the receptor exists on the endoplasmic reticulum. Thus, it is demonstrated that the peroxisome proliferator acts on the endoplasmic reticulum of hepatocytes to proliferate peroxisomes.

Standard methods of electron microscopic radioautography

The standard methods of making electron microscopic radioautograms and quantitation of silver grains in our laboratory were presented. Specialized techniques for insoluble or soluble compounds should be employed for demonstrating the synthesis of various macromolecules and the incorporation of various labeled substances. Specimens for electron microscopic radioautography processed under the same conditions can be employed for the quantitation of radioactive substances in subcellular compartments using image analyzers.

Quantitative Analysis of Histochemical Reactions: Image Analysis of Light and Electron Microscopic Radioautograms.

In order to quantify histochemical reactions, light and electron microscopic radioautograms obtained from mouse organs were analyzed by means of various kinds of available image analyzers. Cultured cells in vitro or epithelial cells of the stomachs, small and large intestines, the livers and the pancreases of mice aged from fetal day 19 to postnatal 2 years were labeled with RI-labeled precursors for macromolecules, e. g. 3H-thymidine for DNA, 3H-uridine for RNA, 3H-amino acid for proteins, 3H-glucosamines and 35SO4 for mucosubstances, 3H-glycerol for lipids and all the cells and tissues were fixed in glutaraldehyde and osmium tetroxide, embedded in Epon, sectioned either thick (2μm) or thin (0.2μm), coated with either Konica NR-M2 or-H2 radioautographic emulsions, exposed for several weeks or months, developed in either SDX-1 or GL-phenidon developers and stained with either toluidine blue or lead citrate for either light or electron microscopy.Light microscopic radioautograms (LMRAG) were observed with Olympus Vanox AHB-LB or Nikon Optiphot-2 and analyzed quantitatively with IBAS-II (Carl-Zeiss) or Quadra 900 (Macintosh) for labeling indices and grain counts. Electron microscopic radioautograms (EMRAG) were observed in Hitachi H-700 or JEOL JEM-4000EX electron microscopes at accelerating voltages of 200 or 400kV, and enlarged photographs were analyzed with several kinds of image analyzers, PPA-250 (Rhesca), MOP (Kontron), Digigramer-G (Mutoh Kogyo) and Quadra 900 (Macintosh) for grain counting or measuring the sizes and numbers of cell organelles. Some of EMRAG were analyzed in analytical electron microscopes, combined with Hitachi H-700 Horiba EMAX-1800E, JEOL JEM-200CX Kevex 7000-77, and JEM-4000EX Tracor Northern TN-5400.From the results, it was demonstrated that correct data can, to some extent, be obtained by controlling both full automatic and semi-automatic analyzers by operating them manually.

LOCALIZATION OF ANTI-ALLERGIC AGENT IN RAT MAST CELLS DEMONSTRATED BY LIGHT AND ELECTRON MICROSCOPIC RADIOAUTOGRAPHY

In order to demonstrate the intracellular localization of anti-allergic agent in rat mast cells, 3H-labeled Tranilast, N-(3', 4'-dimethoxycinnamoyl) anthranilic acid was studied by both light and electron microscopic radio-autography.The peritoneal mast cells of several Wistar strain rats were collected and incubated in vitro with Tranilast (100μM) after sensitization with IgE and challenging with an antigen, demonstrating the inhibition of degranulation by type I allergic reaction. Then, the peritoneal mast cells were incubated in vitro with 3H-Tranilast (100μCi/ml) for varying time intervals from 0 to 60min and the cell pellets were fixed, embedded and processed for light and electron microscopic radioautography. Light microscopic radioautograms revealed that silver grains were localized over the cytoplasm. Electron microscopic radioautograms revealed that silver grains were localized over and around the granules. The number of silver grains increased according to the prolongation of incubation time.Several rats were administered orally with 3H-Tranilast (53μCi/gm body weight). They were sacrificed at various time intervals and the back skins were taken out, fixed, embedded and processed for light and electron microscopic radioautography. Silver grains were observed to localize over the subcutaneous tissues, especially concentrating over the mast cells.From the results, it is concluded that 3H-Tranilast was rapidly taken into mast cells in both in vitro and in vivo and it inhibited degranulation of type I allergic reaction.

ELECTRON MICROSCOPIC RADIOAUTOGRAPHY USING A COMBINATION OF PHENIDON DEVELOPER AND DOMESTIC EMULSION

A routine procedure for electron microscopic radioautography in combination with phenidon developer and the domestic emulsion, Sakura NR-H2, was developed. Good stable radioautograms have not been obtained by simply developing the emulsion coated grids with phenidon developer at any temperature, but the development of these grids at 18°C for 1 min after 30-45 sec gold latensification in freshly prepared gold thiocyanate gives satisfactory results. Some electron microscopic radioautograms developed by this method are presented. The addition of the anionic surfactant, dioctyl sodium sulphosuccinate, to the emulsion was found to be very effective not only for preventing the burst of emulsion but also in obtaining a uniform spread of the emulsion for the wire loop method. Radioautograms obtained with this method are suitable for quantitative electron microscopic radioautographic studies. Details of this method are introduced.

Chemical and autoradiographic analysis of γ-aminobutyric acid transport in Purkinje cells of the cerebellum

The transport of 3 H-GABA in Purkinje cells of rat cerebellum was studied by chemical, light microscopic, and electron microscopic techniques. 3 H-GABA was applied to the exposed cerebellar surface in vivo . One hour later the animals were killed and the radioactive content of the cerebellar cortex and deep cerebellar nuclei examined. In unfixed tissue most of the label corresponded chromatographically with GABA, and in fixed tissue all was in a single ninhydrin-negative peak which corresponded chromatographically to an unidentified derivative formed by treating 3 H-GABA with the fixing solution. Much more radioactivity appeared in the deep cerebellar nuclei than in the liver or the caudate-putamen. Light microscopic radioautographic sections of cerebellum showed significantly higher grain counts in the deep cerebellar nuclei as compared with the surrounding white matter. These findings are consistent with 3 H-GABA moving to the deep nuclei by the process of axoplasmic flow but not by diffusion or transport via the blood stream. Electron microscopic radioautograms of the deep nuclei showed that 42% of the grains were in axons and 20% in nerve endings which showed by far the highest relative grain density (relative grain density = % grains/% area) at 3.39. Dendrites had 16% of the grains (relative grain density = 1.93), while glia had 15% of the grains (relative grain density = 0.98). The labelled boutons all made symmetrical synaptic contacts with dendrites and contained elongated pleomorphic vesicles.

Radioautographic studies of choline incorporation into peripheral nerve myelin.

This radioautographic study was designed to localize the cytological sites involved in the incorporation of a lipid precursor into the myelin and the myelin-related cell of the peripheral nervous system. Both myelinating and fully myelinated cultures of rat dorsal root ganglia were exposed to a 30-min pulse of tritiated choline and either fixed immediately or allowed 6 or 48 hr of chase incubation before fixation. After Epon embedding, light and electron microscopic radioautograms were prepared with Ilford L-4 emulsion. Analysis of the pattern of choline incorporation into myelinating cultures indicated that radioactivity appeared all along the length of the internode, without there being a preferential site of initial incorporation. Light microscopic radioautograms of cultures at varying states of maturity were compared in order to determine the relative degree of myelin labeling. This analysis indicated that the myelin-Schwann cell unit in the fully myelinated cultures incorporated choline as actively as did this unit in the myelinating cultures. Because of technical difficulties, it was not possible to determine the precise localization of the incorporated radioactivity within the compact myelin. These data are related to recent biochemical studies indicating that the mature myelin of the central nervous system does incorporate a significant amount of lipid precursor under the appropriate experimental conditions. These observations support the concept that a significant amount of myelin-related metabolic activity occurs in mature tissue; this activity is considered part of an essential and continuous process of myelin maintenance and repair.