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Morphologic Techniques for the Study of Brown Adipose Tissue and White Adipose Tissue

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Adipose Tissue Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 155))

Abstract

Light microscopy (LM) allows study of the anatomy of the adipose organ. For instance, it permits investigation of its microscopic organization (possible lobular subdivision) and the arrangement in the tissue of vessels, nerves, and main cell types (1). The unilocular or multilocular organization of adipocytes is already clearly visible at low magnification (×2.5), but the identification of other cell types, such as mast cells (particularly frequent in brown tissue), histiocytes (particularly frequent in white tissue, especially in fasting animals and in animals under diet restrictions), and inflammatory cells, requires magnification of at least ×40–100. Other cell types such as fibroblasts, preadipocytes and pericytes, are not easily recognized at LM. This technique also provides data on the functional state of the adipose tissue (AT): the size of white adipose tissue (WAT) adipocytes is indicative of the animal’s nutritional state and areas of capillary bed expansion appear in fasting animals. In brown adipose tissue (BAT), multilocularity is observed in various degrees, depending on the functional state of the organ. When this is stimulated by cold exposure or diet, lipid vacuoles are small and numerous, at rest (i.e., at warm environmental temperature or in fasting animals), they increase in size and gradually merge into fewer vacuoles, and eventually into a single vacuole. The functional activation of mitochondriogenesis in BAT also determines increased eosinophily of adipocyte cytoplasm. LM observation requires specimen processing by the following steps: fixation, dehydration, embedding, sectioning, and staining (2).

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References

  1. Cinti, S. (1999) The Adipose Organ, Editrice Kurtis, Milan, Italy.

    Google Scholar 

  2. Bloom, W. and Fawcett, D. W. (1994) Textbook of Histology, 12th ed., (Fawcett, D. W., ed.), Chapman and Hill, New York.

    Google Scholar 

  3. Polak, J. M. and Van Noorden, S. (1986) Immunohistochemistry: Modern Method and Applications, 2nd ed., Wright PGS, Bristol, UK.

    Google Scholar 

  4. Nonradioactive in situ hybridization. Application Manual. (1996) Boehringher Mannheim, 2nd ed., pp. 44–56, 126–135.

    Google Scholar 

  5. Dagerlind, A., Friberg, K., Bean, A. J., and Hokfelt, T. (1992) Sensitive mRNA detection using unfixed tissue: combined radioactive and non radioactive in situ hybridization histochemistry. Histochemistry 98, 39–49.

    Article  PubMed  CAS  Google Scholar 

  6. Wilkinson, D. G., Bailes, J. A., Champion, J. E., and McMahon, A. P. (1987) Molecular analysis of mouse development from 8 to 10 days post coitum detects changes only in embryonic globin expression. Development 99, 493–501.

    PubMed  CAS  Google Scholar 

  7. Sassoon, D. A., Garner, I., and Buckingham, M. (1988) Transcripts of alpha-cardiac and alpha-skeletal actins are early markers for myogenesis in the mouse embryo. Development 104, 155–164.

    PubMed  CAS  Google Scholar 

  8. Cinti, S. (1992) Morphological and functional aspect of brown adipose tissue. Pediatric. Adolesc. Med. 2, 125–132.

    Google Scholar 

  9. Cinti, S., Cigolini, M., Bosello, O., and Björntorp, P. (1984) Morphological study of the adipocyte precursors. J. Submicrosc. Cytol. 16, 243–151.

    PubMed  CAS  Google Scholar 

  10. Rossouw, D. J., Cinti, S., and Dickersin, G. R. (1986) Liposarcoma: an ultrastructural study of 15 cases. Am. J. Clin. Pathol. 85, 649–667.

    PubMed  CAS  Google Scholar 

  11. De Mey, J. (1983) Colloidal gold probes in immunocytochemistry, in Immunocytochemistry, Practical Application in Pathology and Biology (Polak, J. M. and Van Noorden, S., eds.), Wright-PGS, Bristol, UK.

    Google Scholar 

  12. Cinti, S., Zancanaro, C., Sbarbati, A., Cigolini, M., Vogel, P., Ricquier, D., and Fakan S. (1989) Immunoelectron microscopical identification of the uncoupling protein in brown adipose tissue mitochondria. Biol. Cell 67, 359–362.

    Article  PubMed  CAS  Google Scholar 

  13. Cousin, B., Cinti, S., Morroni, M., Raimbault, S., Ricquier, D., Pénicaud, L., and Casteilla, L. (1992) Occurence of brown adipocytes in rat white adipose tissue: molecular and morphological characterization. J. Cell Sci. 103, 931–942.

    PubMed  CAS  Google Scholar 

  14. Hayat, M. A., ed. (1974). Biological application, in Principles and Techniques of Scanning Electron Microscopy, vol. 2. Van Nostrand Reinhold, London.

    Google Scholar 

  15. Barbatelli, G., Heinzelmann, M., Ferrara, P., Morroni, M., and Cinti, S. (1994) Quantitative evaluations of gap junctions in old rat brown adipose tissue after cold acclimation: a freeze-fracture and ultrastructural study. Tiss. Cell 26, 667–676.

    Article  CAS  Google Scholar 

  16. Cinti, S., Cigolini, M., Gazzanelli, G., and Bosello, O. (1985) Ultrastructural study of adipocyte precursors from epididymal fat pads of adult rats in culture. J. Submicrosc. Cytol. 14, 631–636.

    Google Scholar 

  17. Cigolini, M., Cinti, S., Bosello, O., Brunetti, L., and Björntorp P. (1986) Isolation and ultrastructural features of brown adipocytes in culture. J. Anat. 145, 207–216.

    PubMed  CAS  Google Scholar 

  18. Giger, H. and Riedwyl, H. (1970) Bestimmung der Grössenverteilung von kugeln aus schnittkreisradien. Biometr. Zschr. 12, 156.

    Article  Google Scholar 

  19. Williams, M. A. (1977) Quantitative methods in biology, in Practical Methods in Electron Microscopy, vol. 6, (Glauert, A. M., ed.), North-Holland, Amsterdam, The Netherlands.

    Google Scholar 

  20. Hayat, M. A. (1986) Basic Techniques for Transmission Electron Microscopy, Academic Press, New York.

    Google Scholar 

  21. Thompson, S. W. (1966) Selected Histochemical and Histopathological Methods, Thomas Publisher, Springfield, IL.

    Google Scholar 

  22. Hsu, S. M., Raine, L., and Fanger, H. (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase technique: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577–580.

    Article  PubMed  CAS  Google Scholar 

  23. Barbatelli, G., Morroni, M., Vinesi, P., Cinti, S., and Michetti, F. (1993) S-100 protein in rat brown adipose tissue under different functional conditions: a morphological, immunocytochemical and immunochemical study. Exp. Cell Res. 208, 226–231.

    Article  PubMed  CAS  Google Scholar 

  24. Zancanaro, C., Pelosi, G., Accordini, C., Balercia, G., Sbabo, L. and Cinti, S. (1994) Immunohistochemical identification of the uncoupling protein in human hibernoma. Biol. Cell 80, 75–78.

    Article  PubMed  CAS  Google Scholar 

  25. Giordano, A., Morroni, M., Santone, G., Marchesi, G. F., and Cinti, S. (1996) Tyrosine hydroxylase, neuropeptide Y, substance P, calcitonin gene-related peptide and vasoactive intestinal peptide in nerves of rat periovarian adipose tissue: an immunohistochemical and ultrastructural investigation. J. Neurocytol. 25, 125–136.

    Article  PubMed  CAS  Google Scholar 

  26. Cinti, S., Frederich, R. C., Zingaretti, M. C., De Matteis, R., Flier, J. S., and Lowell, B. (1997) Immunohistochemical localization of leptin and uncoupling protein in white and brown adipose tissue. Endocrinology 38, 797–804.

    Article  Google Scholar 

  27. De Matteis, R., Dashtipour, K., Ognibene, A., and Cinti, S. (1998) Localization of leptin receptor splice variants in mouse peripheral tissues by immunohistochemistry. Proc. Nutr. Soc. 57, 441–448.

    Article  PubMed  Google Scholar 

  28. Giordano, A., Morroni, M., Carle, F., Gesuita, R., Marchesi, G. F., and Cinti, S. (1998) Sensory nerves affect the recruitment and differentiation of rat periovarian brown adipocytes during cold acclimation. J. Cell Sci. 111, 2587–2594.

    PubMed  CAS  Google Scholar 

  29. Cancello, R., Zingaretti, M. C., Sarzani, R., Ricquier, D., and Cinti, S. (1998) Leptin and UCP1 genes are reciprocally regulated in brown adipose tissue. Endocrinology 139, 4747–4750.

    Article  PubMed  CAS  Google Scholar 

  30. Tonello, C., Giordano, A., Cozzi, V., Cinti, S., Stock, M. J., Carruba, M., and Nisoli, E. (1999) Role of sympathetic activity in controlling the expression of vascular endothelial growth factor in brown fat cells of lean and genetically obese rats. FEBS Lett. 442, 167–172.

    Article  PubMed  CAS  Google Scholar 

  31. Shaeren-Wiemers, N. and Gerfin-Moser, A. (1993) Single protocol to detect transcripts of various types and expression levels in neuronal tissue and cultured cells: in situ hybridization using digoxigenin-labeled cRNA probes. Histochemistry 100, 431–440.

    Article  Google Scholar 

  32. Braissant, O. and Wahli, W. (1998) Simplified in situ hybridization protocol using non-radioactively labeled probes to detect abundant and rare mRNAs on tissue sections. Biochemica 3, 10–16.

    Google Scholar 

  33. Blanchette-Mackie, E. J. and Scow, R. O. (1981) Membrane continuities within cells and intercellular contacts in white adipose tissue of young rats. J. Ultrastruct. Res. 77, 277–294.

    Article  PubMed  CAS  Google Scholar 

  34. Johannessen, J. V., ed. (1978) Instrumentation and techniques, in Electron Microscopy in Human Medicine, vol. 1. McGraw-Hill, London, UK.

    Google Scholar 

  35. Sbarbati, A., Zancanaro, C., Cigolini, M., and Cinti, S. (1987) Brown adipose tissue: a scanning electron microscopy study of tissue and cultured adipocytes. Acta Anat. 128, 84–88.

    Article  PubMed  CAS  Google Scholar 

  36. Riva, A., Congiu, T., and Faa, G. (1993) Application of the OsO4 maceration method to the study of human bioptic material. A procedure avoiding freeze-fracture. Microsc. Res. Tech. 26, 526–527.

    Article  PubMed  CAS  Google Scholar 

  37. Willison, J. H. M. and Rowe, A. T. (1980) Replica, shadowing and freeze-etching techniques, in Practical Methods in Electron Microscopy, vol. 8. (Glauert, A. M., ed.), North-Holland, Amsterdam, The Netherlands.

    Google Scholar 

  38. Cigolini, M., Cinti, S., Brunetti, L., Bosello, O., Osculati, F., and Björntorp P. (1985) Human brown adipose cells in culture. Exp. Cell Res. 159, 261–266.

    Article  PubMed  CAS  Google Scholar 

  39. Cinti, S., Cigolini, M., Sbarbati, A., and Zancanaro, C. (1986) Ultrastructure of brown adipocytes mitochondria in cell culture from explants. J. Submicrosc. Cytol. 18, 625–627.

    PubMed  CAS  Google Scholar 

  40. Cinti, S., Enzi, G., Cigolini, M., and Bosello, O. (1983) Ultrastructural features of cultured mature adipocytes precursors from adipose tissue in multiple symmetric lipomatosis. Ultrastruct. Pathol. 5, 145–152.

    Article  PubMed  CAS  Google Scholar 

  41. Cinti, S., Cigolini, M., Sbarbati, A., Zancanaro, C., and Björntorp, P. (1987) Effects of noradrenaline exposure on rat brown adipocytes in cultures. An ultra-structural study. Tissue Cell 19, 809–816.

    Article  PubMed  CAS  Google Scholar 

  42. Cinti, S., Eberbach, S., Castellucci, M., and Accili, D. (1998) Lack of insulin receptors affects the formation of white adipose tissue in mice. A morphometric and ultrastructural analysis. Diabetologia 41, 171–177.

    Article  PubMed  CAS  Google Scholar 

  43. Hirsh, J. and Gallian E. (1968) Methods for the determination of adipose cell size in man and animals. J. Lipid Res. 9, 110–119.

    Google Scholar 

  44. Morroni, M., Barbatelli, G., Zingaretti, M. C., and Cinti, S. (1995) Immunohistochemical, ultrastructural and morphometric evidence for brown adipose tissue recruitment due to cold acclimation in old rats. Int. J. Obesity 19, 126–131.

    CAS  Google Scholar 

  45. Sbarbati, A., Morroni, M., Zancanaro, C., and Cinti, S. (1991) Rat interscapular brown adipose tissue at different ages: a morphometric study. Int. J. Obesity 15, 581–588.

    CAS  Google Scholar 

  46. Weibel, E. R. (1979) Stereological methods in practical methods for biological morphometry. Vol. 1., Academic Press, London.

    Google Scholar 

  47. Lehr, H. A., Mankoff, D. A., Corwin, D., Santeusanio, G., and Gown, A. M. (1997) Application of Photoshop-based image analysis to quantification of hormone receptor expression in breast cancer. J. Histochem. Cytochem. 45, 1559–1565.

    Article  PubMed  CAS  Google Scholar 

  48. De Matteis, R., Ricquier, D., and Cinti, S. (1998) TH-, NPY-, SP-, and CGRP-immunoreactive nerves in interscapular brown adipose tissue of adult rats acclimated at different temperatures: an immunohistochemical study. J. Neurocytol. 27, 877–886.

    Article  PubMed  Google Scholar 

  49. Loncar, D., Afzelius, B. A., and Cannon, B. (1988) Epididymal white adipose tissue after cold stress in rats. II Mitochondrial changes. J. Ultrastruct. Mol. Struct. Res. 101, 109–122.

    Article  PubMed  CAS  Google Scholar 

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Cinti, S., Zingaretti, M.C., Cancello, R., Ceresi, E., Ferrara, P. (2001). Morphologic Techniques for the Study of Brown Adipose Tissue and White Adipose Tissue. In: Ailhaud, G. (eds) Adipose Tissue Protocols. Methods in Molecular Biology™, vol 155. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-231-7:021

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  • DOI: https://doi.org/10.1385/1-59259-231-7:021

  • Publisher Name: Springer, Totowa, NJ

  • Print ISBN: 978-0-89603-747-2

  • Online ISBN: 978-1-59259-231-9

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